Latest Articles Include:
- What's wrong with correlative experiments?
- Nat Cell Biol 13(9):1011 (2011)
Article preview View full access options Nature Cell Biology | Comment What's wrong with correlative experiments? * Marco Vilela1 * Gaudenz Danuser1 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Page:1011Year published:(2011)DOI:doi:10.1038/ncb2325Published online02 September 2011 Here, we make a case for multivariate measurements in cell biology with minimal perturbation. We discuss how correlative data can identify cause-effect relationships in cellular pathways with potentially greater accuracy than conventional perturbation studies. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Marco Vilela and Gaudenz Danuser are in the Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA Corresponding authors Correspondence to: * Marco Vilela or * Gaudenz Danuser Author Details * Marco Vilela Contact Marco Vilela Search for this author in: * NPG journals * PubMed * Google Scholar * Gaudenz Danuser Contact Gaudenz Danuser Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Visualizing branched actin filaments in lamellipodia by electron tomography
- Nat Cell Biol 13(9):1012-1013 (2011)
Article preview View full access options Nature Cell Biology | Correspondence Visualizing branched actin filaments in lamellipodia by electron tomography * Changsong Yang1 * Tatyana Svitkina1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1012–1013Year published:(2011)DOI:doi:10.1038/ncb2321Published online02 September 2011 To the editor: Urban et al.1 recently challenged the dendritic nucleation model of lamellipodia protrusion based on their inability to detect branched actin filaments in lamellipodia by electron tomography. We have carefully analysed the primary data that was provided as Supplementary Information by Urban et al., and report that there are numerous branches that have been overlooked by the authors. Lamellipodia are thin veil-like protrusions of the cell edge that have important roles in cell migration. Their advance is driven by the polymerization of actin filaments. The exact mechanisms of lamellipodia protrusion are still debated, owing in part to controversy regarding the structural organization of the actin filaments in lamellipodia. Initial structural data obtained by Small et al.2 using negative-staining electron microscopy suggested that lamellipodia contained a network of long diagonally oriented actin filaments. Based on this structure, the authors proposed a treadmilling model of lamellipodia protrusion stating that actin filaments continuously elongate at their distal barbed (plus) ends, thus pushing the membrane, and continuously depolymerize from the proximal pointed (minus) ends, thus maintaining actin turnover. A large body of structural, biochemical, kinetic and functional data accumulated over the subsequent three decades has led to a revised model of lamellipodia protrusion, termed the dendritic nucleation model3. A key additional point of this model is that the actin filaments in lamellipodia are constantly nucleated by the Arp2/3 complex as branches on pre-existing filaments. However, despite the compelling evidence, Small and colleagues have questioned the dendritic nucleation model in a recent paper1. In their study, they challenge key evidence supporting the dendritic nucleation model; namely, visualization of branched actin filaments in lamellipodia by platinum-replica electron microscopy4, 5. Urban et al. argue1 that the branched configuration of the actin filaments in these samples are an artefact of critical-point drying, a part of the sample preparation. To prove their hypothesis that the actin filaments in lamellipodia are long and unbranched, they analysed the struct! ure of lamellipodia by cryo-electron microscopy, and did not detect branched actin filaments in lamellipodia1. Because the data from platinum-replica electron microscopy that demonstrate branched actin filaments in lamellipodia have been reported primarily by our group, and because we have received multiple requests from the scientific community to comment on the results of Urban et al., we would like to share our opinion with a broader audience through this commentary. Several points, including comparison of potential artefacts produced by the two electron microscopy techniques and evaluation of evidence for and against the dendritic nucleation model, have already been addressed6, 7. However, one critical point has not been discussed; namely, the fundamental consistency between our results and those of Urban et al. in spite of the different interpretations. We have carefully analysed the primary data provided as Supplementary Information by Urban et al. Based on this analysis, we argue that a subset of their results provides strong supporting evidence for the dendritic nucleation model by showing branched actin filaments in lamellipodia. We have found that Supplementary Movie S6, showing an electron tomogram of the lamellipodium in a 3T3 cell, is of particularly good quality to illustrate this point. Several examples of branched actin filaments from this movie are shown in Fig. 1a as a montage of z planes. In these examples, one of two actin filaments never re-emerges on the other side of the second filament in adjacent movie frames, despite the fact that both filaments are in the same z plane in at least one frame of the series. These features demonstrate that the end of the former filament makes a contact with the side of the latter filament, but does not cross it, which is the definition of a branch. In the zoomed region of ! the movie that shows a cell area of ~0.53 μm2, we have found a total of 147 branches (Fig. 1b), which is in contrast to the authors' statement that "branches at the sides of actin filaments were extremely rare". Branched actin filaments can be also found in other Supplementary movies, but fewer branches can be clearly detected in these movies owing to their insufficient quality and selection of regions corresponding to filopodia or filopodial precursors, which contain long unbranched filaments. Figure 1: Branched actin filaments in the lamellipodium of 3T3 fibroblast. The figure is adapted from Supplementary Movie S6 by Urban et al.1 that shows a scan through an electron tomogram generated from a two-axis tilt series of electron microscopy images. () Examples of individual branches (numbered at left). For each branch, a montage of adjacent z planes in the tomogram is shown. Some frames contain two branches (numbered 4,5 and 6, 7). Note a distinct blob at each branch. () Single plane of the full-field tomogram with box indicating the zoomed region shown in , which we used for identification of branches. () Positions of all detected actin branches marked by Y symbols are shown on the background of the maximum projection image of the region of the tomogram shown in the boxed region in . Numbered Y symbols in dark blue indicate positions of branches shown in with corresponding numbers. Scale bars are 100 nm, as deduced from Supplementary Fig. S4 by Urban et al. showing a fragment of this cell. * Full size image (738 KB) Remarkably, all branches identified in our analysis invariably contained a blob at the branch point that probably corresponds to the Arp2/3 complex. Furthermore, the angle between branched filaments was 70 ± 7 degrees (n = 57), consistent with the conventional angle of ~70° produced by the Arp2/3 complex in vitro8. The actin filament branches found in data from the Urban et al. paper are virtually indistinguishable by appearance from those obtained by Hanein using cryo-electron microscopy of actin branches reconstituted in vitro from the Arp2/3 complex and actin, and could even be fitted into the three-dimensional branch model developed in these studies9. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Department of Biology, University of Pennsylvania, 415 South University avenue, Philadelphia, Pennsylvania 19104, USA * Changsong Yang & * Tatyana Svitkina Contributions C.Y. and T.S. analysed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tatyana Svitkina Author Details * Changsong Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Tatyana Svitkina Contact Tatyana Svitkina Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Reply: Visualizing branched actin filaments in lamellipodia by electron tomography
- Nat Cell Biol 13(9):1013-1014 (2011)
Article preview View full access options Nature Cell Biology | Correspondence Reply: Visualizing branched actin filaments in lamellipodia by electron tomography * J. Victor Small1 * Christoph Winkler2 * Marlene Vinzenz1 * Christian Schmeiser2, 3 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1013–1014Year published:(2011)DOI:doi:10.1038/ncb2322Published online02 September 2011 : Cell migration is initiated by the extension of cytoplasmic sheets, termed lamellipodia. In a currently accepted model of lamellipodial dynamics, the cell front is pushed by short and stiff actin filaments branching from the sides of other filaments in a dendritic array1. The model is based on the finding that the Arp2/3 complex induces actin branching in vitro and the observation of high frequency branching of actin filaments in anterior regions of lamellipodia cytoskeletons prepared for conventional transmission electron microscopy by the critical-point drying/platinum-replica procedure2. Using electron tomography, we recently presented the first three-dimensional images of lamellipodia3 in cells in vitreous ice and in fixed cells contrasted by the negative-staining method. Focusing attention on the front of lamellipodia, we reported that putative branch points were seldom when compared with filament overlaps, and attributed the reported high concentration of branch points! , within 20–50 nm of each other at the front of keratocyte lamellipodia2, to the difficulty of distinguishing incidences of branching from filament overlaps in images obtained by the platinum-replica method. In the accompanying letter, Yang and Svitkina report a re-analysis of one of our published tomograms of a negatively stained lamellipodium of a 3T3 cell spreading on poly-L-lysine, and note the presence of actin branches similar in morphology to those described in negatively stained preparations of actin branches formed in vitro by the Arp2/3 complex4. In light of their analysis and our ongoing studies we agree with them that actin branches are present in the lamellipodium network, but at a very different frequency and distribution from that envisioned in the current dendritic model1. Our original analysis of the cited tomogram (Supplementary Movie S6; ref. 3) was restricted to a relatively small anterior region of the lamellipodium and involved the manual tracking of filaments back from the front edge. Our observation that putative branch junctions are rare relative to filament overlaps is correct; what we missed however was the low-density (relative to filament density) spread of branch junctions throughout the lamellipodium network. We have now re-analysed the entire tomogram (total area 1.6 μm2) and identified 225 branch junctions (yellow points, Fig. 1) with the filaments subtending an average angle of 71.6 ± 7° and with extra material at the junction resembling a triangular foot (Fig.1, inset), as seen in vitro4. In our re-analysis of the high-resolution tomogram we have tracked more filaments manually, and in Fig. 1 highlight three interconnected sets of filaments (in white) to illustrate the large variation in both branch junction separation al! ong single filaments and the distance from branch junctions to filament plus ends at the lamellipodium front (up to 670nm in this set). Our data indicate that branching is important for generating a network, but confirm3 that pushing is not dependent on short filaments bearing on branch points, as previously suggested1. As we have demonstrated, actin filaments in lamellipodia can be extremely long3, up to several microns, and if each filament were initiated from a branch, then the branches would be spread relatively widely throughout the network, as also suggested by Insall5. This is in fact what is observed in Fig. 1. Whether or not all filaments in lamellipodia originate from a branch junction remains to be established. As we have shown3, lamellipodia also contain varied numbers of filaments in parallel pairs that serve as filopodia precursors and whose origin remains to be defined. Figure 1: Projection of three-dimensional model of the published tomogram3 of a lamellipodium of a 3T3 cell spread on poly-L-lysine. Green and white lines mark subsets of actin filaments that were tracked manually through the tomogram (total, 203 filaments). Red points mark filament plus ends (total, 208). Yellow points mark positions of branch junctions (total, 225). Interconnected filament subsets highlighted in white illustrate the variable spacing between branch points along single filaments and between branch points and the lamellipodium tip. Area boxed in blue outlines region in which filaments were tracked in the original publication3. Inset; typical branch junction in a z section of the tomogram. Scale bar, 200 nm; inset, 50 nm. * Full size image (574 KB) Although an important detail was overlooked in our recent study, new standards have been set for visualizing actin filament organization in lamellipodia3. The resolution of our tomograms allows the visualization of the helical substructure of actin filaments in lamellipodia networks and, as confirmed by Yang and Svitkina, the direct morphological identification of branch junctions. The superior resolution achievable by negative-stain electron tomography likewise facilitates the tracking of actin filaments in three-dimensional space. We have already shown the feasibility of combining live-cell imaging with negative-stain electron tomography3 and have controlled our preparative methods using cryo-electron microscopy. Accordingly, we are pursuing this approach with a higher yield of tomograms to shed new light on the roles of different proteins in generating, maintaining, reorganizing and disassembling lamellipodial networks. Included are efforts to develop automatic tracking p! rotocols and image analysis of actin polarity6 and branch conformation in situ. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr Bohr Gasse 3–7, Vienna 1030, Austria * J. Victor Small & * Marlene Vinzenz * RICAM, Austrian Academy of Sciences Vienna, Altenbergstrasse 69, Linz 4040, Austria * Christoph Winkler & * Christian Schmeiser * Faculty of Mathematics, University of Vienna, Nordbergstrasse 15, Vienna 1090, Austria * Christian Schmeiser Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * J. Victor Small Author Details * J. Victor Small Contact J. Victor Small Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph Winkler Search for this author in: * NPG journals * PubMed * Google Scholar * Marlene Vinzenz Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Schmeiser Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The AMPK signalling pathway coordinates cell growth, autophagy and metabolism
- Nat Cell Biol 13(9):1016-1023 (2011)
Nature Cell Biology | Review The AMPK signalling pathway coordinates cell growth, autophagy and metabolism * Maria M. Mihaylova1 * Reuben J. Shaw1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1016–1023Year published:(2011)DOI:doi:10.1038/ncb2329Published online02 September 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg One of the central regulators of cellular and organismal metabolism in eukaryotes is AMP-activated protein kinase (AMPK), which is activated when intracellular ATP production decreases. AMPK has critical roles in regulating growth and reprogramming metabolism, and has recently been connected to cellular processes such as autophagy and cell polarity. Here we review a number of recent breakthroughs in the mechanistic understanding of AMPK function, focusing on a number of newly identified downstream effectors of AMPK. View full text Figures at a glance * Figure 1: The AMPK signalling pathway. AMPK is activated when AMP and ADP levels in the cells rise owing to a variety of physiological stresses, or the presence of pharmacological inducers. LKB1 is the upstream kinase activating AMPK in response to AMP or ADP increase, whereas CAMKK2 activates AMPK in response to calcium increase. Activated AMPK directly phosphorylates a number of substrates to acutely affect metabolism and growth, as well as long-term metabolic reprogramming. Shown are the best-established substrates to date; those needing further in vivo validation are italicized. Substrates in red have been reported to serve as substrates of other AMPK family members (SIK1, SIK2, MARKs, SADs) in vivo in addition to being substrates of AMPK. Asterisks denote substrates that may be indirectly regulated. * Figure 2: The Ras/PI3K/mTOR pathways intersect the LKB1/AMPK pathway at multiple points. The gene encoding LKB1, the upstream kinase for AMPK, is the tumour-suppressor gene mutated in Peutz–Jeghers syndrome, as well a significant fraction of sporadic lung cancers and cervical cancers. Peutz–Jeghers syndrome patients share a number of clinical features with patients inheriting defective PTEN or TSC tumour suppressors, perhaps owing to the control of common biochemical pathways by these suppressors and LKB1, of which the mTORC1 pathway is currently the best understood. Extensive cross-regulation of the LKB1/AMPK pathway by the oncogenic Ras and PI3K pathways has been discovered, which may explain how these commonly mutated oncogenes circumvent this endogenous tumour suppressor pathway. The ULK1 kinase complex has emerged recently as a central node receiving inputs from both AMPK and mTORC1. A number of kinases that can phosphorylate specific residues in LKB1 or AMPK have been identified (upper inset), although the context in which most of these regulatory even! ts occur is poorly defined at present, as is the functional impact of these phosphorylation events on AMPK signalling. The BHD tumour suppressor and its partner FNIP1, as well as the sestrin family of proteins, have also been implicated as being upstream or downstream of AMPK and mTOR depending on the context (bottom inset). Inhibitory phosphorylation events shown in yellow, activating phosphorylations shown in red. * Figure 3: AMPK acts as a mitochondrial 'Cash for Clunkers' salvage mechanism. Activated AMPK acutely triggers the destruction of existing defective mitochondria through ULK1-dependent mitophagy and simultaneously promotes the biogenesis of new mitochondria through effects on PGC-1α-dependent transcription. These dual processes controlled by AMPK have the net effect of replacing existing defective mitochondria with new functional mitochondria. This two-pronged control of mitochondria homeostasis by AMPK is relevant in a number of physiological and pathological conditions, and several such cases are illustrated here. * Figure 4: AMPK controls transcription. AMPK regulates several physiological processes through phosphorylation of transcription factors and co-activators. It shares substrates with its AMPK-family-related kinases to negatively regulate gluconeogenesis in the liver by phosphorylation and inhibition of CRCT2 and class IIa HDACs. These phosphorylation events induce binding of CRCT2 and HDACs to 14-3-3 scaffold proteins and sequestration of these transcription regulators into the cytoplasm. AMPK also regulates transcription factors through induction of their degradation (for example Cry1), preventing their proteolytic activation and translocation to nucleus (for example, Srebp1), and by disrupting protein–protein (for example, p300) or protein–DNA interactions (for example, AREBP and HNF4α). AMPK has also been shown to directly control phosphorylation of histone 2B on Ser 36 as well as indirectly controlling SIRT1 activity by increasing NAD+ levels. Author information * Abstract * Author information Affiliations * Maria M. Mihaylova and Reuben J. Shaw are in the Howard Hughes Medical Institute, Molecular & Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Reuben J. Shaw Author Details * Maria M. Mihaylova Search for this author in: * NPG journals * PubMed * Google Scholar * Reuben J. Shaw Contact Reuben J. Shaw Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Wnt: What's Needed To maintain pluripotency?
- Nat Cell Biol 13(9):1024-1026 (2011)
Article preview View full access options Nature Cell Biology | News and Views Wnt: What's Needed To maintain pluripotency? * Hitoshi Niwa1Journal name:Nature Cell BiologyVolume: 13,Pages:1024–1026Year published:(2011)DOI:doi:10.1038/ncb2333Published online02 September 2011 A precise role for the canonical Wnt pathway in maintaining pluripotency in mouse embryonic stem cells (mESCs) has been debated. Four recent reports add pieces to the puzzle and together these results may help establish a robust model. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Hitoshi Niwa is in the Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 6500047, Japan Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Hitoshi Niwa Author Details * Hitoshi Niwa Contact Hitoshi Niwa Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Coupling mitochondrial and cell division
- Nat Cell Biol 13(9):1026-1027 (2011)
Article preview View full access options Nature Cell Biology | News and Views Coupling mitochondrial and cell division * Koji Yamano1 * Richard J. Youle1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1026–1027Year published:(2011)DOI:doi:10.1038/ncb2334Published online02 September 2011 The mitochondrial network fragments during mitosis to allow proper segregation of the organelles between daughter cells. Two mitotic kinases, the cyclin B–CDK1 complex and Aurora A, are now shown to cooperate with the small G protein RALA and its effector RALBP1 to promote DRP1 phosphorylation and mitochondrial fission. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Koji Yamano and Richard J. Youle are in the Biochemistry Section, National Institute for Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Richard J. Youle Author Details * Koji Yamano Search for this author in: * NPG journals * PubMed * Google Scholar * Richard J. Youle Contact Richard J. Youle Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements
- Nat Cell Biol 13(9):1029-1039 (2011)
