Latest Articles Include:
- Focus on Membrane dynamics
- Nat Cell Biol 14(1):1 (2012)
Nature Cell Biology | Editorial Focus on Membrane dynamics Journal name:Nature Cell BiologyVolume: 14,Page:1Year published:(2012)DOI:doi:10.1038/ncb2415aPublished online22 December 2011 Cellular membranes in eukaryotes are dynamic structures; this is a key property for their roles in numerous cellular processes. In this issue, we present a series of review articles that highlight recent developments in membrane trafficking, and provide an overview of the importance of trafficking events in development and disease. Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Proteins and lipids follow prescribed trafficking pathways in the cell. Nascent proteins shuttle from the endoplasmic reticulum (ER) to the Golgi and onwards to their ultimate cellular destinations, whereas plasma-membrane proteins are internalized and then degraded or recycled back to the membrane. Clathrin-mediated endocytosis is a major mechanism by which cell-surface proteins are internalized, and many fundamental advances in understanding this key trafficking pathway were made in yeast. Sandra Lemmon and colleagues review the process of clathrin-mediated endocytosis in yeast, and discuss similarities and differences between this process in yeast and mammalian cells. Many transport carriers are enrobed by coat proteins, which have been ascribed diverse roles from membrane bending or stabilization of deformed membranes to cargo and adaptor recruitment. In the yeast secretory pathway, transport between the ER and Golgi is mediated by COPII-coated vesicles, and conserved COPII machinery also supports trafficking from the ER in mammalian systems. Randy Schekman and colleagues discuss recent advances in COPII-mediated transport in mammals, including the process of COPII carrier formation and the regulation and function of ER exit sites, where these carriers are generated. The actin cytoskeleton regulates membrane dynamics to deform the membrane, promoting invagination, tubulation and scission of transport carriers in the secretory and endocytic pathways. Cytoskeletal tracks then support subsequent motor-based transport of these carriers. Mihaela Anitei and Bernard Hoflack discuss how the cytoskeleton and trafficking machinery coordinately regulate transport carrier biogenesis and transport. Peter Cullen and Hendrik Korswagen outline the many roles of sorting nexins in the endocytic network. Sorting nexins are key components of the retromer complex that rescues internalized proteins from lysosomal degradation by diverting them to the retrograde endosome-to-Golgi trafficking pathway. As part of the retromer complex, the multifaceted sorting nexin proteins have been suggested to bend membranes and recruit cargo, supporting their key role in several developmental processes. The ESCRT machinery regulates the biogenesis of multivesicular bodies, intermediate compartments in the endocytic pathway that facilitate sorting of proteins destined for degradation by lysosomal proteolysis. Emerging evidence also points to an important role for the ESCRT complexes in development. Harald Stenmark and colleagues review recent data that indicate the importance of ESCRTs in regulating polarity, autophagy and other cellular processes. This Focus issue on membrane dynamics offers a unique perspective on the cellular apparatus involved in the secretory and endocytic pathways, and provides a current view of the intersection between membrane dynamics and other fundamental cellular processes. An accompanying online library presenting selected research papers and reviews from Nature Cell Biology and other Nature journals can be found on the Focus on Membrane dynamics homepage. We are pleased to bring this series of review articles to our readers, and thank our authors and reviewers for their contributions. Additional data - Limited stay for foreign scientists in the UK?
- Nat Cell Biol 14(1):1 (2012)
Nature Cell Biology | Editorial Limited stay for foreign scientists in the UK? Journal name:Nature Cell BiologyVolume: 14,Page:1Year published:(2012)DOI:doi:10.1038/ncb2415bPublished online22 December 2011 New immigration proposals restricting the period of stay for highly skilled migrants in the UK could undermine the future of British science. Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The UK government's new proposals to severely limit the path to settlement or permanent residency for skilled migrants from non-EU countries have renewed concerns about the impact of current immigration policies on British science. Details outlined in a Home Office consultation document suggest that the majority of skilled migrants (also known as 'Tier 2 migrants') and their dependents would be expected to leave the country after a maximum stay of five years. The consultation document outlines opportunities that may arise for Tier 2 migrants to switch to a path that would lead to settlement in the UK, including, for example, an automatic route that is at present available for "ministers of religion, elite sportspeople and those earning over £150,000". However, the vast majority of scientists, including senior investigators, will fail to meet the current salary criterion. For senior researchers, the prospect of uprooting a research programme in five years, often the leng! th of time taken to simply get a programme underway, is likely to be a powerful deterrent to accepting positions in the UK. Similarly, restrictions on the length of stay could force postdoctoral researchers to leave the country before wrapping up a project. A letter organized by CaSE (Campaign for Science & Engineering in the UK), signed by twenty influential representatives drawn from science and technology, makes these and other crucial points. New immigration policies imposing annual caps on the entry of skilled migrants, finalized earlier this year, prioritized applicants to "shortage occupations", including several that are science related. In addition, the government announced this summer that the Royal Society could nominate up to 300 scientists of "exceptional talent" to enter the UK. Any gains from these marginally more scientist-friendly immigration policies could be quickly wiped out with the passage of these new measures. Science is a global enterprise. Draconian measures that would make the UK a less attractive destination for the world's brightest cannot serve to secure the future of British science. Now is the time for concerned scientists to reach out to their elected representatives. Additional data - Lessons from yeast for clathrin-mediated endocytosis
- Nat Cell Biol 14(1):2-10 (2012)
Nature Cell Biology | Review Lessons from yeast for clathrin-mediated endocytosis * Douglas R. Boettner1 * Richard J. Chi1 * Sandra K. Lemmon1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:2–10Year published:(2012)DOI:doi:10.1038/ncb2403Published online22 December 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Clathrin-mediated endocytosis (CME) is the major pathway for internalization of membrane proteins from the cell surface. Half a century of studies have uncovered tremendous insights into how a clathrin-coated vesicle is formed. More recently, the advent of live-cell imaging has provided a dynamic view of this process. As CME is highly conserved from yeast to humans, budding yeast provides an evolutionary template for this process and has been a valuable system for dissecting the underlying molecular mechanisms. In this review we trace the formation of a clathrin-coated vesicle from initiation to uncoating, focusing on key findings from the yeast system. View full text Figures at a glance * Figure 1: Yeast endocytic factors are comprised of many conserved modular domains. Shown are the major endocytic factors with domain structures. Each factor is listed in the order of its recruitment during endocytosis. A red asterisk indicates the EH ligand NPF motifs and red double dagger indicates the acidic motif needed for actin nucleation promoting activity. * Figure 2: The endocytic pathway in yeast. (,) Early factors (clathrin, Syp1 and Ede1) are recruited during the immobile phase (), which is followed by the ordered assembly of the mid/late coat (; Sla2, Yap1801/2, Ent1/2, Pan1 and Sla1). Las17 is also recruited around this time. (,) Shortly before the mobile phase, Syp1 and Ede1 depart from the cortex (), rapidly followed by the WASp/myosin/actin slow mobile invagination phase (; actin, Abp1, Arp2/3, Myo3/5 and Vrp1). () Once the extended tubule forms, the vesicle scission apparatus (Rvs161/167 and Vps1) narrows the neck of the vesicle forming at the invagination tip to promote scission. () After release, the nascent vesicle is immediately uncoated by synaptojanin and Prk1/Ark1 activity. () The vesicle then moves rapidly inwards, shedding its actin shell through the action of Cof1, Aip1, Srv2 and Crn1. Mid/late coat factors are reactivated by Scd5/PP1(Glc7) dephosphorylation and recruited back to the membrane for new rounds of CME. * Figure 3: Regulation of Arp2/3-complex-mediated actin assembly during endocytosis. NPFs are indicated in green, positive regulators of NPFs in blue and negative regulators in red. The colour of the negative regulators changes from red to pink and then to white to indicate that their inhibitory activity is partial or relieved. F-actin is shown as a grey cloud surrounding the endocytic site. Initially, actin assembly is blocked by Syp1, Sla1 and Sla2, which prevents early activation of Las17 and Pan1 (left). Myosin 1 is inhibited by calmodulin in the cytosol. Next, the activator Bzz1 arrives, and Vrp1 is recruited by Las17 (middle). This may allow early F-actin seeding and the beginning of membrane invagination, as inhibitors start to lose their influence (pink). Release of Syp1 from the cortex and recruitment of myosins (by Vrp1) and Arp2/3 complex lead to robust actin assembly and invagination (right). Another