Latest Articles Include:
- Combating scientific misconduct
- ncb 13(1):1 (2011)
Nature Cell Biology | Editorial Combating scientific misconduct Journal name:Nature Cell BiologyVolume: 13,Page:1Year published:(2011)DOI:doi:10.1038/ncb0111-1Published online21 December 2010 Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. The pressures of an increasingly competitive research environment can lead to scientific misconduct. Journals, academic institutions and individual scientists should commit to promoting best practice in research and education in research ethics. View full text Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Out of Africa and into epigenetics: discovering reprogramming drugs
- ncb 13(1):2 (2011)
Nature Cell Biology | Turning Points Out of Africa and into epigenetics: discovering reprogramming drugs * Peter Jones1 Contact Peter Jones Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature Cell BiologyVolume: 13,Page:2Year published:(2011)DOI:doi:10.1038/ncb0111-2Published online21 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. The unsolicited letter from the reviewer of our manuscript submitted to the European Journal of Cancer arrived at the Department of Biochemistry at the University of Rhodesia in March 1972 as I was completing my PhD under the guidance of Professor Arthur Hawtrey. The University was a multiracial island in a segregated country suffering under UN sanctions and the beginnings of a civil war. My advisor Joseph Taderera, who was black, had been arrested in front of me in the lab for smuggling AK47s into the country and the future of my science career looked bleak. The letter, which is the only one I have ever received from an anonymous reviewer, was from Bill Benedict in the US, complimenting me on our work describing how DNA-synthesis inhibitors used to treat cancer could cause oncogenic transformation themselves. By breaking the wall of reviewer anonymity, he gave me the chance of a lifetime. Going to graduate school in Rhodesia as a PhD student of the University of London gave me a different perspective on science, namely one of self reliance. There was no other University within hundreds of miles and there were only two seminars by outside speakers during my entire degree course. You had to make most things like fetal calf serum for yourself and you relied on surface mail for the scientific journals to arrive six months behind everyone else. Without hesitation, I seized this unforeseen opportunity and wrote back to Bill asking if I might come over to the US as a Postdoc. Maybe I would not, after all, be trapped in Britain's rebel colony and possibly be sent to the bush to fight against Joe Taderera and his AK47s. View full text 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 Affiliations * Peter Jones is in the USC Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA. pjones@med.usc.edu Competing financial interests The author declares no competing financial interests. Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Reducing background fluorescence reveals adhesions in 3D matrices
- ncb 13(1):3-5 (2011)
Nature Cell Biology | Correspondence Reducing background fluorescence reveals adhesions in 3D matrices * Kristopher E. Kubow1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alan Rick Horwitz1 Contact Alan Rick Horwitz Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:3–5Year published:(2011)DOI:doi:10.1038/ncb0111-3Published online21 December 2010 To the editor: Adhesion complexes in cells growing on planar substrates have been studied for over three decades. From these studies, several classes of adhesions have been described based on size, location, morphology, dynamics or molecular composition, and their dual role as signalling centres and linkages that connect the extracellular matrix (ECM) with the cytoskeleton has also emerged1. In contrast to this large and growing understanding of adhesion on two-dimensional (2D) substrates, little is known about the adhesions formed during three-dimensional (3D) growth of cells, including whether they even exist. Immunostaining and microscopy of fixed samples2, 3, 4, 5, 6, 7, 8 and dynamic imaging at low spatiotemporal resolution9 have shown the presence of large, elongated adhesions on cells in various 3D model systems. However, attempts to visualize adhesions in living cells growing in 3D with resolution similar to that routinely used in 2D have not been successful10. This has led to the ! recent conclusion that the adhesions defined and characterized in 2D cultures either do not exist in cells in 3D, or are too small or short-lived to be observed11. View full text Figures at a glance * Figure 1: Cell-matrix adhesions are detected in 3D collagen gels. () Z-projection of a multi-polar U2OS cell expressing EGFP–paxillin under the control of a truncated promoter, with adhesions on the distal sections of the protrusions (arrows). See Supplementary Information, Fig. S1a–f for more images. () Z-projection images of protrusions from cells expressing the indicated construct. The cell expressing a low level of normal EGFP–paxillin (upper left) has a diffuse cytoplasmic fluorescence, similar in appearance to the cell expressing the fluorescent protein TagRFP-T alone (upper-right). Adhesions (arrows) are detectable in U2OS cells (bottom left) and HT-1080 cells (bottom right) transfected with a plasmid encoding promoter-truncated EGFP–paxillin, which expresses at a lower level than EGFP–paxillin and decreases background cytoplasmic fluorescence. () Z-projection of a U2OS cell expressing promoter-truncated EGFP–vinculin. Scale bars, 5 μm. * Figure 2: Dynamics of cell-matrix adhesions in 3D culture. Frames are taken from Supplementary Information, Video S1. The top row shows Z-projections of a protrusion end from a U2OS cell that was transfected with a plasmid encoding promoter-truncated EGFP–paxillin. The bottom row shows the image from the top row (green) overlayed with a reflectance image of the collagen fibres (magenta). Time indicates the min:s since the beginning of the movie. At 0:20, a small adhesion (arrowhead) moves rearward, pulling a collagen fibre. At 2:30, a new protrusion (boundary shown by dotted line) pauses, two new adhesions form at the leading edge (arrows), and the earlier adhesion (arrowhead) has elongated while travelling rearward. At 3:10, the two new adhesions (arrows) continue to grow and translocate with the attached collagen fibres. By 4:50, the new adhesions have continued to grow and move rearward while the early adhesion (arrowhead in previous panels) is no longer visible. Scale bar, 2 μm. Author information * 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 Affiliations * Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA. * Kristopher E. Kubow & * Alan Rick Horwitz Contributions K.E.K. performed the experiments and analysed the data. K.E.K. and A.R.H. designed the experiments and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alan Rick Horwitz Supplementary information * Author information * Supplementary information Movies * Supplementary Information (5M) Supplementary Information, Video S1 * Supplementary Information (7M) Supplementary Information, Video S2 PDF files * Supplementary Information (320K) Supplementary Information Additional data - Reply: reducing background fluorescence reveals adhesions in 3D matrices
- ncb 13(1):5-7 (2011)
Nature Cell Biology | Correspondence Reply: reducing background fluorescence reveals adhesions in 3D matrices * Stephanie I. Fraley1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Yunfeng Feng2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Denis Wirtz1, 2 Contact Denis Wirtz Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory D. Longmore2, 3, 4 Contact Gregory D. Longmore Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:5–7Year published:(2011)DOI:doi:10.1038/ncb0111-5Published online21 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Fraley et al. reply: Kubow and Horwitz present evidence supporting the presence of focal adhesion complexes in the mesenchymal cell lines U2OS and HT-1080 when these cells are embedded in a three-dimensional (3D) collagen matrix. The initial adhesions were approximately 0.4 μm in diameter and achieved a median length of 1 μm. They also report that the formation and rearward movement of these adhesions were similar to that observed for these cells in two-dimensional (2D) systems. In contrast, in our recent article1, we reported that we did not observe any structured organization of focal adhesion proteins of this size in migrating HT-1080 cells or non-motile U2OS cells that were completely embedded in a similar 3D collagen gel. The conditions of our experiments and the imaging techniques employed were similar to those used by Kubow and Horwitz, but for one technical difference (see below), and thus should be able to detect focal adhesions of 0.4 μm or greater (our resolution is 0.21 μm or gre! ater). Kubow and Horwitz suggest that the discrepancy between their results and ours was because of the high background fluorescence resulting from overexpression of GFP (green fluorescent protein)-tagged FA proteins in our experiments, which obscures the observation of focal complexes. To circumvent this problem, Kubow and Horwitz used a 'crippled' CMV (cygtomegalovirus) promoter that expresses less protein in cells2. View full text Figures at a glance * Figure 1: Focal adhesion visualization in live HT-1080 cells. (–) (left) and microscopy images (right) of a YFP–zyxin-expressing cell on a 2D collagen-coated substrate (), a GFP–paxillin-expressing cell cultured by the 2.5D method1 (), a YFP–zyxin-expressing cell located 20–30 μm away from the dish bottom () and a GFP–paxillin expressing cell located 236–260 μm from the dish bottom (). Fluorescence, phase, and reflection confocal micrographs are shown on the right for each case. Arrows on micrographs and red stars on the schematics indicate focal adhesions. Numbered red lines in the schematic shown in indicate Z-slices and correspond to micrographs. Panel 5 in is a cross-sectional view of four thin, finger-like protrusions extending upward in the gel (see schematic, slice 5). The intensity of these four protrusions matches that of the cell body, and the 'auto-detect ROI' tool in the image-analysis software did not detect brighter regions (that is, defined as focal adhesions) within these protrusions. () Quantification o! f the number of focal adhesions detected per cell in the various conditions described in –. In each case 20 cells were analysed. nd; none detected. () Quantification of the average area of focal adhesions per cell in the various conditions described in –. In each case 20 cells were analysed. nd; none detected. Data are means ± s.d. Two-tailed unpaired t tests were conducted to determine significance (double asterisks; P < 0.01 and asterisk; P < 0.05). Scale bars; 10 μm in whole-cell images or 1 μm in zoomed images. * Figure 2: Properties of cells on or near the bottom versus fully embedded in 3D collagen gel. () Phase contrast microscopy image of a U2OS cell located near the bottom of a 2mg ml−1 collagen gel formed in a tissue culture dish. Protrusions are wide, flat and in the same plane. () Phase contrast microscopy image of a U2OS cell fully embedded (> 200 μm from any edge) in a 2mg ml−1 collagen gel. Protrusions are thin, tapered and in different focal planes. Arrows identify protrusions. Scale bar; 10 μm. 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 Affiliations * Departments of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA. * Stephanie I. Fraley & * Denis Wirtz * Johns Hopkins Physical Sciences in Oncology Centre, Johns Hopkins University, Baltimore, MD 21218, USA. * Stephanie I. Fraley, * Yunfeng Feng, * Denis Wirtz & * Gregory D. Longmore * Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA. * Yunfeng Feng & * Gregory D. Longmore * BRIGHT Institute, Washington University School of Medicine, St. Louis, MO 63110, USA. * Yunfeng Feng & * Gregory D. Longmore Contributions Y. F. generated YFP-paxillin and GFP-zyxin constructs. S. F. performed all experiments and analysis and co-wrote the manuscript; G. L. and D. W. co-supervised the project and co-wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Denis Wirtz or * Gregory D. Longmore Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Chaperoning the SNAREs: a role in preventing neurodegeneration?
- ncb 13(1):8-9 (2011)
Nature Cell Biology | News and Views Chaperoning the SNAREs: a role in preventing neurodegeneration? * Robert D. Burgoyne1 Contact Robert D. Burgoyne Search for this author in: * NPG journals * PubMed * Google Scholar * Alan Morgan1 Contact Alan Morgan Search for this author in: * NPG journals * PubMed * Google Scholar * AffiliationsJournal name:Nature Cell BiologyVolume: 13,Pages:8–9Year published:(2011)DOI:doi:10.1038/ncb0111-8Published online21 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Despite their potential importance as therapeutic targets, the initial events in neurodegenerative diseases are poorly understood. Emerging evidence suggests that presynaptic dysfunction might be an early event in these pathologies, and three papers now link dysregulation of SNARE protein levels and function caused by the absence of synuclein or cysteine string protein (CSP) to activity-dependent neurodegeneration. View full text 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 Affiliations * Robert D. Burgoyne and Alan Morgan are in the Department of Cellular and Molecular Physiology, The Physiological Laboratory, Institute of Translational Medicine, Crown Street, University of Liverpool, Liverpool, L69 3BX, UK. burgoyne@liv.ac.uk Competing financial interests The authors declare no competing financial interests. Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Neuronal transport: myosins pull the ER
- ncb 13(1):10-11 (2011)
Nature Cell Biology | News and Views Neuronal transport: myosins pull the ER * Michael Stiess1 Search for this author in: * NPG journals * PubMed * Google Scholar * Frank Bradke1 Contact Frank Bradke Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:10–11Year published:(2011)DOI:doi:10.1038/ncb2147Published online12 December 2010 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Whether class V myosins can work as point-to-point transporters in animal cells is highly debated. Myosin-Va is now shown to function as a point-to-point transporter that pulls the endoplasmic reticulum (ER) into dendritic spines, with important consequences for dendritic development and cerebellar motor learning. View full text 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 Affiliations * Michael Stiess and Frank Bradke are in the Axonal Growth and Regeneration Group, Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Frank Bradke Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - Research highlights
- ncb 13(1):12 (2011)
Nature Cell Biology | Research Highlights Research highlights Journal name:Nature Cell BiologyVolume: 13,Page:12Year published:(2011)DOI:doi:10.1038/ncb0111-12Published online21 December 2010 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. In yeast, the HOPS tethering complex regulates vacuolar fusion events. HOPS localizes to late endosomes, where it mediates endosome–vacuole fusion. However, when present on vacuoles, HOPS can regulate fusion of AP-3 vesicles derived from the trans-Golgi network. The mechanism underlying this localization-dependent specificity remained unclear. Ungermann and colleagues now show that phosphorylation of the HOPS component Vps41 affects its affinity for vesicles, and thus regulates fusion specificity (J. Cell Biol.191, 845–859; 2010). The kinase Yck3 was previously shown to phosphorylate Vps41, and inhibition of Vps41 phosphorylation blocked AP-3 vesicle fusion and caused Vps41 to accumulate at endosomes. An intact Vps41 ALPS motif, which is known to associate with highly curved membranes and contains the Yck3 phosphorylation site, was essential for association with small vesicles. This raises the possibility that ALPS motif phosphorylation attenuates Vps41 binding to endosomes. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Additional data - LIF-independent JAK signalling to chromatin in embryonic stem cells uncovered from an adult stem cell disease
- ncb 13(1):13-21 (2011)