Nature Cell Biology | Article The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements * Constance Richter1 * Katarzyna Oktaba2 * Jonas Steinmann1 * Jürg Müller2 * Juergen A. Knoblich1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1029–1039Year published:(2011)DOI:doi:10.1038/ncb2306Received20 December 2010Accepted24 June 2011Published online21 August 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In Drosophila, defects in asymmetric cell division often result in the formation of stem-cell-derived tumours. Here, we show that very similar terminal brain tumour phenotypes arise through a fundamentally different mechanism. We demonstrate that brain tumours in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by derepression of target genes in the Salvador–Warts–Hippo (SWH) pathway. We use ChIP-sequencing to identify L(3)mbt binding sites and show that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting expression of the insulator protein gene mod(mdg4) results in upregulation of SWH pathway reporters. As l(3)mbt tumours are rescued by mutations in bantam or yorkie or by overexpression of Expanded, the deregulation of SWH pathway target genes is an essential step in brain tumour formation. Therefore, very different primary defects result in the formation of brain tumours, which behave quite si! milarly in their advanced stages. View full text Figures at a glance * Figure 1: l(3)mbt is necessary to prevent tumorous overproliferation of the larval central nervous system. () Brains of control and l(3)mbt76/Df(3R)D605 larvae raised at 32 °C stained for Deadpan (anterior). Scale bars, 50 μm. () Central brain (CB) neuroblasts of control and l(3)mbt76 mutant larvae stained for Miranda and PH3 (for overview, see Supplementary Fig. S1b). Scale bars, 20 μm. () Brains of control and l(3)mbtts1 mutant larvae expressing GAL41407>CD8–GFP and stained for Deadpan and E-cadherin. Top, surface views (anterior side) with optic lobe neuroepithelia (NE) outlined. Middle, cross-sections through brain; optic lobe tissues are outlined (neuroepithelia of inner optic anlagen (IOA), neuroepithelia of outer optic anlagen (OOA) and optic lobe neuroblasts (OL NBs)). The white rectangles outline the areas shown in the close-up views (bottom). The white arrow points to folding of an inner optic anlage. Scale bars, 50 μm (top and middle) and 20 μm (bottom). () Schematic illustration of mid–late third instar larval brain (lateral view). Only outer optic ! anlagen are shown, which consist of neuroblasts (NB, dark blue), neuroepithelial cells (red) and lamina cells (La, grey). Central brain and ventral nerve cord (VNC) neuroblasts are depicted as larger circles (light blue) and central brain neurons are shown in green. () Schematic illustration of neurogenesis in outer optic anlagen. Neuroepithelial cells (red) give rise to medulla neuroblasts (blue) and lamina cells (grey). Neuroblasts give rise to medulla neurons (green). () Quantification of optic lobe neuroepithelial volume for genotypes: l(3)mbt76/TM3 (N=16), l(3)mbt76 (N=5) and Df(3R)D605/l(3)mbt76 (N=4). N, number of brain hemispheres quantified. Error bars, s.e.m. () First and second instar stage optic lobes of control and l(3)mbt76 mutant brains stained for PH3 (left panel, L1) and actin. Late L2 inner and outer optic anlagen are clearly detectable in control brains, whereas they are indistinguishable in l(3)mbt76 mutants. Scale bars, 20 μm. () Outer optic anlagen ! (top and middle) and inner optic anlagen (bottom) of third ins! tar brains expressing GAL4C855a>l(3)mbtshmiR>CD8–GFP at 29 °C (top and bottom left) and 32 °C (middle and bottom right; see Methods) stained for Miranda. Scale bars, 50 μm. * Figure 2: Candidate screen reveals a function for the SWH pathway in optic lobe proliferation. () Optic lobes of control and l(3)mbtts1 mutant brains expressing GAL41407>CD8–GFP and stained for aPKC and Cnn (same channel, surface and cross-section views). Note the mitotic GAL41407>CD8–GFP-positive cell within the mutant epithelium (white arrow). Scale bars, 20 μm. () Optic lobe epithelium in cross-section view of control and l(3)mbt76 mutant brains stained for actin. Scale bars, 20 μm. () Larval brains expressing a dominant-active (DA) form of Egfr (λ-Top) show strong overproliferation of inner optic anlagen (IOA) neuroepithelia, whereas a dominant-negative (DN) form of Bsk has no phenotype. Brains express GAL4C855a and are stained for actin, Miranda and Prospero. Scale bars, 50 μm. () Larval brains expressing a dominant-active form of JAK (HopTum) show overproliferation of outer optic anlagen (OOA, outline), whereas a dominant-active form of FGF receptor λ-Htl-H3 has no phenotype. All brains express GAL4C855a and are stained for actin, Miranda and Pros! pero. Scale bars, 50 μm. () Larval brains expressing Hpo and anti-apoptotic P35 permanently activate the SWH pathway and show underproliferation of optic lobe neuroepithelia (NE, outlined). All brains express GAL4C855a and are stained for actin, Miranda and Prospero. Scale bars, 50 μm. () Larval brains expressing dominant-active (DA) YkiS168A–RFP show strong overproliferation of optic lobe neuroepithelia (outlined) but no overproliferation of neuroblasts (NB). All brains express GAL4C855a and are stained for actin and Miranda. Scale bars, 50 μm. () Quantification of optic lobe neuroepithelial volume of control (N=11) and YkiS168A–RFP-expressing (N=4) brains. N, number of brain hemispheres quantified. Error bars, s.e.m. () Larval brains expressing ban miRNA show overproliferation of optic lobe neuroepithelia (outlined, arrows). All brains express GAL4C855a and are stained for actin and Miranda. Scale bars, 50 μm. () Schematic illustration of the SWH pathway. * Figure 3: SWH target genes are misregulated in l(3)mbt mutants. () Simulated time course of optic lobe development from second to third larval instar stage in wild-type brains expressing diap1–GFP4.3 (outlined) and stained for Miranda and actin. Note the changing expression pattern of diap1–GFP4.3 in optic lobes. Optic lobes consist of neuroblasts (NB), neuroepithelial cells (NE) and lamina cells (La). Scale bars, 50 μm. () Top, diap1–GFP4.3 expressed in control and l(3)mbt76 mutant brains stained for Miranda (optic lobe neuroepithelia are outlined). Bottom, diap1–GFP4.3 expressed in control and l(3)mbt76 mutant wing discs stained for DAPI (maximum intensity projections). Each genotype contains one copy of diap1–GFP4.3. Scale bars, 50 μm. () Top, ex–LacZ expressed in control and l(3)mbtE2/Df mutant brains stained for LacZ (optic lobe neuroepithelia are outlined). Bottom, ex–LacZ expressed in control and l(3)mbtE2/76 mutant wing discs stained for LacZ (maximum intensity projections). Each genotype contains one copy of ! ex–LacZ. Scale bars, 50 μm. () ban sensor–GFP expressed in control and l(3)mbt76 mutant wing discs. Note that the level of ban sensor–GFP is decreased in l(3)mbt76 mutants, reflecting an increase in ban activity. Each genotype contains one copy of ban sensor–GFP. Scale bars, 50 μm. () Wing disc expressing GAL4en>l(3)mbtshmiR and diap1–GFP4.3 and stained for L(3)mbt (maximum intensity projection). Scale bars, 50 μm. () Wing disc expressing GAL4en>l(3)mbtshmiR and ban sensor–GFP and stained for L(3)mbt (maximum intensity projection). Scale bars, 50 μm. () Wing disc expressing GAL4en>l(3)mbtshmiR and ex–LacZ and stained for L(3)mbt and LacZ (maximum intensity projection). Scale bars, 50 μm. * Figure 4: The SWH pathway shows genetic interaction with l(3)mbt. () Genetic interaction of ban1 and l(3)mbt76: ban1/+, l(3)mbt76 mutant brain and ban1, l(3)mbt76 double-mutant brain stained for actin, Miranda and Prospero. Double mutants show the ban1 phenotype (see also Supplementary Fig. S4a). Scale bars, 50 μm. () Quantification of optic lobe neuroepithelial (NE) volume of ban1, l(3)mbt76/TM6 (control) (N=5), ban1/+, l(3)mbt76 (N=5) and ban1, l(3)mbt76 (N=6) brains. N, number of brain hemispheres quantified. Error bars, s.e.m. () Genetic interaction of Ex and l(3)mbt76: GAL4C855a>Ex, l(3)mbt76/+ control brains, Ex, l(3)mbt76 mutant brains and GAL4C855a>Ex, l(3)mbt76 rescued brains stained for actin, Deadpan and Prospero. Scale bars, 50 μm. () Quantification of optic lobe neuroepithelial volume of GAL4C855a>Ex, l(3)mbt76/+ control brains (N=5), Ex, l(3)mbt76 mutant brains (N=6) and GAL4C855a>Ex, l(3)mbt76 rescued brains (N=5). N, number of brain hemispheres quantified. Error bars, s.e.m. () Genetic interaction of l(3)mbtE2 and yki! B5: l(3)mbtE2 single-mutant brain and ykiB5/+; l(3)mbtE2 double-mutant brain stained for Miranda, E-cadherin and Prospero. Halving Yki levels in double mutants suppressed the l(3)mbtE2 phenotype (see also Supplementary Fig. S4b–d for further double-mutant brains as well as quantification). Scale bars, 50 μm. * Figure 5: L(3)mbt localizes to the nucleus. () Larval brain (anterior) stained for L(3)mbt and Miranda. Overview and close-up of central brain neuroblasts (CB NBs, outlined) in inter- and anaphase are shown. Note dispersion of L(3)mbt in mitotic neuroblasts. Scale bars, 50 μm in overview and 10 μm in close-up. NE, neuroepithelium, outlined in overview. () Stills of time-lapse imaging of mitotic central brain neuroblasts expressing GAL41407>CD8–GFP and RFP–L(3)mbt. Note that the level of RFP–L(3)mbt is decreased and dispersed in the cytoplasm during mitosis. Arrow points to RFP–L(3)mbt dots in interphase nuclei. Scale bars, 10 μm. () Close-up view of polytene chromosome expressing GAL41407>GFP–L(3)mbt and stained for DAPI. Arrowheads point to DAPI-rich bands at which GFP–L(3)mbt is absent. Scale bars, 1 μm. (See also Supplementary Fig. S5.) * Figure 6: L(3)mbt binds at the TSS and regulates SWH target genes and JAK–STAT pathway activity. () ChIP analysis at the diap1, CycE, Ubx and control loci in wild-type larval tissues for dSfmbt and L(3)mbt. ChIP signals at Polycomb response elements (green blocks) and other regions are presented as the percentage of input chromatin (distances from TSS are indicated in kilobases). Error bars, s.d. of three ChIP experiments. () diap1–GFP5.1 (containing TSS1 of diap1) expressed in control and l(3)mbt76 mutant brains stained for Miranda (top, single plane; bottom, maximum intensity projections). Scale bars, 50 μm. () Histogram of L(3)mbt binding frequency relative to the closest TSS. () KEGG pathway analysis of L(3)mbt target genes (3% FDR). Purple bars, percentage of L(3)mbt target genes in a particular KEGG pathway. Grey bars, percentage expected on the basis of all annotated genes. () 10xStat92E–GFP expressed in wing discs (top, maximum intensity projections) and brains (bottom, single planes) of l(3)mbt76/+ control and l(3)mbt76 mutant larvae stained for Miranda ! (bottom alone). Scale bars, 50 μm. () ChIP-seq tracks for L(3)mbt (purple) and ChIP–chip tracks for the PcG proteins Ph, Pho and dSfmbt (grey) at the diap1 and ban loci. Purple and grey blocks represent bound regions (L(3)mbt-bound regions at 0.5% and 3% FDR). Green lines indicate L(3)mbt low-occupancy peak at −23 kb, and Hth and Yki binding site at −14 kb (ref. 32) relative to the ban miRNA. (BDGP Release 5 (UCSC dm3) assembly.) () Overlap of genes specifically deregulated in l(3)mbt mutants36 (also called MBTS genes) with genes bound within ±2 kb and ±5 kb by L(3)mbt. The number of L(3)mbt-bound genes versus the total number of genes in each category as well as corresponding percentages are shown. () ChIP-seq tracks for L(3)mbt (purple) at germline genes vas, bam and bgcn. Rectangles outline L(3)mbt binding sites at the TSS. Purple blocks indicate L(3)mbt-bound regions at 0.5% and 3% FDR. * Figure 7: L(3)mbt binds at insulator-bound regulatory domains and influences Abd-B expression () De-novo-identified sequence motifs significantly enriched in L(3)mbt-bound regions (0.5% FDR) correspond to motifs for the insulator-associated proteins CP190, BEAF-32, CTCF and Su(Hw) (refs 51, 52). Discovered motifs are depicted as sequence logos generated from a PWM. Histograms show motif enrichment in L(3)mbt-bound regions, computed as the ratio of the PWM match frequency in the data set (purple) and the background (grey) frequency (*** P<0.001, Fisher's exact test). () Venn diagrams showing the overlap of regions bound by L(3)mbt (0.5% FDR, purple) and regions bound by insulator-associated proteins (grey) CP190, BEAF-32 and CTCF (class I insulator proteins), and Su(Hw) (class II insulator protein)51. () ChIP-seq tracks for L(3)mbt (purple) and ChIP–chip tracks for insulator-associated proteins (grey) CP190, BEAF-32, CTCF and Su(Hw) (ref. 51) at the Bithorax complex locus. Purple and grey blocks represent bound regions. For L(3)mbt, bound regions at 0.5% and 3% FD! R are indicated. (BDGP Release 5 (UCSC dm3) assembly; see also Supplementary Fig. S7.) () Micrographs, ventral nerve cord (VNC) of control (Canton S) and l(3)mbt76 mutant brains stained for Deadpan and Abd-B. Scale bars, 50 μm. Graph, quantification of the Abd-B mean fluorescence intensity normalized to Deadpan in control (N=4) and l(3)mbt76 (N=6) animals. Error bars, s.e.m., P=0.041 (one-tailed) (N, number of ventral nerve cords quantified). () Wing disc expressing GAL4en>mod(mdg4)-RNAi and ban sensor–GFP and stained for Mod(mdg4). Scale bars, 50 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Dr. Bohr-Gasse 3, 1030 Vienna, Austria * Constance Richter, * Jonas Steinmann & * Juergen A. Knoblich * European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, 69117 Heidelberg, Germany * Katarzyna Oktaba & * Jürg Müller Contributions C.R. conceived and conducted experiments, coordinated the project and wrote the manuscript. K.O. conducted the bio-informatics analysis and conducted experiments. J.S. wrote the peakfinder software and mapped the Solexa reads. J.M. supervised the project. J.A.K. initiated, designed and supervised the project, conceived experiments and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Juergen A. Knoblich Author Details * Constance Richter Search for this author in: * NPG journals * PubMed * Google Scholar * Katarzyna Oktaba Search for this author in: * NPG journals * PubMed * Google Scholar * Jonas Steinmann Search for this author in: * NPG journals * PubMed * Google Scholar * Jürg Müller Search for this author in: * NPG journals * PubMed * Google Scholar * Juergen A. Knoblich Contact Juergen A. Knoblich Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Table 1 (1M) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information * Supplementary Table 2 (150K) Supplementary Information Additional data - MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis
- Nat Cell Biol 13(9):1040-1050 (2011)
Nature Cell Biology | Article MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis * Catarina P. Samora1, 3 * Binyam Mogessie1, 3 * Leslie Conway2 * Jennifer L. Ross2 * Anne Straube1 * Andrew D. McAinsh1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1040–1050Year published:(2011)DOI:doi:10.1038/ncb2297Received28 March 2011Accepted14 June 2011Published online07 August 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Correct positioning of the mitotic spindle is critical to establish the correct cell-division plane. Spindle positioning involves capture of astral microtubules and generation of pushing/pulling forces at the cell cortex. Here we show that the tau-related protein MAP4 and the microtubule rescue factor CLASP1 are essential for maintaining spindle position and the correct cell-division axis in human cells. We propose that CLASP1 is required to correctly capture astral microtubules, whereas MAP4 prevents engagement of excess dynein motors, thereby protecting the system from force imbalance. Consistent with this, MAP4 physically interacts with dynein–dynactin in vivo and inhibits dynein-mediated microtubule sliding in vitro. Depletion of MAP4, but not CLASP1, causes spindle misorientation in the vertical plane, demonstrating that force generators are under spatial control. These findings have wide biological importance, because spindle positioning is essential during embryogen! esis and stem-cell homeostasis. View full text Figures at a glance * Figure 1: MAP4 is required for correct spindle architecture. () Immunoblots of whole-cell lysates transfected with control or MAP4-4, MAP4-3 or MAP4-1 siRNA and probed with antibodies against MAP4 and α-tubulin. () Immunofluorescence microscopy images of metaphase cells and a magnified single microtubule stained with 4,6-diamidino-2-phenylindole (DAPI; DNA, blue), MAP4 (green) and α-tubulin (microtubules, red) antibodies. The images of metaphase cells correspond to z-projections and the image of the single microtubule is from a single focal plane. () Quantification of MAP4 levels in cells transfected with control and MAP4 siRNA. Levels were determined from three-dimensional reconstructions of cells and calculated relative to α-tubulin, after background correction. () Representative immunofluorescence microscopy images of cells treated with MAP4 siRNA and stained with α-tubulin (red), γ-tubulin (green) and CREST (blue) antisera showing an elliptical cell and a cell with hyper-focused poles. () Percentages of cells with bipolar (bl! ack), monopolar (light grey) or multipolar (red) spindles in cells transfected with control or MAP4-3, MAP4-1 or MAP4-4 siRNA. () Percentages of elliptical- (black) or aberrant- (red) shaped spindles in cells treated with siRNAs as indicated. (,) Distribution of spindle length () and width () in metaphase cells treated with control (black) or MAP4 (red) siRNA. () Distribution of protein levels of α-tubulin in cells transfected with control and MAP4-1 siRNA measured relative to γ-tubulin, after background correction. I–III: representative images of α-tubulin for control siRNA, MAP4 siRNA elliptical and MAP4 siRNA aberrant cells, respectively. () Percentages of cells with elliptical bipolar spindles after complementation of MAP4 siRNA cells with mouse eGFP–MAP4. In all experiments, n=150 cells from three independent experiments. Triangles in histograms represent mean values. Error bars represent s.e.m. Scale bars, 10 μm. Uncropped images of blots are shown in Supple! mentary Fig. S5. * Figure 2: Loss of MAP4 leads to spindle position and orientation defects. () Successive frames from live-cell movies of HeLa cells expressing H2B–eGFP/mRFP– α-tubulin (to mark chromosomes and microtubules respectively) in control siRNA cells (top row) and MAP4 siRNA cells (lower rows). (–) Frequency of xy rotation () xy displacement () and z rotation () in cells transfected with control and MAP4 siRNA. The quantification was derived from live-cell movies (n=100cells per condition). () Different zsections of fixed cells stained for α-tubulin, γ-tubulin and CREST illustrating rotations in the z plane in MAP4-depleted cells. Arrowheads indicate the position of the spindle poles. () Schematic representation of mitotic-spindle angle α measurement in HeLa and hTERT-RPE1 cells. () Statistical box diagrams of mitotic-spindle angle α in control, MAP4 siRNA and mouse eGFP–MAP4-complemented HeLa cells. The box spans from 25 to 75% of the data and the whiskers from 5 to 95% of the data. The means are shown as white squares. () Representative immu! nofluorescence microscopy images of HeLa cells treated with control or MAP4 siRNA and stained for α-tubulin, γ-tubulin and CREST, illustrating spindle displacements during metaphase. Displacement d was calculated as the distance between the centre of the cell (green cross) and the middle of the spindle axis (white circle). () Distribution of spindle position relative to the cell centre in HeLa cells after control and MAP4 siRNA treatment or MAP4 siRNA complementation with mouse eGFP–MAP4. () Immunoblots of whole hTERT-RPE1 cell lysates transfected with control or MAP4 siRNA and probed with antibodies against MAP4 and α-tubulin. () Statistical box diagrams of mitotic-spindle angle α in hTERT-RPE1 cells treated with control and MAP4 siRNA. Diagrams were plotted as explained in . () Representative immunofluorescence microscopy images of hTERT-RPE1 cells treated with control or MAP4 siRNA and stained for α-tubulin, γ-tubulin and CREST, illustrating spindle displacements! during metaphase. () Distribution of spindle position relativ! e to cell centre in hTERT-RPE1 cells after control and MAP4 siRNA treatment. Displacement d was calculated as described in . Triangles in histograms represent mean values. Error bars represent s.d. In all experiments, n=90 cells from three independent experiments (–) and n=50 cells from two independent experiments (–). Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 3: MAP4 depletion increases cortical astral-microtubule and lateral spindle-pole movements. () Image showing lines used for kymograph analyses of astral microtubule (MT) number and dynamics. To assess astral microtubule growth and static dwell, a line (blue) was added to images from the pole to the cortex (dashed yellow line). To assess cortical movement of astral microtubules, a line was drawn across half the cell perimeter (green), and for analyses of pole movement a line was aligned with the pole (red). () Two possible outcomes, catastrophe or movement after transition from end-on to side-on attachment, of an EB3 comet arriving at the cell cortex. () Kymographs of pole (bottom)-to-cortex (top) line scans of control and MAP4-depleted cells. The maximum projection of a 20-pixel-wide blue line (illustrated in ) was plotted at 500 ms time intervals from left to right. Microtubule growth results in diagonal lines and static dwells (white stars) at the cortex in horizontal lines. () Distribution of cortical-movement speeds in cells treated with control and MAP4 siRN! A where poles are near the cortex. Movements of all run lengths were analysed from six cells from three independent experiments for each siRNA. Triangles in histograms represent mean values. () Parameters used in the analyses of astral-microtubule and lateral pole movements. () Relationship between pole-to-cortex distance (p) and frequency of astral-microtubule () or lateral pole () movement in cells treated with control and MAP4 siRNA. All movement frequencies and pole-to-cortex distances were measured from 42–56 cells and seven experiments. () Probability of lateral pole movement in cells treated with MAP4 siRNA for different values of angle σ measured as illustrated in . 218 values of σ were measured for 34 astral-microtubule movement events from seven cells and two experiments. (,) Kymographs of astral-microtubule and lateral pole movements in cells treated with control () and MAP4 () siRNA where the poles are near the cortex. Kymographs were generated by the maximu! m projection of five-pixel-wide green and red lines (illustrat! ed in ) at 500 ms time intervals. Schematic illustrations of movements were constructed by overlaying kymographs with red (poles) and green (astral microtubules) lines. Only astral-microtubule movements greater than 1 μm in length are shown. * Figure 4: MAP4 suppresses dynein-dependent force generation. () Validation of DHC siRNA and efficiency of double siRNA of DHC and MAP4 by immunoblotting. The dynactin subunit p150Glued and α-tubulin were used as loading controls. () Statistical box diagrams (presented as in Fig. 2g) of mitotic-spindle angle α in control, MAP4, DHC and MAP4+DHC depleted cells. Measurements were made as described in Fig. 2f. () Distribution of spindle position relative to cell centre d in HeLa cells after control, DHC, MAP4 and MAP4+DHC siRNA treatment. Displacement was calculated as described in Fig. 2h. Triangles in histograms represent mean values. () Representative images of metaphase cells treated with control and DHC siRNA with no or little spindle displacement. Note the pole-focusing defect in DHC-depleted cells. –, n=90 cells from three independent experiments. Scale bar, 10 μm. () Immunoprecipitation (IP) of dynactin subunit p150Glued with MAP4. Whole-cell lysates were immunoprecipitated with anti-p150Glued and anti-MAP4 antibodies. Two ! different exposures of a western blot are shown. () Immunoprecipitation of MAP4 with dynactin subunit p150Glued. Whole-cell lysates were immunoprecipitated with anti-p150Glued and anti-MAP4 antibodies. Two different exposures of a western blot are shown. n=2 experiments. () SDS–polyacrylamide gel electrophoresis and western blotting analyses of mouse His–MAP4 following protein purification on Ni-NTA (nitrilotriacetic acid) and ion-exchange columns. () Statistical box diagrams (presented as in Fig. 2g) of average speeds measured in dynein gliding assays with microtubules in the presence of varying amounts of His6–MAP4. n=33–76 microtubules from two independent experiments. () Kymograph of a typical dynein-mediated microtubule gliding event without or in the presence of 50 nM His6–MAP4. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 5: Depletion of CLASP1 leads to spindle-positioning defects in the xy plane, but does not affect spindle orientation in z. () Immunoblot of whole-cell extracts from cells transfected with control, MAP4, CLASP1, CLASP2, MAP4+CLASP1, MAP4+CLASP2 and CLASP1+CLASP2 siRNAs and probed with antibodies as indicated. α-tubulin was used as a loading control. () Distribution of spindle position d relative to the cell centre in HeLa cells after control, MAP4, CLASP1, CLASP2, MAP4+CLASP1, MAP4+CLASP2 and CLASP1+CLASP2 siRNA treatment. The displacement was calculated as described in Fig. 2h. Triangles in histograms represent mean values. n=90 cells from three independent experiments. () Representative images of control and CLASP1- and CLASP2-depleted metaphase cells indicating the extent of spindle displacement. () Statistical box diagrams (presented as in Fig. 2g) of mitotic-spindle angle α in cells treated with control, MAP4, CLASP1, CLASP2, MAP4+CLASP1, MAP4+CLASP2 and CLASP1+CLASP2 siRNAs. Measurements were carried out as described in Fig. 2f. n=90 cells from three independent experiments. () Successive! frames from live-cell movies of HeLa cells expressing H2B–eGFP/mRFP– α-tubulin in control siRNA cells (top row), CLASP1 siRNA cells (middle row) and MAP4+CLASP1 siRNA cells (bottom row). (–) Frequency of xy rotation (), xy displacement () and z rotation () in cells transfected with control, MAP4, CLASP1 and MAP4+CLASP1 siRNAs. Control and MAP4 siRNA values were taken from Fig. 2. Quantification was derived from live-cell movies (n=100 cells per condition). Error bars represent s.d. Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 6: CLASP1, but not CLASP2, rescues the spindle mispositioning in CLASP1-depleted cells. () Representative images of cells treated with control or CLASP1 siRNA, transfected with either empty vector, RNA interference-resistant GFP–CLASP1 α or GFP– CLASP2α and stained with anti- γ-tubulin (red). () Quantification of spindle displacement d in CLASP rescue experiments as outlined in . n=60–100 cells from two independent experiments. Triangles in histograms represent mean values. Scale bar, 10 μm. * Figure 7: MAP4- and CLASP1-mediated control of cortical force generators ensures an accurate cell-division axis. () Example of a MAP4-depleted RPE1 H2B–mRFP cell grown on an H pattern. Cells adopt a pseudo-square shape in interphase (cell outline in interphase highlighted with dashed lines) and preferentially align the spindle along the vertical axis (θ=90°). (,) Angular distribution of division axes θ on H patterns () and disc patterns () in the siRNA conditions indicated. Note that distributions for control cells on H patterns are plotted on a different scale. () Model of proposed MAP4 and CLASP function in the context of extracellular cues. FG, force generator; mDia, mammalian diaphanous-related formin. () Schematic representation of proposed functions of MAP4 (green) in limiting accessibility of growing astral microtubules (red arrows) to dynein motors (grey). CLASP1 (brown) functions to capture microtubules and stabilize the end-on state. In the absence of MAP4 or CLASP1, excess motors engage with the microtubule and slide it side-on along the cell cortex. This generates late! ral pulling forces, some of which will result in torque on the spindle pole. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Catarina P. Samora & * Binyam Mogessie Affiliations * Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK * Catarina P. Samora, * Binyam Mogessie, * Anne Straube & * Andrew D. McAinsh * Physics Department, University of Massachusetts, Amherst, Massachusetts 01003, USA * Leslie Conway & * Jennifer L. Ross Contributions This project was co-directed by A.S. and A.D.M. Project conception, planning and data interpretation was carried out by C.P.S., B.M., A.S. and A.D.M. Live- and fixed-cell imaging of spindle geometry, positioning and mitotic progression, as well as co-immunoprecipitation experiments, were carried out and analysed by C.P.S. Live-cell imaging of astral-microtubule dynamics, as well as cloning and purification of MAP4, was carried out and analysed by B.M. Dynein purification and gliding assays were carried out by L.C. and J.L.R. and patterned-substrate experiments by A.S. and A.D.M. The manuscript was prepared by A.S. and A.D.M. with contributions by C.P.S., B.M. and J.L.R. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Anne Straube or * Andrew D. McAinsh Author Details * Catarina P. Samora Search for this author in: * NPG journals * PubMed * Google Scholar * Binyam Mogessie Search for this author in: * NPG journals * PubMed * Google Scholar * Leslie Conway Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer L. Ross Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Straube Contact Anne Straube Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew D. McAinsh Contact Andrew D. McAinsh Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (400K) Supplementary Information * Supplementary Movie 2 (400K) Supplementary Information * Supplementary Movie 3 (300K) Supplementary Information * Supplementary Movie 4 (700K) Supplementary Information * Supplementary Movie 5 (15M) Supplementary Information * Supplementary Movie 6 (13M) Supplementary Information * Supplementary Movie 7 (16M) Supplementary Information * Supplementary Movie 8 (20M) Supplementary Information * Supplementary Movie 9 (15M) Supplementary Information * Supplementary Movie 10 (30M) Supplementary Information * Supplementary Movie 11 (140K) Supplementary Information * Supplementary Movie 12 (150K) Supplementary Information * Supplementary Movie 13 (300K) Supplementary Information * Supplementary Movie 14 (22M) Supplementary Information * Supplementary Movie 15 (17M) Supplementary Information PDF files * Supplementary Information (1600K) Supplementary Information Additional data - In vitro generation of human cells with cancer stem cell properties