negative regulator, Bbc1, arrives in the late phase, preventing excessive endocytic actin accumulation. It is not known when the Pan1 inhibitory fun! ction of Sla2 is relieved, as they move together into the coat. Sla2 is shown here losing its influence as the endocytic site invaginates. * Figure 4: Translating actin assembly into membrane invagination is regulated by clathrin light chain. () Sla2 is recruited to the cortex before actin assembly. () As F-actin is assembled, Sla2 at the perimeter of the clathrin coat binds to the filaments through its C-terminal THATCH domain. () This binding is used to tether the membrane to actin for invagination. () As the clathrin coat continues to form at the tip, Sla2 is bound by clathrin light chain causing a conformational shift (to a closed state) that releases the hold of Sla2 hold on F-actin. This may direct the force of actin assembly towards the formation of a tubule, as well as promote vesicle scission at the neck. Author information * Abstract * Author information Affiliations * Douglas R. Boettner, Richard J. Chi and Sandra K. Lemmon are at the Department of Molecular and Cellular Pharmacology (R-189), Miller School of Medicine, University of Miami, PO Box 016189, Miami, Florida 33101, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sandra K. Lemmon Author Details * Douglas R. Boettner Search for this author in: * NPG journals * PubMed * Google Scholar * Richard J. Chi Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra K. Lemmon Contact Sandra K. Lemmon Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Bridging membrane and cytoskeleton dynamics in the secretory and endocytic pathways
- Nat Cell Biol 14(1):11-19 (2012)
Nature Cell Biology | Review Bridging membrane and cytoskeleton dynamics in the secretory and endocytic pathways * Mihaela Anitei1 * Bernard Hoflack1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:11–19Year published:(2012)DOI:doi:10.1038/ncb2409Published online22 December 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Transport carriers regulate membrane flow between compartments of the secretory and endocytic pathways in eukaryotic cells. Carrier biogenesis is assisted by microtubules, actin filaments and their associated motors that link to membrane-associated coats, adaptors and accessory proteins. We summarize here how the biochemical properties of membranes inform their interactions with cytoskeletal regulators. We also discuss how the forces generated by the cytoskeleton and motor proteins alter the biophysical properties and the shape of membranes. The interplay between the cytoskeleton and membrane proteins ensures tight spatial and temporal control of carrier biogenesis, which is essential for cellular homeostasis. View full text Figures at a glance * Figure 1: Models for carrier biogenesis at the plasma membrane and trans-Golgi network. () The plasma membrane is initially deformed by F-BAR domain protein FCHo and clathrin coat proteins which bind actin nucleation factors. HIP1R connects clathrin coats with pre-existing F-actin and may prevent actin polymerization on the coat surface (upper panel). BDPs bind to tubular membranes, and together with N-WASP and CDC42 sustain ARP2/3-mediated actin polymerization towards the membrane (lower panel). F-actin may thus push against the membrane (arrows) and enhance its deformation. Myosins may also provide mechanical forces that contribute to membrane deformation (arrows). () At the TGN, clathrin coats or alternative mechanisms (not shown) bend membranes (arrows). Clathrin heavy chains bind CYFIP. HIP1R may play similar roles as in endocytosis (upper panel). Rac1 may bind to CYFIP to activate ARP2/3-dependent actin polymerization towards the membrane at the edges of the coats (arrows). During tubule elongation, CDC42 and N-WASP may sustain ARP2/3-dependent actin poly! merization, possibly assisted by BDPs (lower panel). Membrane-associated microtubule motors can further pull and elongate tubules along microtubules (arrows). () Myosin 18A is recruited by the PtdIns(4)P-binding protein GOLPH3 onto TGN membranes, where it could bind pre-existing F-actin and pull the membrane (arrow). Myosin 1b is recruited on clathrin/AP-1-coated as well as non-coated TGN membranes, where it sustains the assembly of ARP2/3 actin foci and provides forces (arrow) for carrier formation. () Model of membrane scission. On tubular membranes, BDP scaffolds promote F-actin polymerization. F-actin may generate pulling forces acting in opposite directions or push against the membrane (large arrows), and/or induce lipid phase separation (dotted line). Myosin 2 may provide similar forces, as is seen on Golgi membranes (1). Microtubule-based motors may pull Golgi membranes (arrows) and create tension (2). Dynamin assembled on tubules constricts membranes, and together w! ith BDPs may sustain ARP2/3-dependent actin polymerization. BD! Ps and/or dynamin may protect lipids from hydrolysis. Tug-of-war between motors and dynamin depolymerization could lead to lipid phase separation and membrane scission (dotted line) (3). * Figure 2: Early endosomal sorting and connections to the cytoskeleton. The early endosome maintained at the cell periphery by KIF16B binding to Rab5 () and dynein/dynactin () is a sorting station from where proteins are sorted to different destinations: fast recycling to the plasma membrane () as shown by SNX27 and WASH controlling the incorporation of the beta-adrenergic receptor into retromer-coated tubules; slow recycling to the plasma membrane by recycling endosomes, dependent on dynein binding to the BDP SNX4 by KIBRA (); or retrograde transport to the TGN mediated by the retromer complex, whose BDP subunits SNX5 and SNX6 bind the dynein–dynactin complex (). The retromer subunits SNX1 and SNX2, as well as FAM21, bind WASH (). On early endosomal membranes, annexin A2, Spire1 and ARP2/3-mediated polymerization is necessary for transport to late endosomes (). The AP-3 coat, required for lysosomal transport, can bind KIF3A (). * Figure 3: Coordinated function of lipids and proteins in membrane bending, elongation and fission. () Locally synthesized PtdInsP contributes to the recruitment of membrane-bending protein machineries. Bending can be induced by coats, ARF GTPases, by the insertion of ENTH/ANTH domains in the outer membrane layer or by F-BAR proteins (for example FCHo1/2). Myosins can also deform membranes, probably pulling them along pre-existing actin filaments. These events increase membrane curvature and tension and lead to lipid sorting. Synthesis of conical lipids, for example diacylglycerol (DAG) may influence membrane curvature, as seen on Golgi membranes after phosphatidate phosphohydrolase (PAP) inhibition. PA, phosphatidic acid; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; DGK, diacylglycerol kinase. () Membrane elongation. Modulation of PtdIns(4,5)P2 levels by PtdInsP-5-kinases (PtdInsP-5-K)129 and phosphatases (oculo-cerebro-renal syndrome of Lowe or OCRL)36 may activate actin polymerization. PtdIns(4,5)P2 may also contribute to BDP bindin! g to curved membranes. Coats may recruit factors that promote actin polymerization and myosins that provide forces for carrier elongation. Membrane tension may influence myosin function. Membrane-binding BDPs can sense and may further enhance membrane curvature and actin polymerization. Microtubule-associated motors can also provide forces for carrier elongation (Golgi and endosomes). The local synthesis of conical lipids, for example LPA, may contribute to membrane curvature. Enzymes such as phospholipase A2 (PLA2)130 and lysophosphatidic acid acyltransferase 3 (LPAAT3)127 modulate tubulation of Golgi carriers. () Membrane fission. PtdIns(4,5)P2 can be hydrolysed by phospholipase C (PLC) to form DAG. Activated phospholipase D (PLD) catalyses PC to form PA. Both PA and DAG can induce negative curvature at the neck. On the Golgi, DAG can contribute to the recruitment of protein kinase D (PKD), a regulator of scission. BDPs may protect PtdIns(4,5)P2 from hydrolysis and contri! bute to the recruitment of hydrolysing enzymes (for example sy! naptojanin). Increased membrane curvature and tension and BDPs may recruit dynamin that constricts membranes and may sustain actin polymerization. Dynamin depolymerization renders the membrane unstable. Forces provided by F-actin and myosins, and by microtubule-based motors, further stretch the membrane. These factors may contribute to create a lipid phase separation and line tension, leading to membrane scission. Author information * Abstract * Author information Affiliations * Mihaela Anitei and Bernard Hoflack are at the Biotechnology Centre, Technische Universität Dresden, Tatzberg 47/49, 01307 Dresden, Germany Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bernard Hoflack Author Details * Mihaela Anitei Search for this author in: * NPG journals * PubMed * Google Scholar * Bernard Hoflack Contact Bernard Hoflack Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - COPII and the regulation of protein sorting in mammals
- Nat Cell Biol 14(1):20-28 (2012)