Nature Cell Biology | Article LIF-independent JAK signalling to chromatin in embryonic stem cells uncovered from an adult stem cell disease * Dean S. Griffiths1 Contact Dean S. Griffiths Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Li1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark A. Dawson1, 2, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew W. B. Trotter3 Search for this author in: * NPG journals * PubMed * Google Scholar * Yi-Han Cheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Aileen M. Smith1 Search for this author in: * NPG journals * PubMed * Google Scholar * William Mansfield4 Search for this author in: * NPG journals * PubMed * Google Scholar * Pentao Liu5 Search for this author in: * NPG journals * PubMed * Google Scholar * Tony Kouzarides6 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer Nichols4 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew J. Bannister6 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony R. Green1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Berthold Göttgens1 Contact Berthold Göttgens Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:13–21Year published:(2011)DOI:doi:10.1038/ncb2135Received18 December 2010Accepted20 October 2010Published online12 December 2010 Abstract * Abstract * Accession codes * 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 Activating mutations in the tyrosine kinase Janus kinase 2 (JAK2) cause myeloproliferative neoplasms, clonal blood stem cell disorders with a propensity for leukaemic transformation. Leukaemia inhibitory factor (LIF) signalling through the JAK-signal transducer and activator of transcription (STAT) pathway enables self-renewal of embryonic stem (ES) cells. Here we show that mouse ES cells carrying the human JAK2V617F mutation were able to self-renew in chemically defined conditions without cytokines or small-molecule inhibitors, independently of JAK signalling through the STAT3 or phosphatidylinositol-3-OH kinase pathways. Phosphorylation of histone H3 tyrosine 41 (H3Y41) by JAK2 was recently shown to interfere with binding of heterochromatin protein 1α (HP1α). Levels of chromatin-bound HP1α were lower in JAK2V617F ES cells but increased following inhibition of JAK2, coincident with a global reduction in histone H3Y41 phosphorylation. JAK2 inhibition reduced levels of the! pluripotency regulator Nanog, with a reduction in H3Y41 phosphorylation and concomitant increase in HP1α levels at the Nanog promoter. Furthermore, Nanog was required for factor independence of JAK2V617F ES cells. Taken together, these results uncover a previously unrecognized role for direct signalling to chromatin by JAK2 as an important mediator of ES cell self-renewal. View full text Subject terms: * Stem cells Figures at a glance * Figure 1: JAK2V617F sustains ES cells in a self-renewing state without any additional factors. () Targeting strategy to insert patient cDNA containing JAK2V617F mutation into the jak2 allele by homologous recombination. () JAK2V617F ES cells are made factor-independent by transferring ES cells growing in N2B27 plus LIF and BMP4 into N2B27 alone. The ES cells then undergo a crisis, detaching and forming spheres. These spheres can then be reattached by transferring into fresh N2B27 on freshly gelatinized flasks. () ES cells were plated at 1 × 103 cells per well of a 12 well plate; 6 days later the cells were fixed and stained for alkaline phosphatase to identify ES cell colonies. Only factor-independent JAK2V617F ES cells (JAK2V617F) can form colonies in N2B27 alone; this ability is lost following the addition of AG490. () Parental ES cells grown in N2B27 plus LIF and BMP4 have a similar pattern of staining for the key ES cell transcription factors Nanog and OCT4 to that of factor-independent JAK2V617F ES cells in N2B27 alone. Factor-independent JAK2V617F cells have th! e characteristic variable levels of Nanog seen with wild-type ES cells. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 20 μm. () Microarray analysis of the three ES cell lines demonstrates that the majority of expressed genes are common to all three ES cell lines. Correlation coefficients were calculated using the mean of each two-way comparison and show a strong correlation coefficient between all three data sets (red values). () Genes known to be critical for ES cell self-renewal are expressed at similar levels in all data sets, and there is no upregulation of genes expressed in more differentiated cell types. Values are mean of three biological replicates; error bars represent s.e.m. * Figure 2: Factor-independent JAK2V617 ES cells are capable of multilineage differentiation in vitro and in vivo. () Factor-independent JAK2V617F ES cells give rise to embryoid bodies containing red blood cells and differentiate into neurons when transferred into appropriate differentiation conditions. () JAK2V617F ES cells generate FLK-1-positive mesodermal cells with delayed differentiation kinetics. Wild-type and factor-independent JAK2V617F ES cells were differentiated to haematopoietic lineages, and the proportion of FLK-1 and SSEA-1-positive cells were measured by fluorescence-activated cell sorting (FACS) at days 3, 5 and 7 of differentiation. () Teratocarcinoma formation from JAK2V617F ES cells. Parental AB2.2 ES cells maintained in N2B27 plus LIF and BMP4 and factor-independent JAK2V617F ES cells were injected into the kidney capsule of 129sv mice and left for 4 weeks. These examples show that teratocarcinomas consisting of cells from all three germ layers formed, but the majority of teratocarcinomas from factor-independent JAK2V617F ES cells remained undifferentiated or were o! f no immediately discernible cell type. Un, undifferentiated; ec, ectoderm; me, mesoderm; en, endoderm. * Figure 3: JAK2V617F does not activate canonical signalling pathways independently of cytokines, but JAK is required for ES cell self-renewal. () Colony forming assay for wild-type and factor-independent ES cells in 2i or N2B27 alone following clonal growth for 6 days in dilution series of JAK inhibitors. ES colonies were confirmed by positive staining for alkaline phosphatase (AP). There was a significant reduction (P < 1 × 10−7) for all inhibitors on all cell types except JAK2V617F ES cells in N2B27 alone at the lowest concentrations of TG101209. Error bars represent s.e.m. () Immunoblot analysis of steady-state levels or levels following 8 h of treatment of cells with LIF or AG490 for P-STAT3 (phosphorylated on Tyr 705), STAT3, P-AKT (on Ser 473) and tubulin of wild-type ES cells grown in N2B27 plus 2i, JAK2V617F ES cells in N2B27 plus 2i or JAK2V71F ES cells in N2B27 alone. () Colony forming assay for STAT3-null ES cells in 2i performed as in . There was a significant reduction (p < 1 × 10−16) using all inhibitors. () Immunocytochemistry of STAT3-null JAK2V617F factor-independent ES cells confirms continu! ed expression of key ES cell genes OCT4 and Nanog. Scale bar, 20 μm. For details of GLM, see Supplementary Information, Table S1. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 4: JAK2 is present in the nucleus of ES cells and dynamically regulates HP1α access to chromatin by phosphorylating histone H3Y41. () Immunohistochemistry for phosphorylated JAK2 in wild-type ES cells growing in 2i. Orthogonal view confirms the presence of phosphorylated JAK2 in the nucleus. () Immunohistochemistry confirms that HP1α was present at lower levels in JAK2V617F ES cells than in parental cells when they are maintained in multiple ES cell conditions, but OCT4 remains unchanged. () Immunohistochemistry for HP1α and Nanog in steady-state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 h. There was a significant increase in the level of HP1α and decrease in Nanog following inhibitor treatment. n, cell number. () Immunohistochemistry for H3Y41ph and Nanog in steady-state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 h. There was a significant decrease in the levels of H3Y41ph and Nanog following inhibitor treatment. In c and d, two independent experiments are combined in a box and whisker plot, and the difference was determi! ned by Student's t-test. n is cell number. Scale bars in all panels, 20 μm. * Figure 5: JAK2 regulates H3Y41ph at the Nanog promoter and Nanog is critical for factor-independent self-renewal. () A 8 kb window surrounding the Nanog transcriptional start site (bottom) was interrogated using ChIP for H3Y41ph in steady-state factor-independent JAK2V617F ES cells (top) or following treatment with TG101209 for 6 h (middle). () The Nanog promoter was interrogated using ChIP for histone H3 methylated three times on lysine 4 (H3K4me3), H3Y41ph and HP1α in wild-type ES cells growing in N2B27 plus 2i or following treatment with AG490 for 16 h. In , , data were normalized to H3 occupancy and representative plots of two independent experiments are shown; error bars represent s.e.m. () Colony forming assay for Nanog overexpressing ES cells, performed as described in Fig. 3a. Although JAK inhibition caused a decrease in ES cell self-renewal of Nanog overexpressing (o/e) ES cells, this was significantly less than the decrease in wild-type ES cell self-renewal. *P < 0.05, **P < 0.01, ***P < 0.001; see Supplementary Information, Table S1 for details of t-test. Error bars represen! t s.e.m. () Growth of JAK2V617F ES cells in N2B27 alone is compromised following transduction with a lentivirus co-expressing shRNA against Nanog, and enhanced green fluorescent protein (eGFP). Exp, experiment. * Figure 6: JAK2 is not the only JAK that can phosphorylate H3Y41. () Immunohistochemistry of JAK2-null ES cells growing in 2i medium demonstrated that H3Y41 is phosphorylated in the absence of JAK2, which in turn was dynamically regulated by JAK inhibition treatment with AG490 for 16 h. There was a significant decrease in the levels of H3Y41ph and OCT4 following inhibitor treatment. Two independent experiments were combined in a box and whisker plot, and the difference was determined by Student's t-test. n, cell number. () Orthogonal view of immunohistochemistry for phosphorylated JAK1 in wild-type ES cells maintained in N2B27 plus 2i. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * E-MTAB-416 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Haematology and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK. * Dean S. Griffiths, * Juan Li, * Mark A. Dawson, * Yi-Han Cheng, * Aileen M. Smith, * Anthony R. Green & * Berthold Göttgens * Department of Haematology, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. * Mark A. Dawson & * Anthony R. Green * The Anne McLaren Laboratory for Regenerative Medicine and Department of Surgery, West Forvie Building, Robinson Way, Cambridge CB2 0SZ, UK. * Matthew W. B. Trotter * Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK. * William Mansfield & * Jennifer Nichols * Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. * Pentao Liu * Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, UK. * Mark A. Dawson, * Tony Kouzarides & * Andrew J. Bannister Contributions D.S.G. designed the experiments and performed most of the experiments. B.G. conceived the study and wrote the paper. J.L., P.L. and A.R.G. designed and made the JAK2V617F ES cells. M.A.D., A.J.B. and T.K. performed ChIPs, western for H3Y41ph and in vitro kinase assay. M.W.B.T. analysed microarray data. W.M. and J.N. generated teratocarcinomas and derived JAK2-null ES cells. Y.-H.C. and A.M.S. generated Fig. 2b. D.S.G., A.R.G and B.G. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Berthold Göttgens or * Dean S. Griffiths Supplementary information * Abstract * Accession codes * Author information * Supplementary information Movies * Supplementary Movie 1 (4M) Supplementary Information * Supplementary Movie 2 (4M) Supplementary Information PDF files * Supplementary Information (773K) Supplementary Information Additional data - Fates-shifted is an F-box protein that targets Bicoid for degradation and regulates developmental fate determination in Drosophila embryos
- ncb 13(1):22-29 (2011)
Nature Cell Biology | Article Fates-shifted is an F-box protein that targets Bicoid for degradation and regulates developmental fate determination in Drosophila embryos * Junbo Liu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jun Ma1, 2 Contact Jun Ma Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:22–29Year published:(2011)DOI:doi:10.1038/ncb2141Received30 April 2010Accepted17 November 2010Published online19 December 2010 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 Bicoid (Bcd) is a morphogenetic protein that instructs patterning along the anterior–posterior (A–P) axis in Drosophila melanogaster embryos. Despite extensive studies, what controls the formation of a normal concentration gradient of Bcd remains an unresolved and controversial question. Here, we show that Bcd protein degradation is mediated by the ubiquitin-proteasome pathway. We have identified an F-box protein, encoded by fates-shifted (fsd), that has an important role in Bcd protein degradation by targeting it for ubiquitylation. Embryos from females lacking fsd have an altered Bcd gradient profile, resulting in a shift of the fatemap along the A–P axis. Our study is an experimental demonstration that, contrary to an alternative hypothesis, Bcd protein degradation is required for normal gradient formation and developmental fate determination. View full text Subject terms: * Developmental biology Figures at a glance * Figure 1: Bcd is degraded through the proteasome-dependent pathway. () Effects of protease inhibitors on the total amount of Bcd in Drosophila S2 cells. Cells stably expressing HA–Bcd were treated with the indicated inhibitors and western blotting was performed using the HA antibody to determine the total amount of HA–Bcd (top). β-actin (bottom) represents the loading control. () Time course of Bcd degradation in embryonic extracts. HA–Bcd, synthesized in vitro, was analysed in embryonic extracts in the presence of MG132 or DMSO (as a control). Aliquots were taken from reaction tubes at the indicated times, and HA–Bcd was detected by western blotting. () Bcd degradation kinetics. Intensity of the HA–Bcd bands in were quantified and calculated as a percentage of the intensity of the band at time zero for each treatment. The lines represent exponential fitting to the experimental data. Throughout this study, all kinetic experiments shown were performed through side-by-side comparison of western blots (as shown in Fig. 1b, c) and onl! y such side-by-side results within a single panel can be compared. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 2: Bcd is ubiquitylated. () Time course of Bcd degradation in embryonic extracts with or without ATP addition (), ATP depletion () or UM-N0K addition (). Hexokinase (together with glucose) was used to deplete ATP. For each experiment, aliquots were taken from the reaction tubes at the indicated times. () HA–Bcd was detected by western blotting. () Fraction of remaining Bcd protein (quantified from intensity of the bands in the western blots) plotted against reaction time. In each case the lines represent exponential fitting to the experimental data. () Bcd ubiquitylation detected in cells. HEK 293T cells were transfected with plasmids expressing HA–Bcd and were treated with MG132 as indicated. HEK 293T cells were co-transfected with plasmids encoding Flag-Ubiquitin to increase ubiquitylated products. Whole-cell extracts were immunoprecipitated using an anti-HA antibody and analysed by western blotting using the anti-ubiquitin antibody. Ubiquitin-modified Bcd products are indicated, and molecular! weight standards are shown on the left. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 3: Fsd has a role in Bcd protein degradation. () CHX assay using cells stably expressing HA–Bcd. Cells were treated with CHX, and harvested at indicated times to detect the total amount of Bcd in cells by western blotting using anti-HA (left). Western blotting of β-actin (right) was used as a loading control. Top, control cells without dsRNA treatment; bottom cells incubated with fsd dsRNA before CHX. () Fraction of remaining Bcd protein in control cells and cells incubated with fsd dsRNA (quantified from intensity of bands in and normalized to β-actin intensities) plotted against time after CHX addition. The lines represent exponential fitting to the experimental data. () Drosophila S2 cells were transiently transfected with a plasmid encoding HA–Bcd. Cells were co-transfected with control vector (pAc5.1; vector used for protein expression), plasmid encoding Fsd (pAc-Fsd), or a control F-box protein (pAc-CG12402). For each lane, the loading amount was adjusted according to the activity of β-galactosidase express! ed from a lacZ control plasmid transfected at the same time as the indicated plasmids (to measure transfection efficiency). Relative intensity of the bands is indicated at the bottom. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 4: Fsd interacts with both Skp1 and Bcd. () A co-immunoprecipitation experiment detecting the interaction between Fsd and Skp1. HEK 293T cells were co-transfected with the indicated combinations of plasmids expressing Flag–Fsd and HA–Skp1. Anti-Flag antibody was used to immunoprecipitate (IP) Flag–Fsd from whole-cell extracts, followed by western blotting analyses using anti-HA (top) and anti-Flag (bottom) antibodies to detect HA–Skp1 and Flag–Fsd, respectively. () Experiments were performed as in , except with the use of a plasmid expressing HA–Bcd (in place of HA–Skp1) to detect the interaction between Bcd and Fsd. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 5: Posterior shift of fatemap along the A–P axis in fsd embryos. () Schematic representation of the fsd gene (not to scale). The arrows under the genes show the direction of transcription and the boxes represent annotated exons. The blue sections of the boxes represent the annotated fsd coding sequences, whereas the unfilled sections represent untranslated regions. The position of the KG02393 insertion and the two F-box domains are marked. Parts of the two annotated neighbouring genes are also shown. () Midsagittal images of living embryos from w1118 () and fsdKG02393() flies at 25 °C, imaged under halocarbon oil. Arrowheads represent the cephalic furrow positions. Scale bar, 100 μm. () Average normalized fluorescence intensities from FISH on whole-mount embryos, detecting the transcripts of eve (), hb () and kni () in wild-type (blue) and fsd (red) embryos. See Supplementary Information, Fig. S6 for raw data, extracted from individual embryos. The eve expression stripes 1 to 7 have the following posterior shifts in their respective pos! terior boundary positions in fsd embryos: 2.2%, 1.9%, 2.9%, 2.8%, 3.4%, 1.5% and 1.1% of the embryo length. * Figure 6: Bcd gradient profiles in wild-type and fsd embryos. () Whole-mount wild-type () and fsd () embryos were immunostained with anti-Bcd antibodies, imaged by immunofluorescence microscopy, and the fluorescence intensity of Bcd was quantified along the A–P axis. Each colour represents data from an individual embryo. The line at the bottom represents background intensities. Data are means ± s.d. B, Bcd intensity. () ln (B/Bmax) plotted against x/L for average Bcd profiles from wild-type () and fsd () embryos. Both B and Bmax are background-subtracted as necessary, without any further adjustments. The solid lines represent linear fits for Bcd profiles from wild-type embryos (y = −7.02x + 0.61, Adjusted R2 = 0.997) and fsd embryos (y = −6.31x + 0.45, Adjusted R2 = 0.998). () λ values calculated from Bcd intensity profiles of individual wild-type and fsd embryos from and . Individual data points (unfilled circles) and means ± s.d. (boxes with error bars) are indicated, with P value from Student's t-test indicated at the top. ! () The average intensity of Bcd along the A–P axis was quantified for wild-type and fsd embryos shown in and . The x/L positions at which the Bcd intensities (without adjustments) from the wild-type and fsd embryos cross a concentration threshold are shown. The measured average hb boundary positions in these embryos are marked with solid arrowheads. Data from wild-type embryos are shown in blue, and fsd embryos in red. Author information * Abstract * Author information * Supplementary information Affiliations * Division of Biomedical Informatics, Cincinnati Children's Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. * Junbo Liu & * Jun Ma * Division of Developmental Biology, Cincinnati Children's Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. * Jun Ma Contributions J.L. and J.M. conceived and designed the study. J.L. performed all experiments and analysis. J.L. and J.M. interpreted the data, J.L. generated all figures and J.L. and J.M. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jun Ma Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (5M) Supplementary Information Additional data - CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity
- ncb 13(1):30-39 (2011)