- Nat Cell Biol 13(9):1051-1061 (2011)
Nature Cell Biology | Article In vitro generation of human cells with cancer stem cell properties * Paola Scaffidi1 * Tom Misteli1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1051–1061Year published:(2011)DOI:doi:10.1038/ncb2308Received28 June 2010Accepted27 June 2011Published online21 August 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cancer stem cells (CSCs) have been implicated in the maintenance and progression of several types of cancer. The origin and cellular properties of human CSCs are poorly characterized. Here we show that CSC-like cells can be generated in vitro by oncogenic reprogramming of human somatic cells during neoplastic transformation. We find that in vitro transformation confers stem-cell properties to primary differentiated fibroblasts, including the ability to self-renew and to differentiate along multiple lineages. Tumours induced by transformed fibroblasts are hierarchically organized, and the cells that act as CSCs to initiate and maintain tumour growth are marked by the stage-specific embryonic antigen SSEA-1. Heterogeneous lineages of cancer cells in the bulk of the tumour arise through differentiation of SSEA-1+ fibroblasts, and differentiation is associated with loss of tumorigenic potential. These findings establish an experimental system to characterize cellular and molecul! ar properties of human CSCs and demonstrate that somatic cells have the potential to de-differentiate and acquire properties of CSCs. View full text Figures at a glance * Figure 1: Differentiation of in vitro-transformed fibroblasts. () Haematoxylin and eosin (H&E) staining (upper left panel) and immunohistochemical detection of the indicated proteins (brown) in paraffin sections of tumours generated by in vitro-transformed fibroblasts. Cells immunopositive for hCD34 and β III-tubulin are indicated by arrows. Wide areas of myosin-positive cells were detected. Scale bar, 100 μm. () Semiquantitative RT–PCR analysis of expression levels of the indicated germ-layer markers in transformed fibroblasts and four primary tumours induced by two independent transformed fibroblast cell lines (tumours 3 and 4 from cell line Transformed-1 and tumours 1 and 2 from cell line Transformed-2). ACTB was used as a control for the RNA amount. Full scans of the gels, including molecular-weight markers, are shown in Supplementary Fig. S7. (,) In vitro differentiation of Transformed-1 and Transformed-2 cell lines and the corresponding hTERT-immortalized control cells after 5 days of adipogenic differentiation () or 7 day! s of osteogenic differentiation (). Transformed cells were plated at densities of 1×104 cells cm−2 for adipogenesis and 3×103 cells cm−2 for osteogenesis. Scale bar, 100 μm. (,) Quantitative analysis of incorporated Oil Red O (OD, optical density) after elution with isopropanol () or of alkaline phosphatase (ALP) activity by luminescent analysis (). Transf., Transformed; undiff., undifferentiated; diff., differentiated. Values represent means±s.d. from three biological replicates. The statistical significances of the differences between uninduced and induced transformed cells are indicated. * Figure 2: The presence of the stage-specific embryonic antigen SSEA-1 in populations of differentiated cells correlates with acquisition of tumorigenicity on transformation. () Relative abundance of cells immunopositive for the indicated antigens in two independent cell lines of in vitro-transformed fibroblasts when compared with the corresponding hTERT-immortalized control cell lines. The average percentage of positive cells in the two transformed cell lines for each antigen measured by flow cytometric analysis is indicated. The red line indicates a relative abundance of unity and values above the line indicate enrichment of the immunopositive cells in transformed fibroblasts when compared with hTERT-immortalized cells. (,) Immunodetection of SSEA-1 in the Transformed-2 cell line and the corresponding hTERT-immortalized control cell line by flow cytometry () and fluorescence microscopy (). The percentage of SSEA-1+ cells and the significance of the difference between the two populations determined by Kolmogorov–Smirnov test are indicated. The signal intensities and the percentage of SSEA-1+ cells in the hTERT-immortalized cell lines were not ! different (P>0.05) from the background measured in unstained cells or cells stained with an isotype control antibody (Supplementary Fig. S2a). Scale bar, 40 μm. () Flow cytometric analysis of SSEA-1 in primary and in vitro-transformed human mammary epithelial cells (HMECs). The percentage of SSEA-1+ cells is indicated. () Immunohistochemical detection of SSEA-1 in clinical samples from one inflammatory myofibroblastoma (top left panel) and three malignant fibrohistiocytomas. Representative images of varying abundance and distribution of SSEA-1+ cells observed in the tissue microarray are shown. No SSEA-1+ cells were detected in normal control tissues (bottom panels). Scale bar, 150 μm. () Abundance of SSEA-1+ cells in a polyclonal population of primary fibroblasts transformed by serial introduction of transforming factors under conditions of high infection efficiency or in four clones (cl.) obtained by simultaneous introduction of the transforming factors under condit! ions of low infection efficiency. Injection of the clones mark! ed by an asterisk induced tumour formation in mice. * Figure 3: SSEA-1 identifies a biologically distinct subpopulation of transformed fibroblasts. () Abundance of SSEA-1+ cells in populations of sorted SSEA-1+ (top) and SSEA-1− fibroblasts (bottom) over time in culture in a representative time course experiment. About 1% of SSEA-1+ cells were present in the unsorted population. (,) Immunodetection of SSEA-1 in untreated cells () or cells treated with 25 mM blebbistatin for 24 h () by fluorescence microscopy. Pairs of daughter cells were identified as described in Supplementary Fig. S3 and Methods. Scale bars, 20 μm. The percentage of cells undergoing each type of cell division is indicated. () Immunodetection of SSEA-1 in an isolated single SSEA-1−-sorted fibroblast and in its daughter cells generated after 3 weeks of culture by fluorescence microscopy. Scale bar, 80 μm. () Flow cytometric analysis of SSEA-1 in three independent clonal populations resulting from single SSEA-1− fibroblasts after three weeks of culture. Variable percentages of SSEA-1+ cells in each clone suggest that conversion to SSEA-1+! fibroblasts occurs in a stochastic manner. () Heat map representing relative expression levels of genes showing at least a 1.5-fold difference between SSEA-1+ and SSEA-1− fibroblasts with a P value <0.05. Two biological replicates for each subpopulation were analysed. Red and blue represent the highest and lowest values of each gene among all samples, respectively. * Figure 4: SSEA-1+ fibroblasts are responsible for tumour initiation. () Experimental design to track cells responsible for tumour initiation among in vitro-transformed fibroblasts and possible outcomes. A higher percentage of GFP+ cells in the tumours when compared with that in the injected cells indicates greater tumorigenicity of labelled SSEA-1+ cells than competing unlabelled SSEA-1− cells. () Percentages of GFP+ cells in tumours induced by the indicated combinations of unlabelled and labelled cells measured by flow cytometric analysis. Dots represent individual tumours at 6 weeks after injection. Crosses are injections that failed to form tumours. Solid lines indicate median values of GFP+ cells for each group. The statistical significance of the differences between the experimental values and the expected values under the hypothesis of equal tumorigenicities of the injected unlabelled and labelled cells (see Methods) is indicated for each group. () Immunohistochemical detection of GFP (brown) in tumours resulting from injection of the! indicated cells. Pictures of four adjacent fields were combined for each tumour. Scale bar, 150 μm. * Figure 5: SSEA-1+ fibroblasts differentiate into non-fibroblastic cells during tumour growth. (,) Flow cytometric analysis () and immunohistochemical detection (brown) () of SSEA-1 in primary tumours. Human H-2Kd− cells were gated and plotted. Pictures of four adjacent fields were combined. Scale bar, 150 μm. () Immunohistochemical detection (brown) of the indicated proteins in primary tumours resulting from injection of GFP-labelled SSEA-1+ fibroblasts mixed with unlabelled SSEA-1− fibroblasts. Cells immunopositive for hCD34 and β III-tubulin are indicated by arrows. Wide areas of myosin-positive cells were detected. Scale bars, 80 μm (top and middle panels) and 50 μm (bottom panels). () Flow cytometric analysis of CD166 and SSEA-1 in transformed fibroblasts, a primary tumour and fibroblasts isolated from the tumour. PE, phycoerythrin. () Abundance of cells expressing high levels of the indicated antigens measured by flow cytometric analysis in transformed fibroblasts, three representative primary tumours and two representative tumour fibroblast popu! lations. The gate was set on the basis of the values measured in the transformed fibroblasts. () Quantitative RT–PCR measuring expression levels of the mesenchymal markers CDH2 and FGF2 in the indicated samples. Values are normalized to the housekeeping gene PPIA and represent means from two replicates. Statistical significance of the differences when compared with the tumour cells (P<0.001) is indicated by an asterisk. () Abundance of fibroblasts in primary tumours resulting from injection of the indicated number of transformed fibroblasts. The values are relative to the number of viable tumour cells after dissociation and represent the mean±s.d. from three tumours. () Immunodetection of SSEA-1 in tumour fibroblasts isolated from primary tumours by fluorescence microscopy. Scale bar, 40 μm. * Figure 6: Hierarchical organization of tumours induced byin vitro-transformed fibroblasts. () Soft-agar assay using unsorted cells and SSEA-1+ tumour fibroblasts from a primary tumour. Scale bar, 400 mm. Similar results were obtained with cells from five other tumours. (,) Quantitative analysis of soft-agar assay () and non-adherent sphere formation assay () using the indicated cells from a primary tumour resulting from injection of 3,000 transformed fibroblasts. The frequency of clonogenic cells is indicated. Values represent means±s.d. from three replicates. Statistical significance of the differences when compared with the unsorted population is indicated by one (P<0.005) or two (P<0.001) asterisks. () Limiting-dilution transplantation assay into NSG mice using the indicated cells from two primary tumours resulting from injection of 3,000 transformed fibroblasts. The frequency of tumorigenic cells (estimate with upper–lower limits) was calculated by limiting-dilution analysis as described in Methods. The statistical significance of the differences when com! pared with the unsorted population is indicated. () Summary of the differences in gene expression profiles between SSEA-1+ tumour fibroblasts and SSEA-1− cells from three primary tumours. The numbers of genes differentially expressed (≥2-fold) in each tumour and of those significantly different in all tumours (≥2-fold, P<0.05) are plotted. The range of fold differences of gene expression levels between SSEA-1+ tumour fibroblasts and SSEA-1− cells in each tumour is indicated above each bar. () Heat map representing relative expression levels of the subset (70%) of differentially expressed genes in all tumours (SSEA-1+ tumour fibroblasts versus SSEA-1− cells, P<0.05, ≥2-fold) that are expressed at similar levels in SSEA-1+ tumour fibroblasts and transformed fibroblasts (P>0.05, <2-fold). Values from two replicates of transformed fibroblasts and three tumours are represented. Red and blue represent the highest and lowest values of each gene among all samples, respe! ctively. * Figure 7: Self-renewal ability and evolution of SSEA-1+-transformed fibroblasts. () Serial transplantation of SSEA-1+ fibroblasts in BALB/cAnNCr-nu/nu mice. 3×106 cells were injected for all passages. () Percentages of SSEA-1+ cells in the indicated samples measured by flow cytometric analysis. The values represent the mean±s.d. from two to six tumours. The statistical significances of the differences are indicated. () Quantitative analysis of soft-agar assay using unsorted cells from primary, secondary and tertiary tumours. The frequency of clonogenic cells is indicated. The values represent the mean±s.d. from three replicates. The statistical significances of the differences are indicated. * Figure 8: Model for reprogramming of somatic cells to CSCs. Inhibition of tumour-suppressor cellular mechanisms and concomitant oncogenic activation convert somatic differentiated cells into pre-malignant SSEA-1− cells. This cellular intermediate is not tumorigenic. Further, stochastic modifications lead to full reprogramming of somatic cells to SSEA-1+ CSCs, conferring self-renewal and differentiation ability. These cellular properties enable reprogrammed somatic cells to initiate and maintain tumours. During tumour growth, SSEA-1+ CSCs differentiate into phenotypically diverse, non-tumorigenic cancer cells, thereby generating heterogeneous and hierarchically organized tumours. Author information * Abstract * Author information * Supplementary information Affiliations * National Cancer Institute, NIH, Bethesda, Maryland 20892, USA * Paola Scaffidi & * Tom Misteli Contributions P.S. conceived the study and carried out the experimental work. P.S. and T.M. designed the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Paola Scaffidi Author Details * Paola Scaffidi Contact Paola Scaffidi Search for this author in: * NPG journals * PubMed * Google Scholar * Tom Misteli Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Table 1 (38K) Supplementary Information * Supplementary Table 2 (37K) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information * Supplementary Table 3 (360K) Supplementary Information Additional data - miRNA-mediated feedback inhibition of JAK/STAT morphogen signalling establishes a cell fate threshold
- Nat Cell Biol 13(9):1062-1069 (2011)
Nature Cell Biology | Letter miRNA-mediated feedback inhibition of JAK/STAT morphogen signalling establishes a cell fate threshold * Wan Hee Yoon1 * Hans Meinhardt2 * Denise J. Montell1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1062–1069Year published:(2011)DOI:doi:10.1038/ncb2316Received08 September 2010Accepted24 June 2011Published online21 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Patterns of cell fates generated by morphogens are critically important for normal development; however, the mechanisms by which graded morphogen signals are converted into all-or-none cell fate responses are incompletely understood. In the Drosophila ovary, high and sustained levels of the secreted morphogen Unpaired (Upd) specify the migratory border-cell population by activating the signal transducer and activator of transcription1, 2 (STAT). A lower or transient level of STAT activity specifies a non-migratory population of follicle cells3, 4. Here we identify miR-279 as a component of a feedback pathway that further dampens the response in cells with low levels of JAK/STAT activity. miR-279 directly repressed STAT, and loss of miR-279 mimicked STAT gain-of-function or loss of Apontic (Apt), a known feedback inhibitor of STAT. Apt was essential for miR-279 expression in non-migratory follicle cells, whereas another STAT target, Ken and Barbie (Ken), downregulated miR-279! in border cells. Mathematical modelling and simulations of this regulatory circuit including miR-279, Apt and Ken supported key roles for miR-279 and Apt in generating threshold responses to the Upd gradient. View full text Figures at a glance * Figure 1: STAT is a target of miR-279. () Effect of miRNAs on expression of a Renilla luciferase reporter carrying the STAT 3′-UTR in S2 cells. Error bars indicate s.e.m. P values were calculated using an analysis of variance (ANOVA) test. *P<0.05. Effects of miR-284, miR-277, miR-92a and miR-280 were not significantly different from the control (P>0.3). () Top, schematic representation of miR-279 pairing with the STAT 3′-UTR. Lines indicate canonical pairings and double dots indicate non-canonical (G:U) pairings. The seed pairings are underlined. Bottom, schematic representation of STAT 3′-UTR reporter with and without the miR-279-seed-binding site (blue). The yellow rectangle represents the Renilla luciferase coding sequence and the white rectangle represents the 3′-UTR. () Effect of miR-279 on expression of a Renilla luciferase reporter carrying the STAT 3′-UTR with or without the miR-279-seed-binding sequence. Error bars represent s.e.m. Pvalues were calculated using Student's t-test. () Derepress! ion of the STAT 3′-UTR reporter by 2′-O-methyl miR-279 antagomir in S2 cells. Error bars represent s.e.m. P values were calculated using ANOVA. () Effect of miR-279 on expression of a Renilla luciferase reporter carrying the Upd 3′-UTR in S2 cells. The error bar indicates the s.e.m. Relative luciferase activity is the ratio of Renilla luciferase activity to a firefly luciferase control in , , and . (–) Confocal micrographs of egg chambers of indicated stages carrying a mir-279 expression reporter (mir-279–GAL4; UAS–GFP; ref. 17). DAPI (blue) labels nuclei and Armadillo (red) labels membranes enriched in adherens junction proteins. Arrowheads indicate the border-cell cluster, arrows indicate non-migratory anterior epithelial follicle cells and asterisks indicate polar cells. Scale bars, 50 μm (,,,) and 10 μm (,,,). * Figure 2: Loss-of-function of miR-279 phenocopies gain-of-function of STAT. (–) Confocal micrographs of stage-10 egg chambers of the indicated genotypes. (,) MARCM analysis of control () and mir-279 mutant () follicle cells. Homozygous mutant cells express GFP (green). Scale bar, 50 μm. () miR-279 knockdown in follicle cells. Arrows indicate extra invasive cells in and . () A stage-10 egg chamber in which STAT was overexpressed in border cells using slbo–GAL4. () Mosaic analysis of the mir-279S036207 allele. Homozygous mutant cells lack GFP and fail to migrate. () miR-279 knockdown in border cells. Arrowheads indicate border-cell clusters (–). () Quantification of extra invasive cells in egg chambers mosaic for the indicated mir-279 alleles in the presence (+) or absence (−) of a transgene containing the wild-type mir-279 gene. White, grey and black bars indicate the percentage of egg chambers with zero, one or two or more extra invasive cells, respectively. () Effect of miR-279 sponge (miR-279SP) expression on the percentage of egg chamb! ers containing extra invasive cells. Pvalues were calculated using Student's t-test. NS, not significant. () Effect of the indicated mir-279 alleles on the number of cells in the border-cell cluster (blue) and the number of extra invasive cells (red) as depicted in the schematic diagram. () Histogram, effect on border-cell migration of expressing UAS–DsRed or UAS–STAT with slbo–GAL4. Schematic diagram, representation of a stage-10 egg chamber showing the approach used for quantification of border-cell migration. Red shading indicates the region of the egg chamber in which border cells appear when they fail to migrate. Yellow and blue indicate incomplete migration, and green indicates complete migration. () Migration of border-cell clusters composed entirely of homozygous mutant cells of the indicated mir-279 alleles in the presence (+) or absence (−) of a transgene containing the wild-type mir-279 gene. () Effect on border-cell migration of expressing UAS–DsRed ! or UAS–miR-279SP with slbo–GAL4. All error bars represent ! s.e.m. * Figure 3: STAT is a critical target of miR-279 in vivo. () Comparison of nuclear STAT levels in wild-type versus homozygous mir-279Δ1.2 mutant border cells in mosaic clusters (see Methods). The Pvalue was calculated using Student's t-test. (–) Confocal micrographs of stage-10 egg chambers of the indicated genotypes. GFP expression (green) reflects STAT activity. DAPI (blue) labels nuclei and Armadillo (red) labels membranes. Arrowheads indicate border cells and arrows indicate non-migratory anterior follicle cells. Scale bars, 50 μm. (–) Genetic interactions between mir-279 and Stat. () Quantification of egg chambers possessing extra invasive cells following induction of mir-279 mutant clones in the presence of a rescuing transgene or homozygous for a hypomorphic stat allele (Statep3391). White, grey and black bars indicate the percentage of egg chamber with zero, one or two or more extra invasive cells, respectively. (–) Quantification of border-cell migration defects in stage-10 egg chambers. Red indicates no migrat! ion; yellow and blue indicate incomplete migration; green indicates complete migration, as in Fig. 2. () Egg chambers containing border-cell clusters composed entirely of homozygous mir-279 mutant cells in the presence of a rescuing transgene or homozygous for a hypomorphic stat allele (Statep3391). () All egg chambers carry slbo–GAL4, with or without UAS–miR-279 sponge (miR-279SP) and/or Statep3391/+, as indicated. () All egg chambers carry slbo–GAL4, with or without UAS–miR-279 in combination with UAS–STAT wild-type (wt) 3′-UTR, which includes both coding and 3′-UTR sequences or UAS–STAT mutant (mut) 3′-UTR in which the miR-279 seed was deleted. All error bars represent s.e.m. * Figure 4: Two STAT targets, Apt and Ken, feedback through miR-279. (–) Confocal micrographs of stage-8 wild-type (,) or apt mutant background (aptKG05830/apt167; ,) egg chambers carrying a miR-279 expression reporter (mir-279–GAL4; UAS–GFP). DAPI (blue) labels nuclei and Armadillo (red) labels membranes enriched in adherens junction proteins. Arrowheads indicate the border-cell cluster and arrows indicate non-migratory anterior follicle cells. Scale bars, 20 μm. () Quantification of egg chambers of the indicated genetic backgrounds with extra invasive cells. Error bars represent s.e.m. Pvalues were calculated using ANOVA. *P<0.05, ***P<0.001. (–) Expression patterns of Apt and Ken in early stage-9 egg chambers. (–) Confocal micrographs of early stage-9 egg chambers carrying a ken reporter expressing β-galactosidase under the control of the ken locus (PZken1) (,, green) and also stained for Apt (,, red). Scale bars, 20 μm. () Graph, relative levels of nuclear staining intensity of each STAT target relative to the DAPI staini! ng intensity plotted as a function of distance from the polar cells. The border cells develop immediately next to the polar cells. Fc2 indicates the cell next to the border cells, fc3 the next cell, as indicated in the schematic diagram (n=4, mean±s.e.m.). () The ratio of miR-279 expression in border cells to that in follicle cells in control and ken mutant (kenk11035/ken1) egg chambers at stage 8. Error bars represent s.e.m. (–) Confocal micrographs of stage-8 egg chambers carrying a ken expression reporter (ken–lacZ; green) in the wild type (,) or Stat mutant (Stat397/Statts; ,). DAPI (blue) labels nuclei and STAT staining is shown in red. Arrows indicate border cells. () ken–lacZ expression in control versus stat mutant border cells at the non-permissive temperature. Error bars represent s.e.m. The Pvalues were calculated using Student's t-tests in and . * Figure 5: A model of the gene regulatory circuit required to specify non-migratory anterior follicle cell and migratory border-cell fate. (,) Schematic model of the gene regulatory circuit including miR-279, STAT, Apt, Ken and SLBO. () The blue oval highlights the negative feedback loop that represses STAT expression/activity in non-migratory follicle cells. () The yellow circle highlights the positive feedback loop amplifying STAT expression/activity in migratory border cells. Mutual repression of SLBO and Apt, and the effect of EYA on Apt are also indicated, as previously reported4, 21. (–) Computer simulations based on the differential equations in the Methods. () Initial condition: the polar cells (green) are specified. Fc1 indicates the follicle cell next to the polar cell (pc), fc2 the next cell, and so on. () Schematic representation of stable steady-state protein distributions within the field of cells; pixel density corresponds to concentrations (for equations and parameters see Methods and Supplementary Table S1). (–) Representations of protein distributions in the wild type and indicated mutants! . The height of the bars is proportional to the protein concentration. () The distributions in the wild type at an early time point. () Wild-type distributions at a late time point, that is steady state. STAT, SLBO and Ken have all reached high levels near the polar cells and have decayed in the rest of the field. (,) This sharpening does not occur if either mir-279 () or apt () is removed. () Loss of ken does not prevent the threshold response. Author information * Author information * Supplementary information Affiliations * Department of Biological Chemistry, Center for Cell Dynamics, Johns Hopkins University School of Medicine, 855 North Wolfe Street, Suite 450, Baltimore, Maryland 21205, USA * Wan Hee Yoon & * Denise J. Montell * Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, D-72076 Tübingen, Germany * Hans Meinhardt Contributions W.H.Y. planned the experimental design, conducted the experiments and analysed data. H.M. developed and tested the mathematical model. D.J.M. conceived of the project, participated in experimental design, discussions of results and interpretations, and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Denise J. Montell Author Details * Wan Hee Yoon Search for this author in: * NPG journals * PubMed * Google Scholar * Hans Meinhardt Search for this author in: * NPG journals * PubMed * Google Scholar * Denise J. Montell Contact Denise J. Montell Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (1600K) Supplementary Information * Supplementary Movie 2 (5400K) Supplementary Information PDF files * Supplementary Information (850K) Supplementary Information Additional data - Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells
- Nat Cell Biol 13(9):1070-1075 (2011)
Nature Cell Biology | Letter Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells * Derk ten Berge1, 2 * Dorota Kurek1 * Tim Blauwkamp2 * Wouter Koole2 * Alex Maas3 * Elif Eroglu2 * Ronald K. Siu2 * Roel Nusse2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1070–1075Year published:(2011)DOI:doi:10.1038/ncb2314Received19 April 2011Accepted05 July 2011Published online14 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks2. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types3, 4, 5, 6. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation o! f new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor. View full text Figures at a glance * Figure 1: ESC self-renewal requires Wnt signals. (–) The 7xTcf–eGFP reporter is active in a subset (arrow) of ESCs cultured for 2 days on MEFs (,); Wnt3a protein activates the reporter in all cells (,), whereas Fz8CRD extinguishes it (,). (,,) Phase-contrast microscopy; (,,) eGFP. () The ability of 7xTcf–eGFP cells to form alkaline phosphatase-positive (AP+) colonies in the absence of MEFs correlated with the level of eGFP, and was enhanced by the presence of Wnt3a protein (mean±s.e.m., n=3). () The expansion of R1 ESCs able to form alkaline phosphatase-positive colonies on MEFs was progressively repressed by increasing concentrations of the Wnt antagonist Fz8CRD. This effect was counteracted by simultaneous addition of Wnt3a protein (mean+s.e.m., n=3). () The expansion of R1 ESCs able to establish alkaline phosphatase-positive colonies on MEFs was repressed by IWP2. This repression was relieved by simultaneous addition of Wnt3a protein (240 ng ml−1) (mean+s.e.m., n=3). (–) Axin2LacZ ESCs cultured in the abs! ence of MEFs, untreated () or treated for 3 days with IWP2 (,), 2 μg ml−1 Fz8CRD (,) and/or 200 ng ml−1 Wnt3a (,,) and stained with X-gal and Nuclear Red. (–) CGR8 ESCs cultured in the absence of MEFs, untreated () or treated for three passages with IWP2 (,), 2 μg ml−1 Fz8CRD (,) and/or 200 ng ml−1 Wnt3a (,,) and stained for alkaline phosphatase. () The expansion of CGR8 ESCs able to form alkaline phosphatase-positive colonies in the absence of MEFs was repressed by IWP2 or 500 ng ml−1 Fz8CRD, and promoted by 200 ng ml−1 Wnt3a protein. Scale bars, 100 μm (–, –), 500 μm (–). * Figure 2: Wnt signals are required to inhibit the differentiation of ESCs into EpiSCs. () R1 ESCs cultured for 3 days on MEFs in the presence of IWP2 form flattened colonies that reduce alkaline phosphatase (AP) and Nanog expression, and increase Claudin6 and Otx2 expression. () On passaging of R1 ESCs in the presence of IWP2, only alkaline phosphatase-low but Oct4-positive colonies expand (mean+s.e.m., n=3). () Gene expression profiles of R1 ESCs cultured on MEFs in the presence of IWP2, and of EpiSCs, relative to untreated ESCs (mean±s.e.m., n=3). () After two passages in the presence of IWP2 or vehicle on MEFs, the cells were passaged in the presence of 2 μM SB431542, and the number of alkaline phosphatase- and Oct4-positive colonies determined (mean±s.e.m., n=3). (,) Female ESCs were cultured on MEFs for two passages in the presence of vehicle () or IWP2 () and immunostained for H3K27me3 (red) and Oct4 (green). A yellow focus indicates the presence of an inactive X chromosome. () In the presence of IWP2, either no or virtually all cells of a colony sh! owed the inactive X focus. The number of colonies showing no focus (2X) or X inactivation (Xin) was determined over multiple passages. The absolute numbers are plotted within the bars. Scale bars, 50 μm (,,). * Figure 3: Wnt3a protein is sufficient to inhibit the differentiation of ESCs into EpiSCs. () FACS plots of EpiSCs and of R1 cells passaged every 3 days in N2B27 supplemented with LIF, bFGF and ActivinA, in the presence of Wnt3a protein (240 ng ml−1) or IWP2 (2 μM) as indicated, and stained with anti-SSEA1-PE (phycoerythrin) and anti-Pecam1-FITC (fluorescein isothiocyanate) antibodies (10,000 cells/plot). () Gene expression profiles of EpiSCs and R1 ESCs cultured in N2B27 supplemented with LIF, bFGF, ActivinA and either Wnt3a (LFAW) or IWP2 (LFAI), relative to untreated ESCs (mean±s.e.m., n=3). (–) The Axin2LacZ/+ reporter indicates Wnt activity in the E3.5 () and E4.5 () ICM, but is inactive in E5.5 implanted embryos (). Scale bars, 25 μm. * Figure 4: LIF and Wnt3a are sufficient to support ESC self-renewal. (,) Expansion over multiple passages of alkaline phosphatase-positive (AP+) R1 ESC colonies in medium containing serum and LIF () or in N2B27 containing LIF () (mean+s.e.m., n=3). (,) Alkaline phosphatase-stained R1 ESCs cultured on MEFs () or maintained for six passages in N2B27 medium supplemented with LIF and Wnt3a (). () Gene expression profile of R1 ESCs following four passages in the indicated conditions. () Expansion of alkaline phosphatase-positive R1 ESC colonies in N2B27 medium. Where indicated, Wnt3a was added at 200 ng ml−1. () Expansion of alkaline phosphatase-positive R1 ESC colonies in N2B27 medium containing LIF and PD0325901. () MEK activity (indicated by the presence of phospho-ERK (p-ERK; extracellular signal-regulated kinase) on western blot) in R1 ESCs cultured in N2B27 with LIF, was repressed by PD0325901 (PD) but not by Wnt3a, or by withdrawal of Wnt3a for 9 h (−9 h). Uncropped images of blots are shown in Supplementary Fig. S4g. Scale bar,! 200 μm (,). * Figure 5: Wnt3a supports derivation of non-permissive ESCs. (–) Alkaline phosphatase, Oct4 and Nanog stainings, respectively, of the newly derived FVB/N ESC line FN3. Scale bar, 200 μm. () Chimaera (black–white spotted) obtained from injection of C57Bl/6 blastocysts with passage 7 ESC line FN3 together with FVB mate and pups showing germline transmission (white pups). () Expansion of alkaline phosphatase-positive (AP+) colonies from newly derived FVB/N ESCs in N2B27 medium with LIF (mean+s.e.m., n=3). PD, PD0325901. () Expansion of alkaline phosphatase-positive colonies from FVB/N ESCs on MEFs in medium containing serum and LIF (mean+s.e.m., n=3). Author information * Author information * Supplementary information Affiliations * Erasmus MC Stem Cell Institute, Department of Cell Biology, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands * Derk ten Berge & * Dorota Kurek * Howard Hughes Medical Institute, and Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA * Derk ten Berge, * Tim Blauwkamp, * Wouter Koole, * Elif Eroglu, * Ronald K. Siu & * Roel Nusse * Department of Cell Biology, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands * Alex Maas Contributions D.t.B., D.K. and T.B. designed and carried out experiments, analysed data and wrote the paper. W.K., A.M., R.S. and E.E. designed and carried out experiments and analysed data. R.N. designed experiments and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Derk ten Berge Author Details * Derk ten Berge Contact Derk ten Berge Search for this author in: * NPG journals * PubMed * Google Scholar * Dorota Kurek Search for this author in: * NPG journals * PubMed * Google Scholar * Tim Blauwkamp Search for this author in: * NPG journals * PubMed * Google Scholar * Wouter Koole Search for this author in: * NPG journals * PubMed * Google Scholar * Alex Maas Search for this author in: * NPG journals * PubMed * Google Scholar * Elif Eroglu Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald K. Siu Search for this author in: * NPG journals * PubMed * Google Scholar * Roel Nusse Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1200K) Supplementary Information Additional data - Phagocytic activity of neuronal progenitors regulates adult neurogenesis
- Nat Cell Biol 13(9):1076-1083 (2011)
Nature Cell Biology | Letter Phagocytic activity of neuronal progenitors regulates adult neurogenesis * Zhenjie Lu1 * Michael R. Elliott2 * Yubo Chen1, 5 * James T. Walsh1 * Alexander L. Klibanov3 * Kodi S. Ravichandran2, 4 * Jonathan Kipnis1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1076–1083Year published:(2011)DOI:doi:10.1038/ncb2299Received19 November 2010Accepted15 June 2011Published online31 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Whereas thousands of new neurons are generated daily during adult life, only a fraction of them survive and become part of neural circuits; the rest die, and their corpses are presumably cleared by resident phagocytes. How the dying neurons are removed and how such clearance influences neurogenesis are not well understood. Here, we identify an unexpected phagocytic role for the doublecortin (DCX)-positive neuronal progenitor cells during adult neurogenesis. Our in vivo andex vivo studies demonstrate that DCX+ cells comprise a significant phagocytic population within the neurogenic zones. Intracellular engulfment protein ELMO1, which promotes Rac activation downstream of phagocytic receptors, was required for phagocytosis by DCX+ cells. Disruption of engulfment in vivo genetically (in Elmo1-null mice) or pharmacologically (in wild-type mice) led to reduced uptake by DCX+ cells, accumulation of apoptotic nuclei in the neurogenic niches and impaired neurogenesis. Collectively, ! these findings indicate a paradigm wherein DCX+ neuronal precursors also serve as phagocytes, and that their phagocytic activity critically contributes to neurogenesis in the adult brain. View full text Figures at a glance * Figure 1: DCX-expressing neuronal progenitor cells exhibit phagocytic activity in vivo and ex vivo. () Wild-type mice (LV, lateral ventricle) intracranially injected with fluorescent PtdSer liposomes examined immunohistochemically in the SVZ for DCX+ and GFAP+ cells with phagocytosed liposomes. DAPI, 4,6-diamidino-2-phenylindole. Representative confocal micrographs are shown (n>10 fields analysed per experiment; five independent experiments carried out). Scale bar, 10 μm (5 μm in inset). () Wild-type mice intracranially injected with fluorescently labelled ultraviolet (UV)-irradiated NPCs examined immunohistochemically in the SVZ for DCX+ cells with phagocytosed cells. A representative micrograph is shown in orthogonal projections of confocal z stacks (n>12 fields analysed per experiment; three independent experiments carried out). Scale bar, 50 μm (2 μm in inset). () Wild-type mice intracranially injected with fluorescent PtdCho liposomes exhibit poor engulfment by DCX+ cells in the SVZ (n>10 fields analysed per experiment; three independent experiments carrie! d out). Scale bar, 5 μm. () Wild-type mice injected intravenously with annexin V before and immediately after intracranial injection with fluorescent PtdSer liposomes. Brain tissue examined immunohistochemically for DCX+ and GFAP+ cells containing liposomes in the SVZ. The bar graph represents a quantification of the volume of DCX+ cells containing PtdSer liposomes (mean±s.e.m.). Student's t-test statistical analysis was carried out for n=4 mice in each group with at least five slices analysed for each mouse (*P<0.05). () Representative confocal microscopy images (with n>30 cells analysed), demonstrating the formation of the phagocytic cup (phalloidin staining) in DCX+ cells around the carboxylate-modified 3 μm beads (simplified apoptotic targets), are shown in top, middle and bottom planes. Scale bar, 3 μm. () Representative confocal microscopy images of phagocytosis by a newly differentiated neuron showing orthogonal projections of confocal z stacks (n>20 fiel! ds analysed per experiment; seven independent experiments carr! ied out). Scale bar, 5 μm. () Newly differentiated NPC cultures were fed with ultraviolet-irradiated NPCs ex vivo and washed after 6 h. Cells were analysed after a further seven days of differentiation. Representative confocal microscopy orthogonal views of maturated neurons are shown. White arrowheads point to remnants of phagocytosed cells in neurons and grey arrows those in other cells (n>20 fields analysed per experiment; four independent experiments carried out). Scale bar, 5 μm. * Figure 2: Inhibition of phagocytosis in the neurogenic niche in vivo impairs adult neurogenesis. () Schematic representation of short-term (7 days) and long-term (28 days) annexin V treatment to block apoptotic cell clearance, coupled with BrdU injection to monitor proliferating cells within the neurogenic zones. () Representative fluorescent microscopy images of accumulated apoptotic nuclei through TUNEL staining (indicated by white arrowheads) in the SGZ after short-term annexin V treatment. The bar graph represents a quantification of apoptotic (TUNEL-positive) nuclei (mean±s.e.m.). Student's t-test statistical analysis was carried out for n=4 mice in each group, with at least five slices analysed for each mouse (*P<0.05). Scale bars, 50 μm. () Representative fluorescent microscopy images of the SGZ from control and short-term annexin V-treated mice examined for DCX expression and BrdU incorporation. The bar graphs (means±s.e.m.) represent quantifications of BrdU+ cells (per hippocampus), DCX+ cells (per 200 μm of subgranular layer) and the fraction of BrdU! + that were also DCX+ (indicative of neuronal differentiation; per hippocampus). Student's t-test statistical analysis was carried out for n=4 mice in each group with at least ten slices analysed for each mouse (*P<0.05; ***P<0.001). Scale bars, 50 μm. () Representative fluorescent microscopy images of control and long-term annexin V-treated brain slices examined for BrdU and NeuN immunoreactivity in the SGZ (BrdU+NeuN+ cells are indicated by white arrowheads). The bar graph represents quantification of BrdU+NeuN+ cells per hippocampus of vehicle- and annexin V-treated mice (mean±s.e.m.). Student's t-test statistical analysis was carried out for n=7 mice in each group with at least ten slices analysed for each mouse (*P<0.05). Scale bars, 50 μm. () Representative confocal microscopy images of the SVZ immunolabelled for DCX from control, short-term and long-term annexin V-treated mice (LV, lateral ventricle). The bar graph represents quantification of the areas oc! cupied by DCX+ cells per field (mean±s.e.m.). Student's t-t! est statistical analysis was carried out between the groups for n=5 mice in each group with at least ten slices analysed for each mouse (*P<0.05). Scale bars, 50 μm. * Figure 3: ELMO1-dependent phagocytosis of DCX+ cells in vitro. () Representative images of primary hippocampal neuronal cultures grown for 2 or 6 days in vitro immunolabelled for DCX and ELMO1 (for n>30 fields). Scale bars, 5 μm. () Primary hippocampal neuronal cultures grown for 2 or 6 days in vitro (DIV) were immunolabelled for DCX and ELMO2. Representative confocal microscopy images (for n>30 fields) are shown. The expression level of ELMO2 was not altered under these conditions in the DCX+ cells after 2 or 6 days in culture. Scale bars, 5 μm. () Cultures of neurons from were fed with targets (carboxylate-modified beads) and assessed for engulfment. The bars represent quantifications of phagocytosed targets by primary DCX+ cells from wild-type and Elmo1−/− mice after 2 or 6 days in culture. A one-way analysis of variance with a Newman–Keuls multiple comparison test was carried out for n=100 DCX+ cells per group (**P<0.01; ***P<0.001). () Accumulation of apoptotic nuclei in PBS- or long-term annexin V-treated Elmo1−/− ! mice, compared with wild-type littermates, was examined by TUNEL immunolabelling (indicated by white arrowheads) of the SGZ. Representative images from wild-type and Elmo1−/− mice are shown. The bar graph represents a quantification of TUNEL-positive nuclei (mean±s.e.m.). Student's t-test was carried out for n=5 mice per group, with at least ten slices analysed for each mouse (**P<0.01). Scale bars, 50 μm. * Figure 4: Effect of ELMO1-dependent phagocytosis of DCX+ cells on neurogenesis in vivo. () Representative images of the SGZ from wild-type and Elmo1−/− mice examined for DCX and BrdU immunoreactivity 7 days after BrdU injection. The brackets indicate the width of the granular layer. Scale bars, 20 μm. () The bar graphs (means±s.e.m.) represent the quantification of total numbers of BrdU+ cells (proliferating cells), total numbers of all BrdU+DCX+ cells (neuronal progenitors), total numbers of DCX+ cells per 200 μm of the SGZ (neuronal progenitors, alternative quantification) and percentages of DCX+BrdU+ out of all BrdU+ cells (neuronal differentiation of proliferating cells) per hippocampus in wild-type and Elmo1−/− mice. Student's t-test statistical analysis was carried out for n=6 mice in each group with at least ten slices analysed for each mouse (*P<0.05; **P<0.01). () Mean (±s.e.m.) width of a granular cell layer. Student's t-test was carried out for n=6 mice per group with at least ten slices analysed for each mouse (***P<0.001). () Re! presentative images of the SVZ immunolabelled for DCX from wild-type and Elmo1−/− mice (LV, lateral ventricle). The bar graph represents quantification of the areas occupied by DCX+ cells per field (mean±s.e.m.). Student's t-test statistical analysis was carried out between the groups for n=6–8 mice in each group with at least ten slices analysed for each mouse (*P<0.05). Representative experiment out of three independently carried out (n>10 fields analysed in each experiment). Scale bars, 50 μm. () High magnification of the SGZ immunolabelled for DCX+ cells in wild-type and Elmo1−/− mice. The bar graph represents a quantification of dendritic arborization of DCX+ cells in wild-type and Elmo1−/− mice (mean±s.e.m.). Student's t-test statistical analysis was carried out for at least n=100 DCX+ cells in each group with at least n=5mice per group analysed (**P<0.01). Scale bars, 40 μm. * Figure 5: ELMO1 deficiency impairs phagocytic activity of DCX+ cells in vivo. () Representative images of neurospheres from wild-type and Elmo1−/− mice. The bar graphs (means±s.e.m.) show the diameter of neurospheres (indicates the ability of cells to grow and divide), the neurosphere self-renewal capacity (number of neurospheres divided by number of plated cells×100) of the first-generation (P1) neurospheres and the initial number of neurospheres obtained from the adult brains of wild-type and Elmo1−/− mice (n=12 mice in each group, with hundreds of neurospheres analysed). Scale bars, 50 μm. () Neurospheres established from NPCs obtained from Elmo1−/− or wild-type mice were cryosectioned and examined for apoptotic (cleaved caspase 3 (CC3)-positive) cells in vitro. Representative images (from n>5 neurospheres) are shown. The bar graph indicates cleaved caspase 3-positive cells (percentage of all DAPI-positive cells) in the neurosphere (mean±s.e.m.). Multiple fields for n=3 neurospheres from each group were analysed. Scale bars, 20 ! μm. () Neurospheres established from NPCs obtained from Elmo1−/− or wild-type mice were examined for differentiation in vitro. Representative images of differentiated NPCs after 10 days in culture, immunolabelled for βIII-tubulin (neurons) and GFAP (astrocytes). The bar graphs (means±s.e.m.) indicate neuronal differentiation (percentage of βIII-tubulin cells) and astrocyte differentiation (percentage of GFAP cells) from wild-type and Elmo1−/− neurospheres (n=10 fields analysed in each experiment). Scale bars, 20 μm. () Wild-type and Elmo1−/− mice were injected with fluorescently labelled PtdSer liposomes and examined immunohistochemically for DCX+ and GFAP+ cells containing phagocytosed liposomes in the SVZ. Representative confocal micrographs are shown. The bar graph indicates a quantification of the volume of DCX cells with PtdSer liposomes in wild-type and Elmo1−/− mice (mean±s.e.m.). Student's t-test statistical analysis was carried out for n=4! mice in each group with at least five slices analysed for eac! h mouse (***P<0.001). Scale bars, 10 μm. Author information * Author information * Supplementary information Affiliations * Department of Neuroscience, University of Virginia, Charlottesville, Virginia 22901, USA * Zhenjie Lu, * Yubo Chen, * James T. Walsh & * Jonathan Kipnis * Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22901, USA * Michael R. Elliott & * Kodi S. Ravichandran * Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22901, USA * Alexander L. Klibanov * Center for Cell Clearance, University of Virginia, Charlottesville, Virginia 22901, USA * Kodi S. Ravichandran * Present address: Key Laboratory for NeuroInformation of Ministry of Education, University of Electonic Science and Technology of China, ChengDu, 610054, China * Yubo Chen Contributions Z.L. participated in the experimental design, carried out most of the experiments, analysed the data and participated in manuscript preparation; M.R.E. assisted with phagocytic assays and participated in experimental design; Y.C. assisted with immunofluorescent experiments; J.T.W. assisted with intracranial injections; A.L.K. supplied all the liposomes used in this study; K.S.R. helped with design of experiments and prepared the manuscript; J.K. designed the experiments, assisted with data analysis and prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Kodi S. Ravichandran or * Jonathan Kipnis Author Details * Zhenjie Lu Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R. Elliott Search for this author in: * NPG journals * PubMed * Google Scholar * Yubo Chen Search for this author in: * NPG journals * PubMed * Google Scholar * James T. Walsh Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander L. Klibanov Search for this author in: * NPG journals * PubMed * Google Scholar * Kodi S. Ravichandran Contact Kodi S. Ravichandran Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan Kipnis Contact Jonathan Kipnis Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data - The T-box transcription factor Eomesodermin acts upstream of Mesp1 to specify cardiac mesoderm during mouse gastrulation
- Nat Cell Biol 13(9):1084-1091 (2011)