Nature Cell Biology | Review COPII and the regulation of protein sorting in mammals * Giulia Zanetti1 * Kanika Bajaj Pahuja1 * Sean Studer2 * Soomin Shim3 * Randy Schekman1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:20–28Year published:(2012)DOI:doi:10.1038/ncb2390Published online22 December 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Secretory proteins are transported to the Golgi complex in vesicles that bud from the endoplasmic reticulum. The cytoplasmic coat protein complex II (COPII) is responsible for cargo sorting and vesicle morphogenesis. COPII was first described in Saccharomyces cerevisiae, but its basic function is conserved throughout all eukaryotes. Nevertheless, the COPII coat has adapted to the higher complexity of mammalian physiology, achieving more sophisticated levels of secretory regulation. In this review we cover aspects of mammalian COPII-mediated regulation of secretion, in particular related to the function of COPII paralogues, the spatial organization of cargo export and the role of accessory proteins. View full text Figures at a glance * Figure 1: Structure of COPII components. () Model of the complex between Sar1A (yellow), Sec23A (light grey) and Sec24A (dark grey) bound to a VSV-G peptide (red), obtained by superimposing the structures of Sec23–24–VSV-G peptide (PDB 3EGD) and human Sar1A (PDB 2GAO) with the structure of the yeast Sec23–Sar1 complexed with a Sec31 active fragment (PDB 2QTV). () Similarly obtained atomic model of the Sar1A–Sec23–Sec24C complex, bound to a syntaxin5 peptide (PDB 3EFO). () Atomic model of the yeast Sec13–31 heterotetramer. The X-ray structure of the Sec13–Sec31 vertex element (PDB 2PM9) was superimposed with that of the Sec13–Sec31 edge element (PDB 2PM6) by overlapping the Sec13 atomic coordinates. Sec31 is depicted in blue and orange, and Sec13 in cyan and red. () Cryoelectron microscopy single-particle reconstruction of in vitro assembled mammalian Sec13–31 cuboctahedral cages (EMDB 1232). () Cryoelectron microscopy single-particle reconstruction of in vitro assembled mammalian Sec13–31–Sec2! 3–24 icosidodecahedral cages (EMDB 1511). Scale bar for panels and , 50 nm. * Figure 2: ERES morphology. A schematic representation of mammalian ERESs and juxtaposed ERGIC. COPII-coated vesicles form in delimited cup-shaped ER regions that are associated with the plus end of microtubules. GTP hydrolysis drives vesicle fission and depolymerization of the Sec13–31 cage, whereas the Sec23–24 complex is retained. Vesicles travel to the ERGIC (also known as the vesicular-tubular cluster, VTC) in a microtubule-independent manner, and are tethered through the interaction between Sec23 and the TRAPPI complex. Cargo can proceed towards the Golgi or be recycled back to the ER membrane in COPI-coated vesicles. The role of Sec23 interaction with the microtubule-binding motor protein dynactin is not known. * Figure 3: Adaptor proteins mediate cargo sorting in COPII vesicles at ER exit sites. () Soluble cargoes exit by receptor-mediated transport. () Escort transmembrane receptor protein exits along with transmembrane cargo and also assists regulated transport of its cargo. () Transmembrane accessory proteins or ER luminal chaperones help to concentrate cargo at ERESs but are retained in the ER. () Cytoplasmic accessory proteins are required as adaptors for exit of certain cargoes. Transmembrane proteins are shown with single membrane-spanning domain for simplicity. * Figure 4: Basis for cage flexibility and incorporation of large cargoes. () A schematic representation of a standard COPII vesicle (60–100 nm). In the inset the interaction between the Sec31 C-terminal peptide and the Sec23–Sar1 complex is represented by a dotted line. () A model to explain vesicle size variation based on the intrinsic flexibility of the Sec13–31 outer cage, which originates from variations in the angles that underlie the cage geometry (panels and ). () Atomic model of the yeast Sec13–31 heterotetramer (Fig. 1c) fitted into the edge element of the mammalian COPII cuboctahedral cage (EMDB 1232). Variations in the angle at the Sec31 dimerization interface are illustrated by the misfit of one of the Sec13–31 dimers. () Comparison of the icosidodecahedral COPII cage (EMDB 1511, dark blue mesh) with the cuboctahedral map (cyan surface), showing the variation in the angle at the vertices. Scale bar, 50 nm. (e) A model to explain vesicle size variation based on the difference between human Sar1A and B paralogues. The interacti! on of Sec31 with Sar1B might be weaker than that between Sec31 and Sar1A, allowing for cage expansion. The question mark indicates that the structural details of such an interaction have not yet been resolved. (f) Atomic model of the human Sar1A–Sec23A–Sec24C complex with a C-terminal fragment of yeast Sec31, obtained as in Fig. 1b. The residues of Sar1A that differ in Sar1B are highlighted in red. Author information * Abstract * Author information Affiliations * Giulia Zanetti, Kanika Bajaj Pahuja and Randy Schekman are at the Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, California 94720, USA * Sean Studer is at the Scripps Research Institute, Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, California 92037, USA * Soomin Shim is at the Hanwha Chemical Bio Business Unit, 1 Janggyodong Junggu, Seoul 100-797, South Korea Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Randy Schekman Author Details * Giulia Zanetti Search for this author in: * NPG journals * PubMed * Google Scholar * Kanika Bajaj Pahuja Search for this author in: * NPG journals * PubMed * Google Scholar * Sean Studer Search for this author in: * NPG journals * PubMed * Google Scholar * Soomin Shim Search for this author in: * NPG journals * PubMed * Google Scholar * Randy Schekman Contact Randy Schekman Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Sorting nexins provide diversity for retromer-dependent trafficking events
- Nat Cell Biol 14(1):29-37 (2012)
Nature Cell Biology | Review Sorting nexins provide diversity for retromer-dependent trafficking events * Peter J. Cullen1 * Hendrik C. Korswagen2 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 14,Pages:29–37Year published:(2012)DOI:doi:10.1038/ncb2374Published online22 December 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Sorting nexins are a large family of evolutionarily conserved phosphoinositide-binding proteins that have fundamental roles in orchestrating cargo sorting through the membranous maze that is the endosomal network. One ancient group of complexes that contain sorting nexins is the retromer. Here we discuss how retromer complexes regulate endosomal sorting, and describe how this is generating exciting new insight into the central role played by endosomal sorting in development and homeostasis of normal tissues. View full text Figures at a glance * Figure 1: Sorting itineraries in the mammalian endosomal network and the distinct SNX-BAR-retromer and SNX3-retromer complexes. () Internalized cargo destined for degradation, such as the epidermal growth factor receptor (EGFR), are first sorted into intraluminal vesicles (ILVs) and by a process of endosomal maturation arrive in late endosomes/multivesicular bodies (MVBs)3. Fusion of MVBs with the lysosomal compartment brings about degradation of cargo associated with ILVs96. In contrast, retrieval from the degradative pathway of, for example the nutrient sensing Transferrin receptor (TfR), occurs back to the plasma membrane via fast or slow recycling routes, while retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) occurs back to the trans-Golgi network (TGN). The latter route, often referred to as retrograde transport, comprises a number of distinct pathways, which include those dependent on Rab9- and SNX-BAR-retromer4. Enrichment of different phosphoinositides within compartments that make up the endosomal network is shown, including the early endosomal PtdIns(3)P. () The dis! tinct cargo-selective and membrane deformation subcomplexes that together form the yeast and mammalian SNX-BAR-retromers. The mammalian genome contains two VPS26 genes. () The evolutionarily conserved mammalian SNX3-retromer complex. Gene duplication has resulted in two orthologues of C. elegans snx-3, SNX3 and SNX12. * Figure 3: Spatial segregation model to account for differential cargo sorting through SNX3-retromer and SNX-BAR-retromer pathways. () A model based on spatial segregation to describe differential sorting of Wntless and CI-MPR by SNX3-retromer and SNX-BAR-retromer complexes, respectively. Briefly, Wntless bound to Wnt morphogens is secreted to the cell surface where release of Wnt establishes short- and long-range morphogenic gradients. Internalized Wntless initially enters the early endosome where it engages the SNX3-retromer for retrieval back to the TGN. In contrast, CI-MPR bound to newly synthesized hydrolase enzymes exits the TGN and is principally trafficked directly to the endosomal network, entering at a stage of maturation downstream of SNX3-retromer. The acidic environment of this compartment leads to dissociation of the hydrolases, allowing retrieval of the CI-MPR via the tubular SNX-BAR-retromer. It is important to note that this model does not suggest exclusive transport of Wntless and CI-MPR through the SNX3-retromer and SNX-BAR-retromer pathway respectively. It simply reflects that steady-! state transport of these cargoes seems to be dependent on specific retromer complexes. () As discussed in the text, in yeast recycling of Fet3p–Ftr1p requires recognition of the cytoplasmic domain of Ftr1p by Grd19p (also known as Snx3p)9. Grd19p physically associates with the SNX-BAR-retromer thereby allowing recycling of Fet3p–Ftr1p9. The function of SNX3 in Wntless retrieval and Wnt secretion is therefore fundamentally different to the role of Grd19p. * Figure 2: A proposed pathway for SNX-BAR-retromer-mediated sorting. Activation and cargo capture is initiated by association of the VPS26–VPS29–VPS35 subcomplex to early-to-late endosomes through interaction with the GTP-loaded form of Rab7, thus forming the nucleation complex. Not depicted are the possible roles of clathrin and various clathrin-adaptors and binding proteins in cargo clustering before formation of the nucleation complex47. Incorporation of other cargo into the nucleation complex is achieved through specific cargo adaptors, and the SNX-BAR dimer enters the nucleation complex either by direct binding from the cytosol and/or lateral movement on the early-to-late endosomal surface. This increases the effective concentration of retromer SNX-BARs resulting in their switch to curvature-inducing mode to re-model the membrane into tubular profiles. An area of concentrated VPS26–VPS29–VPS35 and SNX-BAR-binding sites is therefore formed, onto which accessory proteins are sequestered, leading to further re-modelling of the matur! ing tubular profile. Generation of opposing forces through actin polymerization and motor activity aids carrier scission through either line tension and/or mechano-enzymatic fission possibly catalysed by dynamin-II and/or EHD1. Uncoating of the isolated carrier, possibly requiring ATP hydrolysis and/or Rab7–GTP hydrolysis, occurs either immediately after scission or on tethering to the recipient compartment. See text for more details. Author information * Abstract * Author information Affiliations * Peter J. Cullen is at the Henry Wellcome Integrated Signalling Laboratories, School of Biochemistry, Medical Sciences Building, University Walk, University of Bristol, Bristol, BS8 1TD, UK * Hendrik C. Korswagen is at the Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Peter J. Cullen or * Hendrik C. Korswagen Author Details * Peter J. Cullen Contact Peter J. Cullen Search for this author in: * NPG journals * PubMed * Google Scholar * Hendrik C. Korswagen Contact Hendrik C. Korswagen Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Shaping development with ESCRTs