Nature Cell Biology | Article CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity * Manu Sharma1 Contact Manu Sharma Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline Burré1 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas C. Südhof1, 2 Contact Thomas C. Südhof Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:30–39Year published:(2011)DOI:doi:10.1038/ncb2131Received15 April 2010Accepted28 October 2010Published online12 December 2010Corrected online21 December 2010 Abstract * Abstract * Change history * 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 A neuron forms thousands of presynaptic nerve terminals on its axons, far removed from the cell body. The protein CSPα resides in presynaptic terminals, where it forms a chaperone complex with Hsc70 and SGT. Deletion of CSPα results in massive neurodegeneration that impairs survival in mice and flies. In CSPα-knockout mice, levels of presynaptic SNARE complexes and the SNARE protein SNAP-25 are reduced, suggesting that CSPα may chaperone SNARE proteins, which catalyse synaptic vesicle fusion. Here, we show that the CSPα–Hsc70–SGT complex binds directly to monomeric SNAP-25 to prevent its aggregation, enabling SNARE-complex formation. Deletion of CSPα produces an abnormal SNAP-25 conformer that inhibits SNARE-complex formation, and is subject to ubiquitylation and proteasomal degradation. Even in wild-type mouse terminals, SNAP-25 degradation is regulated by synaptic activity; this degradation is decreased by CSPα overexpression, and enhanced by CSPα deletion. Thu! s, SNAP-25 function is maintained during rapid SNARE cycles by equilibrium between CSPα-dependent chaperoning and ubiquitin-dependent degradation, revealing unique protein quality-control machinery within the presynaptic compartment. View full text Figures at a glance * Figure 1: CSPα knockout selectively decreases SNAP-25 levels and increases SNAP-25 protease sensitivity. (, ) SNAP-25 levels in brains from wild-type (WT) and CSPα-knockout (CSPα−/−) mice at postnatal days P5, P20 and P40, and in cultured neurons at 9 days in vitro (9 DIV). Lysates of the indicated cells were analysed by immunoblot () and SNAP-25 levels were quantified by quantitative immunoblotting (n = 5; ). () Snap25 mRNA levels measured by qRT–PCR in brains from indicated mice at 20 days old. mRNA levels were normalized to those of BiP (n = 9). (, ) Brain homogenates from wild-type mice, CSPα−/− mice and CSPα−/− mice with transgenic overexpression of α-synuclein (+ tSyn) at P40 were digested with the indicated trypsin concentrations. Levels of SNAP-25, synaptobrevin-2 (Syb2) and syntaxin-1 (Synt-1) were assessed by immunoblots () and the concentration of trypsin that causes 50% reduction (EC50) in these proteins was quantified by quantitative immunoblotting (n = 5; ). See also Supplementary Information, Fig. S1e. (, ) Brain lysates from wild-type, CSPα�! �/− and CSPα−/− + tSyn mice (at P40) were incubated in SDS-sample buffer at the indicated temperatures for 15 min. The levels of remaining SDS-resistant SNARE-complexes were assessed by immunoblots (), and TM, the temperature where 50% of SNARE-complexes melt, was quantified by quantitative immunoblotting (n = 3; ). See also Supplementary Information, Fig. S1f. () Cortical neurons were isolated from wild-type (left) and CSPα−/− (right) mice, cultured for 9 days, treated with cycloheximide at the indicated temperatures, and lysates were immunoblotted. () Quantitative immunoblotting was used to quantify the time required for 50% degradation of the indicated proteins (t1/2). See also Supplementary Information, Fig. S1g. () t1/2 ratios in wild-type:CSPα−/− neurons assessed from data from experiments performed as in and (n = 6). All data are means ± s.e.m. (asterisks, P < 0.05; double-asterisks, P < 0.01; triple-asterisks, P < 0.001 using Student's t-test). Unc! ropped images of blots are shown in Supplementary Information,! Fig. S7. * Figure 2: CSPα deletion increases SNAP-25 ubiquitylation and proteasomal degradation. () Brain proteins from wild-type mice, CSPα-knockout mice, and CSPα-knockout mice expressing transgenic α-synuclein (CSPα−/− + tSyn) at P40 were immunoprecipitated (IP) with antibodies against SNAP-25, synaptobrevin-2 (Syb2), and syntaxin-1 (Synt-1), using pre-immune serum as a control (P-Imm). Top: bound proteins (and 5% of the input from wild-type samples) were immunoblotted with antibodies against ubiquitin (upper panels) or the immunoprecipitated proteins (lower panels). Bottom: the levels of the ubiquitylated proteins was assessed by quantitative immunoblotting. Levels of ubiquitylated proteins were normalized to the respective immunoprecipitated non-ubiquitylated proteins and calculated as percentage of levels in wild-type brains (n = 5). () Cortical neurons (at 9 DIV) from wild-type and CSPα-knockout mice were incubated for 36 h (wild-type) or 22 h (CSPα−/−; shorter time was chosen to correct for shorter half-life of SNAP-25) with cycloheximide and the i! ndicated protease inhibitors (EtOH, ethanol vehicle control; Leu, leupeptin; Pep, pepstatin; L + P, combined leupeptin and pepstatin; MG, MG132; Lac, clasto-lactacystin β-lactone). Top: lysates from neurons were analysed by immunoblotting. Bottom: remaining SNAP-25 in the cortical neurons was assessed by quantitative immunoblotting (0 h = neurons before treatment). SNAP-25 levels were normalized to β-actin and then to SNAP-25 levels before treatments (n = 4). () Cortical neurons (at 9 DIV) from wild-type and CSPα-knockout mice were incubated for 36 h (wild-type) or 22 h (CSPα−/−; SNAP-25 degrades faster in neurons from CSPα-knockout mice) with indicated protease inhibitors. SNAP-25 was then immunoprecipitated. Top: immunoprecipitated proteins (and 10% of the input) were analysed by SDS–PAGE and immunoblotting with antibodies against ubiquitin (upper panels) or SNAP-25 (lower panels). Bottom: SNAP-25 was quantified by quantitative immunoblotting. Levels of ubiquit! ylated SNAP-25 were normalized to the immunoprecipitated non-u! biquitylated SNAP-25, and further normalized to the levels of ubiquitylated SNAP-25 present in neurons incubated in ethanol only (n = 3). All data are means ± s.e.m. (Asterisk, P < 0.05; double-asterisks, P < 0.01; triple-asterisks, P < 0.001, using Student's t-test to compare experimental samples to wild-type controls). Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 3: Neuronal activity controls SNAP-25 levels in a CSPα-dependent manner. (, ) Cortical neurons from littermate wild-type and CSPα-knockout mice were cultured for 10 DIV, and then incubated in control medium (–), tetrodotoxin (TTX), a combination of CNQX and AP5 (C/A), K+, or Ca2+, in absence or in presence of cycloheximide. Neurons were incubated for 36 h (wild-type) or 22 h (CSPα−/−; shorter time to account for the destabilization of SNAP-25 in neurons from CSPα-knockout mice). Levels of SNAP-25, syntaxin-1 (Synt-1), synaptobrevin-2 (Syb2), CSPα, Hsc70 and SGT were assessed by immunoblotting () and quantified by quantitative immunoblotting (normalized to guanine nucleotide dissociation inhibitor, GDI; ). All data are means ± s.e.m. (asterisk indicates P < 0.05; double-asterisks indicate P < 0.01 using Student's t-test comparing experimental samples to control treatments; n = 5). Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 4: CSPα overexpression stabilizes SNAP-25. () Fluorescence microscopy images of cortical neurons that were infected with lentiviruses expressing EGFP–Myc–CSPα (top) or EGFP–Myc–SSPα (bottom) at 2 DIV, and analysed by immunofluorescence microscopy staining with antibodies against synapsin at 9 DIV (note that SSPα lacks the palmitoylated cysteines of CSPα). Individual (left, middle) and merged images (right) obtained for the EGFP signal (green) and the synapsin signal (red) are shown. () Quantification of synaptic protein levels in neurons overexpressing EGFP, EGFP–CSPα or EGFP–SSPα. Top left: lysates were immunoblotted with antibodies against the indicated proteins. Top right: level of EGFP–CSPα or EGFP–SSPα expression relative to endogenous CSPα, quantified by quantitative immunoblotting. Bottom: relative expression levels of indicated proteins in neurons expressing either EGFP alone, EGFP–CSPα or EGFP–SSPα, quantified by quantitative immunoblotting. Neurons were analysed at 21 days pos! t infection (Synt-1, syntaxin-1; Syb2, synaptobrevin-2; α-Syn, α-synuclein; n = 4). (, ) Measurements of synaptic protein degradation after overexpression of EGFP alone or EGFP–CSPα. Hippocampal neurons were infected with lentiviruses expressing EGFP only or EGFP–CSPα at 2 DIV, and subjected to treatment with cycloheximide at 16 DIV. The amount of remaining SNAP-25, Synt-1, Syb2, Hsc70, SGT, and SNAP-23 at indicated times after cycloheximide treatment was assessed by immunoblotting (), and half-lives were analysed from quantitative immunoblotting (n = 5; ). See also Supplementary Information, Fig. S4b–d. All data are means ± s.e.m. (Asterisk, P < 0.05; double asterisks, P < 0.01, using Student's t-test). Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 5: CSPα binds to monomeric SNAP-25 through Hsc70. () Immunoprecipitation analysis of protein complexes in the brain of wild-type mice. Solubilized brain proteins were immunoprecipitated with antibodies indicated on top (P-Imm, pre-immune serum; α-Syn, α-synuclein; mS-25, antibody that recognizes only monomeric SNAP-25, but not SNAP-25 in SNARE complexes; Syb2, synaptobrevin-2; Synt-1, syntaxin-1), and analysed by immunoblotting with antibodies against the indicated proteins. () GST pulldown experiments from wild-type brain lysates using purified GST–fusion proteins (shown on Coomassie-stained gels; top). Brain lysate was incubated in the presence of non-hydrolyzable ADPβS with immobilized GST, GST–Syb2, GST–SNAP-25 (GST-S-25) or GST–Synt-1, and with GST, GST–Hsc70, GST–CSPα, GST–SGT or GST–CSPα. Bound proteins were analysed by immunoblotting (bottom). () Binding of purified recombinant Hsc70, SGT or CSPα to an immobilized GST–SNAP-25 fusion protein (left) or GST alone (right). Binding was carried out! in presence of non-hydrolyzable ATPγS or ADPβS, and analysed by SDS–PAGE and Coomassie-blue staining (top) or immunoblotting for Hsc70 (bottom). For the reverse binding experiment, see Supplementary Information, Fig. S5b. Representative data from n = 3 independent experiments are shown. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 6: The CSPα–Hsc70–SGT complex chaperones SNAP-25 and enhances its incorporation into SNARE complexes in vitro. () Immunoblots of in vitro SNAP-25 aggregation (left) and SNARE-complex assembly assays (right). Purified SNAP-25 was incubated at 37 °C for the indicated times in presence of Mg2+–ATP with CSPα, SGT and Hsc70 (all at equimolar concentration), as indicated on the left. Mg2+–ATP was also replenished at the times indicated at the top. At the end of an incubation, SNAP-25 aggregation was analysed by SDS–PAGE of non-boiled samples and SNAP-25 immunoblotting (left). At the same time, part of each sample was incubated with equimolar amounts of syntaxin-1 (Synt-1) and synaptobrevin-2 (Syb2) for 16 h at 4 °C, and SNARE-complex assembly was analysed by SDS–PAGE and syntaxin-1 immunoblotting of non-boiled samples (right; the left lane in all gels corresponds to a control incubation lacking SNAP-25 to show that SNARE-complex assembly is dependent on SNAP-25). ( Quantification of in vitro SNAP-25 aggregation (left) and SNARE-complex assembly assays (right) by quantitative imm! unoblotting. For each assay, the time required for a 50% effect was calculated (see Supplementary Information, Fig. S6b, d). Data in are means ± s.e.m. (asterisk; P < 0.05; double asterisk; P < 0.01, using Student's t-test; n = 5). Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 7: Model of the function of the CSPα–Hsc70–SGT chaperone complex. After monomeric SNAP-25 is released from SNARE complexes by NSF and SNAPs (ref. 29), it is in an equilibrium between a SNARE-complex assembly-competent conformer (SNAP-25), and a misfolded conformer (SNAP-25*) that inhibits SNARE-complex assembly. SNAP-25* generated by synaptic activity is ubiquitylated (Ub) and degraded by the proteasome. The CSPα–Hsc70–SGT complex refolds SNAP-25* into SNARE-complex competent SNAP-25, preventing its degradation and boosting SNARE-complexes. Change history * Abstract * Change history * Author information * Supplementary informationCorrigendum 21 December 2010In the version of this article initially published online and in print, grant information was missing from the acknowledgements. Author information * Abstract * Change history * Author information * Supplementary information Affiliations * Department of Molecular and Cellular Physiology, Stanford University, SIM1, 265 Campus Drive, Palo Alto, CA 94304-5453, USA. * Manu Sharma, * Jacqueline Burré & * Thomas C. Südhof * Howard Hughes Medical Institute, Stanford University, SIM1, 265 Campus Drive, Palo Alto, CA 94304-5453, USA. * Thomas C. Südhof Contributions M.S. and T.C.S. designed the study. M.S. and J.B. performed and analysed the experiments. M.S., J.B. and T.C.S. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Manu Sharma or * Thomas C. Südhof Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Information (961K) Supplementary Information Additional data - Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons
- ncb 13(1):40-48 (2011)
Nature Cell Biology | Article Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons * Wolfgang Wagner1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan D. Brenowitz2 Search for this author in: * NPG journals * PubMed * Google Scholar * John A. Hammer III1 Contact John A. Hammer III Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:40–48Year published:(2011)DOI:doi:10.1038/ncb2132Received15 January 2010Accepted17 November 2010Published online12 December 2010 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 Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va. We show here that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into Purkinje neuron spines. Specifically, myosin-Va accumulates at the ER tip as the organelle moves into spines, and hydrolysis of ATP by myosin-Va is required for spine ER targeting. Moreover, myosin-Va is responsible for almost all of the spine ER insertion events. Finally, attenuation of the ability of myosin-Va to move along actin filaments reduces the maximum velocity of ER movement into spines, providing direct evidence that myosin-Va drives ER motility. Thus, we have established that an actin-based motor moves ER within animal cells, and have uncovered the mechanism for ER localization to Purkinje neuron spines, a prerequisite for synaptic plasticity. View full text Figures at a glance * Figure 1: The delayed mGluR1-dependent Ca2+ transient, but not the fast AMPA receptor-dependent Ca2+ transient, is absent in dl20J/dl20J Purkinje neuron spines. () Images of a Purkinje neuron (upper panel; scale bar, 30 μm) and a Purkinje neuron dendrite and spines (lower panel; scale bar, 1 μm). Purkinje neurons in cerebellar slices were voltage clamped at −70 mV, loaded with Ca2+ indicator Fluo-4 and red fluorophore Alexa-594, and imaged by two-photon laser scanning microscopy. The white circle in the lower panel indicates the location of two-photon laser uncaging of MNI-glutamate, and the yellow line indicates the line scan region. () Ca2+ transients in littermate control dv/dl20J Purkinje neuron spines (red traces) and adjacent dendritic shafts (blue traces) evoked by glutamate uncaging at the spine head at the time indicated (arrowhead), in the presence of the AMPA receptor inhibitor DNQX (left and middle panels), or in the additional presence of the mGluR1 antagonist CPCCOEt (right panel). Left panel: traces from a single trial from the spine depicted in . The middle and right panels: group data for 23 spines (4 Purkinje n! eurons) and 20 spines (3 Purkinje neurons), respectively (solid traces indicate mean, shaded areas indicate s.e.m.). () CPCCOEt-sensitive Ca2+ transients are not observed in dl20J/dl20J Purkinje neuron spines following glutamate uncaging in the presence of DNQX (compare with , middle panel). Mean Ca2+ traces (± s.e.m.) from group data for 45 spines (4 Purkinje neurons) are shown. () Average Ca2+ transient peak magnitudes (ΔF/F mean ± s.e.m.) in spines (red bars) and dendrites (blue bars) of dv/dl20J (with and without CPCCOEt) and dl20J/dl20J Purkinje neurons. (, ) Fast Ca2+ transients in dv/dl20J () and dl20J/dl20J () Purkinje neuron spines (red traces) and adjacent dendritic shafts (blue traces) evoked by two-photon laser uncaging of MNI-glutamate at the spine head at the time indicated (arrowhead), in the presence of CPCCOEt (upper panels), and in the additional presence of DNQX (lower panels). Ca2+ traces from group data for 13 spines (3 Purkinje neurons, ) and 5 spin! es (2 Purkinje neurons, ) are shown (solid traces indicate mea! n, shaded areas indicate s.e.m.). The peaks of the fast Ca2+ transient in the presence of CPCCOEt are ΔF/F = 39 ± 5% (dv/dl20J) and 109 ± 32% (dl20J/dl20J). * Figure 2: Models of how myosin-Va might function to localize ER to Purkinje neuron spines. () Mechanism 1 (non-cell autonomous model): myosin-Va present in another cerebellar cell type, such as a granule neuron, facilitates the release of a diffusible factor that confers on the Purkinje neuron the ability to target ER to its spines. Of relevance to this model, myosin-Va is a synaptic vesicle-associated protein that has a role in regulating exocytosis5, 26, 27, and is widely expressed throughout the brain, including within granule neurons14. () Mechanism 2 (tethering model): myosin-Va delivers a tethering factor to the spine tip that links the ER to the spine tip after myosin-Va-independent transport of ER into the spine. In this case, ER motility might be microtubule-based, as microtubules transiently enter the spines of hippocampal neurons31, 32, and microtubule plus-end-directed motors are known to transport ER18, 33. Alternatively, a direct interaction between the microtubule plus-end tracking proteins EB1/3 and the integral ER membrane protein STIM1 might driv! e the movement of ER tubules into spines18, 34. With regard to myosin-Va-dependent tethering, candidates for tethering proteins that might link the ER to the spine tip, such as Homer, are present in Purkinje neuron spines28, 29, 30. Alternatively, myosin-Va itself could be an integral component of the tethering mechanism4, 5, 6, 7. () Mechanism 3 (transport model): myosin-Va associates with the ER and transports it along actin filaments into spines. In the simplest case, the ER is then maintained at the spine tip by the continued effort of myosin-Va to carry it to the barbed end of actin filaments. We note, however, that the myosin-Va-mediated transport of ER into spines envisioned by Mechanism 3 could be followed by the maintenance of the ER in spines through a secondary tethering mechanism that is, by definition, myosin-Va-dependent either because it occurs only after the myosin has delivered the ER to the vicinity of the spine tip, or because once there, the myosin is an! essential component of the tethering mechanism. * Figure 3: Translocation of ER into spines is disrupted in dl20J/dl20J Purkinje neurons. Shown are cultured, live Purkinje neurons from wild-type mice at 15 DIV (, ) and at 10 DIV (, , , ), and from dl20J/dl20J mice at 15 DIV (, ) and at 10 DIV (, ). Cultures were transfected with pL7-mRFP–ER-IRES–EGFP to visualize cell volume and ER. Superimpositions are also shown (overlay). The upper panels in , , , and are images reconstructed from confocal Z-stacks (scale bar, 20 μm), whereas the lower panels show magnified images of a single confocal plane of Purkinje neuron dendrites (scale bar, 2 μm). The latter images were taken from a time series (Supplementary Information, Movies 1, 3). Panels , , and show montages of 30 frames of a time series and depict the spines in the white boxes in , , and , respectively, at higher magnification (scale bar, 2 μm; see also Supplementary Information, Movies 2, 4). Panels and show