Nature Cell Biology | Letter The T-box transcription factor Eomesodermin acts upstream of Mesp1 to specify cardiac mesoderm during mouse gastrulation * Ita Costello1 * Inga-Marie Pimeisl2 * Sarah Dräger2 * Elizabeth K. Bikoff1 * Elizabeth J. Robertson1 * Sebastian J. Arnold2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1084–1091Year published:(2011)DOI:doi:10.1038/ncb2304Received26 January 2011Accepted23 June 2011Published online07 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Instructive programmes guiding cell-fate decisions in the developing mouse embryo are controlled by a few so-termed master regulators. Genetic studies demonstrate that the T-box transcription factor Eomesodermin (Eomes) is essential for epithelial-to-mesenchymal transition, mesoderm migration and specification of definitive endoderm during gastrulation1. Here we report that Eomes expression within the primitive streak marks the earliest cardiac mesoderm and promotes formation of cardiovascular progenitors by directly activating the bHLH (basic-helix-loop-helix) transcription factor gene Mesp1 upstream of the core cardiac transcriptional machinery2, 3, 4. In marked contrast to Eomes/Nodal signalling interactions that cooperatively regulate anterior–posterior axis patterning and allocation of the definitive endoderm cell lineage1, 5, 6, 7, 8, formation of cardiac progenitors requires only low levels of Nodal activity accomplished through a Foxh1/Smad4-independent mechanism. ! Collectively, our experiments demonstrate that Eomes governs discrete context-dependent transcriptional programmes that sequentially specify cardiac and definitive endoderm progenitors during gastrulation. View full text Figures at a glance * Figure 1: Fate mapping of EomesCre-expressing cells reveals selective contributions to definitive endoderm and cardiovascular cell lineages. () Targeting strategy used to generate the EomesCre reporter allele. Cre recombinase coding sequences were inserted into exon 1 of the Eomes locus. RV, EcoRV; H, HpaI; S, SphI; E, EagI; Cre, Cre recombinase; TK, thymidine kinase. () Embryonic stem cell clones were screened by Southern blot on EcoRV-digested DNA using a 3′ probe (red line in ) to detect wild-type and targeted alleles. () Correctly targeted embryonic stem cells were transiently transfected with Cre to excise the phosphoglycerate kinase–neomycin (PGK-neo) selection cassette and generate the reporter allele, as shown by Southern blot. (,) Fate-mapping experiments demonstrate that descendants of EomesCre-expressing cells contribute to the myocardium and endocardium of the heart (He), the head mesenchyme (Hm), vasculature and the endoderm of the primary gut tube (Gt), but rarely colonize other mesoderm tissues formed from paraxial and lateral plate mesoderm. Sections were counterstained with eosin to highlight! non-labelled cells. The black lines indicate the plane of section. * Figure 2: Eomes functional loss disrupts specification of cardiovascular progenitors. () Whole-mount in situ hybridization analysis of cardiac mesoderm (Myl7, Wnt2, Nkx2.5) and vascular (Agtrl1, Rasgrp3, Klhl6) markers in control and EomesN/CA;Sox2.Cre-mutant embryos. Eomes mutants entirely lack expression of cardiac marker genes and show significantly reduced expression of vascular markers. In contrast, in wild-type embryos vascular markers broadly delineate the embryonic and extra-embryonic vasculature at E8.5 and E9.5. () Schematic representation of the protocol for generation of chimaeric embryos. Eomes-null embryonic stem cells were introduced into wild-type ROSA26LacZ blastocysts. (,) Histological sections of two independent LacZ-stained E9.5 chimaeric embryos were counterstained with eosin to identify Eomes-mutant cell populations (pink). The myocardium and endocardium of the heart (He) and endoderm of the gut tube (Gt) exclusively consist of LacZ-positive wild-type cells. Relatively few Eomes-null cells colonize the head mesenchyme (Hm), whereas the m! ajority of other tissues consist of mixed populations of Eomes-null and wild-type cells. Scale bar, 500 μm. * Figure 3: Eomes-null embryonic stem cells fail to give rise to definitive endoderm and cardiomyocytes. () Wild-type, Eomes−/− and Smad2−/− embryonic stem cells were cultured in the presence of high doses of ActivinA. Semi-quantitative RT–PCR analysis shows that Eomes−/− and Smad2−/− cultures strongly express mesoderm marker genes such as Brachyury and Mixl1, but lack expression of the definitive endoderm marker genes Foxa2 and Sox17. () qRT–PCR analysis confirms the markedly reduced definitive endoderm marker transcript levels in Eomes−/− at day 4 of ActivinA-induced differentiation. () Wild-type and Eomes-mutant embryoid bodies were induced to form cardiomyocytes in hanging-drop cultures. Semi-quantitative RT–PCR analysis reveals that cardiac-specific transcription factors (Mesp1, Mesp2, Gata6, Nkx2.5, Mef2c and Myocardin), as well as structural proteins (Myl7 and Myl2) are significantly downregulated in Eomes-null embryoid bodies whereas expression of the pan-mesodermal marker Brachyury is unaffected. () Clusters of beating cardiomyocytes are readi! ly detectable in wild-type embryoid body outgrowths but absent from Eomes-null cultures at day 7. Error bars represent the standard error of the mean (s.e.m.) of three independent experiments. () At day 8 TnI-positive cardiomyocytes are detectable in wild-type outgrowths but are entirely absent from Eomes-mutant cultures. Higher magnification reveals characteristic cross-striation of myofibrils. Scale bar, 100 μm for the overview and 10 μm for the higher-magnification image. DAPI, 4,6-diamidino-2-phenylindole. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 4: Eomes directly binds conserved T-box sites within the Mesp1 locus to activate expression. () Mesp1, robustly expressed in wild-type embryos at E7.0, is absent from EomesN/CA;Sox2Cre mutants. At slightly later stages (E7.25) EomesN/CA;Sox2Cre embryos occasionally show weak expression that probably reflects activity of Tbx6, known to be expressed in E7.5 Eomes mutants1. () As judged by qRT–PCR, Mesp1 and Mesp2 transcripts are markedly reduced in E7.25 Eomes-mutant embryos. Error bars represent the standard deviation (s.d.), n=5 per genotype. () Schematic representation of cis-regulatory elements in the Mesp1/2 locus. The positions of previously identified T-box sites within the Mesp1/2 EME and Mesp2 PSME (nomenclature according to refs 26, 27) and a putative T-box site identified near the Mesp1 TSS are indicated. The Mesp2 PSME contains three binding elements (sites B, G, D) that contain T-box binding motifs. T-box consensus sequences are indicated in red. Red bars indicate areas amplified by qPCR after ChIP using different antibodies. Ex1, exon 1; Ex2, exon 2; P! SME, presomitic mesoderm enhancer; Tbx, T-box site. () ChIP analysis of P19Cl6 cells treated for 4 days with DMSO using antibodies specific for Eomes, RNA polymerase II (PolII) or an IgG control. Specific enrichment for genomic loci containing T-box sites (Mesp1_TSS, Mesp1_Tbx, Mesp2_Tbx) was observed using the Eomes-specific antibody. The PolII antibody gave specific enrichment of the Mesp1_TSS, but not other tested regions of the Mesp1/2 locus. Specific binding to T-box sites (indicated by asterisks) in the Mesp1/2 EME, the Mesp2 PSME and the Mesp1 TSS was detectable after 4 days, but not at day 0. d0, day 0; d4, day 4 of DMSO differentiation of P19Cl6 cells. () Cells expressing a tamoxifen-inducible Eomes fusion construct (P19EoER cells) also show Eomes binding to these T-box sites at day 4 of tamoxifen treatment. Con, non-tamoxifen-induced control P19EoER cells; Tam, day 4 tamoxifen-treated P19EoER cells. The most representative plots of three independent experiments ar! e shown. * Figure 5: Eomes and dose-dependent Nodal/Smad2/3 signalling levels control cardiac mesoderm and definitive endoderm specification during gastrulation. () Smad4 and Foxh1 are critical Nodal pathway components for transducing high levels of signalling6, 12, 13. Mesp1 is expressed normally in E6.5 and E7.5 Foxh1-null embryos and in embryos lacking Smad4 in the epiblast only (Smad4Δ). Mesp1 expression is also efficiently induced in Lhx1-mutant embryos, which show definitive endoderm and midline mesoderm defects. However, the failure of anterior–posterior axis rotation results in induction of Mesp1 throughout the proximal epiblast. () Eomes activity regulates formation of both cardiac mesoderm and definitive endoderm progenitors during gastrulation. Eomes+ epiblast cells confined to the posterior side of the embryo before overt streak formation are exposed to low levels of Nodal signalling. Eomes-dependent activation of Mesp1/2 marks the earliest cardiac progenitors induced in the forming primitive streak. Mesp1/2 expression leads to activation of the Nodal antagonist Lefty2 (ref. 23) and direct repression of definitive endo! derm genes2. As cells begin to migrate away from the primitive streak, Mesp1/2 expression is downregulated through a negative-feedback loop2, 23, 25. In contrast, the Eomes expression domain extends distally and overlaps with increased Nodal signalling levels as the primitive streak elongates. Eomes, acting cooperatively with Nodal/Smad4/Foxh1-dependent signals in the APS, induces definitive endoderm. () At later stages from E7.5 onwards, Tbx6 expression in the presomitic mesoderm activates a second wave of Mesp1/2 expression in the presomitic mesoderm through occupancy of the conserved T-box regulatory elements. Author information * Author information * Supplementary information Affiliations * Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK * Ita Costello, * Elizabeth K. Bikoff & * Elizabeth J. Robertson * University Medical Centre, Renal Department, Centre for Clinical Research, Breisacher Strasse 66, 79106 Freiburg, Germany * Inga-Marie Pimeisl, * Sarah Dräger & * Sebastian J. Arnold Contributions I.C., E.K.B., E.J.R. and S.J.A. designed experiments, I.C., I-M.P., S.D., E.J.R. and S.J.A. carried out research, I.C., E.J.R. and S.J.A. analysed data and I.C., E.K.B., E.J.R. and S.J.A. wrote and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Elizabeth J. Robertson or * Sebastian J. Arnold Author Details * Ita Costello Search for this author in: * NPG journals * PubMed * Google Scholar * Inga-Marie Pimeisl Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah Dräger Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth K. Bikoff Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth J. Robertson Contact Elizabeth J. Robertson Search for this author in: * NPG journals * PubMed * Google Scholar * Sebastian J. Arnold Contact Sebastian J. Arnold Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (500K) Supplementary Information * Supplementary Movie 2 (1M) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - FOXO1 is an essential regulator of pluripotency in human embryonic stem cells
- Nat Cell Biol 13(9):1092-1099 (2011)
Nature Cell Biology | Letter FOXO1 is an essential regulator of pluripotency in human embryonic stem cells * Xin Zhang1 * Safak Yalcin1 * Dung-Fang Lee1 * Tsung-Yin J. Yeh2 * Seung-Min Lee1 * Jie Su1 * Sathish Kumar Mungamuri1 * Pauline Rimmelé1 * Marion Kennedy3 * Rani Sellers4 * Markus Landthaler5, 8 * Thomas Tuschl5 * Nai-Wen Chi2 * Ihor Lemischka1, 6 * Gordon Keller3 * Saghi Ghaffari1, 6, 7 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1092–1099Year published:(2011)DOI:doi:10.1038/ncb2293Received09 December 2010Accepted06 June 2011Published online31 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Pluripotency of embryonic stem cells (ESCs) is defined by their ability to differentiate into three germ layers and derivative cell types1, 2, 3 and is established by an interactive network of proteins including OCT4 (also known as POU5F1; ref. 4), NANOG (refs 5, 6), SOX2 (ref. 7) and their binding partners. The forkhead box O (FoxO) transcription factors are evolutionarily conserved regulators of longevity and stress response whose function is inhibited by AKT protein kinase. FoxO proteins are required for the maintenance of somatic and cancer stem cells8, 9, 10, 11, 12, 13; however, their function in ESCs is unknown. We show that FOXO1 is essential for the maintenance of human ESC pluripotency, and that an orthologue of FOXO1 (Foxo1) exerts a similar function in mouse ESCs. This function is probably mediated through direct control by FOXO1 of OCT4 and SOX2 gene expression through occupation and activation of their respective promoters. Finally, AKT is not the predominant r! egulator of FOXO1 in human ESCs. Together these results indicate that FOXO1 is a component of the circuitry of human ESC pluripotency. These findings have critical implications for stem cell biology, development, longevity and reprogramming, with potentially important ramifications for therapy. View full text Figures at a glance * Figure 1: FOXO1 is essential for the expression of hESC pluripotency markers. () qRT–PCR analysis of expression of FOXO genes in pluripotent undifferentiated hESCs and during mesodermal induction. The expression levels of FOXO3A and FOXO4 are relative to that of FOXO1 in undifferentiated H1 cells under self-renewal conditions. Note, downregulation of FOXO1 and upregulation of FOXO3A during differentiation of hESCs. FOXO6 expression was not detectable in hESCs. EBs, embryoid bodies. () FOXO1 expression was analysed by qRT–PCR in parental H1 hESCs and in H1 cells expressing shRNA targeting FOXO1 (H1/FOXO1 shRNA) cultured in the absence or presence of doxycycline (Dox) for 4 days under undifferentiated self-renewal conditions. H1 cells expressing vector control (H1/VC) or scrambled FOXO1 shRNA (H1/scr) were used as controls. () qRT–PCR analysis carried out as in at the indicated times in cells treated with or without doxycycline. Quantification of the target genes was relative to the endogenous ACTB (β-actin) transcript levels. Results are mean±s! .e.m. of three independent experiments, each carried out in triplicate (–); *P<0.05, **P<0.01, ***P<0.001 (,). () An aliquot of cells from was subjected to western blot analysis of the indicated proteins; relative intensities of bands are shown below each panel relative to that measured at time 0 in H1 cells (uncropped scanned gels are shown in Supplementary Fig. S12). () hESCs were cultured with or without doxycycline for the indicated times and immunostained for surface markers of pluripotency, TRA-1-60 and TRA-1-81, and counterstained with DAPI. Scale bars, 100 μm. * Figure 2: Reversible effect of loss of FOXO1 on pluripotency and differentiation of hESCs. () qRT–PCR analysis of pluripotency (left), mesoderm (middle) and endoderm (right) gene expression in H1/FOXO1-shRNA hESCs. Cells were maintained under pluripotency self-renewing conditions and treated with or without doxycycline for up to 4 days, after which cells were washed extensively and maintained in the absence of doxycycline. Quantification of the genes was relative to the endogenous β-actin transcript levels. Error bars indicate s.e.m. of three independent experiments, each carried out in triplicate. *P<0.05, **P<0.01. () In H1 cells, ectopic expression of FOXO1 and not FOXO3A induces expression of pluripotency genes. GFP-positive hESCs lentivirally transduced with an empty control vector or a vector containing FOXO1 or FOXO3A were FACS sorted 72 h after transduction and analysed by qRT–PCR for FOXO1 or FOXO3A and pluripotency marker gene expression; results shown are relative to endogenous ACTB and normalized to untransduced H1 cells under self-renewal condi! tions. Error bars indicate s.e.m. of three independent experiments each, carried out in triplicate; *P<0.05, **P<0.01, ***P<0.001. NT, not transduced. NS, not significant. () FOXO1-knockdown-mediated loss of pluripotency and induction of differentiation markers in H1/FOXO1-shRNA cells after several passages. Morphology (left) and alkaline phosphatase staining (right) of hESCs cultured in doxycycline for five passages. () Expression of surface markers of pluripotency, SSEA4, TRA-1-60 and TRA-1-81, by immunostaining and DAPI counterstaining of hESCs was analysed after five passages in the absence or presence of doxycycline (Dox). Scale bar, 100 μm. One representative of two independent experiments is shown. () qRT–PCR analysis of FOXO1 and pluripotency genes (left), mesoderm (middle) and endoderm (right) markers in H1 cells maintained in pluripotency self-renewal conditions with or without doxycycline for five passages. Results shown are relative to the endogenous ACTB. ! Error bars indicate s.e.m. (n=3). *P<0.05, ***P<0.001. * Figure 3: Foxo1 and Foxo3 regulate pluripotency of mESCs. () Alkaline phosphatase staining in Foxo1-, Foxo3- or control-knockdown mESCs. Scale bar, 50 μm. (,) qRT–PCR analysis of pluripotency (top) and developmental genes (bottom) in mESCs expressing one of two distinct shRNA targeting Foxo1 () or Foxo3 (). shRNA-mediated knockdown of luciferase was used as a control. The gene expression levels of four lineages, including trophectoderm (Cdx2 and Mash2, also known as Ascl2), endoderm (Gata6 and Foxa2), ectoderm (Cxcl12, Mash1, also known as Ascl1, and Fgf5) and mesoderm (brachyury) were examined after knockdown of Foxo1 or Foxo3. All graphs show mean±s.e.m. for n=3; *P<0.05, **P<0.01. P values are from comparing Foxo1 shRNA 4 or Foxo3 shRNA 4 with Luc shRNA 1 (,). Ectopic expression of a resistant form of the targeted FoxO1-rescued FoxO1-knockdown-mediated phenotype in both hESCs and mESCs. () hESCs were transfected with a lentivirus expressing FOXO1 shRNA III (targeting the 3′ untranslated region of the FOXO1 mRNA); GFP-pos! itive cells were FACS sorted 3 days later (>50–60% GFP positive), transduced with lentiviral vector (pLEIGW-FOXO1) that contains only the FOXO1 coding region and cultured for another 2 days before gene expression analysis. All results are relative to the endogenous ACTB. Error bars indicate s.e.m. (n=3). *P<0.05. () Endogenous Foxo1 was targeted by Foxo1 shRNA 4 in mESCs and rescued by re-expressing the shRNA-resistant Foxo1-m4 construct (EF1α-Foxo1-m4). EF1α promoter empty vector (EF1α) was used as a control. Gene expression analysis of Foxo1 and pluripotency genes was carried out by qRT–PCR and all results in Foxo1-shRNA 4 cells are expressed relative to the sample with Luc shRNA and EF1α (set as 1). Error bars indicate s.e.m. (n=3). P<0.05 for all genes in rescued samples when compared with controls. * Figure 4: FOXO1 knockdown does not impact hESC proliferation or redox status. () Left, H1, H1/VC, H1/scr and H1/FOXO1-shRNA cells maintained in pluripotency self-renewing conditions were cultured with (top) or without (bottom) doxycycline (Dox) for 96 h, after which cell proliferation was analysed by BrdU staining. BrdU incorporation versus DNA content were used to detect percentage of cells that are in G1, S or G2/M phase of cell cycle. Right, quantification of results of two independent experiments is shown. () qRT–PCR analysis of anti-oxidant enzymes and anti-oxidant response genes in parental H1 and in H1/FOXO1-shRNA cells maintained with or without doxycycline for 96 h. Quantification of the target genes is relative to the endogenous ACTB transcript levels and normalized to untreated H1 cells under self-renewal conditions. Results are shown as mean±s.e.m. of triplicate experiments; one representative of two independent experiments is shown. () Anti-oxidant treatment does not impact the expression of pluripotency genes in hESCs. qRT–PCR a! nalysis of FOXO1 and pluripotency genes in H1, H1/scr and H1/FOXO1-shRNA cells maintained under pluripotency conditions in the presence or absence of NAC (100 μM) and treated or not with doxycycline for 96 h. Results shown are relative to the endogenous ACTB and normalized to untreated H1/scr or H1 cells under self-renewal conditions. n=2. * Figure 5: FOXO1 activates the expression of OCT4 and SOX2 pluripotency genes in hESCs by binding directly to their regulatory regions. () Sequence alignment of human OCT4 and SOX2 regulatory regions containing putative FoxO-binding sites. () Endogenous FOXO occupation of sites shown within arrows was analysed by ChIP in H1 cells. DNA co-immunoprecipitated with either anti-FOXO1 (H-128), anti-FOXO3A (N-15) or a control pre-immune immunoglobulin was amplified by qPCR. Binding of FOXO1 to p27KIP1 promoter and binding of FOXO1 to a conserved upstream region with no FOXO-binding sequences (in −2.7 kb of human OCT4 (NEG Seq O) and in −4 kb of human SOX2 (NEG Seq S)) were used, respectively, as positive and negative controls. Results of three independent experiments (mean±s.e.m.) are shown as relative fold enrichment when compared with control immunoglobulin after normalization to the values obtained from the input samples. () ChIP analysis of FOXO1 in undifferentiated H1/FOXO1-shRNA hESCs treated or not with doxycycline (Dox) for 3 days. Results of three independent experiments (mean±s.e.m.) are shown. ! () pcDNA3, pcDNA3–FOXO1 or pcDNA–FOXO3A were co-transfected into HEK293T cells with a pGL3 containing the O1 or O2 site of human OCT4, or the S1, S2 or S3 site of SOX2 or their mutants driving the luciferase gene; luciferase activity was measured 48 h later. Results are from three independent experiments (mean±s.e.m.). () Luciferase activity measured 48 h after co-transfection of hESCs (H1) with a lentiviral vector control or containing FOXO1 or FOXO3A and a human OCT4 reporter plasmid containing a wild-type or mutant O2 sequence. Results are the mean of two independent experiments. () H1 hESCs were maintained continuously (Contin.) in bFGF for 72 h (lane 1) or starved overnight and stimulated or not with bFGF (40 ng ml−1 for 10 min) and/or PI(3)K inhibitor LY294002 (10 μM) for 45 min before stimulation, and preparing the whole-cell lysate (lanes 2–5). One representative immunoblot of two experiments is shown. () H1 cells maintained in undifferenti! ated conditions and treated or not with LY294002 (10 μM) fo! r 2 h before double immunostaining of FOXO1 and phospho-AKT (Ser 473) and counterstaining with DAPI. One representative of three independent experiments is shown (uncropped scanned gels are shown in Supplementary Fig. S12). Author information * Author information * Supplementary information Affiliations * Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York 10029, USA * Xin Zhang, * Safak Yalcin, * Dung-Fang Lee, * Seung-Min Lee, * Jie Su, * Sathish Kumar Mungamuri, * Pauline Rimmelé, * Ihor Lemischka & * Saghi Ghaffari * Research Service, VA San Diego Healthcare System, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA * Tsung-Yin J. Yeh & * Nai-Wen Chi * McEwen Center for Regenerative Medicine, University Health Network, Toronto, Ontario M5G 1L7, Canada * Marion Kennedy & * Gordon Keller * Department of Pathology, Albert Einstein College of Medicine, New York, New York 10461, USA * Rani Sellers * Howard Hughes Medical Institute, Laboratory for RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA * Markus Landthaler & * Thomas Tuschl * Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, New York 10029, USA * Ihor Lemischka & * Saghi Ghaffari * Department of Medicine Division of Hematology, Oncology, Mount Sinai School of Medicine, New York, New York 10029, USA * Saghi Ghaffari * Present address: Berlin Institute for Medical Systems Biology at the Max-Delbrück-Center for Molecular Medicine, 13092 Berlin, Germany * Markus Landthaler Contributions X.Z. and S.G. designed experiments and analysed data; X.Z. carried out most of the experiments, with significant help from S.Y. and some assistance from S-M.L., S.K.M. and P.R.; M.K. helped with the set-up of some critical techniques; R.S. analysed data; D-F.L. and J.S. designed and carried out experiments involving mESCs; N-W.C. designed and carried out antibody calibration experiments with the help of T-Y.J.Y.; M.L. and T.T. contributed key reagents; I.L. and G.K. provided valuable reagents and advice; S.G. conceived the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Saghi Ghaffari Author Details * Xin Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Safak Yalcin Search for this author in: * NPG journals * PubMed * Google Scholar * Dung-Fang Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Tsung-Yin J. Yeh Search for this author in: * NPG journals * PubMed * Google Scholar * Seung-Min Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Jie Su Search for this author in: * NPG journals * PubMed * Google Scholar * Sathish Kumar Mungamuri Search for this author in: * NPG journals * PubMed * Google Scholar * Pauline Rimmelé Search for this author in: * NPG journals * PubMed * Google Scholar * Marion Kennedy Search for this author in: * NPG journals * PubMed * Google Scholar * Rani Sellers Search for this author in: * NPG journals * PubMed * Google Scholar * Markus Landthaler Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Tuschl Search for this author in: * NPG journals * PubMed * Google Scholar * Nai-Wen Chi Search for this author in: * NPG journals * PubMed * Google Scholar * Ihor Lemischka Search for this author in: * NPG journals * PubMed * Google Scholar * Gordon Keller Search for this author in: * NPG journals * PubMed * Google Scholar * Saghi Ghaffari Contact Saghi Ghaffari Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Excel files * Supplementary Table 1 (80K) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Cleavage of E-cadherin by ADAM10 mediates epithelial cell sorting downstream of EphB signalling
- Nat Cell Biol 13(9):1100-1107 (2011)