- Nat Cell Biol 14(1):38-45 (2012)
Nature Cell Biology | Review Shaping development with ESCRTs * Tor Erik Rusten1 * Thomas Vaccari2 * Harald Stenmark1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:38–45Year published:(2012)DOI:doi:10.1038/ncb2381Published online22 December 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Originally identified for their involvement in endosomal sorting and multivesicular endosome (MVE) biogenesis, components of the endosomal sorting complex required for transport (ESCRT) are now known to control additional cellular functions such as receptor signalling, cytokinesis, autophagy, polarity, migration, miRNA activity and mRNA transport. The diverse cell biological functions of ESCRT proteins are translated into a pleiotropic set of developmental trajectories that reflect the wide repertoire of these evolutionarily conserved proteins. View full text Figures at a glance * Figure 1: The ESCRT machinery in endosomal sorting and MVE biogenesis. () Receptors (dark red) in the limiting membrane of the MVE are capable of signalling (yellow signal bars). () Many receptors are ubiquitinated in response to ligand binding. () The ubiquitin moieties are recognized by ESCRT-0, which sequesters cargo into specific domains of the limiting membrane. HRS binds to membrane PtdIns(3)P (black hexagon). () ESCRT-0 also recruits ESCRT-I, presumably in conjunction with the transfer of ubiquitinated cargo. () Together with ESCRT-II, ESCRT-I mediates invagination of the limiting membrane of the MVE. VPS36 binds to membrane PtdIns(3)P (black hexagon). () ESCRT-III is recruited by binding to ESCRT-II, and cargo is deubiquitinated by ESCRT-III-associated deubiquitinating enzymes (DUBs). () Spiral-shaped ESCRT-III filaments assemble around the neck of the forming vesicle to promote its abscission from the limiting membrane, forming an ILV. () The ATPase VPS4, recruited by ESCRT-III, mediates disassembly of ESCRT-III oligomers so that the s! ubunits are recycled. Note that several parallel nomenclature systems exist for ESCRT subunits7. Throughout this manuscript we have used a yeast-centric nomenclature. * Figure 2: Cell biological processes regulated by ESCRTs. ESCRT components mediate endosomal sorting of ubiquitinated receptors and MVE biogenesis, micro-autophagy, macro-autophagy, cytokinetic abscission, mRNA transport and RNAi-mediated mRNA silencing. In addition (and outside the scope of this review), the ESCRT machinery is also required for budding of HIV-1 and many other enveloped viruses from the plasma membrane91. * Figure 3: The ESCRT machinery in developmental processes. An illustration of how the various cell biological functions of ESCRT components can be translated into developmental programmes. Author information * Abstract * Author information Affiliations * Tor Erik Rusten and Harald Stenmark are at the Centre for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, Montebello, 0310 Oslo, Norway and also in the Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, 0310 Oslo, Norway * Thomas Vaccari is at the Istituto FIRC di Oncologia Molecolare, via Adamello 16, 20139, Milano, Italy Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Harald Stenmark Author Details * Tor Erik Rusten Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Vaccari Search for this author in: * NPG journals * PubMed * Google Scholar * Harald Stenmark Contact Harald Stenmark Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Navigating the ERAD interaction network
- Nat Cell Biol 14(1):46-47 (2012)
Article preview View full access options Nature Cell Biology | News and Views Navigating the ERAD interaction network * Thibault Mayor1Journal name:Nature Cell BiologyVolume: 14,Pages:46–47Year published:(2012)DOI:doi:10.1038/ncb2412Published online22 December 2011 The endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, which orchestrates the degradation of ER proteins by the proteasome, involves a plethora of proteins with diverse functions. Using a combination of proteomic and genetic approaches, a recent study provides fresh insights into the organization of the mammalian ERAD interaction network and the functions of its components. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Thibault Mayor is at the Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, University of British Columbia, Vancouver, V6T 1Z4 British Columbia, Canada Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Thibault Mayor Author Details * Thibault Mayor Contact Thibault Mayor Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - c-Cbl targets active Src for autophagy
- Nat Cell Biol 14(1):48-49 (2012)
Article preview View full access options Nature Cell Biology | News and Views c-Cbl targets active Src for autophagy * Francesco Cecconi1Journal name:Nature Cell BiologyVolume: 14,Pages:48–49Year published:(2012)DOI:doi:10.1038/ncb2413Published online22 December 2011 Autophagy can promote both cancer cell survival and death, and the mechanisms by which it mediates these disparate processes are under intense investigation. Autophagosomes are now shown to entrap and promote degradation of the active tyrosine kinase Src, enabling tumour cell survival. The E3 ubiquitin ligase c-Cbl acts as an autophagosome cargo receptor for Src. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Francesco Cecconi is at the Dulbecco Telethon Institute, Department of Biology, University of Tor Vergata, 00133 Rome, Italy, and IRCCS Santa Lucia Foundation, 00143 Rome, Italy Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Francesco Cecconi Author Details * Francesco Cecconi Contact Francesco Cecconi Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling
- Nat Cell Biol 14(1):51-60 (2012)
Nature Cell Biology | Article Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling * Emma Sandilands1, 5 * Bryan Serrels1, 5 * David G. McEwan2 * Jennifer P. Morton3 * Juan Pablo Macagno3 * Kenneth McLeod1 * Craig Stevens1 * Valerie G. Brunton1 * Wallace Y. Langdon4 * Marcos Vidal3 * Owen J. Sansom3 * Ivan Dikic2 * Simon Wilkinson1 * Margaret C. Frame1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 14,Pages:51–60Year published:(2012)DOI:doi:10.1038/ncb2386Received01 August 2011Accepted24 October 2011Published online04 December 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src–LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity. View full text Figures at a glance * Figure 1: Localization of active Src is altered in the absence of FAK in SCCs. () Left, cells were fixed and stained for anti-pTyr-416-Src (green) and with DAPI (blue). Solid arrows indicate cells with active Src in adhesions; dashed arrows show cells with active Src in puncta. Scale bars, 20 μm. Right, percentage of cells with active Src localizing to intracellular structures was quantified. Data are presented as mean±s.d. and significance is P<0.001 (n=3). () Cells were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Merged and higher-magnification images of the outlined areas are also shown. Solid and dashed arrows show Src localization to adhesions or to puncta, respectively. Scale bars, 20 μm. () Lysates from cells were immunoblotted with anti-FAK, anti-Src, anti-Yes, anti-Fyn, anti-pTyr-416-Src and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 2: Active Src co-localizes with Atg proteins. () Cells were fixed and stained for anti-pTyr-416-Src (red) and co-stained for anti-LC3B, anti-Atg7 or anti-Atg12 (all green). Scale bars, 20 μm. () Higher-magnification images of the areas outlined in . Solid arrows indicate co-localization in focal adhesions and dashed arrows show puncta in the cytoplasm. () LC3B was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-LC3B antibodies. () Cells were transfected with 40 nM scrambled, Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti-Atg5, anti-Atg12 and anti-actin antibodies (lower panels) or fixed and stained for anti-pTyr-416-Src (green), anti-paxillin (red) and with DAPI (blue). Dashed arrows point to intracellular phospho-Src-containing puncta; solid arrows point to peripheral phospho-Src. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 3: Rate of autophagy is enhanced and active Src is turned over in a lysosomal-dependent manner. () Top, cells were transiently transfected with an RFP–GFP tandem fluorescent-tagged LC3 (RFP–GFP–LC3). Scale bars, 20 μm. Bottom, the number of yellow puncta and the number of RFP LC3-positive puncta in the merged images were counted and the total number of puncta per cell was calculated. Data are presented as mean±s.d. (n=3). () Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti-pTyr-416-Src, anti-Src, anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. () Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti-pTyr-416-Src, anti-Src, anti-p53 and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 4: Src activity is responsible for targeting to intracellular puncta. () Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P<0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. () Top, FAK−/− cells were transiently transfected with SrcY527F–GFP or with Src-251–GFP (green) and stained for anti-LC3B (red). Scale bars, 20 μm! . Bottom, quantification of cells with co-localization between Src and LC3B. Data are presented as mean±s.d. and significance is P<0.001 (n=3). () Left, FAK−/− cells stably re-expressing wild-type FAK, FAKY397F or FAKY4F-Y9F were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate active Src in autophagosomes. Scale bars, 20 μm. Right, lysates from these cells were also immunoblotted with anti-FAK and anti-actin antibodies. () Left, wild-type FAK, FAKY397F or FAKY4F-Y9F cells were transfected with RFP–GFP–LC3. Solid arrows indicate co-localization and dotted arrows indicate its absence. Scale bars, 20 μm. () Lysates were immunoblotted with anti-LC3B and actin antibodies in the absence and presence of chloroquine. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type after treatment with chloroquine and is! presented as a percentage increase for the immunoblot shown. ! Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 5: Loss of adhesion is sufficient to promote the autophagic targeting of Src in the presence of FAK. () Wild-type FAK cells were suspended in 1.4% methylcellulose solution in growth media and plated on agarose for 3 days. Top and bottom right, cells were recovered and cytospins prepared that were stained for anti-pTyr-416-Src (red), LC3B (green) and with DAPI (blue) or for paxillin (red), LC3B (green) and with DAPI (blue). Higher-magnification images of the outlined areas are also shown. Solid arrows indicate localization of pTyr-416-Src in LC3B-positive puncta. Scale bars, 20 μm. Bottom left, immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti-pTyr-397 FAK, anti-FAK, anti-pTyr-416 and anti-Src antibodies. () FAK+/+ cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti-pTyr-416-Src (green) and with DAPI (blue). A higher-magnification image of the area outlined is also shown. Scale bar, 20 μm. () LC3B was immunoprecipitated (IP) from adherent or suspended FAK+/+ an! d then immunoblotted with anti-Src and anti-LC3B antibodies. Lysates were also immunoblotted with anti-pTyr-397-FAK and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 6: The autophagic targeting of Src prevents cancer cell death. () Top, FAK−/− cells were sparsely plated and then treated with 3-MA (for 48 h), chloroquine or dasatinib (both for 24 h). Cells were then left at 37 °C for 1 week to allow colony formation, fixed and then stained with crystal violet. Bottom, the number of colonies formed was then quantified and normalized against untreated controls. Data are presented as mean±s.d. and significance is P<0.001 for 3-MA, P<0.01 for chloroquine and P>0.5 for dasatinib (n=3). () Top, wild-type FAK and FAK−/− cells were transiently transfected with two individual Atg5 shRNAs (1 and 2), selected in puromycin and then the clonogenic assay was carried out as described above. Bottom, quantification; data are presented as mean±s.d. and significance is P>0.1 for Atg5 A and B in wild-type FAK cells and P<0.01 for Atg5 A and P<0.05 for Atg5 B in FAK−/− cells (n=3). () Cells were also stained with TUNEL. The positive control was DNase1-treated cells and the negative control lacked TUN! EL reaction mix. Quantification is shown and data are presented as mean±s.d. and significance is P<0.005 for 3-MA and P<0.001 for Atg5 siRNA (n=3). () FAK−/− cells were untreated or treated with dasatinib, 3-MA, chloroquine or bafilomycin A (BFA) for 24 h, or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti-PARP antibody. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 7: Src trafficking into intracellular puncta requires c-Cbl. () Cells untreated, or treated with chloroquine for 24 h, were fixed and stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate co-localization in autophagosomes. Scale bars, 20 μm. () c-Cbl was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-c-Cbl antibodies. (,) Cells were transfected with scrambled siRNA or with c-Cbl siRNA for 72 h and then immunoblotted with anti-c-Cbl and actin antibodies () or stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green; , left). Dashed arrows indicate co-localization and solid arrows indicate Src at focal adhesions. Scale bars, 20 μm. Quantification is shown (, right). Data are presented as mean±s.d. and significance is P>0.5 for FAK+/+ cells and P<0.001 for FAK−/− cells (n=3). () LC3B was immunoprecipitated from FAK−/− cells expressing either scrambled or c-Cbl s! iRNA and then immunoblotted with anti-Src and anti-LC3B antibodies. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 8: A c-Cbl LIR domain, but not E3 ligase activity, mediates Src autophagic targeting. () c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA resistant, HA-tagged wild-type c-Cbl or c-Cbl mutants with defective E3 ligase activity (c-CblC381A or c-Cbl-70Z). Left, cells were stained for anti-p-Tyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows show co-localization in intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P>0.5 (n=3). () Schematic illustration of the structure of c-Cbl and alignment of the potential LIR motif with those in known LC3B-interacting proteins. () GST-pulldown assay of HA-tagged wild-type c-Cbl, c-CblAA (W802A, L805A) and c-CblAAAA (W802A, L803A, S804A, L805A) expressed in HEK293T cells was carried out using GST–LC3. () c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA-resistant HA-tagged wild-type c-Cbl or c-CblAAAA. Left, cells were stained for a! nti-pTyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows indicate pTyr-416 localization to adhesions and dashed arrows indicate localization to intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P<0.01 (n=3). Uncropped images of blots are shown in Supplementary Fig. S9. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Emma Sandilands & * Bryan Serrels Affiliations * Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XR, UK * Emma Sandilands, * Bryan Serrels, * Kenneth McLeod, * Craig Stevens, * Valerie G. Brunton, * Simon Wilkinson & * Margaret C. Frame * Frankfurt Institute for Molecular Life Sciences, Goethe University, Theodor-Stern-Kai 7, Frankfurt arn Main D-60590, Germany * David G. McEwan & * Ivan Dikic * Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK * Jennifer P. Morton, * Juan Pablo Macagno, * Marcos Vidal & * Owen J. Sansom * School of Pathology and Laboratory Medicine, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia * Wallace Y. Langdon Contributions E.S. and B.S. contributed equally to experimental work, project planning and data analysis. D.G.M., K.M., J. P. Morton and J. P. Macagno contributed to the experiments described in this manuscript. C.S., V.G.B., M.V., O.J.S. and I.D. provided intellectual input. W.Y.L. provided c-Cbl reagents. S.W. carried out electron microscopy and contributed to project planning and interpretation of data. M.C.F. was the grant holder and principal investigator under whom this work was carried out. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Simon Wilkinson or * Margaret C. Frame Author Details * Emma Sandilands Search for this author in: * NPG journals * PubMed * Google Scholar * Bryan Serrels Search for this author in: * NPG journals * PubMed * Google Scholar * David G. McEwan Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer P. Morton Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Pablo Macagno Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth McLeod Search for this author in: * NPG journals * PubMed * Google Scholar * Craig Stevens Search for this author in: * NPG journals * PubMed * Google Scholar * Valerie G. Brunton Search for this author in: * NPG journals * PubMed * Google Scholar * Wallace Y. Langdon Search for this author in: * NPG journals * PubMed * Google Scholar * Marcos Vidal Search for this author in: * NPG journals * PubMed * Google Scholar * Owen J. Sansom Search for this author in: * NPG journals * PubMed * Google Scholar * Ivan Dikic Search for this author in: * NPG journals * PubMed * Google Scholar * Simon Wilkinson Contact Simon Wilkinson Search for this author in: * NPG journals * PubMed * Google Scholar * Margaret C. Frame Contact Margaret C. Frame Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2300K) Supplementary Information Additional data - A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain
- Nat Cell Biol 14(1):61-72 (2012)