montages of 15 frames of a time series and depict nascent spine-like protrusions (scale bar, 2 μm). * Figure 4: Quantification of ER dynamics indicates that ER movement into Purkinje neuron spines depends critically on myosin-Va. Movies recorded at 1 frame per second (fps) at 10 DIV from wild-type (WT), dv/dl20J, and dl20J/dl20J Purkinje neurons expressing mRFP–ER and free GFP to visualize ER and cell volume were analysed. All data shown in , , and are the mean ± s.e.m. (n = 3 experiments, except wild-type, where n = 4 experiments). () Steady state presence of spine ER. The relative times that spines were filled, partially filled, or devoid of ER were determined from recorded movies and are plotted in the graphs. () The graph shows the frequency of ER insertion events towards the spine tip when observing individual spines that are devoid or only partially filled (that is, not fully loaded) with ER. Only motility events where the ER translocated for more than 0.3 μm towards the spine tip were counted. During a 2-min recording, ER insertions took place, on average, in 14.0% ± 1.5 of all WT spines and 20.9% ± 0.3 of all dv/dl20J spines, indicating that ER insertions are not restricted to a small s! ubset of spines. () The graph shows the frequency of spine ER retractions when observing individual spines that are fully or partially filled with (that is, not devoid of) ER. Only motility events where the ER retracted for more than 0.3 μm towards the dendritic shaft were counted. * Figure 5: ER insertion into control dv/dl20J Purkinje neuron spines does not depend significantly on microtubule entry into spines. () Upper left panel: dendrite of a 10 DIV dv/dv Purkinje neuron expressing GFP-α2-tubulin and mCherry to visualize microtubules and cell volume, respectively. Right panels: the spine indicated by the white line in the left panel was subjected to kymograph analysis. Lower left panel: graph showing the fraction of wild-type spines with microtubule entry after nocodazole (NOCO) or vehicle (DMSO) addition (mean ±s.e.m.; DMSO, n = 3; NOCO; n = 4; *N/D, not detected). () Steady state presence of spine ER in dv/dl20J and dl20J/dl20J Purkinje neurons after addition of NOCO or DMSO. The graphs show the relative times that spines were filled, partially filled, or devoid of ER (mean ± s.e.m.; n = 3 experiments; **data from a single experiment). () Frequency of ER insertion events towards the spine tip in individual spines that are devoid or only partially filled with ER after addition of NOCO or DMSO (mean ± s.e.m.; n = 3 experiments). No difference was detected between NOCO-treate! d, DMSO-treated and untreated dv/dl20J (Fig. 4b) samples (P >0.4, Student t-test). *N/D, no ER movement into spines detected in three experiments; **data from a single experiment (frequency similar to mean of untreated dl20J/dl20J Purkinje neurons; Fig. 4b). () Frequency of spine ER retractions from individual spines that were fully or partially filled with ER (mean ± s.e.m.; n = 3 experiments). No difference was detected between NOCO-treated, DMSO-treated and untreated dv/dl20J (Fig. 4c) samples (P >0.3, Student t-test). () Graph showing the fraction of spines with microtubule entry (mean ± s.e.m.; n = 23 dv/dv Purkinje neurons and 15 dl20J/dl20J Purkinje neurons, respectively; microtubules and cell volume visualized as in ). () Upper panel: dendrite of a dv/dl20J Purkinje neuron expressing mRFP–ER and GFP–α2-tubulin to visualize ER and microtubules, respectively. The spine subjected to kymograph analysis is indicated by the white line. Lower panels: kymographs; spi! ne ER extensions that coincide (+MT) and do not coincide (–M! T) with a spine microtubule visit are indicated. * Figure 6: mGFP–myosin-Va expressed in dl20J/dl20J Purkinje neurons rescues ER targeting and accumulates at the tip of the spine ER. (–) Live dl20J/dl20J Purkinje neurons transfected with pL7-mRFP-ER and pL7-mGFP–myosin-Va to visualize the ER and the myosin (MVaWT), respectively. Scale bars, 2 μm. () Maximum projection of a confocal Z-stack of a section of a Purkinje neuron dendrite at 15 DIV (see Supplementary Information, Movie 6 for the corresponding three-dimensional reconstruction). () Images corresponding to single confocal planes of Purkinje neuron dendrites at 10 DIV and 15 DIV. Three examples are shown for each time point. The images were taken from time series (Supplementary Information, Movies 7, 8). () Kymograph images of the spines indicated by the white boxes in , showing that the dot of mGFP–myosin-Va fluorescence remains present at the distal tip of the ER tubule as the tubule extends or shortens over time (x axis, time; y-axis, size). () Live dl20J/dl20J Purkinje neurons transfected with pL7-mRFP–ER to visualize the ER and either pL7-mGFP–myosin-Va (MVaWT), pL7-mGFP–myosin-Va! R219A (MVaR219A), pL7–mGFP–myosin-VaG440A (MVaG440A) or pL7-mGFP–myosin-VaE442A (MVaE442A). Images were reconstructed from confocal Z-stacks and show cells at 13 DIV. Superimposition of images is also shown (overlay). Scale bar, 20 μm. Each insert in the overlay images shows a single confocal plane image of a dendrite from a Purkinje neuron transfected with the respective plasmids and depicts ER (red) and myosin (green). Images were taken from a time series (Supplementary Information, Movie 9). Scale bar, 2 μm. * Figure 7: Myosin-Va does not colocalize with the PSD marker PSD-93 and is present at the leading tip of the ER tubule as the organelle translocates into a spine. () Two examples of live wild-type Purkinje neurons expressing mCherry-Myosin-Va (MVa) and PSD-93–mGFP (PSD-93) at 10 DIV. Superimposition of the images is also shown (overlay). The images are single confocal sections and were taken from a time series (see Supplementary Information, Movie 11). Scale bar, 2 μm. () Live dl20J/dl20J Purkinje neurons transfected with pL7–mCerulean, pL7–mGFP–myosin-Va and pL7–mRFP–ER to visualize cell volume, myosin-Va (MVa) and ER, respectively. Superimpositions of volume and myosin-Va and myosin-Va and ER are also shown. Images show a single confocal plane and were taken from a time series recorded at a rate of 0.5 fps at DIV 8 (see also Supplementary Information, Movie 12). Scale bar, 1 μm. * Figure 8: Decreasing the step size or ATPase activity of myosin-Va reduces the efficiency of ER targeting to Purkinje neurons spines and the maximum velocity of ER movement into spines. () Wild-type (WT), dv/dl20J, and dl20J/dl20J Purkinje neurons expressing mRFP–ER to visualize ER and free GFP to visualize cell volume, as well as dl20J/dl20J Purkinje neurons expressing GFP-tagged versions of WT myosin-Va, myosin-Va4IQ, myosin-Va2IQ or myosin-VaS217A, in addition to mRFP–ER and free GFP were observed using confocal live microscopy. The graph shows the fraction of spines (mean ± s.e.m.) that contain ER, as determined from confocal images recorded at 15 DIV. The numbers of analysed Purkinje neurons were 15 (WT), 18 (dv/dl20J), 16 (dl20J/dl20J), 21 (MVaWT), 6 (MVa4IQ), 22 (MVa2IQ), and 17 (MVaS217A) (on average, 23.5 spines per Purkinje neurons were analysed; *P <0.0001; Student t-test; see Supplementary Information, Movie 10 for examples). () Instantaneous velocities of ER insertion movements into spines were measured at DIV 10 (see Methods and Supplementary Information, Fig. S7). WT Purkinje neurons expressing mRFP–ER and free GFP, as well as dl20J/dl! 20J Purkinje neurons expressing GFP-tagged versions of WT myosin-Va, myosin-Va4IQ, myosin-Va2IQ or myosin-VaS217A, in addition to mRFP–ER and free GFP were analysed. The graph shows the maximum velocity calculated by averaging the fastest 10% of instantaneous velocities directed towards the spine tip (error bars are s.e.m.). The total numbers of instantaneous movements analysed were 91 (WT), 207 (dl20J/dl20J), 264 (MVa4IQ), 350 (MVa2IQ), and 133 (MVaS217A) (*P <0.05; **P <0.0005, Student t-test). () Images of live dl20J/dl20J Purkinje neurons expressing mRFP–ER and either mGFP–myosin-Va2IQ or mGFP–myosin-VaS217A. Images depict single confocal sections of Purkinje neuron dendrites. Overlay is also shown (overlay). Scale bar, 2 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, MD 20892, USA. * Wolfgang Wagner & * John A. Hammer III * Section on Synaptic Transmission, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, MD 20892, USA. * Stephan D. Brenowitz Contributions W.W. and J.A.H. designed the project; W.W. carried out the experiments, except the Ca2+ imaging, which was carried out by S.D.B.; J.A.H. contributed new reagents and W.W., S.D.B. and J.A.H. analysed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * John A. Hammer III Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (10M) Supplementary Information * Supplementary Movie 2 (474K) Supplementary Information * Supplementary Movie 3 (11M) Supplementary Information * Supplementary Movie 4 (352K) Supplementary Information * Supplementary Movie 5 (4M) Supplementary Information * Supplementary Movie 6 (1M) Supplementary Information * Supplementary Movie 7 (13M) Supplementary Information * Supplementary Movie 8 (12M) Supplementary Information * Supplementary Movie 9 (12M) Supplementary Information * Supplementary Movie 10 (6M) Supplementary Information * Supplementary Movie 11 (2M) Supplementary Information * Supplementary Movie 12 (521K) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - Collective cell migration requires suppression of actomyosin at cell–cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6
- ncb 13(1):49-58 (2011)
Nature Cell Biology | Article Collective cell migration requires suppression of actomyosin at cell–cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6 * Cristina Hidalgo-Carcedo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Steven Hooper1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shahid I. Chaudhry1 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Williamson2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin Harrington3 Search for this author in: * NPG journals * PubMed * Google Scholar * Birgit Leitinger4 Search for this author in: * NPG journals * PubMed * Google Scholar * Erik Sahai1 Contact Erik Sahai Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:49–58Year published:(2011)DOI:doi:10.1038/ncb2133Received18 March 2010Accepted02 November 2010Published online19 December 2010 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 Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell–cell junctions. Here we show that collective invasion by cancer cells depends on decreasing actomyosin contractility at sites of cell–cell contact. When actomyosin is not downregulated at cell–cell contacts, migrating cells lose cohesion. We provide a molecular mechanism for this downregulation. Depletion of discoidin domain receptor 1 (DDR1) blocks collective cancer-cell invasion in a range of two-dimensional, three-dimensional and 'organotypic' models. DDR1 coordinates the Par3/Par6 cell-polarity complex through its carboxy terminus, binding PDZ domains in Par3 and Par6. The DDR1–Par3/Par6 complex controls the localization of RhoE to cell–cell contacts, where it antagonizes ROCK-driven actomyosin contractility. Depletion of DDR1, Par3, Par6 or RhoE leads to increased actomyosin contactility at cell–cell contacts, a loss of cell–cell cohesion! and defective collective cell invasion. View full text Subject terms: * Cancer Figures at a glance * Figure 1: DDR1 is required for collective cell migration. () Top left: A431 SCC cells collectively invading a three-dimensional matrix (red, F-actin; cyan, reflectance). Other left-hand panels show a higher magnification of the indicated area: blue, β-catenin; red, F-actin; green, GFP–MLC. Right: A431 cells on a two-dimensional substrate; colours as in the left-hand panels. Scale bars, 10 μm. () DDR1 western blots showing efficacy of siRNA (left) and shRNA (right); tubulin was used as a loading control. () Representative images of F-actin organization, pS19-MLC, myosin IIa or E-cadherin in A431 cells transfected with control siRNA (left) or with two different siRNA oligonucleotides against DDR1: DDR1 siRNA #3 (middle) and DDR1 siRNA #4 (right). Scale bars, 20 μm. () (A) Top: snapshots from phase-contrast movies of control and DDR1 shRNA-transfected A431 cells moving on collagen gels. Scale bars, 20μm. Blue shading shows the position of cell groups at t = 0; red shading shows the position of the same cell groups at t = 6 h. (B! , C) Cell dispersion index (B; means and s.d.) and the speed of cells within groups (C; median, quartiles and 10th and 90th centiles shown). Data were calculated from tracking multiple cell groups from multiple experiments (analysis of seven to ten colonies from three independent experiments). Asterisk, P < 0.01 (Student's t-test). () Three-dimensional reconstruction of 'spheroid' SCC invasion assays in control and DDR1-depleted A431 cells. Grid spacing, 50 μm. () Haematoxylin/eosin stain sections of control or DDR1 siRNA-transfected SCC cells in an organotypic assay. The number in each bottom left corner shows the invasion index of three independent experiments as a percentage of the non-transfected cells. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 2: DDR1 does not require kinase activity or collagen binding to regulate actomyosin at cell contacts. () Representative pictures in which DDR1 resistant to siRNA oligonucleotides 3 and 4 was expressed in two DDR1 stable knockdown clones (DDR1 shRNA #3 and DDR1 shRNA #4) or control cells (Control shRNA). F-actin is shown in red and DDR1 in green. The bottom panels show shDDR1 3 cells transfected with Cherry as a control. WT, wild type. Scale bars, 10 μm. () Top: quantification of recovery of normal actin organization in two DDR1 stable knockdown clones (DDR1 shRNA #3 and DDR1 shRNA #4) or control cells (Control shRNA) transfected with Cherry as a control or with a DDR1 construct resistant to siRNA oligonucleotides 3 and 4. The left graphs show when DDR1 (or Cherry) was expressed in only one of the two cells in contact; the right graphs show when DDR1 (or Cherry) was expressed in both cells in contact. Bottom: percentage of cells with normal actin organization in the cell–cell contacts in DDR1 shRNA #3 and DDR1 shRNA #4 cells transfected with Cherry as a control or with dif! ferent DDR1 constructs resistant to siRNA oligonucleotides 3 and 4 (WT, K618A, R105A or ΔDS1). Means and s.e.m. are shown for three experiments; 20–30 cells were counted in each experiment. () F-actin and pS19-MLC organization in A431 cells transfected with ECTM–GFP (wild-type in top panels and R105A in bottom panels). Scale bar, 20 μm. () Example of DDR1 staining (green) in apical (top) and basal (bottom) membranes of A431 cells. F-actin; red. Scale bars, 20 μm. () F-actin and DDR1 staining is shown in A431 cells transfected with control or E-cadherin siRNA. Scale bar, 20 μm. () Western blot showing E-cadherin and DDR1 levels in A431 cells transfected with control or E-cadherin siRNA. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 3: DDR1 interacts with Par3 and Par 6. () Comparison of the last C-terminal amino acids of DDR1 in human, rat, mouse, claudin 4, 5 and 6 and DDR2. Residues in red are conserved amino acids. () Negative staining of glutathione S-transferase (GST) pulldown, using GST alone as control (left), DDR1 amino acids 801–876 (centre) and DDR1 amino acids 801–875 (right). Asterisks show bands present only in the 801–876 lane. Bottom panel: Par3 western blot (WB) showing Par3 pulled down only by DDR1 801–876. () (A) Non-transfected (left), GFP-transfected (centre) or Par3–GFP-transfected (right) A431 cell lysates were incubated with anti-GFP antibody and the amount of endogenous DDR1 bound was determined by western blotting. The Par3 western blot is shown in the middle. The amount of DDR1 in the starting lysates is shown in the bottom blot. IP, immunoprecipitation. (B) Non-transfected (left), empty PRK5.1-transfected (centre) or Par6–Flag-transfected (right) A431 cell lysates were incubated with anti-Flag antibody! and the amount of endogenous DDR1 bound was determined by western blotting. The amount of DDR1 in the starting lysates is shown in the bottom blot. () Flag-tagged PDZ domains of Par3 (third to fifth lanes) and Par6 (sixth lane) were immunoprecipitated from 293 cells also expressing DDR1. Non-transfected cells (first lane) and DDR1 alone (second lane) were used as controls. () Representative pictures showing Par3, DDR1 and F-actin staining (blue in merge) in A431 cells in apical and basal membranes. Scale bar, 10 μm. () Representative pictures showing actin (red) and Par3 (green) localization in two clones stably knocked down for DDR1 (DDR1 shRNA #3 or DDR1 shRNA #4) or control A431 cells (Control shRNA). Scale bars, 10 μm. () Representative images of Par3 (top) and DDR1 (bottom) in A431 cells that were mock transfected (left panels) or transfected with siRNA against Par3 (right panels). Scale bar, 10 μm. Uncropped images of blots are shown in Supplementary Information, ! Fig. S7. * Figure 4: Par3 and Par6 are required for efficient collective invasion. () (A) Representative images showing pS19-MLC (green) and F-actin (red) localization in siRNA control, Par3 or Par6 transfections with two different siRNA oligonucleotides. Scale bars, 10 μm. (B) Quantification of F-actin organization at cell–cell contacts. A431 cells were transfected with the indicated siRNA; 60 h after transfection they were fixed and stained for F-actin. The proportion of cells with tight F-actin at cell–cell contacts was scored (n = 2; more than 200 cells for each data point). () Left, Par3 and DDR1 levels in A431 cells transfected with control or Par3 siRNA; β-tubulin is shown as a loading control. Right, quantitative RT–PCR confirmation of Par6 depletion by two siRNA oligonucleotides in A431 cells (normalized to glyceraldehyde-3-phosphate dehydrogenase). Means and s.d. are shown of triplicates. () Dispersion index of cells moving on collagen gels in control, Par3 or Par6 siRNA-transfected A431 cells. Analysis of seven to ten colonies from three! independent experiments is shown. Asterisk, P < 0.01 (Student's t-test). () Representative images of haematoxylin/eosin-stained sections of control or Par3 or Par6 siRNA-transfected SCC cells in an organotypic assay. The number in the bottom right corner of each micrograph indicates the invasion index after quantification of three independent experiments as a percentage of the control cells. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 5: DDR1 controls RhoE localization at cell–cell contacts. () Representative images of F-actin organization or pS19-MLC in A431 cells transfected with control siRNA or with two different siRNA oligonucleotides against RhoE or p190RhoGAP. Scale bars, 10 μm. () RhoE (top) and p190RhoGAP (bottom) western blots showing efficacy of siRNA; β-tubulin was used as loading control. () Dispersion index of cells moving on collagen gels in control or RhoE siRNA-transfected A431 cells. Analysis of five to ten colonies from two independent experiments is shown. Asterisk, P < 0.01 (Student's t-test). () Three-dimensional reconstruction of 'spheroid' SCC invasion assays in control and RhoE-depleted A431 cells. Grid spacing, 50 μm. () Immunofluorescence for Myc (green) and actin (red) showing RhoE–Myc localization in two DDR1 stable knockdown clones (DDR1 shRNA #3 and DDR1 shRNA #4) or control cells. Scale bars, 10 μm. () Percentage of cells with RhoE localized to the plasma membrane. Means and s.d. are shown for three experiments; 20–30 cell! s were counted in each experiment. () Representative images showing DDR1 localization in A431 cells transfected with either control or RhoE siRNA. Scale bar, 10 μm. () Representative images of F-actin and pS19-MLC in control and DDR1 siRNA-transfected cells treated with 5 μm Y27632 (ROCK inhibitor). Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 6: Actomyosin organization in collective cell migration. Schematic model showing actomyosin organization in collective movement in normal conditions () or when DDR1, Par3 or RhoE have been depleted (). Author information * Abstract * Author information * Supplementary information Affiliations * Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. * Cristina Hidalgo-Carcedo, * Steven Hooper, * Shahid I. Chaudhry & * Erik Sahai * St George's Hospital, Tooting, London SW17 0QT, UK. * Peter Williamson * Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. * Kevin Harrington * National Heart and Lung Institute, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. * Birgit Leitinger Contributions C.H.C. and E.S. conceived and designed the experiments. C.H.C. performed all experiments except Figs 1a, e and 5c, d, f. Supplementary Information, Figures S1, S2 and S5c were performed by E.S. Fig 4b and various molecular cloning procedures were performed by S.H. The clinical samples used in Supplementary Information, Figures S1 and S6 were collected by S.I.C., P.W. and K.H. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Erik Sahai Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - The E3 ubiquitin ligase Wwp2 regulates craniofacial development through mono-ubiquitylation of Goosecoid