Nature Cell Biology | Letter Cleavage of E-cadherin by ADAM10 mediates epithelial cell sorting downstream of EphB signalling * Guiomar Solanas1 * Carme Cortina1, 3 * Marta Sevillano1 * Eduard Batlle1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1100–1107Year published:(2011)DOI:doi:10.1038/ncb2298Received09 July 2010Accepted15 June 2011Published online31 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The formation and maintenance of complex organs requires segregation of distinct cell populations into defined territories (that is, cell sorting) and the establishment of boundaries between them. Here we have investigated the mechanism by which Eph/ephrin signalling controls the compartmentalization of cells in epithelial tissues. We show that EphB/ephrin-B signalling in epithelial cells regulates the formation of E-cadherin-based adhesions. EphB receptors interact with E-cadherin and with the metalloproteinase ADAM10 at sites of adhesion and their activation induces shedding of E-cadherin by ADAM10 at interfaces with ephrin-B1-expressing cells. This process results in asymmetric localization of E-cadherin and, as a consequence, in differences in cell affinity between EphB-positive and ephrin-B-positive cells. Furthermore, genetic inhibition of ADAM10 activity in the intestine of mice results in a lack of compartmentalization of Paneth cells within the crypt stem cell niche! , a defect that phenocopies that of EphB3-null mice. These results provide important insights into the regulation of cell migration in the intestinal epithelium and may help in the understanding of the nature of the cell sorting process in other epithelial tissues where Eph–ephrin interactions play a central role. View full text Figures at a glance * Figure 1: Asymmetrical E-cadherin distribution downstream of EphB signalling is mediated by metalloproteinase activity. (–) Representative confocal micrographs of co-cultures of EphB3/E-cadherin–GFP with E-cadherin–Cherry (,) and EphB3/E-cadherin–GFP with ephrin-B1/E-cadherin–Cherry (,,,) MDCK cells. – show higher-magnification pictures. MDCK cell populations were co-cultured for 48 h either without inhibitors (,,,) or treated with 10 μM TAPI-1 for 4 h before fixation (,). Areas of co-localization of E-cadherin–GFP with E-cadherin–Cherry have been pseudo-coloured in white (white arrowheads). Interfaces between E-cadherin–GFP and E-cadherin–Cherry in EphB-positive/ephrin-B-positive co-cultures are devoid of co-localization (arrows; ,). (–) Analysis of cell sorting in Co115 colorectal cancer cells. Cells expressing EphB3 and labelled with GFP were co-cultured with cells labelled with RFP (,) or cells expressing ephrin-B1 and RFP (,,,). Note the robust cell sorting and compartmentalization of EphB-positive and ephrin-B1-positive cells (,). In these experiments, TAPI-! 1 (10 μM) was added at the moment of seeding. The medium was replaced with fresh inhibitor every 12 h and pictures were taken 48 h after plating (,). () Quantification of cell distribution in Co115 co-cultures. Cells forming homogeneous GFP clusters were quantified and categorized according to cluster size. Shown here is the percentage of cells scattered (that is forming groups of less 10 cells) and the percentage of cells clustered in big groups (in >50 cells per group). The complete distribution of cells in different cluster sizes is shown in Supplementary Fig. S2. Student's t-test was carried out to assess the significance of the effects of TAPI-1 on cell sorting, taking EphB3/ephrin-B1 co-cultures as a reference (see Methods for details). Error bars, s.d. (n=5random fields per condition; ***P<0.0005, *P<0.05 by Student's t-test). The arrows in point to sharp boundaries of EphB3/GFP clusters, whereas the arrowheads in and indicate the lack of well-established ! boundaries between EphB3/GFP and ephrin-B1/RFP populations. Sc! ale bars: 10 μm (–), 5 μm (–), 50 μm (–), 20 μm (–). * Figure 2: ADAM metalloproteinases are required for EphB/ephrin-B-mediated cell sorting. () Normalized raw expression values of ADAM metalloproteinase family members obtained from microarray gene expression analysis of Co115 and DLD-1 colorectal cancer cells. () Quantification of messenger RNA of Adam9, Adam10, Adam15 or Adam17 by quantitative PCR in Co115 EphB3/GFP cells expressing each indicated shRNA. The values are relative to expression levels in control non-targeting shRNA-expressing cells (CV shRNA). Error bars, s.d. () Quantification of unsorted (GFP-positive cells in clusters of <10 cells) and sorted cells (GFP-positive cells forming clusters >50 cells) on co-culture of cells knocked down for ADAM family members. The complete distribution of cells in different cluster sizes is shown in Supplementary Fig. S2. Student's t-test was carried out to evaluate the effects of ADAM downregulation on cell sorting, with EphB3 CV shRNA/ephrin-B1 co-cultures as a reference. Error bars, s.d. (n=5random fields per condition; ***P<0.0005, *P<0.05 by Student's t-test! ). (–) Representative confocal micrographs of cell-sorting experiments using Co115 EphB3/GFP cells expressing shRNAs targeting different ADAM family members (scale bars: 50 μm). (–) Examples of the morphology of EphB3-positive clusters (scale bars: 20 μm). The arrows in ,, point to refined boundaries of EphB3/GFP clusters. The arrowheads in ,, indicate a lack of well-established boundaries between EphB3/GFP and ephrin-B1/RFP populations. * Figure 3: ADAM10 metalloproteinase activity is required for EphB/ephrin-B-mediated cell sorting and E-cadherin remodelling. (–) Representative confocal micrographs of EphB3/GFP control Co115 cells (,,,,,) and EphB3/GFP/ADAM10ΔMP (,,,) co-cultured with RFP cells (,), ephrin-B1 cells (,,,) or ephrin-B1 ADAM10ΔMP cells (,,,). Scale bars: 50 μm (–), 20 μm (–). The arrow in points to the compact morphology of homogeneous EphB-positive cell clusters. The arrowheads in – indicate examples of non-refined boundaries as a result of intermingling of EphB3 and ephrin-B1 cells. () ADAM10 immunodetection by western blotting (WB) of Co115 EphB3/GFP and ephrin-B1/RFP cells expressing an ADAM10ΔMP construct or an empty control vector. Note the two forms of endogenous ADAM10 (pro-protein, p) and mature (m) and the exogenous dominant-negative form (ΔMP) are recognized by the antibody. Western blotting for anti-actin was used as a loading control. () Quantification of cell distribution in the above-mentioned experiments. Mixed (<10 cells per cluster) and sorted (>50 cells per cluster) cells are rep! resented here. The complete distribution of cells in different cluster sizes is shown in Supplementary Fig. S2. Student's t-test was carried out to check the significance of the reduction of cell sorting by expression of ADAM10ΔMP on the different populations, with EphB3/ephrin-B1 co-cultures as a reference. Error bars, s.d. (n=5random fields per condition; **P<0.005, *P<0.05 by Student's t-test). (–) E-cadherin co-localization analysis in MDCK EphB2/E-cadherin–GFP cells (,,,) and EphB2/E-cadherin–GFP/ADAM10ΔMP cells (,,,) co-cultured with ephrin-B1/E-cadherin–Cherry (,,,) or control E-cadherin–Cherry cells (,,,) for 48 h. Areas of co-localization between GFP and Cherry are pseudo-coloured in white in – and represented in yellow in –. The arrowheads in ,,,,, depict areas of co-localization between both fluorophores. The arrows in and point to areas showing lack of cell adhesion at the boundary between EphB2/E-cadherin–GFP and ephrin-B1/E-cadherin–C! herry cells. The open arrowheads mark cell–cell adhesions be! tween cells of the same population. Scale bars: 10 μm. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 4: ADAM10, EphB2 and E-cadherin form a complex that induces shedding of E-cadherin at sites of EphB–ephrin-B interaction. (–) Representative confocal micrographs of in situ zymography of MDCK EphB2 (,,,,,) and EphB2/ADAM10ΔMP cells (,,,,,) co-cultured with E-cadherin–Cherry cells (,,,) or with ephrin-B1/E-cadherin–Cherry cells (–,–) and ephrin-B1/E-cadherin–Cherry culture (,). For these experiments, EphB2-expressing cells were not labelled with E-cadherin–GFP, to avoid interference with visualization of gelatin–FITC. EphB2-positive cells can be distinguished by the lack of E-cadherin–Cherry cell groups, marked in the images by the plus symbol or EphB2+ in dashed circles (scale bars: 20 μm). () EphB3 activation by ephrin-B1 leads to E-cadherin cleavage in an ADAM10-dependent manner. Extracellular (EC) E-cadherin fragments present in the medium of EphB3/GFP and EphB3/GFP/ADAM10ΔMP cells co-cultured with RFP control cells (−) or with ephrin-B1 cells (+) were detected by immunoprecipitation followed by western blotting (top). A band with a relative molecular mass of 80,000 ! (Mr 80(K)) corresponding to extracellular E-cadherin was detected. Bottom, the black bars in the histogram show quantification of bands of three independent experiments as a fold increase in supernatants from each condition relative to the control situation (EphB3/GFP-RFP co-cultures). Total E-cadherin and actin from whole-cell extracts were used as loading controls. The ratio of full-length E-cadherin versus actin in each experiment is represented with grey bars in the histogram (total E-cadherin). Error bars, s.d. (n=3; *P=0.01 by Student's t-test). () Association of EphB2 with ADAM10 and E-cadherin in the SW403 colorectal cancer cell line. Complexes co-immunoprecipitating with endogenous EphB2 were analysed by western blotting (WB) for endogenous ADAM10 and E-cadherin. Goat immunoglobulins were used as mock immunoprecipitate (IP). () EphB2–HA was immunoprecipitated from co-cultures of HEK293T populations expressing E-cadherin–GFP or E-cadherin–Cherry as well as E! phB2–HA or ephrin-B1 as indicated. Top, interaction of EphB2! –HA with E-cadherin–GFP and ADAM10–Flag was analysed (all input lanes belong to the same blot and were cropped to remove irrelevant lanes). Bottom, extracellular proteins were crosslinked using a membrane non-permeable, short-arm crosslinker (DTSSP) to be able to analyse the interaction of EphB2 with E-cadherin expressed by the ephrin-B1 population (E-cadherin–Cherry). ADAM10 pro-protein (p) and mature (m) forms are detected in the western blot. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 5: Transgenic expression of dominant-negative ADAM10 induces mis-positioning of Paneth cells in the intestinal epithelium. () Expression pattern of EphB receptors and ephrin-B1 in the intestinal epithelium. EphB3 is expressed in epithelial cells at the crypt base. EphB2 is present in ISCs and transient amplifying (TA) cells in a decreasing gradient from the base towards the surface. The levels of ephrin-B1 decrease towards the crypt base. (,) Detection of Paneth cells (anti-lysozyme) in wild-type () or EphB3−/− () mice (scale bars: 20 μm). The arrows point to a mis localized Paneth cell and the red arrowheads mark irregular organization of a Paneth cell at the crypt base16. () Vector used to generate transgenic mice expressing Flag-tagged ADAM10ΔMP in Paneth cells. (,) Immunohistochemistry for anti-Flag confirmed expression of the transgene exclusively in Paneth cells (arrowhead in inset). (,) Immunohistochemistry anti-lysozyme in wild-type () and CR2–ADAM10ΔMP () mice. The dotted lines indicate the Paneth cell compartment. The black arrows point to mis positioned Paneth cells and the! red arrowheads to areas of irregular organization of Paneth cell at the crypt base (scale bars: 100 μm). () Quantification of the percentage of mis-positioned Paneth cells in the duodenum of wild-type (WT) and two different CR2–ADAM10ΔMP transgenic mice lines (P=0.02 and <0.000, respectively, by Student's t-test, n=5 ×10 magnification fields counted per condition.) Error bars, s.d. () Quantification of the abundance of differentiated cell types in the intestine of wild-type and CR2–ADAM10ΔMP transgenic mice. Periodic acid Shiff (PAS) staining was used as a marker for goblet cells, synaptophysin (Synapt.) for endocrine cells and Ki67 for proliferative cells (Supplementary Fig. S6). Quantifications of mice derived from founder line #2. (P>0.8in the three cell types, by Student's t-test, n=5 ×10 magnification fields counted per condition). Error bars, s.d. () Model proposed to explain cell sorting induced by EphB/ephrin-B signalling in epithelia. EphB receptors! , E-cadherin and ADAM10 interact at the basolateral membrane. ! EphB–ephrin-B complexes activate ADAM10 locally, which mediates shedding of E-cadherin at the EphB-positive and ephrin-B-positive contacts. Asymmetric E-cadherin at different sides of the cell results in decreased affinity between the two populations (cell sorting). Migration in and out of the ISC niche may be regulated by differential adhesion. AJ,adherens junction; WNT, WNT signalling. Author information * Author information * Supplementary information Affiliations * Oncology program, Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona, Spain * Guiomar Solanas, * Carme Cortina, * Marta Sevillano & * Eduard Batlle * Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys, 23, 08010 Barcelona, Spain * Eduard Batlle * Present address: Center of Regenerative Medicine in Barcelona (CMRB), Dr. Aiguader, 88, 08003 Barcelona, Spain * Carme Cortina Contributions G.S., C.C. and E.B designed the experiments, G.S and C.C. carried out and analysed the experiments, M.S. contributed with technical assistance with histology, G.S. and E.B. prepared the manuscript and E.B. set the conceptual framework and supervised the work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Eduard Batlle Author Details * Guiomar Solanas Search for this author in: * NPG journals * PubMed * Google Scholar * Carme Cortina Search for this author in: * NPG journals * PubMed * Google Scholar * Marta Sevillano Search for this author in: * NPG journals * PubMed * Google Scholar * Eduard Batlle Contact Eduard Batlle Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (3M) Supplementary Information Additional data - RALA and RALBP1 regulate mitochondrial fission at mitosis
- Nat Cell Biol 13(9):1108-1115 (2011)
Nature Cell Biology | Letter RALA and RALBP1 regulate mitochondrial fission at mitosis * David F. Kashatus1 * Kian-Huat Lim1, 3 * Donita C. Brady1, 2 * Nicole L. K. Pershing1 * Adrienne D. Cox2 * Christopher M. Counter1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1108–1115Year published:(2011)DOI:doi:10.1038/ncb2310Received24 March 2011Accepted28 June 2011Published online07 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission1. Equal distribution of mitochondria to daughter cells during mitosis requires fission2. Mitotic mitochondrial fission depends on both the relocalization of the large GTPase DRP1 to the outer mitochondrial membrane and phosphorylation of Ser 616 on DRP1 by the mitotic kinase cyclin B–CDK1 (ref. 2). We now report that these processes are mediated by the small Ras-like GTPase RALA and its effector RALBP1 (also known as RLIP76, RLIP1 or RIP1; refs 3, 4). Specifically, the mitotic kinase Aurora A phosphorylates Ser 194 of RALA, relocalizing it to the mitochondria, where it concentrates RALBP1 and DRP1. Furthermore, RALBP1 is associated with cyclin B–CDK1 kinase activity that leads to phosphorylation of DRP1 on Ser 616. Disrupting either RALA or RALBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and ! a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B–CDK1 converge on RALA and RALBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function. View full text Figures at a glance * Figure 1: Aurora A promotes RALA mitochondrial localization and fission. () HEK-TtH cells stably expressing GFP-tagged RALA, RALAS194D or RALAS194A were incubated with MitoTracker red to visualize mitochondria. GFP–RALA (green) and mitochondria (red) were visualized using confocal microscopy. A merge (yellow, two-dimensional co-localization) and surface rendering (white, three-dimensional co-localization) of the co-localization of GFP–RALA with mitochondria were determined with Imaris software. The percentage of GFP protein co-localized with MitoTracker red was quantified by calculating the total of the intensity sum per object for the co-localized image divided by the total of the intensity sum per object for the GFP image. Scale bars, 5 μm. (–) Immunoblot analysis of RALA and DRP1 levels in highly enriched mitochondrial fractions (mito), whole-cell extracts (WCE) and when investigated, cytoplasmic fractions (cyto) isolated from HEK-TtH cells expressing no transgene (), vector, Aurora AT288D or Aurora AK162R() or scramble or RALA shRNA ! (). Complex Vβ (mitochondria), Calnexin (endoplasmic reticulum), Na+/K+ ATPase (plasma membrane) and Tubulin (cytoplasm) were analysed to assess purity. Representative of three experiments. (–) Micrographs; mitochondrial morphology visualized by MitoTracker red staining of HEK-TtH cells expressing the indicated shRNAs and/or transgenes. Scale bars, 5 μm. The bottom images show magnifications of the areas outlined in the top images. Histograms; quantification of the percentage of cells (mean±s.d.) exhibiting highly fragmented (black), intermediate (grey) or highly interconnected (white) mitochondrial morphologies from three independent experiments (>100 cells). () Mitochondrial network connectivity in HEK-TtH cells expressing scramble or RALA shRNA and transfected with mito-YFP. The normalized and photobleach-corrected mobile fractions represent the mean±s.e.m. of 30 individual FRAP curves (**P=2.02×10−8). Uncropped images of blots are shown in Supplementary Fig! . S4. * Figure 2: RALBP1 promotes mitochondrial fragmentation. (–) Immunoblot analysis of RALA, RALBP1 or DRP1 levels in highly enriched mitochondrial fractions (mito) and whole-cell extracts (WCE) isolated from HEK-TtH cells expressing vector, Aurora AT288D or Aurora AK162R (), scramble control, RALA shRNA or RALBP1 shRNA (), Aurora AT288D plus either scramble control or RALBP1 shRNA complemented with vector (V) or shRNA-resistant RALBP1 (BP1) () or Myc–RALBP1–RALAS194A or Myc–RALBP1–RALAS194D (). Complex Vβ (mitochondria) was analysed to assess purity. Representative of three experiments. Histogram in ; quantification of the mean RALBP1 signal intensity from three experiments ±s.d. (–) Micrographs: mitochondrial morphology visualized by MitoTracker red staining of HEK-TtH cells (), HEK-TtH cells expressing active Aurora AT288D and a scramble sequence or RALBP1 shRNAs alone or in conjunction with shRNA-resistant wild-type RALBP1 () or HEK-TtH cells expressing Myc–RALBP1–RALAS194A or Myc–RALBP1–RALAS194D (). The bo! ttom images show magnifications of the areas outlined in the top images. Scale bars, 5 μm (), 25 μm (,). Histograms: quantification of the percentage of cells (mean±s.d.) exhibiting highly fragmented (black), intermediate (grey) or highly interconnected (white) mitochondrial morphologies from three independent experiments (>100 cells). () Overlap (merge, yellow) of the distribution of GFP–RALBP1–RALAS194A or GFP–RALBP1–RALAS194D, as detected by immunofluoresence microscopy of GFP (green) and MitoTracker red-positive mitochondria (red) in HEK-TtH cells. Surface rendering of the co-localization of GFP–RALBP1–RALA fusion proteins with mitochondria (white) was determined with Imaris software. The percentage of GFP protein co-localized with MitoTracker red was quantified by calculating the total of the intensity sum per object for the co-localized image divided by the total of the intensity sum per object for the GFP image. Scale bars, 5 μm for S194D, 3 �! �m for S194A. Uncropped images of blots are shown in Supplemen! tary Fig. S4. * Figure 3: RALA and RALBP1 promote mitochondrial localization of DRP1. () Extracts isolated from HeLa cells expressing Myc–RALA, either unsynchronized (U) or synchronized in mitosis (M) using a double-thymidine block were analysed by immunoblotting for levels of Myc–RALA and cyclin B or subjected to immunoprecipitation (IP) with antibodies against Myc and analysed by immunoblotting for levels of Myc–RALA or phospho-Ser-194-RALA. Representative of three experiments. () Immunoblot analysis of RALA, RALBP1 and DRP1 levels in mitochondrial fractions (mito) and whole-cell extracts (WCE) isolated from HeLa cells either unsynchronized (U) or synchronized in mitosis (M) using a double-thymidine block. Representative of three experiments. (–) Immunoblot analysis of RALA, RALBP1, DRP1, cyclin B, Aurora A and MFF1 levels in crude mitochondrial fractions (mito) and whole-cell extracts (WCE) isolated from HeLa cells, either unsynchronized (U) or synchronized in mitosis (M) using a double-thymidine block, and expressing scramble control, RALA shRNA o! r RaBP1 shRNA (), doxycycline-inducible scramble control or Aurka shRNA (), scramble control or MFF1 shRNA () or PLK1 or Fis1 shRNA (). Representative of three experiments. Uncropped images of blots are shown in Supplementary Fig. S4. * Figure 4: RALBP1 promotes phosphorylation of DRP1. () Blots: immunoblot analysis of Ser-616-phosphorylated DRP1 (p-S616-DRP1), DRP1, RALA, RALBP1 and actin levels in HeLa cells expressing a scramble control, RALA shRNA or RALBP1 shRNA. Histogram: quantification of the mean p-S616-DRP1 signal intensity measured by ImageJ from three experiments ±s.d. (**P=0.008). () Blots: extracts isolated from HeLa cells, unsynchronized (U) or synchronized in mitosis (M) using a double-thymidine block, were either analysed by immunoblotting for levels of RALA, DRP1, cyclin B or Ser-616-phosphorylated DRP1, or subjected to immunoprecipitation (IP) with either no antibody or antibodies against cyclin B or RALBP1, followed by incubation with GST–DRP1 and [γ-32P]ATP, and resolved on an acrylamide gel. γ-32P-labelled GST–DRP1 was visualized by a phosphorimager and total GST–DRP1 was visualized by Coomassie brilliant blue staining. Histograms: quantification of the mean [γ-32P]GST–DRP1 signal intensity measured by ImageJ from three exp! eriments ±s.d. (*P=0.028 for cyclin B, 0.027 for RALBP1). () Blots: GST–DRP1 (200 ng) and GST–cyclin B (CycB)–CDK1 (25 ng) were incubated with increasing amounts of either GST–RALBP1 or GST (0, 125, 250, 500, 1,000 ng) in addition to either ATP or [γ-32P]ATP. Reactions were resolved by SDS–PAGE and either subjected to immunoblotting for DRP1, RALBP1 and Ser 585-phosphorylated DRP1 (cold kinase assay) or visualized by phosphorimager (hot kinase assay). GST, GST–DRP1 and GST–RALBP1 were visualized by Coomassie brilliant blue staining (CBB). Histogram: quantification of the mean fold-change in p-S616-DRP1 signal intensity as measured by ImageJ from three experiments ±s.d. (*P=0.020 (bar 3), *P=0.028 (bar 4), **P=0.009). () Immunoblot analysis of RALA, RALBP1, DRP1 and cyclin B levels in crude mitochondrial fractions (mito) and whole-cell extracts (WCE) isolated from HeLa cells synchronized in mitosis (M) using a double-thymidine block, treated with DSP a! nd subjected to immunoprecipitation with an anti-RALA antibody! . Uncropped images of blots are shown in Supplementary Fig. S4. * Figure 5: Knockdown of RALA or RALBP1 prevents mitosis-induced mitochondrial fission. () Mitochondrial morphology as visualized by live-cell imaging of HeLa cells stably expressing mito-RFP and scramble control, RALA shRNA or RALBP1 shRNA after release from a double-thymidine block. Arrows (1, 2) indicate regions of mitochondria retained during telophase and (3, 4) unequal distribution of mitochondria to daughter cells. Scale bars, 10 μm. () Mitochondrial morphology visualized by MitoTracker red staining of HeLa cells expressing a scramble control, RALA shRNA or RALBP1 shRNA at the indicated phases of mitosis on being synchronized in mitosis using a double-thymidine block. DAPI staining was used to assign mitotic phase. For the MitoTracker micrographs, the bottom images show magnifications of the areas outlined in the images above. Scale bars, 25 μm for scramble interphase and metaphase and RALA interphase and prophase, 10 μm for scramble anaphase and telophase and RALBP1 interphase and 7.5 μm for all other images. () Mitochondrial morphology of H! eLa cells expressing RALA shRNA complemented with GFP-tagged, shRNA-resistant RALA in the wild-type or S194A mutant configuration and captured at metaphase (DAPI). Scale bars, 10 μm. () Quantification of the percentage of cells (n≥30, mean±s.d., representative of three independent experiments) exhibiting mitochondrial elongation during metaphase (*P≤0.05, **P≤0.01). () ATP production of HeLa cells stably expressing scramble, RALA or RalPB1 shRNA was determined by measuring ATP-dependent luciferase activity measured at 560 nm on a Victor (ref. 3) luminometer. (n=12, mean±s.d., representative of three independent experiments, **P=1.6×10−7 for RALA, 5.3×10−7 for RALBP1.) () Proliferation of HeLa cells stably expressing scramble (diamond) RALA (square) or RALBP1 (triangle) as measured by an MTT assay. Measurements were carried out over three days using cells with fewer than 5 or greater than 20 population doublings following selection for the transgene. (n≥! 16, mean±s.d., representative of three independent experiment! s; day 2: **P=4.5×10−5 for RALA, 6.3×10−5 for RALBP1, day 3: **P=4.5×10−8 for RALA, 2.7×10−6 for RALBP1.) Author information * Author information * Supplementary information Affiliations * Department of Pharmacology and Cancer Biology, Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710, USA * David F. Kashatus, * Kian-Huat Lim, * Donita C. Brady, * Nicole L. K. Pershing & * Christopher M. Counter * Department of Pharmacology and Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA * Donita C. Brady & * Adrienne D. Cox * Present address: Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA * Kian-Huat Lim Contributions Initial pilot experiments were carried out by K-H.L. and expanded on by D.F.K., D.C.B. and N.L.K.P. All authors contributed to the study design. The manuscript was written by D.F.K. and C.M.C. with contributions by K-H.L., D.C.B., N.L.K.P. and A.D.C. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Christopher M. Counter Author Details * David F. Kashatus Search for this author in: * NPG journals * PubMed * Google Scholar * Kian-Huat Lim Search for this author in: * NPG journals * PubMed * Google Scholar * Donita C. Brady Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole L. K. Pershing Search for this author in: * NPG journals * PubMed * Google Scholar * Adrienne D. Cox Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher M. Counter Contact Christopher M. Counter Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (4M) Supplementary Information * Supplementary Movie 2 (600K) Supplementary Information * Supplementary Movie 3 (5M) Supplementary Information * Supplementary Movie 4 (7M) Supplementary Information * Supplementary Movie 5 (3M) Supplementary Information * Supplementary Movie 6 (4M) Supplementary Information PDF files * Supplementary Information (4M) Supplementary Information Additional data - Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations
- Nat Cell Biol 13(9):1116-1123 (2011)