Nature Cell Biology | Article A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain * Ben Chih1 * Peter Liu2 * Yvonne Chinn2 * Cecile Chalouni3 * Laszlo G. Komuves3 * Philip E. Hass2 * Wendy Sandoval2 * Andrew S. Peterson1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:61–72Year published:(2012)DOI:doi:10.1038/ncb2410Received02 September 2011Accepted23 November 2011Published online18 December 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Using RNAi screening, proteomics, cell biological and mouse genetics approaches, we have identified a complex of nine proteins, seven of which are disrupted in human ciliopathies. A transmembrane component, TMEM231, localizes to the basal body before and independently of intraflagellar transport in a Septin 2 (Sept2)-regulated fashion. The localizations of TMEM231, B9D1 (B9 domain-containing protein 1) and CC2D2A (coiled-coil and C2 domain-containing protein 2A) at the transition zone are dependent on one another and on Sept2. Disruption of the complex in vitro causes a reduction in cilia formation and a loss of signalling receptors from the remaining cilia. Mouse knockouts of B9D1 and TMEM231 have identical defects in Sonic hedgehog (Shh) signalling and ciliogenesis. Strikingly, disruption of the complex increases the rate of diffusion into the ciliary membrane and the amount of plasma-membrane protein in the cilia. The complex that we have described is essential for normal! cilia function and acts as a diffusion barrier to maintain the cilia membrane as a compartmentalized signalling organelle. View full text Figures at a glance * Figure 1: Identification of the B9 protein complex and its localization at the transition zone. () Representative silver-stained gels after tandem affinity purification. () Consistently identified proteins are listed with percentage coverage by identified fragments and a depiction of their domain architecture. () IMCD3 total lysate was analysed by gel filtration chromatography. The eluted fractions were immunoblotted for the indicated proteins. The high-molecular-weight fractions containing all five complex components analysed are outlined in red. The input contains 20 μg of lysate. Molecular weight markers are indicated. (,) Subcellular fractionation was carried out using wild-type MEFs or IMCD3 cells transfected with B9D1-GFP and B9D2-GFP cLAP constructs. Lysates were immunoblotted for the indicated proteins using antibodies directed against either the endogenous proteins or, in the case of B9D1 and B9D2, against the GFP portion of the fusion protein. T, total lysate; M, membrane fraction; S, soluble fraction. Transferrin receptor (TfR1) and GAPDH are controls for! the membrane and soluble fraction, respectively. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 2: The B9 complex is required for receptor localization in the cilia. () SSTR3–GFP (green) labels the ciliary membrane and γ-tubulin (γtub; blue) labels the basal body. TMEM231 (red) is localized in the transition zone, adjacent to the ciliary membrane and distal to the basal body. Scale bar,1 μm. () In super-resolution images of TMEM231 (green) and γ-tubulin (red), both can be seen as ring structures in IMCD3 cells. TMEM231 is localized at the base of the axoneme identified by acetylated tubulin (Ac-tub; blue) and close to γ-tubulin. Scale bar, 0.5 μm. () 10DIV mouse primary hippocampal neurons were stained for TMEM231 (green) and ACIII (red), a neuronal cilia marker. Scale bar,1 μm. (,) IMCD3 cells were transfected with Ift88 siRNA to block ciliogenesis and were then stained for TMEM231 (green), acetylated tubulin (red) and γ-tubulin (blue). TMEM231 still localized at the transition zone in the absence of axonemal microtubules (white arrows). Scale bar, 5 μm. (–) TMEM231 co-localized with members of the B9D1 protein comp! lex. IMCD3 cells were transfected with GFP-tagged versions of B9 complex components (green) and then stained for TMEM231 (red) and acetylated tubulin (blue). (–) IMCD3 cells were transfected with the indicated siRNA for 3 days and stained for TMEM231 (green) or CC2D2A (green) and acetylated tubulin (red). Stable IMCD3 cell lines expressing B9D1–GFP (green) were used to identify B9D1 localization. Knockdown of B9D1, TMEM231, TMEM17 or CC2D2A reduces TMEM231 (–), B9D1–GFP (–) or CC2D2A (–) transition-zone localization. Scale bar, 5 μm. () Transition-zone localization was defined as TMEM231, B9D1–GFP or CC2D2A staining adjacent to γ-tubulin. The percentage of cells with transition-zone localization was quantified. () The percentage of cells with cilia (acetylated-tubulin staining) was quantified. For the data in , more than 1,000 cilia across 10 images were analysed for each condition. In , the number in each column indicates the total number (N) of γ-tubuli! n quantified across 10 images. Data are mean±s.e.m. One-way A! NOVA, compared with the NTC control, **P<0.01, ***P<0.001. (–) B9D1 and TMEM231 are required for B9 complex constitution. B9D1- and TMEM231-knockout embryos were analysed with gel filtration chromatography. The eluted fractions were immunoblotted for indicated proteins. The red arrowheads indicate the protein now eluted at lower-molecular-weight fractions in the absence of B9D1 or TMEM231. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 3: The B9 complex is required for receptor localization in the cilia. (–) SSTR3–GFP (–) or HTR6–GFP (–) IMCD3 cells transfected with the indicated siRNAs and stained for acetylated tubulin (Ac-tub; red), PCTN (blue) and intrinsic SSTR3–GFP (green) are shown. Scale bars, 5 μm. (,) The number of cells analysed for each condition is indicated on the graph and binned into low, medium and high levels of SSTR3–GFP localization to the cilia. One-way ANOVA, compared with the NTC control, ***P<0.001; error bars show ±s.e.m. (–) Mouse hippocampal neurons cultured for 5 days in vitro were transfected with the indicated siRNAs and cultured for 5 additional days. Cells were fixed and stained for SSTR3 (green), a neuronal cilia marker (ACIII, red) and a neuronal marker (NeuN, blue). Scale bar, 5 μm. ( The number of cells analysed is indicated on the graph. The percentage of cilia was determined as the percentage of NeuN-positive cells containing either ACIII-positive or SSTR3-positive cilia. One-way ANOVA, compared with the NTC contro! l, ***P<0.001; error bars show ±s.e.m. * Figure 4: The B9 protein complex acts as a ciliary barrier. () Bleaching of a fraction of the cilia membrane in an HTR6c–GFP-expressing cilium shows rapid recovery from diffusion within the cilia membrane. Scale bar,1 μm. (–) SSTR3–GFP- (–) or HTR6–GFP- (–) expressing IMCD3 cells were transfected with the indicated siRNAs for 3 days and deprived of serum overnight to induce ciliation. Cilia with adequate GFP signal were selected for FRAP analysis. Scale bars,1 μm. () B9d1-siRNA-treated SSTR3–GFP-expressing IMCD3 cells were imaged for 9 min after FRAP. (,) Kinetics of average (±s.e.m., as shown by error bars) FRAP for the entire cilium. t-test, compared with the NTC control, *P<0.05, **P<0.01, ***P<0.001. N is indicated on the graphs. (–) Sept2 regulates B9D1, CC2D2A and TMEM231 localization at the transition zone. IMCD3 and B9D1–GFP stable cell lines were transfected with the indicated siRNAs for 3 days and stained for GFP (B9D1–GFP, green), CC2D2A (green) or TMEM231 (green) and acetylated tubulin (Ac-tub! ; red) and γ-tubulin (γtub; blue). Scale bar, 5 μm. Magnifications of the outlined areas are shown as insets. () The percentage of γ-tubulin with adjacent GFP (B9D1–GFP, green) or CC2D2A (green) staining was quantified. N is indicated on the graph. () TMEM231 coverage is the percentage of acetylated-tubulin staining area that is co-localized with TMEM231. This is binned into low, medium and high levels of coverage with acetylated tubulin. N is indicated in the figure. One-way ANOVA, compared with the NTC control, ***P<0.001; error bars show ±s.e.m. * Figure 5: The B9 complex is required to restrict non-ciliary-membrane protein from the cilia membrane. (–) IMCD3 cells were first transfected with the indicated siRNAs followed 24 h later by transfection with GFP–CEACAM1 (–) or GFP–GPI (–). After two additional days of culture and a subsequent 12 h serum starvation, cells were fixed and stained with anti-GFP (green) and anti-acetylated-tubulin (Ac-tub; red). The top rows (,,, and ; ,,, and ) show the projections of total Z stacks.Scale bar, 10 μm. The bottom rows (,,, and ;,,, and ) show the projections of apical Zsections of the outlined region of the corresponding top panel. Cilia are outlined by circles (GFP negative) or rectangles (GFP positive) in each bottom panel. Scale bar, 5 μm. (,) The percentage of GFP–CEACAM1- () or GFP–GPI- () positive cilia was determined as the percentage of transfected cells containing GFP staining in acetylated-tubulin-positive cilia. N is indicated in each condition on the graphs. One-way ANOVA, compared with the NTC control, ***P<0.001; error bars show ±s.e.m. * Figure 6: The B9 complex is required for Shh signalling in vivo. (–) IMCD3 cells stably expressing SMO–GFP were transfected with the indicated siRNAs for 3 days. Scale bar, 5 μm. The cells in , and were treated with Shh to induce SMO translocation. Intrinsic SMO–GFP (green) and immunofluorescence signal for acetylated tubulin (Ac-tub; red) are shown. Insets show representative cilia. () The percentage of cilia that is positive for SMO–GFP in each condition. One-way ANOVA, compared with NTC control, ***P<0.001; error bars show ±s.e.m. N is indicated on the graph. (,,) E14.5 embryos. Microphthalmia, haemorrhage, edema and polydactyly are apparent in B9D1- and TMEM231-knockout embryos in whole mount. (,,) Microphthalmia of mutant embryos at E14.5. (,,) Hindlimb polydactyly (red arrows) in E14.5 knockout embryos. (,,) Immunostaining of paraffin sections from E10.5 spinal cords showing that Olig2 expression (red) is expanded ventrally in B9D1- and TMEM231-knockout embryos. Scattered cells are displaced dorsally (white arrowheads) i! nto the Pax6 (green) domain. Knockout embryos lack Shh expression (blue) in the floorplate (yellow arrow in ) whereas Shh expression is present in the notochord (white arrows). (,,) Knockout embryos show intermixing of Olig2 (red) and Nkx2.2 (green) neuro-progenitors. There is a reduction of FoxA2 (blue) neuro-progenitors at the floorplate. () A TaqMan assay of Gli1 mRNA induction by Shh shows that Shh responsiveness is reduced in knockout embryos. The average fold-induction of Gli1 by Shh from four independent MEF lines made from E13.5 embryos is quantified. t-test, compared with wild-type MEFs, ***P<0.001; error bars show ±s.e.m. (–) Paraffin sections through the spinal cord of wild-type and knockout embryos, stained for Arl13b (green) and acetylated tubulin (red) to mark cilia, show a severe reduction in cilia in knockout embryos. White arrows indicate a few remaining cilia. Scale bar, 5 μm. * Figure 7: The B9 complex is required for ciliogenesis. MEFs were made from E13.5 littermate embryos. (,,,) Wild-type (,), B9D1-knockout () and TMEM231-knockout () MEFs were cultured for 2 days and serum starved for an additional 24 h to induce cilia formation. Cells were stained for