- ncb 13(1):59-65 (2011)
Nature Cell Biology | Letter The E3 ubiquitin ligase Wwp2 regulates craniofacial development through mono-ubiquitylation of Goosecoid * Weiguo Zou1 Search for this author in: * NPG journals * PubMed * Google Scholar * Xi Chen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jae-Hyuck Shim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhiwei Huang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas Brady1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dorothy Hu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rebecca Drapp1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kirsten Sigrist1 Search for this author in: * NPG journals * PubMed * Google Scholar * Laurie H. Glimcher1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Dallas Jones1, 2 Contact Dallas Jones Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:59–65Year published:(2011)DOI:doi:10.1038/ncb2134Received15 July 2010Accepted28 October 2010Published online19 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Craniofacial anomalies (CFAs) are the most frequently occurring human congenital disease, and a major cause of infant mortality and childhood morbidity. Although CFAs seems to arise from a combination of genetic factors and environmental influences, the underlying gene defects and pathophysiological mechanisms for most CFAs are currently unknown. Here we reveal a role for the E3 ubiquitin ligase Wwp2 in regulating craniofacial patterning. Mice deficient in Wwp2 develop malformations of the craniofacial region. Wwp2 is present in cartilage where its expression is controlled by Sox9. Our studies demonstrate that Wwp2 influences craniofacial patterning through its interactions with Goosecoid (Gsc), a paired-like homeobox transcription factor that has an important role in craniofacial development. We show that Wwp2-associated Gsc is a transcriptional activator of the key cartilage regulatory protein Sox6. Wwp2 interacts with Gsc to facilitate its mono-ubiquitylation, a post-tran! slational modification required for optimal transcriptional activation of Gsc. Our results identify for the first time a physiological pathway regulated by Wwp2 in vivo, and also a unique non-proteolytic mechanism through which Wwp2 controls craniofacial development. View full text Subject terms: * Developmental biology Figures at a glance * Figure 1: Craniofacial patterning defects are present in Wwp2GT/GT mice. () Schematic representation of the position of β-gal insertion into the Wwp2 locus. () Analysis of Wwp2 transcript levels by qPCR in wild-type mice (WT), Wwp2GT/+ (GT/+) and Wwp2GT/GT (GT/GT) mice. Values represent means ± s.d. (n = 6 for each genotype) () Immunoprecipitation and western blot analysis showing that Wwp2 protein is absent in the Wwp2GT/GT mice. () Photograph of 4-week-old male WT and Wwp2GT/GT mice. () Body weight of male and female WT and Wwp2GT/GT at 4 weeks of age. Values represent means ± s.d. (n = 6 for each genotype) () Presence of shortened snout in Wwp2GT/GT mice as shown grossly (left) as well as by alizarin red staining of WT and Wwp2GT/GT skulls (right). () Photograph showing misalignment of the jaw and the overgrown mandibular incisor in Wwp2GT/GT (GT/GT) mice. () μCT analysis of skulls from WT mice (left), and Wwp2GT/GT mice showing the shortened (middle) or twisted (right) nasal bone in the Wwp2GT/GT mice. () Quantitative analysis of the dist! ances between the various landmarks in the skulls of 4-week-old WT and Wwp2GT/GT mice. Values represent means ± s.d. (n = 8 for each genotype; *P <0.001). Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 2: Regulation of Wwp2 expression in the skull. () Whole-mount staining for β-gal in skulls of wild-type (WT) and Wwp2GT/GT mice. (, ) Immunohistochemical and safranin-O staining of skull sections from Wwp2GT/GT and wild-type mice reveal β- gal and cartilaginous regions, respectively. Immunohistochemical staining of a skull section from a wild-type mouse is shown in . Box in indicates area magnified from sections to give images in . In , safranin-O (left) and immunohistochemical (right) staining of indicated regions are shown for sections from wild-type (top) and Wwp2GT/GT mice (bottom). Scale bars, 0.5 mm. (, ) Analysis of Wwp2 by in situ hybridization () and Sox9 by immunostaining () in the skull of WT mice. Scale bar, 0.5mm. The boxed areas in and are magnified in the right panels. () Wwp2 transcript levels were measured by qPCR in ATDC5 cells (left) and C3H10T1/2 cells (right) following infection of cells with control or HA-tagged Sox9-expressing lentivirus. Values represent means ± s.d. (n = 3) () Levels of Sox9, ! Wwp2, Smurf1 and Smurf2 were evaluated by qPCR following infection of ATDC5 cells with a control lenvtivirus or lentivirus containing Sox9-specific shRNAs. Values represent means ± s.d. (n = 3) () Six separate primer sets (1–6) were used in qPCR reactions to scan the Wwp2 intron region that was bound by Sox9 in ChIP experiments in C3H10T1/2 cells, using anti-Sox9 (black bars) or IgG control (grey bars) antibodies. Values represent means ± s.d. (n = 3) () Increasing amounts of Sox9 but not Sox6 led to induction of luciferase levels in C3H10T1/2 cells transfected with a luciferase reporter construct that contains the region surrounding the Sox9 binding site in the Wwp2 intron. Values represent means ± s.d. (n = 3; *P <0.01, #P <0.05, compared with empty vector control). * Figure 3: Gsc interacts with and is ubiquitylated by Wwp2. () Co-immunoprecipitation experiments were conducted in 293T cells transfected with Myc–Wwp2 and Flag–Gsc or Flag–HoxA2 expression constructs. Wwp2 was immunoprecipitated from cell lysates with anti-Flag antibody, followed by western blot analysis with anti-Myc antibody. () Purified recombinant fragments of Wwp2 were used for in vitro interaction analysis. Western blot analysis with an anti-Flag antibody was used to detect Gsc interaction with the various fragments of Wwp2. () Co-immunoprecipitation experiments were conducted in 293T cells with Flag-epitope-tagged Gsc deletion mutants and Myc–Wwp2, as described in . () Interactions between recombinant WW domain fragments of Wwp2 and wild-type Gsc (PPGY) or Gsc bearing a AAGY or PPGA mutation were analysed by GST pulldown followed by western blot analysis. () Immunofluoresence analysis of ATDC5 chondrocyte cell line transfected with YFP-tagged Wwp2 expression construct and a Flag-epitope-tagged Gsc expression construc! t reveals colocalization of these proteins in the nucleus. Scale bar, 10 μm () Analysis of Gsc ubiquitylation was performed in 293T cells transfected with Flag–Gsc, Myc–Wwp2 and His-epitope-tagged ubiquitin. Ubiquitylated Flag–Gsc proteins were detected in cells by precipitating ubiquitylated proteins from denatured lysates with Ni-NTA-agarose, followed by western blot analysis with an anti-Flag antibody. () Additional Gsc ubiquitylation experiments were conducted as described in , using either wild-type (WT) Wwp2 or functionally inert Wwp2 harbouring a Cys to Ala mutation.() In vitro ubiquitylation of Gsc was performed using Flag–Gsc purified from 293T cells transfected with Flag–Gsc. Flag–Gsc was incubated with recombinant Wwp2, His–ubiquitin, UBE1 and Ubch7. Reactions were then subjected to western blot analysis with an anti-Flag antibody to detect Gsc proteins. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 4: Mono-ubiquitylation augments transcriptional activity of Gsc. () Western blot analysis of Gsc and Wwp2 protein levels in cell lysates generated from 293T cells that were transfected with either a Gsc-expression construct, or Gsc and Wwp2-expression constructs. Transfected cells were treated with cyclohexamide (CHX) for 2, 5, 8 and 12 h before cell lysis. () Endogenous Gsc protein levels were assessed by western blot analysis using an anti-Gsc antibody in lysates generated from nasal cartilage cells isolated from wild-type (WT) and Wwp2GT/GT mice. () Ubiquitylation of Gsc was evaluated in 293T cells transfected with expression constructs of Flag–Gsc, Myc–Wwp2, and either His–ubiquitin (WT) or His–ubiquitin (K0). Ubiquitylated Flag–Gsc proteins were detected as described in . Analysis of Gsc ubiquitylation was also assessed using recombinant His–Ub or His–K0 through in vitro ubiquitylation assays similar to those described above. () Schematic representation of the Gsc–Gal4 fusion protein and reporter construct used in sub! sequent luciferase experiments. () Luciferase levels were evaluated following transfection of Gsc-gal4 expresson construct, Gal4–luciferase reporter and increasing amounts of Wwp2 expression construct. Values represent means ± s.d. (n = 3; *P <0.01, compared with empty vector control). Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 5: Ubiquitylation of Gsc by Wwp2 is required for optimal expression of Sox6. () Coronal sections of wild-type (WT) and Wwp2GT/GT skulls were evaluated for expression of β-gal by immunostaining, and for Sox6 and Col1a1 by in situ hybridization. Scale bar, 1 mm () Sox6 mRNA levels were analysed by qPCR in WT nasal cartilage cells transduced with a control lentivirus, Wwp2-expressing lentivirus or lentivirus expressing Wwp2 with a non-functional Hect domain (Wwp2-CA). Values represent means ± s.d. (n = 3, *P <0.01). () 293T cells were transfected with expression constructs for Flag–Myc–Wwp2, His–ubiquitin and Flag-tagged Gsc or Flag-tagged GscK3R, a Gsc protein with the three Lys residues mutated to Arg. Ubiquitylation of the WT Gsc and mutant GscK3R were analysed by immunoprecipitation and western blot analysis. () WT nasal cartilage cells were infected with lentivirus expressing WT Gsc or mutant GscK3R or control lentivirus. Transcript levels of Sox6 in these cell populations were then evaluated by qPCR. Values represent means ± s.d. (n = 3, ! *P <0.01). () 293T cells were transfected with Sox6pro-luc reporter construct and increasing amounts of a Gsc-expression construct. Results were normalized to the expression of the pRL-Tk plasmid. Values represent means ± s.d. (n = 3; *P <0.01). () Gsc DNA binding to the Sox6 promoter in nuclear extracts generated from nasal cartilage cells. Gsc binding to region −235 to −185 (probe no. 1), −187 to −136 (probe no. 2) and −136 to −87 (probe no. 3) was detected by western blotting following oligonucleotide pulldown experiments. () Analysis of Sox6pro-luc transactivation by the GscK3R mutant was evaluated after transfection of 293T cells, and was compared with cells transfected with the Sox6pro-luc and construct expressing WT Gsc. Levels of luciferase in these experiments were normalized to the expression of the pRL-Tk plasmid. Values represent means ± s.d. (n = 3, *P <0.01). () Sox6 transcript levels were analysed by qPCR in nasal cartilage cells from WT or Wwp2! GT/GT mice infected with control or Gsc-expressing lentivirus.! Values represent means ± s.d. (n = 3, *P <0.01) () Model depicting the mechanism through which Wwp2-mediated mono-ubiquitylation of Gsc leads to augmented Sox6 expression. Uncropped images of blots are shown in Supplementary Information, Fig. S7. Author information * Author information * Supplementary information Affiliations * Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, Massachusetts 0211, USA. * Weiguo Zou, * Xi Chen, * Jae-Hyuck Shim, * Zhiwei Huang, * Nicholas Brady, * Dorothy Hu, * Rebecca Drapp, * Kirsten Sigrist, * Laurie H. Glimcher & * Dallas Jones * Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. * Laurie H. Glimcher & * Dallas Jones * Ragon Institute of MIT/MGH and Harvard, Boston, Massachusetts 02129, USA. * Laurie H. Glimcher Contributions W.Z. and D.C.J. designed the research; W.Z. performed most experiments; X.C., J.S., Z.H. and R.D. provided additional technical assistance; N.B. generated and analysed μCT data and images; D.H. provided technical assistance with histological analyses; K.S. generated the Wwp2GT/GT mouse line; W.Z., L.H.G and D.J. analysed the data and wrote the manuscript. Competing financial interests L.H.G. holds equity in and is on the corporate board of directors of Bristol-Myers Squibb. Corresponding author Correspondence to: * Dallas Jones Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (820K) Supplementary Information Additional data - Direct reprogramming of fibroblasts into epiblast stem cells
- ncb 13(1):66-71 (2011)
Nature Cell Biology | Letter Direct reprogramming of fibroblasts into epiblast stem cells * Dong Wook Han1 Search for this author in: * NPG journals * PubMed * Google Scholar * Boris Greber1 Search for this author in: * NPG journals * PubMed * Google Scholar * Guangming Wu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Natalia Tapia1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marcos J. Araúzo-Bravo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kinarm Ko2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Christof Bernemann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Stehling1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hans R. Schöler1, 4 Contact Hans R. Schöler Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:66–71Year published:(2011)DOI:doi:10.1038/ncb2136Received15 April 2010Accepted28 October 2010Published online05 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Epiblast stem cells (EpiSCs) derived from epiblast tissue of post-implantation embryos are pluripotent and can give rise to all three germ layers in teratoma assays1, 2. Introduction of the four transcription factors Oct4, Sox2, Klf4 and c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs) that are very similar to embryonic stem cells (ESCs) in a number of characteristics3, 4, 5, 6. However, generation of EpiSCs by the direct reprogramming of somatic cells using these transcription factors has not been shown to date. Here, we show that these transcription factors can be used to directly generate induced EpiSCs (iEpiSCs) under EpiSC culture conditions. iEpiSCs resemble EpiSCs in morphology, gene expression pattern, epigenetic status and chimaera-forming capability. This study demonstrates that the culture environment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We therefore hypothesize ! that it will eventually be possible to shape the identity of a directly reprogrammed cell simply by modulating culture conditions. View full text Subject terms: * Stem cells Figures at a glance * Figure 1: Direct reprogramming of fibroblasts into iEpiSCs. (, ) Generation of iEpiSCs from GOF18ΔPE fibroblasts. Cells were infected with retrovirus expressing Oct4, Sox2, Klf4, and c-Myc, and were cultured in ESM and formed iPSC colonies (), or cultured in CDM and formed iEpiSC colonies (). Left and right, brightfield microscopy images; middle, fluorescence microscopy images. () Generation of iEpiSCs from X-GFP fibroblasts. X-GFP fibroblasts not expressing GFP were isolated by FACS, infected with retrovirus expressing Oct4, Sox2, Klf4 and c-Myc, and cultured in ESM (left) or CDM (right). Top, brightfield microscopy images; bottom, fluorescence microscopy images. () FACS analysis of GFP expression in cells treated as in . GFP-positive cells were FACS-sorted from iEpiSCs in CDM (left) and iPSCs in ESM (right). SSC-A; side scatter area. () iEpiSCs exhibit the same morphology as EpiSCs. Morphology of iPSCs (left), iEpiSCs (middle), and EpiSCs (right), as assessed by brightfield microscopy. () EpiSCs, iEpiSCs and iPSCs were assessed fo! r alkaline phosphatase activity. () Immunofluorescence microscopy images of iEpiSCs, EpiSCs and iPSCs, using antibodies against Oct4, Sox2 and Nanog. Scale bars, 100 μm. * Figure 2: Characterization of iEpiSCs. () Heat map from microarray data demonstrating global gene expression pattern in MEFs, ESCs, EpiSCs, four iEpiSC lines, iEpiSC-Rs and two iEpiSC-R lines overexpressing Cre recombinase (iEpiSC-RCs). The colour bar at the top indicates gene expression in the log2 scale. Red and blue represent higher and lower gene expression, respectively. () Hierarchical clustering of the cell lines based on the gene expression profiles in . () Scatter-plot comparison of the global gene expression profiles of two iEpiSC lines (iEPiSC C1 and iEPiSC L7) with ESCs (top) and with T9 EpiSCs (bottom). Black lines indicate boundaries of 2-fold difference in gene expression. Pluripotent and epiblast markers are indicated in orange. The bar to the right indicates the scattering density; the higher the scattering density, the darker the blue color. Gene expression levels are depicted on a log2 scale. The numbers of differentially expressed genes are indicated under each scatter plot. () Promoter region! s of endogenous Oct4, Oct4–GFP, Nanog and Stella genes in iEpiSCs were analysed by bisulphite sequencing PCR. Open and filled circles represent unmethylated and methylated CpGs, respectively. () Teratoma. iEpiSCs (1 × 106 cells) were subcutaneously injected into SCID mice. Differentiation of injected iEpiSCs into all three germ layers was assessed by microscopy analysis of haematoxylin and eosin staining. () Chimaeric contribution of iEpiSCs was assessed by LacZ staining of foetuses at embryonic day (E)12.5. () iEpiSCs isolated from a GOF18ΔPE/Rosa26 double transgenic mouse were