Nature Cell Biology | Letter Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations * Danilo Ritz1, 2, 7 * Maja Vuk1, 2, 7 * Philipp Kirchner1 * Monika Bug1 * Sabina Schütz2 * Arnold Hayer2 * Sebastian Bremer1 * Caleb Lusk3 * Robert H. Baloh3 * Houkeun Lee4 * Timo Glatter4, 5 * Matthias Gstaiger4, 5 * Ruedi Aebersold4, 5, 6 * Conrad C. Weihl3 * Hemmo Meyer1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1116–1123Year published:(2011)DOI:doi:10.1038/ncb2301Received25 March 2011Accepted21 June 2011Published online07 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The AAA-ATPase VCP (also known as p97) cooperates with distinct cofactors to process ubiquitylated proteins in different cellular pathways1, 2, 3. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear4, 5. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds to the plasma membrane protein caveolin-1 (CAV1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP–UBXD1 targets mono-ubiquitylated CAV1 in SDS-resistant high-molecular-weight complexes on endosomes, which are en route to degradation in endolysosomes6. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of CAV1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific caveolin-3 accumulates in sarcoplasmic pools and specifically delocalizes! from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitylated cargo in the endocytic pathway and indicate that impaired trafficking of caveolin may contribute to pathogenesis in individuals with VCP mutations. View full text Figures at a glance * Figure 1: The VCP–UBXD1 chaperone complex binds to caveolin, and this interaction is specifically disrupted by IBMPFD-associated mutations in VCP. () Mass spectrometry-based strategy to identify differences in the interaction pattern of wild-type VCP, the disease-associated mutant RH or the ATPase-deficient mutant EQ. Left, Myc/Strep-tagged VCP variants were doxycycline (DOX)-induced and tandem isolated from stable HEK293 cell lines. Right, associated proteins were detected in a shotgun approach and high-probability (≥99%) candidates are represented in a Venn diagram. For full details, see Supplementary Table S1. FRT, flippase recognition target. () Western blot (WB) confirming differential interaction of CAV1 and UBXD1 with overexpressed VCP variants. The p47 cofactor served as a control. RH1 and RH2 represent two independent cell lines. The arrows indicate endogenous and overexpressed VCP. Consistent results for the R95G and A232E disease mutants are shown in Supplementary Fig. S1c. () Immunoprecipitation (IP) of HA-tagged UBXD1 from cells expressing wild-type VCP or VCPRH. Note the decreased level of binding of VC! PRH (upper arrow) and CAV1 in the VCPRH background. The asterisk indicates the position of the antibody light chain. () Immunoprecipitations of indicated endogenous VCP cofactors with specific antibodies. Note that UBXD1 specifically binds to VCP and CAV1, and is devoid of the p47 or UFD1 cofactors. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 2: VCP targets mono-ubiquitylated CAV1 in SDS-resistant oligomers. () Ubiquitin modification of CAV1 was detected with the anti-ubiquitin FK2 or the anti-Myc antibodies after CAV1–Myc expression and isolation with the CAV1-specific N20 antibody. Note the prominent mono-ubiquitylation (Ub1) and minor multi- and/or polyubiquitylation (Ubn). () Sequential immunoprecipitations show that VCP binds to mono-ubiquitylated CAV1 (arrow). First, doxycycline (DOX)-induced VCP was isolated from cells expressing HA–ubiquitin and CAV1–Myc. Then, 5% of it was loaded (first IP), the remaining 95% was denatured to remove associated proteins and CAV1 was re-isolated with the specific N20 antibody (second IP). Direct immunoprecipitation from denatured lysates served as a reference. The asterisk and the cross indicate the positions of the antibody heavy and light chains, respectively. () Wild-type CAV1–HA or the K*R mutant, whose ubiquitylation is abolished owing to mutation of all 12 lysine residues to arginine, was expressed, and association with VCP ! and UBXD1 was analysed by CAV1–HA immunoprecipitation. () VCP preferentially targets SDS-resistant CAV1 oligomers. Wild-type VCP and VCPRH complexes were isolated from CV-1 cells and separated in SDS gels for western blotting (WB) without prior boiling. CAV1 monomers and SDS-resistant oligomers were detected in inputs. Note the specific enrichment of oligomers in VCP complexes. () The disease-associated and oligomerization-deficient CAV1P132L mutant was overexpressed with HA-tag and immunoprecipitated. Note the decreased level of VCP binding. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 3: Overexpression of VCP mutants or depletion of UBXD1 affect CAV1 transport to endolysosomes. () CAV1 and UBXD1 co-localize on early and late endosomes. UBXD1–mCherry and CAV1–GFP were transiently expressed and visualized in live U2OS cells by spinning-disc confocal microscopy. Arrowheads indicate compartments showing co-localization. Manders' co-localization coefficient was 0.955, compared with 0.079 for mCherry alone (not shown), n=16 cells per condition. See Supplementary Fig. S2a for co-localization with endosome markers. Scale bar, 10 μm. The insets in , and show magnifications of the areas outlined in the main panels. () Expression of wild-type VCP, VCPEQ or VCPRH variants was induced by doxycycline for 24 h in stable U2OS cells transiently expressing CAV1–RFP and LAMP1–GFP. Live cells were visualized by spinning-disc confocal microscopy. The arrowheads indicate CAV1 localizing to late endosome/lysosomes. The arrows indicate CAV1 on enlarged late endosomes. Scale bars, 10 μm. () The number of enlarged LAMP1-positive vesicles (>0.8 μm diamet! er) with CAV1 at the limiting membrane (error bars represent s.d., three independent experiments, >140 cells per condition). () Electron micrographs of stable U2OS cells expressing wild-type VCP or VCPRH. Arrows indicate MVBs. The arrowhead indicates a single intraluminal vesicle. The bottom images are magnifications of the areas outlined in the top images. Scale bars, 500 nm. () Quantification, number of MVBs per square micrometre of cytoplasm. Error bars represent s.d., n=10 cells per condition. **, P<0.05. () Cellular depletion of UBXD1 affects endosomal sorting of CAV1. U2OS cells were transfected with control or UBXD1 siRNA oligonucleotides. CAV1–RFP and RAB5–GFP were visualized in live cells by confocal microscopy. Scale bars, 5 μm. () Number of enlarged CAV1/RAB5-positive vesicles (>1 μm diameter; error bars represent s.d., four independent experiments, >130 cells per condition). () After imaging, cells were lysed and depletion efficiency was confirmed by! western blot (WB) analysis. The asterisk marks the position o! f a non-specific band. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 4: Chemical inhibition of VCP with DBeQ impairs CAV1 trafficking and delays degradation of the EGFR. () Left, laser scanning confocal sections of live U2OS cells transiently expressing CAV1–GFP and LAMP1–RFP after treatment with 10 μM DBeQ or solvent alone (dimethylsulphoxide, DMSO) for 6 h. The insets in the micrographs in and show magnifications of the areas outlined in the main panels. Scale bars, 5 μm. Right, the number of enlarged LAMP1-positive vesicles with CAV1 at the limiting membrane (error bars represent s.d., three independent experiments, >100 cells per condition). () Left, spinning-disc confocal sections of endogenous CAV1 and LAMP1 immunolocalization after treatment as in . Scale bars, 3 μm. Right, quantification of the fraction of enlarged LAMP1-positive vesicles (>0.5 μm diameter) with CAV1 accumulation (error bars represent s.d., n=30 cells per condition). () EGFR and LAMP1 were immunolocalized in CV-1 cells at the indicated times after EGF stimulation. DBeQ treatment, 10 μM for 5 h. Scale bars, 10 μm. () Top, western blot analysi! s of the EGFR in HEK293 cells at indicated times after EGF stimulation with or without DBeQ treatment as in . Bottom, quantification of electrochemiluminescence signals on film. error bars represent s.d., n=5. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 5: Mislocalization of caveolin in fibroblasts and muscle tissue of IBMPFD patients. () Cultured skin fibroblasts from two healthy donors, two patients with sALS and three IBMPFD patients were fixed, immunostained with antibodies to CAV1 and LAMP2 and imaged by epifluorescence microscopy. Only cells of one healthy control, and of IBMPFD patients no. 2 (pt no. 2, harbouring the VCPR155H mutation) and no. 8 (L198W mutation) are shown. See Supplementary Table S2 for patient demographics. Scale bars, 10 μm. () Percentage of cells in with more then one CAV1/LAMP2-positive vesicle. Average and s.d. (error bars) are from four control, and four IBMPFD cell isolates (two of pt no. 2, denoted 2-1 and 2-2, one of no. 7 and no. 8 each), respectively, with >100 cells each. Significance (t-test) is indicated. () Muscle tissue sections of a healthy individual and IBMPFD patients. Immunohistochemistry as indicated. Note the accumulation of CAV3 with LAMP2 in the sarcoplasm (arrowheads) and specific delocalization of CAV3 from the sarcolemma (arrows). The bottom right ima! ge shows a magnification of the area outlined in the bottom left image. See Supplementary Fig. S4 for localization of unaffected control sacrolemmal proteins and additional patient samples. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Danilo Ritz & * Maja Vuk Affiliations * Centre for Medical Biotechnology, University of Duisburg-Essen, 45117 Essen, Germany * Danilo Ritz, * Maja Vuk, * Philipp Kirchner, * Monika Bug, * Sebastian Bremer & * Hemmo Meyer * Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland * Danilo Ritz, * Maja Vuk, * Sabina Schütz, * Arnold Hayer & * Hemmo Meyer * Department of Neurology, Washington University School of Medicine, Saint Louis, Missouri 63110, USA * Caleb Lusk, * Robert H. Baloh & * Conrad C. Weihl * Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland * Houkeun Lee, * Timo Glatter, * Matthias Gstaiger & * Ruedi Aebersold * Competence Center for Systems Physiology and Metabolic Diseases, ETH Zurich, 8093 Zurich, Switzerland * Timo Glatter, * Matthias Gstaiger & * Ruedi Aebersold * Faculty of Science, University of Zurich, 8006 Zurich, Switzerland * Ruedi Aebersold Contributions D.R. generated cells, isolated VCP complexes and carried out the biochemical analyses with help from P.K., S.S. and S.B., and M.V. and M.B. carried out microscopy. A.H. helped design experiments and carried out the co-localization analysis. H.L, T.G., M.G. and R.A. carried out mass spectrometry analysis. C.L., R.H.B. and C.C.W. carried out electron microscopy and analysis of patient material. H.M. conceived the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Conrad C. Weihl or * Hemmo Meyer Author Details * Danilo Ritz Search for this author in: * NPG journals * PubMed * Google Scholar * Maja Vuk Search for this author in: * NPG journals * PubMed * Google Scholar * Philipp Kirchner Search for this author in: * NPG journals * PubMed * Google Scholar * Monika Bug Search for this author in: * NPG journals * PubMed * Google Scholar * Sabina Schütz Search for this author in: * NPG journals * PubMed * Google Scholar * Arnold Hayer Search for this author in: * NPG journals * PubMed * Google Scholar * Sebastian Bremer Search for this author in: * NPG journals * PubMed * Google Scholar * Caleb Lusk Search for this author in: * NPG journals * PubMed * Google Scholar * Robert H. Baloh Search for this author in: * NPG journals * PubMed * Google Scholar * Houkeun Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Timo Glatter Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Gstaiger Search for this author in: * NPG journals * PubMed * Google Scholar * Ruedi Aebersold Search for this author in: * NPG journals * PubMed * Google Scholar * Conrad C. Weihl Contact Conrad C. Weihl Search for this author in: * NPG journals * PubMed * Google Scholar * Hemmo Meyer Contact Hemmo Meyer Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Excel files * Supplementary Table 1 (20K) Supplementary Information * Supplementary Table 2 (10K) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Actin dynamics counteract membrane tension during clathrin-mediated endocytosis
- Nat Cell Biol 13(9):1124-1131 (2011)
Nature Cell Biology | Letter Actin dynamics counteract membrane tension during clathrin-mediated endocytosis * Steeve Boulant1, 2, 4 * Comert Kural1, 2, 4 * Jean-Christophe Zeeh1, 2 * Florent Ubelmann3 * Tomas Kirchhausen1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1124–1131Year published:(2011)DOI:doi:10.1038/ncb2307Received21 January 2011Accepted27 June 2011Published online14 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between 'actin dependent and 'actin independent'. We also find that light-cha! in-bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding. View full text Figures at a glance * Figure 1: Formation of endocytic coated pits and vesicles at the apical and basolateral surfaces of polarized MDCK cells. () Imaging procedures used to visualize the dynamics of pit formation at the apical and basolateral surfaces of polarized MDCK cells. Movies from the dome-like apical surface are from 3D time series acquired at 2 s intervals from 3–5 serial optical sections spaced by 0.5 μm and using 100 ms exposures; 2D time series were created from maximum-intensity z-projection sets. Movies from the basolateral surface are 2D time series acquired at 2 s intervals from a single optical section. () Snapshot from a maximum-intensity projection (left) and representative kymograph (right) of coated-pit formation at the apical and basolateral surfaces from the same polarized MDCK cell; AP-2 labelled with σ2–eGFP; scale bars, 5 μm. () Average fluorescence intensity of AP-2 structures forming at the apical and basolateral surfaces of polarized MDCK cells (n=3), normalized to the lifetime of each individual pit analysed (percentage of lifetime). Each point represents average ± ! s.d. () Scatter plot of individual lifetimes of coated structures from seven polarized MDCK cells. Each data set represents average ±s.d. n is the number of objects analysed. Statistical significances for lifetime differences are shown. () Scatter plot of individual maximum fluorescence intensities for coated structures. () Scatter plots of lifetimes for individual AP-2 spots at the apical and basolateral surfaces of polarized MDCK cells stably expressing σ2–eGFP in the absence or presence of jasplakinolide or latrunculin A. The upper and lower data sets are from distinct time series of 10 and 2.5 min in duration, respectively. Bottom, fraction of AP-2 objects with longer duration than the time series (percentage of arrested pits). () Morphological analysis of clathrin-coated structures on the apical surface of polarized MDCK cells treated with jasplakinolide, latrunculin A, SecinH3 or secramine A. Representative electron microscopy images of the most abundant clathri! n-coated-pit profiles. Scale bars, 100 nm. The coated struct! ures were classified as shallow, U-shaped or nearly-mature Ω- and fully-mature O-shaped vesicles. () Relative frequency of profiles in about 45 cells per condition. * Figure 2: Disruption of apical coat formation by pharmacological interference with the small GTPases Rac1 and Arf6 and interference with the function of clathrin light chains. () Top, scatter plots of lifetimes for individual AP-2 spots. Polarized MDCK cells stably expressing σ2–eGFP were treated for 10 min before imaging with secramine A, NSC-23766 or SecinH3, small molecule inhibitors of Cdc42, Rac1 and Arf6, respectively. Each data set represents average ±s.d. for objects whose duration was fully included in the time series. n is the number of objects analysed. Statistical significances for the differences in lifetimes are shown. Bottom, fraction of AP-2 objects with longer duration than the time series. () Top, scatter plots of lifetimes for individual AP-2 spots imaged at the apical and basolateral (Baso) surfaces of polarized MDCK cells stably expressing σ2–eGFP and transiently expressing wild-type or mutant clathrin light chain B fused to Cherry (left) or depleted of both clathrin light chains by Clta and Cltb siRNA (right). Each data set represents average ±s.d. n is the number of objects analysed. Bottom, fraction of AP-2 object! s with longer duration than the 160 s time series. * Figure 3: Actin dependence for endocytic coat formation in cells swelled by hypo-osmotic treatment. Polarized MDCK or non-polarized BSC1 cells incubated for 10 min in serially diluted medium (from 100% to 50%) of decreasing osmolarity, ranging from 311 to 174 mOsM, were analysed for the effects of altering actin dynamics on the lifetimes of their AP-2-coated structures. Each data set represents average ± s.d.; n is the number of objects analysed.() Top, schematic representation of polarized MDCK cells stably expressing σ2–eGFP exposed to hypo-osmotic medium. Middle, scatter plots of lifetimes for individual basolateral AP-2 spots of cells exposed for 10 min to hypo-osmotic media in the absence and presence of jasplakinolide (Jasp). Bottom, fraction of AP-2 objects with longer duration than the time series. () Top, schematic representation of BSC1 cells stably expressing σ2–eGFP exposed to hypo-osmotic medium. Middle, scatter plots of lifetimes for individual ventral AP-2 spots of cells exposed for 10 min to hypo-osmotic media in the absence and presence of j! asplakinolide. Bottom, fraction of AP-2 objects with longer duration than the time series. * Figure 4: Actin dependence of endocytic coat formation in mechanically stretched cells. () Schematic representation of the device used to image mechanically stretched, non-polarized MDCK cells. An optically clear, stretchable silicon holder made of PDMS, about 50 μm thick, which could make contact with the oil above a ×63 objective lens was placed at the bottom of the stretching device. Cells were grown for 24 h on the PDMS surface pre-coated with fibronectin and then imaged by spinning-disc confocal microscopy; the spherical-aberration-correction device was essential for detecting the diffraction-limited coated structures containing AP-2–eGFP. () Dynamics of coated pits before and after ~25% linear stretching. Top, scatter plots for lifetimes of individual AP-2 spots from the ventral surface of cells subjected to controlled stretching in the presence or absence of jasplakinolide. Each data set represents average±s.d. n is the number of objects analysed. Bottom, fraction of AP-2 objects with longer duration than the time series. * Figure 5: Model depicting the role of actin polymerization during the formation of endocytic clathrin-coated pits. () Under non-stringent conditions and low membrane tension, assembly of the clathrin coat is sufficient to deform the membrane into a tightly constricted coated pit. () Under more stringent conditions of high membrane tension, clathrin assembly is not sufficient and membrane invagination stalls; actin polymerization then provides the additional work needed to complete membrane bending. Hip1R links the assembling clathrin coat to actin polymers, and if sufficient time is allowed, then assembly of short-branched actin rescues the stalled coat. The two main forces resisting membrane deformation are bending and tension. The bending work per unit area depends inversely on the curvature and hence is uniform for a spherical or nearly spherical vesicle. The work done against a constant membrane tension depends on the net increase in membrane area—that is, the area of the invaginated membrane minus the area of the opening it covers. The plot represents the cumulative work required ! to counteract membrane tension, and does not include the work required to create the membrane vesicle. The cumulative work was calculated according to W=πr2T(1−cosα)2, where Tis the membrane tension and α is the angle (in radians) between the pole of the budding pit and the position at which the curved pit intersects the plane of the plasma membrane (see Methods). When the neck begins to constrict (α=π/2), the area of the opening decreases and the net increase in area rises sharply. When the pit is complete, α=π. () Hip1R links actin filaments with the clathrin coat by interactions with F-actin and clathrin light chains. Branched actin filaments grow towards the plasma membrane through new filament assembly at the barb end of the stabilized actin filaments; this growth relies on Arp2/3 stimulation, mediated by cortactin and by small GTPases such as Arf6 and Rac1. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Steeve Boulant & * Comert Kural Affiliations * Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA * Steeve Boulant, * Comert Kural, * Jean-Christophe Zeeh & * Tomas Kirchhausen * Immune Disease Institute and Program in Cellular and Molecular Medicine at Children's Hospital, Boston, Massachusetts 02115, USA * Steeve Boulant, * Comert Kural, * Jean-Christophe Zeeh & * Tomas Kirchhausen * Laboratoire de Morphogénèse et Signalisation Cellulaires, Institut Curie Paris, Paris, Cedex 05, France * Florent Ubelmann Contributions S.B., C.K. and T.K. designed experiments; S.B. and C.K. carried out experiments; S.B. and C.K. analysed data; C.K. developed the analytical tools to analyse the 3D data. F.U. provided expression vectors specific for the experiments with villin-1/villin-2, contributed to the experimental design of the experiments involved in villin-1/villin-2 depletion and created the LLCPK1 cell line. S.B. and T.K. wrote the manuscript. All authors discussed the results and contributed to the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tomas Kirchhausen Author Details * Steeve Boulant Search for this author in: * NPG journals * PubMed * Google Scholar * Comert Kural Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Christophe Zeeh Search for this author in: * NPG journals * PubMed * Google Scholar * Florent Ubelmann Search for this author in: * NPG journals * PubMed * Google Scholar * Tomas Kirchhausen Contact Tomas Kirchhausen Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (12M) Supplementary Information * Supplementary Movie 2 (10M) Supplementary Information * Supplementary Movie 3 (8M) Supplementary Information PDF files * Supplementary Information (8M) Supplementary Information Additional data - aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division
- Nat Cell Biol 13(9):1132-1138 (2011)
Nature Cell Biology | Letter aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division * Matilde Galli1 * Javier Muñoz2, 3 * Vincent Portegijs1 * Mike Boxem1 * Stephan W. Grill4, 5 * Albert J. R. Heck2, 3 * Sander van den Heuvel1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1132–1138Year published:(2011)DOI:doi:10.1038/ncb2315Received16 February 2011Accepted05 July 2011Published online21 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The position of the mitotic spindle controls the plane of cell cleavage and determines whether polarized cells divide symmetrically or asymmetrically1, 2, 3. In animals, an evolutionarily conserved pathway of LIN-5 (homologues: Mud and NuMA), GPR-1/2 (homologues: Pins, LGN, AGS-3) and Gα mediates spindle positioning, and acts downstream of the conserved PAR-3–PAR-6–aPKC polarity complex1, 2, 3, 4, 5, 6. However, molecular interactions between polarity proteins and LIN-5–GPR–Gα remain to be identified. Here we describe a quantitative mass spectrometry approach for in vivo identification of protein kinase substrates. Applying this strategy to Caenorhabditis elegans embryos, we found that depletion of the polarity kinase PKC-3 results in markedly decreased levels of phosphorylation of a cluster of four LIN-5 serine residues. These residues are directly phosphorylated by PKC-3 in vitro. Phospho-LIN-5 co-localizes with PKC-3 at the anterior cell cortex and temporally co! incides with a switch from anterior- to posterior-directed spindle movements in the one-cell embryo. LIN-5 mutations that prevent phosphorylation increase the extent of anterior-directed spindle movements, whereas phosphomimetic mutations decrease spindle migration. Our results indicate that anterior-located PKC-3 inhibits cortical microtubule pulling forces through direct phosphorylation of LIN-5. This molecular interaction between polarity and spindle-positioning proteins may be used broadly in cell cleavage plane determination. View full text Figures at a glance * Figure 1: PKC-3 aPKC directs LIN-5 phosphorylation in vivo. () Analysis of LIN-5 immunoprecipitations from embryo lysates by tandem mass spectrometry revealed 25 phosphorylated residues. () Schematic overview of the quantitative mass spectrometry approach to determine whether LIN-5 phosphorylation is controlled by PKC-3 and PAR-1. () Log2 ratios for all of the quantified 15N/14N peptide pairs as a function of their mass spectrometry intensities in the three LIN-5 immunoprecipitates. LIN-5 phosphopeptides are represented in red, and LIN-5 regular peptides are represented in blue. Peptides belonging to other proteins are shown in grey. Peptide intensities were calculated using the average of the 14N and 15N extracted ion chromatograms. () Example of a downregulated LIN-5 phosphopeptide in the 14N pkc-3(RNAi)/15N gfp(RNAi) experiment. Both precursor ions are shown, in blue (14N) and red (15N) colours (25 nitrogen atoms of mass difference). The inset shows the corresponding extracted-ion chromatograms for both species. MS, mass spectrome! tric, RT, retention time. Th, Thomson (unit of mass-to-charge ratio). () Annotated tandem mass spectrometry spectrum resulting from the collision-induced dissociation fragmentation of the 14N peptide, indicating the presence of a phosphorylated Ser 737 residue. Additional information including post-translational modification scores can be found in Supplementary Tables S1 and S2. * Figure 2: PKC-3 phosphorylates LIN-5 at the anterior cortex of the one-cell embryo. () In vitro kinase assay with recombinant GST–LIN-5 and GFP immunoprecipitations (IP) from control (wild type) embryos or embryos expressing PAR-6::GFP, treated with or without pkc-3 RNAi. Top, autoradiogram; bottom, Coomassie brilliant blue (CBB)-stained gel. () Fluoresence micrographs, images of wild-type, lin-5(RNAi) and pkc-3(RNAi)C. elegans embryos at metaphase of the first division stained with anti-pS737 LIN-5 antibody (red) and anti-tubulin (green). Scale bar, 10 μm. The images on the right show a cortical region at higher magnification and an intensity plot of pS737 staining at the anterior versus posterior. () Quantification of cortical enrichment (relative to cytoplasm) of total LIN-5 (left) and LIN-5 pS737 (right) at different stages of mitosis: prometaphase/metaphase (LIN-5, n=6; pS737, n=11), anaphase (LIN-5, n=13; pS737, n=7) and telophase (LIN-5, n=12; pS737, n=2). See also Supplementary Figs S2 and S3. Error bars, s.e.m. at the 95% confidence level. * Figure 3: PKC-3 phosphorylates LIN-5 in mitosis and requires CDK-1. One-cell embryos at different stages after pronuclear migration immunostained for pS737-LIN-5 (red) and α-tubulin (green). () Wild-type embryos. () air-1(RNAi) embryos. () cdk-1(RNAi) embryos. Scale bars, 10 μm. * Figure 4: PKC-3-mediated phosphorylation of LIN-5 directs mitotic spindle positioning in the one-cell embryo. () Pronuclear centration in wild-type, lin-5(RNAi) or lin-5 phospho-mutant embryos treated with lin-5 RNAi to deplete endogenous LIN-5. DIC images are taken just before NEB. The arrowheads indicate the centrosome position. The right panels show trajectories of pronuclei from pronuclear meeting to NEB. Scale bar, 10 μm. () Quantification of the extent of centration, with the average position indicated as a percentage of embryo length (0% anterior and 100% posterior) and the standard deviation (wild type, n=10; lin-5 RNAi, n=12; lin-5 RNAi+LIN-5 wild type, n=15; lin-5 RNAi+LIN-5 4A, n=7; lin-5 RNAi+LIN-5 4E, n=9). The dotted line shows the average position of centration in wild-type embryos. () Spindle movements during division in wild-type, lin-5(RNAi) or lin-5 phospho-mutant embryos treated with lin-5 RNAi to deplete endogenous LIN-5. DIC images were taken just after completion of the first division. The arrowheads indicate a round anterior centrosome; the asterisks repre! sent the flattened posterior centrosome. Note that in LIN-5 4A mutants both centrosomes are flattened, whereas in LIN-5 4E mutants both centrosomes are round. The right panels show centrosome trajectories of embryos from NEB to completion of cytokinesis. Scale bar, 10 μm. () Quantification of the cleavage plane in different embryos, with the average position indicated as a percentage of embryo length (0% anterior and 100% posterior) and the standard deviation (wild type, n=10; lin-5 RNAi, n=12; lin-5 RNAi+LIN-5 wild type, n=15; lin-5 RNAi+LIN-5 4A, n=7; lin-5 RNAi+LIN-5 4E, n=9). The dotted line shows the average position of cleavage in wild-type embryos. () Mean peak velocities (μm s−1) of anterior and posterior spindle poles measured in a 10 s time frame after spindle severing in one-cell embryos of the indicated genotypes. Error bars, s.e.m. *P<0.05when compared with wild type, **P<0.01 when compared with wild type, NS not significant. * Figure 5: Model illustrating how phosphorylation of LIN-5 by PKC-3 may control the position of the mitotic spindle in the one-cell embryo. Phosphorylation of LIN-5 (blue) by PKC-3 (red) begins during pronuclear centration and proceeds until anaphase. Phosphorylation inhibits cortical microtubule pulling forces at the anterior cortex, allowing the spindle to switch from anterior-directed movements during centration to posterior-directed movements during metaphase and anaphase. Author information * Author information * Supplementary information Affiliations * Developmental Biology, Utrecht University, 3584 CH Utrecht, The Netherlands * Matilde Galli, * Vincent Portegijs, * Mike Boxem & * Sander van den Heuvel * Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands * Javier Muñoz & * Albert J. R. Heck * Netherlands Proteomics Center, The Netherlands * Javier Muñoz & * Albert J. R. Heck * Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany * Stephan W. Grill * Max Planck Institute for the Physics of Complex Systems, 01187 Dresden, Germany * Stephan W. Grill Contributions M.G. and S.v.d.H. designed the project and wrote the manuscript. J.M. and A.J.R.H. helped design the quantitative mass spectrometry experiments, J.M. carried out the mass spectrometry experiments and analysed the data. M.G. carried out all other experiments. V.P. and M.B. provided help in molecular cloning and yeast two-hybrid experiments. S.W.G. guided the spindle severing experiments. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sander van den Heuvel Author Details * Matilde Galli Search for this author in: * NPG journals * PubMed * Google Scholar * Javier Muñoz Search for this author in: * NPG journals * PubMed * Google Scholar * Vincent Portegijs Search for this author in: * NPG journals * PubMed * Google Scholar * Mike Boxem Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan W. Grill Search for this author in: * NPG journals * PubMed * Google Scholar * Albert J. R. Heck Search for this author in: * NPG journals * PubMed * Google Scholar * Sander van den Heuvel Contact Sander van den Heuvel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Excel files * Supplementary Table 1 (65K) Supplementary Information * Supplementary Table 2 (670K) Supplementary Information * Supplementary Table 3 (500K) Supplementary Information PDF files * Supplementary Information (2500K) Supplementary Information * Supplementary Table 4 (200K) Supplementary Information Additional data - DNA-damage response and repair activities at uncapped telomeres depend on RNF8
- Nat Cell Biol 13(9):1139-1145 (2011)
Nature Cell Biology | Letter DNA-damage response and repair activities at uncapped telomeres depend on RNF8 * Marieke H. Peuscher1 * Jacqueline J. L. Jacobs1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1139–1145Year published:(2011)DOI:doi:10.1038/ncb2326Received17 May 2011Accepted25 July 2011Published online21 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Loss of telomere protection causes natural chromosome ends to become recognized by DNA-damage response and repair proteins. These events result in ligation of chromosome ends with dysfunctional telomeres, thereby causing chromosomal aberrations on cell division. The control of these potentially dangerous events at deprotected chromosome ends with their unique telomeric chromatin configuration is poorly understood. In particular, it is unknown to what extent bulky modification of telomeric chromatin is involved. Here we show that uncapped telomeres accumulate ubiquitylated histone H2A in a manner dependent on the E3 ligase RNF8. The ability of RNF8 to ubiquitylate telomeric chromatin is associated with its capacity to facilitate accumulation of both 53BP1 and phospho-ATM at uncapped telomeres and to promote non-homologous end-joining of deprotected chromosome ends. In line with the detrimental effect of RNF8 on uncapped telomeres, depletion of RNF8, as well as of the E3 ligas! e RNF168, reduces telomere-induced genome instability. This indicates that, besides suppressing tumorigenesis by mediating repair of DNA double-strand breaks, RNF8 and RNF168 might enhance cancer development by aggravating telomere-induced genome instability. View full text Figures at a glance * Figure 1: RNF8 contributes to telomere-induced genome instability. () Schematic timeline for analysis of different aspects of the telomere-damage response using TRF2ts. At the permissive temperature of 32 °C TRF2ts protects telomeres from activating DNA-damage responses. At the non-permissive temperature of 39 °C, TRF2-mediated telomere protection is lost. When placing cells back at 32 °C, TRF2ts becomes functional again and telomere protection is restored. () Long-term inactivation of TRF2 by growing TRF2ts cells for 12 days at 39 °C leads to loss of cell viability, but not when TRF2ts cells are generated in a DNA ligase IV-deficient background. () Survival assay of TRF2ts cells infected with pRS control retrovirus or pRS–Lig4 shRNA3 retrovirus, showing that DNA ligase IV knockdown by RNAi can rescue a portion of TRF2ts cells from lethal genome instability induced by growing TRF2ts cells for 12 days at 39 °C. Plates were stained 4 weeks after placing cells back at 32 °C. () Quantitative PCR with reverse transcription (q! RT–PCR) analysis of DNA ligase IV expression levels of cells shown in . () Survival assay of TRF2ts cells (C15 clone) infected with control or Rnf8 shRNA lentiviruses (pLKO) or retroviruses (pRS), stained 4 weeks after cells were grown for 12 days at 39 °C. () Photograph of TRF2ts cells infected with pLKO control or pLKO-Rnf8 shRNA1 lentivirus, grown for 12 days at 39 °C. () qRT–PCR analysis of mouse Rnf8 expression levels of cells shown in and . Scale bars, 100 μm. * Figure 2: RNF8 facilitates telomeric G-overhang degradation and telomere fusion. () Representative metaphase spreads (two examples) of TRF2ts MEFs infected with control or Rnf8 shRNA2 retrovirus, harvested after 24 h at 39 °C and processed for telomere FISH. Scale bar, 10 μm. () Quantification of the percentage of chromosomes fused in TRF2ts MEFs grown at 32 °C or after 24 h of telomere deprotection at 39 °C. 1,000–1,400 chromosomes were evaluated per condition. () Representative example of G-overhang and total telomere signals in TRF2ts MEFs at 32 °C and after 48 h at 39 °C, detected by pulsed-field electrophoresis and in-gel hybridization with a (CCCTAA)4 radioactive probe under native conditions (single-strand, ss, TTAGGG repeats) and after denaturation (total TTAGGG repeat signal). The increase in molecular weight of TTAGGG hybridization signals at 39 °C versus 32 °C is indicative of telomere fusions at 39 °C. () Quantification of relative telomeric G-overhang signals in TRF2ts MEFs grown at 32 °C or for 48 h a! t 39 °C in the absence or presence of Rnf8 knockdown. The P-value was calculated using a two-tailed paired Student t-test on three biological replicate experiments using C15 and B17 TRF2ts MEF clones and Rnf8 shRNA1 or Rnf8 shRNA2 knockdown vectors. Error bars represent s.d. * Figure 3: Impaired 53BP1 and p-ATM accumulation at uncapped telomeres on knockdown of Rnf8. () Representative images of immunofluorescence FISH (IF-FISH) detection of p-ATM Ser 1987, γ-H2AX, 53BP1 and telomere repeats in TRF2ts MEFs grown at 39 °C for 3 h. Scale bar, 10 μm. () Quantification of p-ATM, γ-H2AX and 53BP1 foci at telomeres in TRF2ts MEFs, with or without Rnf8 knockdown, after 3 h at 39 °C to deprotect telomeres. Error bars represent s.d., n=5 independent experiments. () Quantification of 53BP1 foci in p53 shRNA-immortalized MEFs infected with control or Rnf8 shRNA2 retroviruses and subsequently with control retrovirus or Tpp1 shRNA2 retrovirus to inhibit the shelterin component TPP1. Error bars represent s.d., n=5 replicates. () Western blot analysis of DNA-damage response and repair proteins after 0, 3, 24 and 48 h of telomere uncapping in whole-cell lysates of TRF2ts MEFs infected with control retrovirus or Rnf8 shRNA2 retrovirus. The asterisk in the 53BP1 lane marks a non-specific band. The asterisk in the CHK2 lane indicates phosph! orylated CHK2. Immunoblotting for γ-tubulin serves as a loading control. As a positive control for CHK1 phosphorylation we included a lysate of cells treated for 3 h with 2 mM hydroxyurea (HU). Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 4: Telomere uncapping induces ubiquitylation of H2A and H2AX in an RNF8-dependent manner. () Representative images of IF-FISH detection of Ub–H2A at telomeres in TRF2ts MEFs grown at 32 °C or for 3 h at the non-permissive temperature of 39 °C in the absence or presence of Rnf8-shRNA2-mediated knockdown. () Quantification of Ub–H2A foci at telomeres in TRF2ts MEFs grown at 32 °C or for 3 h at 39 °C with or without Rnf8 knockdown. Error bars represent s.d., n=4 independent experiments. () Western blots of whole-cell lysates showing RNF8-dependent induction of γ-H2AX ubiquitylation after 0, 3, 24 and 48 h of telomere uncapping in TRF2ts cells. () IF-FISH detection of transfected haemagglutinin (HA)–RNF8 at telomeres in TRF2ts MEFs grown for 6 h at 39 °C to inactivate TRF2 and uncap telomeres. Scale bars, 10 μm. * Figure 5: 53BP1, p-ATM and Ub–H2A accumulation at uncapped telomeres and telomere-induced genome instability depend on the RNF8 FHA and E3-ligase domains. () Expression of Flag-tagged murine RNF8RR, RNF8C406S RR and RNF8R42A RR proteins after retroviral infection in TRF2ts MEFs. RR refers to silent mutations introduced to confer resistance to RNAi by Rnf8 shRNA1. () Examples of Ub–H2A foci in wild-type TRF2ts cells or cells with knockdown of Rnf8 and complemented with wild-type murine RNF8 or the C406S or R42A RNF8 mutants. Scale bar, 10 μm. () Quantification of IF-FISH detection of 53BP1, p-ATM and Ub–H2A foci at telomeres (TIFs) in TRF2ts MEFs infected with control viruses or infected with Rnf8 shRNA1 lentivirus together with either control retrovirus or RNAi-resistant Flag-tagged RNF8, RNF8C406S or RNF8R42A retrovirus. TIFs were scored after 3 h at the non-permissive temperature to inactivate TRF2ts. () Survival assay of TRF2ts cells infected with control or Rnf8 shRNA1 lentiviruses and control or RNAi-resistant Flag-tagged RNF8, RNF8C406S or RNF8R42A retroviruses. The effect of Rnf8 shRNA1, wild-type RNF8 and ! mutant RNF8 on cell growth in the absence of telomere damage was monitored after 12 days at 32 °C. Their effect on survival from 12 days of telomere deprotection was monitored at 4 weeks of growth at 32 °C. Author information * Author information * Supplementary information Affiliations * Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands * Marieke H. Peuscher & * Jacqueline J. L. Jacobs Contributions M.H.P. and J.J.L.J. designed the experiments. M.H.P. carried out most experimental work. J.J.L.J. supervised the project, helped generate Trf2−/−; p53−/−;TRF2ts clones and carried out all molecular cloning involved and experiments represented in Fig. 3d and Supplementary Fig. S4. J.J.L.J. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jacqueline J. L. Jacobs Author Details * Marieke H. Peuscher Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline J. L. Jacobs Contact Jacqueline J. L. Jacobs Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1400K) Supplementary Information Additional data - E2F transcription factor-1 regulates oxidative metabolism
- Nat Cell Biol 13(9):1146-1152 (2011)
Nature Cell Biology | Letter E2F transcription factor-1 regulates oxidative metabolism * Emilie Blanchet1, 2, 3, 4, 5, 6, 10 * Jean-Sébastien Annicotte1, 2, 3, 4, 5, 6, 10 * Sylviane Lagarrigue1, 2, 3, 4, 5, 6 * Victor Aguilar1, 2, 3, 4 * Cyrielle Clapé1, 2, 3, 4 * Carine Chavey1, 2, 3, 4, 5, 6 * Vanessa Fritz1, 2, 3, 4, 5, 6 * François Casas7 * Florence Apparailly8 * Johan Auwerx9 * Lluis Fajas1, 2, 3, 4, 5, 6 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1146–1152Year published:(2011)DOI:doi:10.1038/ncb2309Received28 April 2011Accepted28 June 2011Published online14 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cells respond to stress by coordinating proliferative and metabolic pathways. Starvation restricts cell proliferative (glycolytic) and activates energy productive (oxidative) pathways. Conversely, cell growth and proliferation require increased glycolytic and decreased oxidative metabolism levels1. E2F transcription factors regulate both proliferative and metabolic genes2, 3. E2Fs have been implicated in the G1/S cell-cycle transition, DNA repair, apoptosis, development and differentiation2, 3, 4. In pancreatic β-cells, E2F1 gene regulation facilitated glucose-stimulated insulin secretion5, 6. Moreover, mice lacking E2F1 (E2f1−/−) were resistant to diet-induced obesity4. Here, we show that E2F1 coordinates cellular responses by acting as a regulatory switch between cell proliferation and metabolism. In basal conditions, E2F1 repressed key genes that regulate energy homeostasis and mitochondrial functions in muscle and brown adipose tissue. Consequently, E2f1−/− mice! had a marked oxidative phenotype. An association between E2F1 and pRB was required for repression of genes implicated in oxidative metabolism. This repression was alleviated in a constitutively active CDK4 (CDK4R24C) mouse model or when adaptation to energy demand was required. Thus, E2F1 represents a metabolic switch from oxidative to glycolytic metabolism that responds to stressful conditions. View full text Figures at a glance * Figure 1: Loss of E2F1 affects energy expenditure, adaptive thermogenesis, mitochondrial function and physical activity. () Energy expenditure (VO2). n=4 animals per group. The black frame indicates the period of time used for calculation in . () Respiratory exchange ratio (VCO2/VO2) during the total, light and dark phases of the experiment. n=5 animals per group. () Rectal temperatures under room temperature, fed (control), cold or fasted conditions. n=4 animals per group. () Mitochondrial DNA (mtDNA) content measured relative to nuclear DNA in BAT tissues of E2f1+/+ and E2f1−/− mice. n=5 animals per group. () Mitochondrial oxygen consumption without (−) and with (+) succinate. n=4 animals per group. () Mitochondrial DNA content in E2f1+/+ and E2f1−/− muscles. n=5 animals per group. () GNM mitochondrial O2 consumption measured after electrotransfer of pCMV or pCMV-E2F1, without (−) and with (+) ADP and succinate. n=4 animals per group. () Relative gene expression levels of GNM MyHC type I, IIa, IIX (also known as MyH1), and IIb. Results were normalized to the expression of mouse 1! 8S RNA. n=7 animals per group. () Immunofluorescence microscopy analysis of serial GNM sections showing expression of MyHC-I, MyHC-IIa and MyHC-IIb (red) in fibres. Nuclei are stained with Hoechst reagent. n=4 animals per group. The percentage of positive stained fibres is indicated. Scale bars, 100 μm. () E2f1+/+ and E2f1−/− mice were tested for physical endurance. Individual animal performances are shown. n=11 E2f1+/+ animals; n=14 E2f1−/− animals. () The effect of E2F1 rescue in E2f1−/− mice was evaluated with an endurance test. Individual performances are represented for E2f1 wild-type mice electroporated with empty vector (E2f1+/+ pCMV), knockout (E2f1−/− pCMV) and knockout-rescued animals (E2f1−/− pCMV-E2F1). n=4 animals for E2f1+/+ pCMV group; n=3 animals for E2f1−/− pCMV group; n=3 animals for E2f1−/− pCMV-E2F1 group. Values represent means±s.e.m. *P<0.05. **P<0.01. * Figure 2: Increased E2f1−/− oxidative metabolic gene expression level. (,) Relative expression of relevant mitochondrial genes in BAT () and GNM (). Results were normalized to mouse 18S RNA expression. n=7 animals per group. () Relative expression levels of oxidative genes in E2f1−/− GNM electroporated with an empty vector (pRNAT-control) or a plasmid expressing shRNA targeting Ucp2, Ppargc1a, Esrra or Pdk4. mRNA levels are represented relative to 18S mRNA. n=3 animals. () At 72 h post-electroporation, O2 consumption was measured in isolated mitochondria from GNM using a Clark electrode. The consumption of O2 was measured in the absence and presence of succinate and ADP. n=3 animals. Values represent means±s.e.m. *P<0.05; **P<0.01; ***P<0.001. * Figure 3: Cold and fasting modulate gene expression through the pRB–E2F1 complex. (,) qPCR quantification of the level of expression of relevant genes involved in oxidative metabolism in BAT () and GNM () of E2f1+/+ and E2f1−/− mice in cold/room temperature and fasted/refed conditions, respectively (see Supplementary Table S1 for sequences). Results were normalized to the expression of mouse 18S RNA and are expressed as means±s.e.m. of three independent experiments. (–) Immunohistochemistry analysis of pRB phosphorylation on Ser 780 (S780p-pRB). For , BAT was obtained from mice placed at 23 °C or 4 °C. For , GNM was obtained from mice fasted for 24 h or fasted for 20 h and refed for 4 h. For , mice were fasted for 4 h, injected i.p. with 0.9% NaCl or 0.9% NaCl+isoproterenol and GNM was collected 30 min after injection. Tissues were subsequently processed for immunohistochemistry as described in the Methods section. Scale bars, 100 μm. () Mitochondrial activity, measured as oxygen consumption in E2f1+/+ embryonic cells in the pres! ence or absence of isoprotenerol and the CDK4 inhibitor PD0332991 as indicated. Results were normalized to protein levels and are expressed as means±s.e.m. of four independent experiments per conditions. (–) ChIP demonstrates binding of E2F1 and pRB to oxidative metabolic gene promoters in BAT obtained at room temperature (23 °C) or after 4 h cold exposure (4 °C; ) or in muscle in 24 h fasting (F) and 4 h refed (R) conditions (,). Values represent means±s.e.m. of six independent experiments. Immunoprecipitates (IP) were analysed by qPCR (,) or classical PCR () with specific primers for the E2F-responsive element identified in these promoters (see Supplementary Table S1 for sequences). Values represent means±s.e.m. *P<0.05; **P<0.01; ***P<0.001. †P<0.05 E2f1+/+ fasted (24 h) versus refed (4 h); ‡P<0.05 E2f1+/+ fasted versus E2f1−/− fasted; #P<0.05 E2f1+/+ refed versus E2f1−/− refed. Uncropped images of blots are shown in Supplementary Fig. ! S7. * Figure 4: Increased O2 consumption, running time and expression of oxidative genes in CDK4R24C/R24C mice. () Mitochondrial oxygen consumption without (−) and with (+) succinate. n=6 animals. () Wild-type and CDK4R24C/R24C mice were tested for physical endurance. Average running time to exhaustion is shown. n=6 animals per group. () qPCR quantification of the expression of relevant genes involved in oxidative metabolism in GNM of wild-type versus CDK4R24C/R24C mice in fasted/refed conditions. Results were normalized to the expression of mouse 18S RNA and are expressed as means±s.e.m. of three independent experiments. n=3 animals per group. () ChIP demonstrates binding of E2F1 and pRB to oxidative metabolic gene promoters in muscle in 24 h fasting and 5 h refed conditions. n=3 animals. Values represent means±s.e.m. *P<0.05; **P<0.01; †P<0.05 E2f1+/+ fasted (24 h) versus refed (4 h); ‡P<0.05 E2f1+/+ fasted versus CDK4R24C/R24C fasted; #P<0.05 E2f1+/+ refed versus CDK4R24C/R24C refed. * Figure 5: DNA methylation of proliferative target genes modulates E2F1 transcriptional activity in muscle. () qPCR quantification of the expression of Dhfr and Tk1 in wild-type mice in fasted/refed conditions (see Supplementary Table S1 for sequences). Results were normalized to the expression of mouse 18S RNA and are expressed as means±s.e.m. n=6 animals per group. () ChIP demonstrates binding of E2F1 relative to IgG to proliferative gene promoters in muscle in 24 h fasting or 20 h fasting/4 h refed conditions. n=3 animals. () Methylation status of Dhfr, Tk1 and Ppargc1a promoters on CpG islands in fasted muscles. PCR analysis of methylated DNA–MBD2b complexes in genomic DNA from muscle. A sample corresponding to genomic DNA before incubation with MBD2b was included in the PCR (Input). As a positive control (+), a PCR reaction using A431 methylated DNA and Dhfr-, Tk1- and Ppargc1a-promoter-specific primers was carried out. A negative control (−) was carried out using unmethylated HeLa genomic DNA as a template and Dhfr-, Tk1- and Ppargc1a-promoter-specific primers. ()! In basal (fed/room temperature) conditions, E2F1–pRB complexes can repress the oxidative gene program (Oxphos) by switching off those genes. External stimuli activate E2F1 transcriptional activity through the release of pRB, which induces the cell to switch to oxidative metabolism. This molecular mechanism enables the transcription of an oxidative metabolic gene programme, allowing the cell to adapt to energy demands and triggers physiological processes such as thermogenesis in BAT or enhanced physical activity in muscle. On the other hand, classical proliferative E2F1 target genes are not subjected to E2F1 regulation because of epigenetic mechanisms. Uncropped images of blots are shown in Supplementary Fig. S7. Author information * Author information * Supplementary information Affiliations * IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier F-34298, France * Emilie Blanchet, * Jean-Sébastien Annicotte, * Sylviane Lagarrigue, * Victor Aguilar, * Cyrielle Clapé, * Carine Chavey, * Vanessa Fritz & * Lluis Fajas * INSERM, U896, Montpellier F-34298, France * Emilie Blanchet, * Jean-Sébastien Annicotte, * Sylviane Lagarrigue, * Victor Aguilar, * Cyrielle Clapé, * Carine Chavey, * Vanessa Fritz & * Lluis Fajas * Université de Montpellier1, Montpellier, F-34298, France * Emilie Blanchet, * Jean-Sébastien Annicotte, * Sylviane Lagarrigue, * Victor Aguilar, * Cyrielle Clapé, * Carine Chavey, * Vanessa Fritz & * Lluis Fajas * CRLC Val d'Aurelle Paul Lamarque, Montpellier F-34298, France * Emilie Blanchet, * Jean-Sébastien Annicotte, * Sylviane Lagarrigue, * Victor Aguilar, * Cyrielle Clapé, * Carine Chavey, * Vanessa Fritz & * Lluis Fajas * IGMM, Institut de Génétique Moléculaire de Montpellier, Montpellier F-34293, France * Emilie Blanchet, * Jean-Sébastien Annicotte, * Sylviane Lagarrigue, * Carine Chavey, * Vanessa Fritz & * Lluis Fajas * CNRS, UMR5535, Montpellier F-34293, France * Emilie Blanchet, * Jean-Sébastien Annicotte, * Sylviane Lagarrigue, * Carine Chavey, * Vanessa Fritz & * Lluis Fajas * INRA, UMR0866, Montpellier F-34060, France * François Casas * INSERM U844, Montpellier F-34295, France * Florence Apparailly * Ecole Polytechnique Fédérale de Lausanne, 1115 Lausanne, Switzerland * Johan Auwerx * These authors equally contributed to this work * Emilie Blanchet & * Jean-Sébastien Annicotte Contributions J-S.A. and L.F. designed the study. E.B., J-S.A., S.L., V.A., V.F., C. Chavey and C. Clapé carried out the experiments. F.A., F.C. and J.A. provided reagents and data. J-S.A. and L.F. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Lluis Fajas Author Details * Emilie Blanchet Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Sébastien Annicotte Search for this author in: * NPG journals * PubMed * Google Scholar * Sylviane Lagarrigue Search for this author in: * NPG journals * PubMed * Google Scholar * Victor Aguilar Search for this author in: * NPG journals * PubMed * Google Scholar * Cyrielle Clapé Search for this author in: * NPG journals * PubMed * Google Scholar * Carine Chavey Search for this author in: * NPG journals * PubMed * Google Scholar * Vanessa Fritz Search for this author in: * NPG journals * PubMed * Google Scholar * François Casas Search for this author in: * NPG journals * PubMed * Google Scholar * Florence Apparailly Search for this author in: * NPG journals * PubMed * Google Scholar * Johan Auwerx Search for this author in: * NPG journals * PubMed * Google Scholar * Lluis Fajas Contact Lluis Fajas Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Excel files * Supplementary Table 1 (12K) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data
No comments:
Post a Comment