acetylated tubulin (Ac-tub; red) to mark cilia and PCTN (green) to mark centrosome. Scale bars, 5 μm. (,) The percentage of ciliation was determined as the percentage of PCTN-positive cells containing a cilium. Nis indicated on the graphs. t-test, compared with wild-type MEFs, ***P<0.001; error bars show ±s.e.m. * Figure 8: The B9 complex regulates ciliogenesis. (–) IMCD3 cells were transfected with the indicated siRNAs for 3 days and deprived of serum to induce ciliation. Serum starvation was initiated at time 0 and cells were fixed and stained for acetylated tubulin (Ac-tub; green) and PCTN (red) at the indicated intervals to follow the time course of ciliation. Scale bar, 5 μm. (,) B9d1-siRNA-transfected cells have an 8 h delay in ciliation. () The percentage of ciliation was determined as the percentage of PCTN-positive cells containing an acetylated-tubulin-positive cilium. t-test, compared with NTC control, ***P<0.001; error bars show ±s.e.m. N is indicated on the graph. () Cilia growth was measured by the length of acetylated-tubulin staining. t-test, compared with NTC control, ***P<0.001; error bars show ±s.e.m. N is indicated on the graph. () A model of how the B9D1 complex functions at the transition zone. TMEM231 and TMEM17 anchor the B9 complex at the transition-zone membrane, tethering the ciliary membrane to t! he microtubules. The B9 complex diffusion barrier retains ciliary-membrane proteins and prevents non-ciliary-membrane proteins from diffusing into the ciliary membrane. () A description of B9-complex-dependent events in ciliogenesis. The basal body is first docked just below the apical plasma membrane. Then the B9 complex is formed at the transition zone. Following transition-zone formation, IFT begins to rapidly build cilia. Without the B9 complex to prevent diffusion and retain ciliary material, IFT is inefficient and thus leads to delayed ciliogenesis and short cilia. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular Biology, Genentech, South San Francisco, California 94080, USA * Ben Chih & * Andrew S. Peterson * Department of Protein Chemistry, Genentech, South San Francisco, California 94080, USA * Peter Liu, * Yvonne Chinn, * Philip E. Hass & * Wendy Sandoval * Department of Pathology, Center for Advance Light Microscopy, Genentech, South San Francisco, California 94080, USA * Cecile Chalouni & * Laszlo G. Komuves Contributions B.C. planned, carried out and analysed experiments. P.L. and W.S. carried out mass spectrometry experiments. Y.C. and P.E.H. collected the gel filtration samples. C.C. and L.G.K acquiredthe super-resolution images. B.C. and A.S.P. designed and interpreted the experiments and wrotethe manuscript. Competing financial interests All authors are employees of Genentech, a for-profit institution. Corresponding author Correspondence to: * Andrew S. Peterson Author Details * Ben Chih Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Yvonne Chinn Search for this author in: * NPG journals * PubMed * Google Scholar * Cecile Chalouni Search for this author in: * NPG journals * PubMed * Google Scholar * Laszlo G. Komuves Search for this author in: * NPG journals * PubMed * Google Scholar * Philip E. Hass Search for this author in: * NPG journals * PubMed * Google Scholar * Wendy Sandoval Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew S. Peterson Contact Andrew S. Peterson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (5M) Supplementary Information Movies * Supplementary Movie 1 (3M) Supplementary Information Excel files * Supplementary Table 1 (28K) Supplementary Information * Supplementary Table 2 (31K) Supplementary Information Additional data - Puma and p21 represent cooperating checkpoints limiting self-renewal and chromosomal instability of somatic stem cells in response to telomere dysfunction
- Nat Cell Biol 14(1):73-79 (2012)
Nature Cell Biology | Letter Puma and p21 represent cooperating checkpoints limiting self-renewal and chromosomal instability of somatic stem cells in response to telomere dysfunction * Tobias Sperka1 * Zhangfa Song1, 5 * Yohei Morita1 * Kodandaramireddy Nalapareddy1 * Luis Miguel Guachalla1 * André Lechel1 * Yvonne Begus-Nahrmann1 * Martin D. Burkhalter1 * Monika Mach2 * Falk Schlaudraff3 * Birgit Liss3 * Zhenyu Ju4 * Michael R. Speicher2 * K. Lenhard Rudolph1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:73–79Year published:(2012)DOI:doi:10.1038/ncb2388Received26 July 2011Accepted27 October 2011Published online04 December 2011 Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The tumour suppressor p53 activates Puma-dependent apoptosis and p21-dependent cell-cycle arrest in response to DNA damage. Deletion of p21 improved stem-cell function and organ maintenance in progeroid mice with dysfunctional telomeres, but the function of Puma has not been investigated in this context. Here we show that deletion of Puma improves stem- and progenitor-cell function, organ maintenance and lifespan of telomere-dysfunctional mice. Puma deletion impairs the clearance of stem and progenitor cells that have accumulated DNA damage as a consequence of critically short telomeres. However, further accumulation of DNA damage in these rescued progenitor cells leads to increasing activation of p21. RNA interference experiments show that upregulation of p21 limits proliferation and evolution of chromosomal imbalances of Puma-deficient stem and progenitor cells with dysfunctional telomeres. These results provide experimental evidence that p53-dependent apoptosis and cell-c! ycle arrest act in cooperating checkpoints limiting tissue maintenance and evolution of chromosomal instability at stem- and progenitor-cell levels in response to telomere dysfunction. Selective inhibition of Puma-dependent apoptosis can result in temporary improvements in maintenance of telomere-dysfunctional organs. View full text Figures at a glance * Figure 1: Puma deletion prolongs lifespan and improves stem- and progenitor-cell-based organ maintenance of telomere-dysfunctional mice. () Kaplan–Meier survival curves of the indicated mouse cohorts. Puma gene status did not affect the lifespan of mTerc+/+ mice during the experimental period but significantly prolonged survival of G3 mTerc−/− Puma−/−mice when compared with G3 mTerc−/− Puma+/+ mice (median survival: 333 days versus 283 days). () Quantification of colon crypt density of 10- to 12-month-old mice of the indicated genotypes (n=5 mice per group). Note that the number of crypts is significantly reduced in G3 mTerc−/−,Puma+/+mice but partially rescued in G3 mTerc−/−,Puma−/− mice. () Representative images of small-intestine sections labelled for the intestinal stem-cell marker olfactomedin-4 (Olfm4) through RNA in situ hybridization (scale bar, 100 μm). () Quantification of Olfm4-labelled stem cells in the intestine of 10- to 12-month-old mice of the indicated genotypes. Note that G3 mTerc−/− mice show a significant reduction in intestinal stem-cell numbers, which is ! partially rescued by Puma deletion. () In vitro cultures of single freshly isolated crypts from 10- to 12-month-old mice of the indicated genotypes. Pictures were taken at day 7 in culture. Note that de novo growth and budding of crypts containing granulated Paneth cells (arrows) was reduced in crypts from G3 mTerc−/− Puma+/+ mice but partially rescued in crypts from G3 mTerc−/− Puma−/− mice (scale bar, 50 μm). () Quantification of organoids containing newly formed crypts after seven days in culture (n=3 independent cultures per group). Error bars represent s.e.m. * Figure 2: Puma deletion reduces apoptosis in stem and progenitor cells with dysfunctional telomeres. (,) Apoptosis detection through TUNEL staining in basal crypts of the small intestine. () Representative staining of 10- to 12-month-old mice of the indicated genotypes, scale bar 30 μm. () Number of TUNEL-positive cells per crypt from 10- to 12-month-old mice of the indicated genotypes (n=6–9 mice per group). () Apoptosis index in basal crypt cells of 10- to 12-month-old G3 mTerc−/−, Puma+/+ mice. Apoptosis was quantified for the indicated position of crypt cells relative to the middle cell on the crypt base. Highest rates of apoptosis occurred at stem-cell position +4 and in transient amplifying progenitor cells (position >4; n=3 mice). () Western blot analysis of Puma expression in whole-intestine lysates from 10- to 12-month-old mice of the indicated genotypes. The unspecific band at a relative molecular mass of 20,000 (Mr 20K) serves as a loading control. () qPCR analysis of Puma mRNA expression relative to hydroxymethylbilane synthase (HMBS) in small intesti! ne of 10- to 12-month-old mice of the indicated genotypes (n=3 mice per group). () The number of proliferating-cell nuclear antigen (PCNA)-positive cells in basal crypts of 10- to 12-month-old mice of the indicated genotypes. Error bars represent s.e.m., n=5 mice per group. A full scan of the Western blot is provided in Supplementary Fig. S7. * Figure 3: Puma deletion accelerates the accumulation of DNA damage and the upregulation of p21 in telomere-dysfunctional stem and progenitor cells. () Quantification of γH2AX-positive cells (containing three or more nuclear γH2AX foci) per crypt in small intestine of 10- to 12-month-old mice (n=5 mice per group). () Anaphase bridges were quantified in haematoxylin and eosin stained sections of the small intestine of 10- to 12-month-old mice (percentage of total number of anaphases; inset, representative anaphase bridge). G3 mTerc−/− mice show an increase in anaphase bridges when compared with mTerc+/+ mice. Note that Puma deletion leads to a significant increase in G3 mTerc−/− crypts whereas p21 deletion has no significant effect (G3 mTerc−/−, Puma−/− group, n=12; G3 mTerc−/−, Puma+/+ and iG4 mTerc−/−, Puma+/+ groups, n=5; other groups, n=4). Note that there is no significant difference between G3 and iG4 mTerc−/− mice. () aCGH analysis was carried out on individual laser-captured crypts of