LacZ-positive. Scale bar, 100 μm. * Figure 3: Induction of naive pluripotency in iEpiSCs. () Top: iEpiSCs were transduced with vector containing Klf4–2A–Td-Tomato (resulting in overexpression of Klf4; Tomato high) and cultured in ESC medium with 2i + LIF, leading to reversion of iEpiSCs (GFP high). Bottom: cells expressing GFP, but with low Tomato expression Iindicating a fewer number of viral insertions) were sorted for Cre-mediated Klf4 excision. The weak expression of Tomato was only detectable with photo overexposure (inset). () Both iEpiSC-Rs and iEpiSC-RCs were stably maintained in 2i + LIF medium, but not in LIF-only medium, as as determined by morphology and alkaline phosphatase activity. () Microscopy images of iEpiSC-Rs cells overexpressing Cre recombinase (iEpiSC-RCs), showing cells with complete loss of Tomato expression. () FACS analysis of Td-Tomato and GFP expression in iEpiSC-RCs (left) and iEpiSC-Rs (right). () Promoter regions of endogenous Oct4, Oct4–GFP, Nanog and Stella genes in iEpiSC-RCs were analysed by bisulphite sequencing PCR. Ope! n and filled circles represent unmethylated and methylated CpGs, respectively. () Scatter plots of global gene expression profiles comparing iEpiSC-Rs with ESCs (left) or iEpiSC-RCs (middle), and ESCs with iEpiSC-RCs (right). Black lines indicate boundaries of 2-fold difference in gene expression. Pluripotent and epiblast markers are indicated in orange. The bar to the right indicates the scattering density; the higher the scattering density, the darker the blue color. Gene expression levels are depicted on a log2 scale. The numbers of differentially expressed genes are indicated under each scatter plot. () iEpiSC-RCs could readily contribute to chimaeras. Somatic contribution in E13.5 foetal (left) and adult mice (right) was confirmed by distribution of LacZ staining and indicated by coat colour, respectively. () Germline contribution of iEpiSC-RCs, as shown by expression of Oct4−GFP in the gonad of a E13.5 embryo.. Scale bars, 100 μm. * Figure 4: Four factors induce a naive or primed pluripotent state in fibroblasts depending on culture conditions. () Expression levels of marker genes in two EpiSC lines and two iEpiSC lines. Note the inverse correlation between media-based reversibility to an ESC-like state and the expression levels of mesendodermal genes. Data are from microarray hybridizations. All cells were grown under the same culture conditions (CDM on MEFs). Data are means ± s.e.m. () The four factors Oct4, Sox2, Klf4 and c-Myc, which have been able to induce a naive pluripotency in fibroblasts, can also induce a primed pluripotency in fibroblasts under specific culture conditions—iPSCs under LIF/STAT3 pathway and iEpiSCs in the presence of bFGF and Activin A with inhibition of LIF activity. A primed pluripotency achieved from fibroblasts by the 4 factors and EpiSC conditions can be further reprogrammed to a naive pluripotent state by Klf4 overexpression in the presence of 2i. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE21516 Author information * Accession codes * Author information * Supplementary information Affiliations * Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany. * Dong Wook Han, * Boris Greber, * Guangming Wu, * Natalia Tapia, * Marcos J. Araúzo-Bravo, * Christof Bernemann, * Martin Stehling & * Hans R. Schöler * Center for Stem Cell Research, Institute of Biomedical Sciences and Technology, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea. * Kinarm Ko * Department of Neuroscience, School of Medicine, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea. * Kinarm Ko * University of Münster, Medical Faculty, Domagkstrasse 3, 48149 Münster, Germany. * Hans R. Schöler Contributions D.W.H. designed and performed most experiments and wrote the manuscript. B.G. performed microarray experiments, analysed the data and edited the manuscript. M.J.A. analysed the microarray data. N.T. carried out lentivirus construction and infection. C.B. analysed the gene expression data. G.W. and K.K. performed morula aggregation and the teratoma assay, respectively. M.S. performed FACS analysis. H.S. conceived the experiments and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hans R. Schöler Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data - HoxA3 is an apical regulator of haemogenic endothelium
- ncb 13(1):72-78 (2011)
Nature Cell Biology | Letter HoxA3 is an apical regulator of haemogenic endothelium * Michelina Iacovino1 Search for this author in: * NPG journals * PubMed * Google Scholar * Diana Chong2 Search for this author in: * NPG journals * PubMed * Google Scholar * Istvan Szatmari1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lynn Hartweck1 Search for this author in: * NPG journals * PubMed * Google Scholar * Danielle Rux1 Search for this author in: * NPG journals * PubMed * Google Scholar * Arianna Caprioli2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ondine Cleaver2 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Kyba1 Contact Michael Kyba Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:72–78Year published:(2011)DOI:doi:10.1038/ncb2137Received13 September 2010Accepted17 November 2010Published online19 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells1, 2 and are referred to as haemogenic endothelium3, 4, 5, 6, 7, 8. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial–haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-funct! ion epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells. View full text Subject terms: * Developmental biology Figures at a glance * Figure 1: Reciprocal expression of HoxA3 and Runx1 in embryonic endothelium. (–) In situ hybridization of E8.25–E10.5 embryonic tissues with HoxA3 (, , , ) and Runx1 (, , , ) probes.) HoxA3, but not Runx1, is expressed in aortic endothelial cells. Note that Runx1, but not HoxA3 is expressed in the yolk sac (black arrowheads) (, ). At E8.5, HoxA3 expression begins to decline in aortic endothelium, but Runx1 is still not detected. Note that the omphalomesenteric artery is negative for HoxA3, but positive for Runx1 (, ). The boxed areas in the left panels are magnified in the right panels (–). In situ hybridization of dissected E9.5 and E10.5 AGMs (–, left panels). At E9.5, HoxA3 expression is barely detectable in aortic endothelial cells, whereas Runx1 expression is now observed (, ). At E10.5, HoxA3 expression is completely abolished in aortic endothelial cells, whereas Runx1 expression has increased (, ). A, aorta; G, gut tube; NT, neural tube; OA, omphalomesenteric artery. Stippled lines in – outline aorta. Scale bar, 50 μm for lower magn! ification panels and AGM explants, and 10 μm for higher magnification panels. * Figure 2: HoxA3 expression in early mesoderm and committed haemogenic endothelium restrains haematopoiesis. () Representative flow cytometric profiles of embryoid bodies at day 6 without doxycycline (No dox) or with 1 μg ml−1 doxycycline (+Dox) to induce HoxA3 expression from day 4–6. VE-cadherin (VE-cad)/Flk-1 antibody staining or c-Kit/CD41 and c-Kit/CD45 staining was performed to identify vascular and haematopoietic progenitor populations. () Frequencies of cells expressing endothelial surface markers (Flk-1+/VE-cadherin+, F/V), haematopoietic markers CD41+ and CD45+ cells during EB differentiation in 7 independent experiments. Data are mean ± s.d.; P = 0.0004, CD41; P = 0.0031, CD45 () 50,000 cells from day 6 embryoid bodies (induced with 1 μg ml−1 dox to express HoxA3 continually from EB day 4–6 or not) were plated in methylcellulose with haematopoietic cytokines. Data are mean ± s.d., n = 3; P = 0.0075, granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM); P = 0.0075, granulocyte/macrophage (GM); P = 0.008, macrophage only (M), P = 0.0009, erythrocyte-megakary! ocyte (Ery-Meg); P = 0.035, definitive erythroid (Ery-D), P = 0.0002, primitive erythroid (Ery-P). () Bright-field and fluorescence images showing both endothelial (+Dox) and haematopoietic colonies (No dox or Dox removal) derived from Flk1+/VE-cadherin+ (F/V) endothelial progenitors from day 6 embryoid bodies. Immunofluorescence for VE-cadherin (red) is shown in adherent cells growing in the presence of doxycycline. Scale bar, 100 μm. () Equivalent analysis of cultures derived from day 6 EB c-Kit+/CD41+ (K/41) haematopoietic progenitors. (, ) Representative flow cytometric profile of 100,000 Flk-1/VE-cadherin double-positive cells () or c-Kit/CD41 double-positive cells () from day 6 uninduced embryoid bodies (left), cultured on OP9 for 5 days, in the presence or absence of doxycycline (1 μg ml−1). Dox-induced cells were cultured for an additional 4 days in the absence of dox to test the effect of HoxA3 downregulation. Haematopoietic surface markers c-Kit, CD41 and CD45! , and endothelial markers Flk-1 and VE-cadherin, are plotted. * Figure 3: Global expression changes on HoxA3 induction. () Venn diagram of regulated genes in endothelial (F/V) and haematopoietic (K/41) progenitor cells. Upward arrows represent upregulated genes, downward arrows represent downregulated genes. () Clustering of genes upregulated in the c-Kit CD41 double-positive cells on HoxA3 induction during embryoid body differentiation on the basis of their baseline (control) expression levels in the two populations from uninduced embryoid bodies. () Clustering of genes downregulated in the Flk-1 VE-cadherin double-positive population on 6-h HoxA3 induction, in day 6 embryoid body cells, on the basis of their baseline (control) levels in the two populations in uninduced embryoid bodies. () Real-time RT–PCR measurements of gene expression changes following HoxA3 induction in sorted endothelial (F/V black bars) or haematopoietic (K/41 grey bars) progenitors; scale is log2; data are mean ± s.e.m.; n = 5 independent experiments. * Figure 4: Global expression changes on reversion of HoxA3 by Runx1 or Gata1. () Bright-field (left) and fluorescent (right) images of HoxA3-induced day 6 embryoid body-derived endothelial progenitors (F/V cells) transduced with control GFP vector or Runx1B–iresGFP retroviral vector and cultured on OP9 for 5 days. Scale bar, 100 μm. () Representative flow cytometric profiles of F/V cells expressing HoxA3 and transduced with retroviral vectors expressing: ires-GFP (HoxA3), Ikaros (HoxA3+Ikaros), PU.1 (HoxA3+PU.1), Runx1-B (HoxA3+Runx1B), Gata1 (HoxA3+Gata1), Gfi1B (HoxA3+Gfi1B). GFP+ gated events are shown. () Expression levels of haematopoietic marker genes (fold of GAPDH), without HoxA3 induction (No dox) or with HoxA3 induction (+Dox) for control or Runx1B-transduced cells on OP9. P = 0.0003, Gfi1B; P = 0.0073, Ikaros; P <0.0001, Phemx; P = 0.0009, PU.1. Data are mean ± s.d.; n = 3 independent experiments. () Genes upregulated by HoxA3 clustered according to their expression in control uninduced, HoxA3+Runx1iresGFP, HoxA3+Gata1 ires-GFP, and Hox! A3+GFP sorted cells. Both Runx1 and Gata1 significantly reverse these HoxA3-induced changes, indicated by their clustering together and near to the non-HoxA3 expressing control. Examples are shown on the heat map below. () Genes downregulated by HoxA3. Runx1 reverses downregulation by HoxA3 more effectively than Gata1, indicated by its clustering closest to control. Among these genes are key endothelial regulatory factors, and genes involved in adhesion and cell polarity. * Figure 5: Regulation of intraembryonic haematopoiesis by HoxA3 and Runx1. () In situ hybridization showing Runx1 expression in HoxA3+/+, HoxA3+/− and HoxA3−/− E8.25 embryos. Runx1 expression is absent in the dorsal aortae of HoxA3+/+ (i) and HoxA3+/− (ii) embryos, but robustly expressed in yolk sac. Runx1 is ectopically expressed in the dorsal aortae of HoxA3−/− (iii) embryos. Stippled red lines outline dorsal aortae. Penetrance of this phenotype is indicated at lower left. (i'–iii') Sections of the embryos shown above. Both endothelial and haematopoietic cells are negative for Runx1 in wild-type or heterozygous embryos, whereas Runx1-expressing cells are found in HoxA3−/− embryos. Arrows indicate Runx1-expressing endothelial cells. A, aorta; YS, yolk sac. Scale bar, 50 μm for whole mounts, 10 μm for sections. () Model for regulation of endothelial haemogenesis by HoxA3 and Runx1. HoxA3 represses a cascade of transcription factors that promote haemogenesis and induces a set of genes that maintain endothelial character. Runx1 is! a positive regulator of most of these transcription factors, and a negative regulator of genes essential for endothelial character, thus transient expression of Runx1 erases the endothelial program and initiates the haematopoietic. Author information * Author information * Supplementary information Affiliations * Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, 312 Church St. SE, Minneapolis MN 55455, USA. * Michelina Iacovino, * Istvan Szatmari, * Lynn Hartweck, * Danielle Rux & * Michael Kyba * Department of Molecular Biology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9148 USA. * Diana Chong, * Arianna Caprioli & * Ondine Cleaver Contributions M.I., O.C. and M.K. designed the experiments, and wrote the manuscript; M.I. performed the experiments; D.C. and A.C. performed in situ hybridization studies; I.S. performed microarray studies; L.H. and D.R. performed chromatin IP studies. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael Kyba Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (411K) Supplementary Information Additional data - Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans
- ncb 13(1):79-86 (2011)
Nature Cell Biology | Letter Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans * Lukas J. Neukomm1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas P. Frei1, 2, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Cabello3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jason M. Kinchen5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ronen Zaidel-Bar6 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhong Ma5 Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa B. Haney5 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeff Hardin6 Search for this author in: * NPG journals * PubMed * Google Scholar * Kodi S. Ravichandran5 Search for this author in: * NPG journals * PubMed * Google Scholar * Sergio Moreno3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael O. Hengartner1 Contact Michael O. Hengartner Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:79–86Year published:(2011)DOI:doi:10.1038/ncb2138Received14 May 2010Accepted23 November 2010Published online19 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Multicellular animals rapidly clear dying cells from their bodies. Many of the pathways that mediate this cell removal are conserved through evolution. Here, we identify srgp-1 as a negative regulator of cell clearance in both Caenorhabditis elegans and mammalian cells. Loss of srgp-1 function results in improved engulfment of apoptotic cells, whereas srgp-1 overexpression inhibits apoptotic cell corpse removal. We show that SRGP-1 functions in engulfing cells and functions as a GTPase activating protein (GAP) for CED-10 (Rac1). Interestingly, loss of srgp-1 function promotes not only the clearance of already dead cells, but also the removal of cells that have been brought to the verge of death through sublethal apoptotic, necrotic or cytotoxic insults. In contrast, impaired engulfment allows damaged cells to escape clearance, which results in increased long-term survival. We propose that C. elegans uses the engulfment machinery as part of a primitive, but evolutionarily con! served, survey mechanism that identifies and removes unfit cells within a tissue. View full text Figures at a glance * Figure 1: Loss of srgp-1 activity reduces the numbers of persistent apoptotic cell corpses in C. elegans. () srgp-1(RNAi) results in positive Acridine Orange straining (indicative of engulfment) of germ-cell corpses in ced-6 mutants. Adult hermaphrodites of the indicated genotypes were stained with Acridine Orange and observed under DIC () and fluorescence microscopy (). All strains carry a gla-1 mutation to increase the overall number of apoptotic germ cells. Arrows point to Acridine Orange-positive germ-cell corpses and arrowheads to Acridine Orange-negative germ-cell corpses. In all pictures, anterior is to the left, dorsal on top. Scale bar, 10 μm. () DIC microscopy images of frshly hatched L1 larvae of the indicated genotypes. Arrowheads indicate persistent cell corpses. Scale bar, 10 μm. () Persistent cell corpses were scored in the head region of freshly hatched L1 larvae of the indicated genotypes. Expression of srgp-1::gfp or srgp-1::mcherry driven by the endogenous srgp-1 promoter rescues the srgp-1(ok300) phenotype. Data are means ± s.d.; n, number of scored indivi! duals. Alleles used: gla-1(op234), ced-6(n1813), srgp-1(ok300), opIs224[Psrgp-1::srgp-1(cDNA)::gfp], opIs228[Psrgp-1::srgp-1(genomic)::gfp] and opEx1424-6[Psrgp-1::srgp-1(genomic)::mcherry]. * Figure 2: SRGP-1 functions in the engulfing cell and antagonizes engulfment activity. () Developmental apoptosis is not defective in srgp-1 mutants. Extra cell nuclei were scored in the pro- and metacorpus heads of L3/L4 larvae with the indicated genotypes. Alleles used: ced-3(n2433), ced-6(n1813) and srgp-1(ok300). () Persistent cell corpses were scored in the head region of freshly hatched L1 larvae of the indicated genotypes. Expression of srgp-1::gfp in the engulfing cell (opEx1373) rescues the srgp-1(ok300) phenotype; expression in apoptotic cells (opIs391) does not. Alleles used: ced-6(n1813), srgp-1(ok300), opEx1373[Pced-1::srgp-1(cDNA)::gfp] and opIs391[Pegl1::srgp-1(cDNA)::gfp]. () Loss of srgp-1 increases engulfment kinetics. The first 13 apoptotic cell deaths in the AB lineage were followed by 4D microscopy and the cell corpse persistence was measured. Each circle represents a single cell. Analysis was performed in three independent individuals in each genetic background (n = 3 × 13 cells). Cells indicated as not engulfed either floated off the ti! ssue into the egg-shell cavity or their lineage could not be followed anymore owing to the beginning of muscle contraction at the 1.5-fold stage (320 min post fertilization or approximately 120 min post-onset of cell death). Alleles used: ced-6(tm1826) and srgp-1(ok300). Asterisk indicates P < 0.005 (Kolmogorov-Smirnov test). () Overexpression of srgp-1::mcherry enhances corpse persistence. Persistent cell corpses were scored in the head region of freshly hatched L1 larvae of the indicated genotypes. Alleles used: ced-1(e1735), ced-2(n1994), ced-5(n1812), ced-6(n1813), ced-7(n1996), ced-10(n1993) and ced-12(k149). Indicated strains carried unc-119(ed3) and the opEx1424[Psrgp-1::srgp-1::mcherry] transgene. All data are means ± s.d. Asterisk indicates P ≤ 0.02, double asterisk indicates P < 10−6 and triple asterisks indicate P < 10−10 (t-test, 1tailed, unequal variances). * Figure 3: SRGP-1 (srGAP1) binds to and modulates CED-10 (Rac1) GTPase activity. () The GAP domain of SRGP-1 specifically binds GTP-loaded CED-10 in vitro. The Q61 and N17 isoforms (resembling GTP- and GDP-bound, respectively) of His-tagged CED-10 were used for GST-fusion pulldown using the GAP domain of SRGP-1, Pak1 p21 binding domain (PBD, positive control) and GST alone (negative control). () The SRGP-1 GAP domain preferentially binds to CED-10 in vitro. The GTP-bound isoforms of His-tagged CED-10, MIG-2 and RHO-1 (Q61, Q65 and Q63, respectively) were used for GST-fusion pulldown using the GAP domain of SRGP-1, the GTP hydrolysis-deficient GAP (SRGP-1R563A) and GST alone. () The SRGP-1 GAP domain promotes Rac1 GTP-hydrolysis in vitro. Human Rac1 was loaded with [γ32P]GTP and GTP hydrolysis was measured through the loss of Rac1 radioactivity through the cleavage of the γ32P from GTP. As controls, loaded Rac1 was treated with buffer alone or the GTP hydrolysis-deficient GAP (SRGP-1R563A). Data are means ± s.d. (n = 4 independent experiments). () srGA! P1 function in corpse removal is evolutionarily conserved. LR73 cells were transiently transfected in triplicate with plasmids encoding the indicated proteins (srGAP1, Flag–Rac1(N17), BAI1–GFP or GFP alone, respectively) and incubated with TAMRA-labelled apoptotic cells. Top: phagocytosis by GFP-positive cells was assessed by two-colour flow cytometry. The percentage of GFP-positive cells that had ingested apoptotic SCI cells is indicated for each condition. Bottom: the expression of the transfected proteins was confirmed by immunoblotting of total cell lysates. () srGAP family members inhibit cell-corpse clearance. Knockdown of all three srGAP family members increases phagocytotic activity in NIH/3T3 cells. () In vivo structure/function analysis of SRGP-1. Persistent cell corpses were scored in the head region of freshly hatched L1 larvae of the indicated genotypes. The rescue ability of SRGP-1::GFP (full length) and GFP fusions of the indicated SRGP-1 mutants were tes! ted by expression in ced-6; srgp-1 mutants. Data are means ± ! s.d. Alleles used: ced-6(n1813) and srgp-1(ok300). * Figure 4: Enhanced engulfment promotes the removal of viable but sick cells in C. elegans. () Loss of srgp-1 promotes killing of cells on the verge of apoptosis in the larval ventral cord. Left: representative fluorescence microscopy images of wild type, ced-3(lf), ced-3(rf) and srgp-1 ced-3(rf) L4 larvae. Alleles used: ced-3(n717lf), ced-3(op149rf) and srgp-1(ok300). nIs96 is present in all backgrounds. White arrowheads indicate surviving Pn.aap cells, P3–P8; green arrowheads indicate extra survival of P1, P2 and P9 – P12 cells. Scale bar, 100 μm. Middle: quantification of Pn.aap extra cell survival monitored in the indicated genotypes using the nIs96 transgene. srgp-1(lf) is not sufficient to kill cells that normally survive (P3–P8). Alleles used: ced-3(n717lf), ced-3(op149rf), ced-3(n2438rf), and srgp-1(ok300). Data are means ± s.d. of three experiments, n ≥ 40 animals per experiment. Asterisk indicates P < 10−10 (t-test, 1tailed, unequal variances). Right: enhanced cell killing in srgp-1 mutants is ced-10-dependent. Extra cells in the ventral cord ! in wild type, ced-10(t1875) and ced-10(t1875) srgp-1(ok300) were scored through expression of the nIs106[Plin11::gfp] transgene34 (n ≥ 100 per genotype). ced-10 animals were generated from heterozygote mothers. () Engulfment activity modulates the removal of viable cells subjected to a neurotoxic insult. Left: DIC and fluorescence microscopy images of wild-type L4 larvae raised at 15 °C containing either 2 or 1 PLM touch neurons labelled with GFP. Middle and right: survival of the PLM touch neurons (labelled with GFP) was quantified in L4 larvae of the indicated genotype grown at either 20 °C (middle) or 15 °C (right). Alleles used: ced-5(tm1949) and srgp-1(ok300). Ismec-10(d) is present in all genetic backgrounds. Scale bar, 20 μm. () Engulfment activity modulates the removal of viable cells subjected to a non-classical toxic insult. The number of surviving Pn.p cells were scored in early L3 larvae with the indicated genotypes. Data are means ± s.d., n = 25. Asteris! k indicates P < 10−4 (t-test, 1tailed, unequal variances). A! lleles used: srgp-1(ok300) and ced-2(e1752). () Simplified model of sick-cell tolerance versus removal. Sick cells within a tissue (red) signal their unhealthy status to their neighbour. Depending on the strength of this signal, the sick cell is either tolerated (light blue), or removed (red, 'eaten alive') through phagocytosis. Author information * Author information * Supplementary information Affiliations * Institute of Molecular Life Sciences, University of Zurich, 8057 Zurich, Switzerland. * Lukas J. Neukomm, * Andreas P. Frei & * Michael O. Hengartner * Ph.D. Program in Molecular Life Sciences (MLS), University of Zurich/ETH Zurich, 8057 Zurich, Switzerland. * Andreas P. Frei * Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, 37007 Salamanca, Spain. * Juan Cabello & * Sergio Moreno * Center for Biomedical Research of La Rioja (CIBIR), C/Piqueras 98, 26006 Logroño, Spain. * Juan Cabello * Center for Cell Clearance and the Department of Microbiology, University of Virginia, Charlottesville, VA 22902, USA. * Jason M. Kinchen, * Zhong Ma, * Lisa B. Haney & * Kodi S. Ravichandran * Department of Zoology, University of Wisconsin – Madison, WI 53706, USA. * Ronen Zaidel-Bar & * Jeff Hardin * Current address: Department of Neurobiology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA. * Lukas J. Neukomm * Current address: Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland. * Andreas P. Frei Contributions L.J.N., A.P.F. and R.Z.B. contributed to the generation of nematode transgenics and fluorescence microscopy studies. A.P.F. and L.J.N. conducted the unbiased screen and the epistasis experiments. J.C. performed the 4D microscopic analysis. J.M.K. performed the mammalian cell culture experiments. Z.M. and L.B.H. performed the pulldowns and the hydrolysis assays. L.J.N. performed the cell-killing assay and wrote the manuscript. A.P.F. and M.O.H. contributed to the data analysis, project planning and writing of the manuscript. All authors contributed to editing the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael O. Hengartner Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells
- ncb 13(1):87-94 (2011)
Nature Cell Biology | Letter CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells * Yongkun Wei1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ya-Huey Chen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Long-Yuan Li2, 3, 4 Contact Long-Yuan Li Search for this author in: * NPG journals * PubMed * Google Scholar * Jingyu Lang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Su-Peng Yeh5 Search for this author in: * NPG journals * PubMed * Google Scholar * Bin Shi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Cheng-Chieh Yang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jer-Yen Yang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Chun-Yi Lin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Chien-Chen Lai6, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Mien-Chie Hung1, 2, 3, 4, 8 Contact Mien-Chie Hung Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:87–94Year published:(2011)DOI:doi:10.1038/ncb2139Received10 June 2010Accepted10 November 2010Published online05 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and catalyses the trimethylation of histone H3 on Lys 27 (H3K27), which represses gene transcription. EZH2 enhances cancer-cell invasiveness and regulates stem cell differentiation. Here, we demonstrate that EZH2 can be phosphorylated at Thr 487 through activation of cyclin-dependent kinase 1 (CDK1). The phosphorylation of EZH2 at Thr 487 disrupted EZH2 binding with the other PRC2 components SUZ12 and EED, and thereby inhibited EZH2 methyltransferase activity, resulting in inhibition of cancer-cell invasion. In human mesenchymal stem cells, activation of CDK1 promoted mesenchymal stem cell differentiation into osteoblasts through phosphorylation of EZH2 at Thr 487. These findings define a signalling link between CDK1 and EZH2 that may have an important role in diverse biological processes, including cancer-cell invasion and osteogenic differentiation of mesenchymal stem cell! s. View full text Figures at a glance * Figure 1: CDK1 negatively regulates H3K27 trimethylation. () Top: 435, SKBr3, 468 and MCF7 cells were treated with CGP74514A as indicated, and the lysates were analysed by immunoblot using antibodies against the specified proteins. Bottom: in vitro kinase assay. CDK1, immunoprecipitated from the cell lines treated with CGP74514A as indicated at the top, was incubated with H1 and [γ-32P]ATP. Reaction products were resolved by SDS–PAGE and visualized by autoradiography (equal loading of H1 was assessed by Coomassie-stained gel shown at the bottom). () Lysates from MCF7 cells infected with lentiviruses expressing control or two different CDK1 shRNA were immunoblotted with antibodies against the indicated proteins. Relative intensities of the H3K27me3 bands are shown, normalized to the H3K27me3 band from parental MCF7 cells. Bottom: in vitro kinase assay, performed as in , with CDK1 immunoprecipitated from cells treated as indicated at the top. () Lysates of 293T cells transfected with plasmids encoding cyclin B, and CDK1 or dominan! t-negative mutant CDK1 (DN-CDK1) were immunoblotted with antibodies against the indicated proteins. Relative intensities of the H3K27me3 bands are shown, normalized to the H3K27me3 band from 293T cells transfected with plasmid encoding CDK1. p-CDK1-T161; CDK1 phosphorylated at Thr 161. Bottom: in vitro kinase assay, performed as in , with CDK1 immunoprecipitated from cells transfected as indicated at the top. () Left top: immunoblot of lysate from HEK293 cells treated with DMSO or CGP using antibodies against the indicated proteins. Left bottom: in vitro kinase assay, performed as in , with CDK1 immunoprecipitated from cells treated as indicated at the top. Right: analysis of mRNA levels of HOXA families by qRT–PCR after treatment of HEK293 cells with DMSO or CGP74514A. Data are means ± s.e.m. (n = 3). Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 2: CDK1 interacts with, and phosphorylates, EZH2 at Thr 487. () Lysates from 293T cells, transfected with plasmids encoding Myc–EZH2 and HA–CDK1 as indicated, were immunoprecipitated (IP) using anti-Myc and analysed by immunoblot using anti-Myc or anti-HA. IgG; immunoglobulin G. () Pulldowns. GST and GST–EZH2 were incubated with lysate from HeLa cells. Bound CDK1 was detected by immunoblotting. () Lysate from MCF7 cells was immunoprecipitated with antibodies against EZH2 (left) or CDK1 (right), and analysed by immunoblotting. () Cyclin B, CDK1 and GST–EZH2 were subjected to an in vitro kinase assay and analysed by mass spectrometry. The spectrum of the charged ion (m/z 724.7217) shows that Thr 487 is phosphorylated (lower case p) in the indicated peptide (top right). b ions, fragmentation ions containing the amino terminus of the peptide; y ions, fragmentation ions containing the carboxy terminus of the peptide. () In vitro kinase assay with CDK1, cyclin B, and wild-type GST–EZH2 (WT) or GST– EZH2T487A. Phosphorylation of ! EZH2 and H1 was visualized by autoradiography, and loading of GST–EZH2 and H1 was assessed by Coomassie-stained gel. () 293T cells were transfected with plasmids encoding wild-type Myc–EZH2 or Myc–EZH2T487A and treated with CDK1 inhibitor CGP74514A or DMSO. Lysates were immunoprecipitated with anti-Myc and analysed by immunoblotting. () MCF7 cells stably expressing wild-type Myc–EZH2 or Myc–EZH2T487A were transfected with control vector or plasmids encoding CDK1 and cyclin B, and infected with lentivirus expressing CDK1 shRNA, as indicated. Cells were labelled with [32P]-orthophosphate, EZH2 was immunoprecipitated from lysates with anti-Myc and analysed by autoradiography. Immunoblotting was used to confirm equal loading of EZH2 (bottom). () HeLa cells expressing Myc–EZH2 or Myc–EZH2T487A were transfected with plasmid encoding CDK1 and cyclin B, or control vector. Cell lysates were immunoprecipitated with anti-Myc and immunoblotted. () Lysates of HeLa cells tr! ansfected with plasmid encoding CDK1 and cyclin B, or control ! vector, were immunoprecipitated with anti-EZH2, and immunoblotted. () Immunoblot of lysates from HeLa cells treated with Nocodazole as indicated. Cell lysates were immunoprecipitated with anti-EZH2. () MCF7 cells stably expressing wild-type Myc–EZH2 were infected with lentivirus expressing control or CDK1 shRNA. Cell lysates were immunoprecipitated with anti-Myc and immunoblotted. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 3: CDK1-mediated phosphorylation of EZH2 promotes disassociation of EZH2 from SUZ12 and EED and suppresses EZH2 HMTase activity. () HeLa cells were transfected with plasmids encoding wild-type EZH2 or EZH2T487A and treated with CGP74514A as indicated. Top: immunoblot. Relative intensity of the H3K27me3 bands is indicated. Bottom: Autoradiograph and immunoblot of in vitro kinase assay using H1 as substrate. () In vitro histone methyltransferase assay. PRC2 complexes were purified from MCF7 cell lines stably expressing wild-type Myc-His–EZH2 or Myc-His–EZH2T487A and 0.5, 1 and 2 μg were used in the assay. Top: immunoblots of purified proteins. Bottom: proteins were incubated with oligonucleosome and 3H-labelled S-adenosylmethionine. Methylation was assessed by autoradiography and oligonucleosome loading by Coomassie-stained gel. Relative intensities of bands are indicated at the top of each panel. () MCF7 cell lines as in were transfected with control vector or plasmids encoding HA–CDK1. Left: immunoblots of experiment performed as in . Right: immunoblot of lysates from MCF7 cells used in protein! purification. () Immunoblot of lysates from stable MCF7 transfectants, established by transfection of cells with control vector or plasmids encoding Myc–EZH2 or Myc–EZH2T487A. () qRT–PCR of HOXA genes in MCF7 cell lines described in . Data are means ± s.d. from three individual experiments. () Quantitative chromatin immunoprecipitation analysis on HOXA7 and HOXA9 promoters in MCF7 cell lines as described in . Data are means ± s.d. from three individual experiments. () Immunoblot of MCF7 cell lines stably expressing wild-type EZH2, treated by serum starvation (to collect cells at G0/G1 phase) or by double thymidine blockage and release (to collect cells at S and G2/M phases). () Immunoblot of HeLa cells treated as in . () qRT–PCR of HOXA gene expression in MCF7 stable cell lines treated as in . Data are means ± s.d. from three individual experiments. () Images and quantification of cell migration of MCF7 stable cell lines. Data are means ± s.e.m. from three indi! vidual experiments. () Images and quantification of cell invas! ion of MCF7 stable cell lines. Data are means ± s.e.m. from three individual experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 4: Phosphorylation of EZH2 by CDK1 promotes osteogenic differentiation of human mesenchymal stem cells. () Osteoblast differentiation medium induces activation of CDK1 in hMSCs. Immunoblot of lysates from undifferentiated cells or cells differentiated into osteoblasts or adipocytes. () hMSCs were left untreated or were treated with osteoblast differentiation medium (OM) and shRNA as indicated. Alizarin Red S staining was performed at day 7. Differentiated stem cells positive for Alizarin Red S are stained red. () Effect of CDK1 knockdown on the expression of osteogenic-specific genes in hMSCs. Cells were cultured in control medium or osteoblast differentiation medium, and infected with lentiviruses expressing control or CDK1 shRNA as indicated. Cell lysates were subjected to immunoblot analysis. () Disruption of PRC2 complex after osteogenic differentiation. Lysates from cells undifferentiated or differentiated into osteoblasts were immunoprecipitated with EZH2 antibody and subjected to immunoblot analysis as indicated. * Figure 5: CDK1 regulates EZH2-target gene expression in human mesenchymal stem cells. () Effect of CDK1 knockdown on the expression of EZH2-target genes in hMSCs. Cells were cultured in control medium or osteoblast differentiation medium with or without CDK1 shRNA. mRNA levels of RUNX2 (left) and TCF7 (right) were measured by qRT–PCR, and are calculated relative to GAPDH expression. Data are means ± s.e.m. (n = 3). () Effect of CDK1 knockdown on the binding of EZH2 to RUNX2 gene promoter in hMSCs. Cells were cultured in control medium or osteoblast differentiation medium with or without CDK1 shRNA infection. Quantitative chromatin immunoprecipitation was performed using antibodies against the indicated proteins on the RUNX2 promoter. Levels of CDK1 are shown by immunoblot (top). (, ) Four genes shown to have differential EZH2 binding after osteogenic differentiation from a genome-wide ChIP-on-chip assay were randomly selected for ChIP assay using antibodies against EZH2 () and H3K27me3 (). hMSCs were cultured in control medium or osteoblast differentiation! medium. () The expression of the four genes analysed in and was assessed by qRT–PCR from hMSCs cultured in control medium or osteoblast differentiation medium. Author information * Author information * Supplementary information Affiliations * Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA. * Yongkun Wei, * Jingyu Lang, * Bin Shi, * Cheng-Chieh Yang, * Jer-Yen Yang & * Mien-Chie Hung * Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan. * Ya-Huey Chen, * Long-Yuan Li, * Chun-Yi Lin & * Mien-Chie Hung * Graduate Institute of Cancer Biology, China Medical University, Taichung 404, Taiwan. * Long-Yuan Li & * Mien-Chie Hung * Asia University, Taichung 413, Taiwan. * Long-Yuan Li & * Mien-Chie Hung * Division of Hematology and Oncology, Department of Medicine, China Medical University and Hospital, Taichung 404, Taiwan. * Su-Peng Yeh * Graduate Institute of Chinese Medical Science, China Medical University, Taichung 404, Taiwan. * Chien-Chen Lai * Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan. * Chien-Chen Lai * Program in Cancer Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA. * Mien-Chie Hung Contributions M.-C.H., Y.W. and L.-Y.L. designed the project and wrote the paper. M.-C.H. and L.-Y.L. supervised the research. Y.W. performed most of the experiments in Figs 1, 2, 3. Y.-H.C. performed mesenchymal stem cell experiments in Figs 4 and 5 and experiments investigating the interaction between EZH2 and CDK1. C.-C.L. performed the mass spectrometry analysis. C.-Y.L. generated and characterized the phospho-EZH2 antibody. S.-P.Y. collected and characterized primary human mesenchymal stem cells. J. L., B.S., C. -C.Y. and J.-Y.Y. assisted with experiments. All authors participated in interpreting the results. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Long-Yuan Li or * Mien-Chie Hung Supplementary information * Author information * Supplementary information Excel files * Supplementary Table 1 (531K) Supplementary Information * Supplementary Table 2 (28K) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information * Supplementary Table 3 (79K) Supplementary Information * Supplementary Table 4 (46K) Supplementary Information Additional data - Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs
- ncb 13(1):95-101 (2011)
Nature Cell Biology | Letter Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs * Yuntao S. Mao1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hongjae Sunwoo1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Bin Zhang1 Search for this author in: * NPG journals * PubMed * Google Scholar * David L. Spector1 Contact David L. Spector Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:95–101Year published:(2011)DOI:doi:10.1038/ncb2140Received29 June 2010Accepted20 October 2010Published online19 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The cell nucleus is a highly compartmentalized organelle harbouring a variety of dynamic membraneless nuclear bodies1, 2, 3, 4. How these subnuclear domains are established and maintained is not well understood5, 6, 7, 8. Here, we investigate the molecular mechanism of how one nuclear body, the paraspeckle, is assembled and organized. Paraspeckles are discrete ribonucleoprotein bodies found in mammalian cells and implicated in nuclear retention of hyperedited mRNAs9, 10, 11. We developed a live-cell imaging system that allows for the inducible transcription of Men ε/β (also known as Neat1; ref. 12) noncoding RNAs (ncRNAs) and the direct visualization of the recruitment of paraspeckle proteins. Using this system, we demonstrate that Men ε/β ncRNAs are essential to initiate the de novo assembly of paraspeckles. These newly formed structures effectively harbour nuclear-retained mRNAs confirming that they are bona fide functional paraspeckles. By three independent approaches! , we show that it is the act of Men ε/β transcription, but not ncRNAs alone, that regulates paraspeckle maintenance. Finally, fluorescence recovery after photobleaching (FRAP) analyses supported a critical structural role for Men ε/β ncRNAs in paraspeckle organization. This study establishes a model in which Men ε/β ncRNAs serve as a platform to recruit proteins to assemble paraspeckles. View full text Figures at a glance * Figure 1: Immobilization of protein components fails to assemble paraspeckles. () Tethering PSP1 protein to a specific locus in the nucleus recruited p54nrb, but not Men ε/β ncRNAs, PSP1, PSF or PSP2, and therefore paraspeckles failed to form. Fluorescence microscopy of C2C12 cells transfected with plasmids encoding ECFP–LacI and EYFP–LacI–PSP1. Arrow indicates localization of EYFP–LacI–PSP1 to tethered foci (assessed by expression of ECFP–LacI), whereas arrowhead indicates an endogenous paraspeckle. Cells were also transfected with plasmids encoding mCherry–PSP1, mCherry–p54nrb or mCherry–PSP2, were labelled with antibodies against PSF, or were labelled with Men ε/β FISH probes. Scale bar, 5 μm. () Quantification of protein-tethering experiments. Fluorescently-tagged LacI–PSP1, p54nrb or PSF were expressed in C2C12 cells. The co-localization of these proteins and the indicated paraspeckle proteins was quantified. n > 50 cells in each condition. () Lysates from C2C12 cells transiently transfected with plasmids encoding PSF were! treated with RNase A and immunoprecipitated with anti-PSF. Immunoprecipitations were analysed by immunoblotting. * Figure 2: Men ε/β transcription induces de novo formation of functional paraspeckles. () Schematic representation of an inducible system to visualize expression of the Men ε/β reporter ncRNAs and recruitment of paraspeckle proteins. A LacO array, a tetracycline response element (TRE) array, and 24 MS2 stem loop repeats were placed upstream of the Men ε/β gene allowing visualization of the locus by expression of ECFP–LacI. Transcription is initiated by the addition of DOX and nascent transcripts are visualized through binding of EYFP–MS2 to the MS2 stem loops. Not drawn to scale. Recruitment of paraspeckle proteins was visualized through expression of mCherry-tagged versions of these proteins. The reporter was stably integrated into a single site in the genome of C2C12 cells, and these cells were used in –. () Live-cell microscopy imaging of cells at indicated times after DOX addition. Transcriptional induction of Men ε/β ncRNAs initiated de novo formation of paraspeckles labelled by PSP1 (arrow). Scale bars, 5 μm. () Quantification of the percent! age of cells with the indicated protein or RNA in the induced paraspeckles. Men ε/β transcription initiated the recruitment of different paraspeckle proteins, but not SC35 and SF2/ASF (nuclear speckle RNA-binding proteins) and resulted in the retention of different inverted repeat containing mRNAs, but not mCat2 mRNA (n > 50 cells in each condition). () Fluorescence microscopy imaging of cells co-transfected with plasmids encoding the indicated fluorescently tagged proteins, and treated with FISH probes against the indicated RNA. De novo formed paraspeckles (arrow) retained inverted repeat-containing mRNAs similarly to the endogenous paraspeckles (arrowhead), confirming that they are bona fide functional paraspeckles. * Figure 3: Maintenance of paraspeckles depends on active Men ε/β transcription. () Left: fluorescence microscopy of cells using DNA FISH to visualize Men ε/β and Ctn RNA gene loci. Paraspeckles were labelled by PSP1 antibody, and were often found localized adjacent to Men ε/β gene loci, but not to Ctn RNA gene loci, in interphase nuclei of C2C12 cells. Scale bars, 5 μm. Right: quantification of the distance between paraspeckles and Men ε/β gene and Ctn RNA gene loci from fluorescence microscopy images (n = 100 paraspeckles in 20 cells each, data are means ± s.e.m.). () Left: cells expressing fluorescently tagged paraspeckle protein PSP1 were imaged by fluorescence microscopy. DRB was added at 5 min and the cells were washed to remove DRB at 95 min. PSP1 redistributed to perinucleolar caps (arrowhead) on transcriptional inhibition by DRB treatment and paraspeckles reassembled at the same location (arrow) after DRB wash-out. Right: Men ε/β transcript level on DRB treatment as assessed by qRT–PCR analysis (n = 4, data are means ± s.e.m.). Note! that 60-min DRB treatment did not affect the Men ε/β transcript level, but paraspeckles already disassembled at this time. () Fluorescence microscopy of cells harbouring the Men ε/β RNA reporter at the indicated times after withdrawal of DOX to switch off Men ε/β transcription. Arrow indicates disassembly of de novo formed paraspeckle. Arrowhead indicates endogenous paraspeckles, which were unaffected. This demonstrates that the maintenance of paraspeckles is coupled with Men ε/β transcription. * Figure 4: Dynamic behaviour of paraspeckles by live-cell imaging. () Fluorescence microscopy images of cells harbouring the Men ε/β RNA reporter during the indicated stages of the cell cycle. Both endogenous (arrowhead) and de novo formed paraspeckles (arrow) disassembled during mitosis, and re-assembled at the Men ε/β loci in the daughter nuclei during next G1 phase. Scale bars, 5 μm. () Cells imaged at indicated times after DOX addition during interphase. A paraspeckle assembled on DOX induction at the Men ε/β locus, increased in size and formed clusters of paraspeckles. * Figure 5: Differential kinetics of Men ε/β ncRNAs and paraspeckle proteins. () FRAP analyses of paraspeckles. Paraspeckle proteins exhibit a rapid exchange rate, whereas Men ε/β ncRNAs exhibit a much slower exchange. Top: images of cells harbouring the Men ε/β RNA reporter and expressing mCherry–PSP1, and EYFP–MS2 to label Men ε/β ncRNAs, at indicated times after bleaching of endogenous (arrowhead) or de novo formed (arrow) paraspeckles. Scale bar, 5 μm. Bottom: kinetics of recovery of paraspeckle proteins after bleaching in nucleoplasm (blue cross), endogenous paraspeckles (open red circle) or de novo formed paraspeckles (solid black circle), and of Men ε/β ncRNAs (green square) (n = 8 for PSP1 and p54nrb, n = 5 for PSF, and n = 12 for Men ε/β ncRNAs, data are means ± s.e.m.). Intensity was normalized so that that the first time point pre-bleach was set as 1 and the first time point post-bleach was set as 0. Note that the scale of the x axis of Men ε/β ncRNAs recovery profile is different from those of paraspeckle proteins. () Pro! tein dynamics during paraspeckle formation in cells harbouring the Men ε/β RNA reporter. Left: the intensity of EYFP–MS2 and the indicated mCherry-fused paraspeckle proteins at the transcription site was quantified and normalized over time. Right: kinetics of first 40 min after DOX induction is shown. Within the temporal resolution (5 min), all paraspeckle proteins examined were found to be recruited to newly formed paraspeckles instantly on the detection of Men ε/β transcription. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Yuntao S. Mao & * Hongjae Sunwoo Affiliations * Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA. * Yuntao S. Mao, * Hongjae Sunwoo, * Bin Zhang & * David L. Spector Contributions Y.S.M., H.S., and D.L.S. designed the experiments. Y.S.M., H.S., and B.Z. performed experiments and analysed data. Y.S.M. and D.L.S. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David L. Spector Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (1M) Supplementary Information * Supplementary Movie 2 (3M) Supplementary Information * Supplementary Movie 3 (427K) Supplementary Information * Supplementary Movie 4 (258K) Supplementary Information * Supplementary Movie 5 (309K) Supplementary Information * Supplementary Movie 6 (112K) Supplementary Information * Supplementary Movie 7 (347K) Supplementary Information * Supplementary Movie 8 (4M) Supplementary Information * Supplementary Movie 9 (1M) Supplementary Information * Supplementary Movie 10 (415K) Supplementary Information * Supplementary Movie 11 (299K) Supplementary Information PDF files * Supplementary Information (845K) Supplementary Information Additional data - TSPYL5 suppresses p53 levels and function by physical interaction with USP7
- ncb 13(1):102-108 (2011)
Nature Cell Biology | Letter TSPYL5 suppresses p53 levels and function by physical interaction with USP7 * Mirjam T. Epping1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lars A.T. Meijer3 Search for this author in: * NPG journals * PubMed * Google Scholar * Oscar Krijgsman4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes L. Bos3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pier Paolo Pandolfi2 Search for this author in: * NPG journals * PubMed * Google Scholar * René Bernards1 Contact René Bernards Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:102–108Year published:(2011)DOI:doi:10.1038/ncb2142Received11 March 2010Accepted01 November 2010Published online19 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We have previously reported a gene expression signature that is a powerful predictor of poor clinical outcome in breast cancer1. Among the seventy genes in this expression profile is a gene of unknown function: TSPYL5 (TSPY-like 5, also known as KIAA1750). TSPYL5 is located within a small region at chromosome 8q22 that is frequently amplified in breast cancer, which suggests that TSPYL5 has a causal role in breast oncogenesis2, 3. Here, we report that high TSPYL5 expression is an independent marker of poor outcome in breast cancer. Mass spectrometric analysis revealed that TSPYL5 interacts with ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease; HAUSP). USP7 is the deubiquitylase for the p53 tumour suppressor4 and TSPYL5 reduces the activity of USP7 towards p53, resulting in increased p53 ubiquitylation. We demonstrate that TSPYL5 reduces p53 protein levels and inhibits activation of p53-target genes. Furthermore, expression! of TSPYL5 overrides p53-dependent proliferation arrest and oncogene-induced senescence, and contributes to oncogenic transformation in multiple cell-based assays. Our data identify TSPYL5 as a suppressor of p53 function through its interaction with USP7. View full text Subject terms: * Cancer Figures at a glance * Figure 1: TSPYL5 is a poor-prognosis marker in breast cancer and binds to USP7. () Kaplan–Meier plot of distant metastasis-free survival of breast cancer patients (n = 143) with low (n = 84) or high (n = 59) TSPYL5 gene expression in the validation set. () Schematic representation of TSPYL5 location on chromosome 8q22, a region of recurrent amplification in breast cancer. () Summary of protocol used to purify TSPYL5 from MCF7 cells with stable expression of 3 × Flag–biotin–TSPYL5. Mass spectrometric analysis identified USP7 as a TSPYL5-binding protein. () MCF7 cells were infected with retrovirus encoding Flag–TSPYL5 or empty vector (EV; as a control). Cell lysates were immunoprecipitated with anti-Flag antibodies and analysed by western blot using anti-USP7 and anti-Flag antibodies. () TSPYL5 was immunoprecipitated from PC3 cells with an antibody against TSPYL5 and the western blots were stained with anti-USP7 and anti-TSPYL5 antibodies. IgG; immunoglobulin G. () Schematic representation of USP7 domains fused to GFP that were used for the inter! action analysis in (FL; full-length). () Lysates of cells transfected with plasmids encoding Flag–TSPYL5 and the indicated GFP–USP7 domains (as defined in ) were immunoprecipitated with anti-Flag antibody and analysed by western blotting. () Lysates of cells transfected with plasmids encoding Flag–p53, Myc-USP7 and either TSPYL5 or empty vector were immunoprecipitated with anti-Flag antibody and analysed by western blotting. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 2: TSPYL5 increases p53 ubiquitylation and suppresses p53 protein levels. () PC3 cells were transfected with plasmid encoding p53 and were co-transfected with plasmids encoding USP7, TSPYL5 (T5) or USP7 and TSPYL5. Cells were treated with proteasome inhibitor MG132 and lysates were analysed for p53 ubiquitylation by western blotting with an anti-p53 antibody. () Lysates of 293 cells transfected with plasmids encoding the indicated proteins were immunoprecipitated with an anti-p53 antibody and analysed for ubiquitylation by western blotting with an anti-p53 antibody. () U2OS cells were transfected with plasmids encoding USP7 or TSPYL5 and lysates were immunoblotted for p53. () U2OS cells were transfected with plasmid encoding Myc–USP7 and increasing concentrations of plasmid encoding Flag–TSPYL5 and were immunoblotted for p53, p21CIP and MDM2. () Western blot analysis of MCF7 cells transfected with TSPYL5 siRNA after treatment with the indicated concentrations of Nutlin-3 for 24 h. Short and longer exposures are shown. () Left: qRT–PCR analys! is of TSPYL5 mRNA levels in MCF7 cells transfected with TSPYL5 siRNA. Relative mRNA levels were normalized to GAPDH (mean ± s.d.; n = 3). Right: western blot analysis of PC3 cells transfected with siRNA against luciferase (Luc siRNA), TSPYL5 or USP7. () Western blot analysis of MCF7 cells transfected with TSPYL5 siRNA, without treatment or after treatment with increasing concentrations of Nutlin-3. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 3: TSPYL5 inhibits p53 transactivation and target gene transcription. () Cytosolic and nuclear fractionation of ST.HdhQ111 cells that were infected with retrovirus encoding Flag–TSPYL5, empty vector or p53 shRNA at the restrictive temperature of 39 °C. () Western blot analysis of ST.HdhQ111 cells cultured at 32 °C or 39 °C. () CDKN1A-promoter luciferase assay in U2OS cells transfected with plasmid encoding TSPYL5 or shRNA against GFP or p53 (mean ± s.d.; n = 3). () PIG3-luciferase reporter assay for p53 transactivation in U2OS cells transfected with plasmid encoding TSPYL5 after treatment with the indicated concentrations of Nutlin-3 for 24 h (mean ± s.d.; n = 3). () qRT–PCR analysis of p53 target genes in ST.HdhQ111 cells, infected with empty vector, retrovirus encoding TSPYL5 or p53 shRNA, at 39 °C. Relative mRNA levels were normalized to β-actin (mean ± s.d.; n = 3). (, ) qRT–PCR analysis of p53 target genes () and p53 mRNA () in human BJ fibroblasts with an inducible RASV12-ER, after treatment with tamoxifen to activate RASV1! 2. The cells were infected with retrovirus encoding Flag–TSPYL5, empty vector or p53 shRNA. Relative mRNA levels were normalized to β-actin (mean ± s.d.; n = 3). Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 4: TSPYL5 inhibits oncogene-induced senescence and allows transformation. () ST.HdhQ111 cells were infected with empty vector, retrovirus encoding Flag–TSPYL5 or p53 shRNA and were cultured at 32 °C or 39 °C. Top: culture dishes were stained with Coomassie blue and photographed after 3 weeks. Bottom: Phase-contrast microscopy images of ST.HdhQ111 cells proliferating at 39 °C. Scale bar, 10 μM. () Proliferation of ST.HdhQ111 cells at 39 °C. () BJ fibroblasts with an inducible RASV12-ER were infected with empty vector, retrovirus encoding Flag–TSPYL5 or p53 shRNA, and were treated with tamoxifen (OHT) to activate RASV12. Top: culture dishes were stained with Coomasie blue and photographed after 3 weeks. Bottom: Phase-contrast microscopy images of BJ-RASV12-ER cells proliferating in the presence of tamoxifen. Scale bar, 10 μM. () Proliferation of BJ-RASV12-ER cells in the continuous presence of tamoxifen. () TIG3/16i cells infected with empty vector, retrovirus encoding Flag–TSPYL5 or p53 shRNA and were subsequently infected with RASV12 r! etrovirus. () TIG3/16i cells were infected with empty vector, retrovirus encoding Flag–TSPYL5, or p53 shRNA and were subsequently infected with BRAFV600E retrovirus. Top: culture dishes were stained with Coomassie blue and photographed after 3 weeks. Bottom: microscopy images of cellls stained for the senescence marker acidic β-galactosidase. Scale bar, 10 μM. () Soft agar transformation assay of BJ cells with stable expression of hTERT, RASV12, p16 shRNA, small t antigen and TSPYL5 or p53 shRNA (empty vector was used as a control; data are means ± s.d.; n = 3). Top: photographs of colony formation. Scale bar, 500 μM. Bottom: quantification of colonies formed. Author information * Author information * Supplementary information Affiliations * Division of Molecular Carcinogenesis, Centre for Biomedical Genetics and Cancer Genomics Centre, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, Netherlands. * Mirjam T. Epping & * René Bernards * Cancer Genetics Program, Beth Israel Deaconess Cancer Center, Departments of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. * Mirjam T. Epping & * Pier Paolo Pandolfi * Department of Physiological Chemistry, Centre for Biomedical Genetics and Cancer Genomics Centre, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, Netherlands. * Lars A.T. Meijer & * Johannes L. Bos * Agendia BV, Science Park 406, 1098 XH Amsterdam, Netherlands. * Oscar Krijgsman * Department of Pathology, VU Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, Netherlands. * Oscar Krijgsman Contributions The experiments were conceived and designed by M.T.E., P.P.P. and R.B. Experiments were performed by M.T.E. Mass spectrometry was performed by L.A.T.M. and supervised by J.L.B. Statistical analysis of gene expression in breast cancer was performed by O.K. The paper was written by M.T.E., P.P.P. and R.B. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * René Bernards Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (812K) Supplementary Information Additional data
No comments:
Post a Comment