the small intestine of 10- to 12-month-old mice (n=7–9 crypts per group). The histogram depicts the! total number of aberrations per crypt for the indicated genotypes. (–) Immunostaining of p21 in crypts of the small intestine of 10- to 12-month-old mice. The group of G3 mTerc−/−, Puma−/− mice was subdivided into animals exhibiting low average weight loss (10–15%) or high average weight loss (>15%) indicating an end of the rescue period mediated by Puma deletion. () Representative p21 staining in basal crypts (arrows point to p21-positive cells; scale bar, 20 μm). () Quantification of p21-positive cells per crypt (n=6 mice per group). () Quantification of p21-positive stem cells at the crypt base (n=6). Inset: Morphological appearance of a stem cell between granular Paneth cells at the crypt base. Note that Puma deletion led to a significant increase in the accumulation of p21-positive cells in G3 mTerc−/−, Puma−/−crypts when compared with G3 mTerc−/−, Puma+/+ crypts, which is further enhanced on increasing weight loss. () qPCR analysis of p21 m! RNA expression in freshly isolated long-term haematopoietic st! em cells (LT-HSCs; CD34lo/−LSK) of 10- to 12-month-old mice (n=3 mice per group). Error bars represent s.e.m. * Figure 4: p21 and Puma represent synergistic checkpoints preventingproliferation and the evolution of chromosomal instability of telomere-dysfunctional stem and progenitor cells. (–) Freshly isolated intestinal basal crypt cells of 10-month-old mice were infected with lentivirus expressing control or two different shRNAs targeting p21. () Representative pictures of primary organoid cultures on day 10 after lentivirus transduction (scale bar, 200 μm). () A quantification of organoid development. Note that deletion of Puma and knockdown of p21 synergistically rescue the organoid-forming capacity of telomere-dysfunctional stem and progenitor cells. (,) Organoids were cultured over a period of 70 days, collected and subjected to aCGH analysis. () Superimposed aCGH profiles showing chromosomal gains and losses (peaks above and below baseline) in G3 mTerc−/−, Puma−/−p21 shRNA organoids. chr, chromosome. () Quantification of chromosomal gains and losses in epithelial organoids (n=9–13 organoids per genotype). CIN, chromosomal instability. (–) Freshly isolated haematopoietic stem and progenitor cells (LSK) of 10-month-old mice were infected ! with lentivirus and transplanted along with non-infected cells into lethally irradiated mice. () Total chimaerism of green fluorescent protein (GFP)-positive cells in the peripheral blood was determined. Note that p21 knockdown had a positive effect on the repopulation capacity of LSK cells from G3 mTerc−/−, Puma+/+ mice (G3W, left) and G3 mTerc−/−, Puma−/− mice (G3P, right, n=2–3 mice per group). () 100 days after transplantation single-sorted GFP+, long-term HSCs (CD34lo/−LSK) were isolated from primary recipients and cultured for two weeks. Knockdown of p21 and Puma deletion synergistically rescued the colony-forming capacity of G3 mTerc−/− HSCs (n=60 single-sorted HSCs per genotype). () Superimposed aCGH profiles showing chromosomal gains and losses in colonies derived from single-cell-sorted G3 mTerc−/−, Puma−/−p21 shRNA HSCs. () Quantification of chromosomal gains and losses in HSC colonies (n=8–9 colonies per genotype). () Model of coope! rating checkpoints in response to telomere dysfunction. Puma-d! ependent apoptosis represents the primary checkpoint to chromosomal fusions, whereas p21 is activated in response to DNA damage. Prolonged survival and organ maintenance of cells with fused chromosomes leads to accumulation of DNA breaks through fusion–bridge–breakage cycles in G3 Terc−/−Puma−/− mice. Accumulating DNA damage leads to increased p21 activation, limiting the rescue period of improved organ maintenance. Error bars represent s.e.m. Author information * Author information * Supplementary information Affiliations * Institute of Molecular Medicine and Max-Planck-Research Department on Stem Cell Aging, University of Ulm, 89081 Ulm, Germany * Tobias Sperka, * Zhangfa Song, * Yohei Morita, * Kodandaramireddy Nalapareddy, * Luis Miguel Guachalla, * André Lechel, * Yvonne Begus-Nahrmann, * Martin D. Burkhalter & * K. Lenhard Rudolph * Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/8, A-8010 Graz, Austria * Monika Mach & * Michael R. Speicher * Institute of Applied Physiology, University of Ulm, 89081 Ulm, Germany * Falk Schlaudraff & * Birgit Liss * Institute of Aging Research, Hangzhou Normal University College of Medicine, 16 Xuelin Road, 310036, Hangzhou, China * Zhenyu Ju * Present address: Department of Colorectal Surgery, Sir Run Run Shaw Hospital, 3 East Qingchun Road, 310016, Hangzhou, China * Zhangfa Song Contributions T.S., Z.S., Y.M., K.N., Y.B-N., M.D.B. and Z.J. carried out, designed and analysed experiments; T.S., A.L., Y.B-N., M.M. and M.R.S. carried out and analysed aCGH experiments; F.S. and B.L. carried out microdissection; Z.S. and L.M.G. generated mouse crosses; K.L.R. and T.S. wrote the manuscript; K.L.R. conceived the study. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * K. Lenhard Rudolph Author Details * Tobias Sperka Search for this author in: * NPG journals * PubMed * Google Scholar * Zhangfa Song Search for this author in: * NPG journals * PubMed * Google Scholar * Yohei Morita Search for this author in: * NPG journals * PubMed * Google Scholar * Kodandaramireddy Nalapareddy Search for this author in: * NPG journals * PubMed * Google Scholar * Luis Miguel Guachalla Search for this author in: * NPG journals * PubMed * Google Scholar * André Lechel Search for this author in: * NPG journals * PubMed * Google Scholar * Yvonne Begus-Nahrmann Search for this author in: * NPG journals * PubMed * Google Scholar * Martin D. Burkhalter Search for this author in: * NPG journals * PubMed * Google Scholar * Monika Mach Search for this author in: * NPG journals * PubMed * Google Scholar * Falk Schlaudraff Search for this author in: * NPG journals * PubMed * Google Scholar * Birgit Liss Search for this author in: * NPG journals * PubMed * Google Scholar * Zhenyu Ju Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R. Speicher Search for this author in: * NPG journals * PubMed * Google Scholar * K. Lenhard Rudolph Contact K. Lenhard Rudolph Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1700K) Supplementary Information Excel files * Supplementary Tables 1–3 (1700K) Supplementary Information Additional data - Polarized cell growth in Arabidopsis requires endosomal recycling mediated by GBF1-related ARF exchange factors
- Nat Cell Biol 14(1):80-86 (2012)
Nature Cell Biology | Letter Polarized cell growth in Arabidopsis requires endosomal recycling mediated by GBF1-related ARF exchange factors * Sandra Richter1 * Lena M. Müller1, 5 * York-Dieter Stierhof2 * Ulrike Mayer1, 2 * Nozomi Takada1 * Benedikt Kost3 * Anne Vieten1 * Niko Geldner1, 5 * Csaba Koncz4 * Gerd Jürgens1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:80–86Year published:(2012)DOI:doi:10.1038/ncb2389Received13 May 2011Accepted31 October 2011Published online04 December 2011 Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Polarized tip growth is a fundamental cellular process in many eukaryotic organisms, mediating growth of neuronal axons and dendrites1 or fungal hyphae2. In plants, pollen and root hairs are cellular model systems for analysing tip growth3, 4, 5. Cell growth depends on membrane traffic. The regulation of this membrane traffic is largely unknown for tip-growing cells, in contrast to cells exhibiting intercalary growth. Here we show that in Arabidopsis, GBF1-related exchange factors for the ARF GTPases (ARF GEFs) GNOM and GNL2 play essential roles in polar tip growth of root hairs and pollen, respectively. When expressed from the same promoter, GNL2 (in contrast to the early-secretory ARF GEF GNL1) is able to replace GNOM in polar recycling of the auxin efflux regulator PIN1 from endosomes to the basal plasma membrane in non-tip growing cells. Thus, polar recycling facilitates polar tip growth, and GNL2 seems to have evolved to meet the specific requirement of fast-growing pol! len in higher plants. View full text Figures at a glance * Figure 1: Polarized tip growth depends on BFA-sensitive GBF1-related ARF GEFs. (–) Root hair development in wild-type (WT; ) and BFA-resistant Myc-tagged GN (GNR–Myc; ) lines and in lines in which GFP-tagged BFA-resistant GNL2 is expressed from the GN promoter (GN::GNL2R–GFP; ). (,,,,,) BFA-treatment interferes with root hair elongation in wild-type (WT; ,) but not in BFA-resistant GN (GNR–Myc; ,) lines or lines in which BFA-resistant GNL2 is expressed from the GN promoter (GN::GNL2R-GFP; ,). (,,) Higher-magnification images of , and . Note, initiation of root hair growth is not affected by BFA. Scale bars, 100 μm. (–) Different stages of germinating pollen expressing GNL2–3×GFP. GNL2 accumulates at the pollen germination site and at the pollen tube tip (arrows). Scale bars, 10 μm. () Germination frequency of pollen from gnl2 heterozygous plants is reduced to about half the wild-type (WT) value. Error bars indicate standard error; numbers of pollen analysed are indicated in brackets (–). () Germination frequency of pollen from wild! -type (WT), BFA-sensitive GNL2–Myc (GNL2S–Myc) and BFA-resistant GNL2–Myc (GNL2R–Myc) lines in the presence and absence of BFA. Only pollen from BFA-resistant GNL2 lines germinate on BFA. () Analysis of pollen tube growth of untreated wild-type (WT) and BFA-treated wild-type as well as BFA-resistant GNL2 (GNL2R–Myc) pollen. BFA arrests pollen tube growth in wild-type (WT) but not in BFA-resistant GNL2 (GNL2R). Error bars indicate s.d. P values (t-test), *<0.0001, **<0.0001. (–) Germination and growth of pollen from wild-type (WT; ,) and BFA-resistant GNL2 (GNL2R; ,) lines in the absence (,) or presence (,) of BFA. Scale bars, 40 μm. * Figure 2: Functional relationship of GBF-related ARF GEFs. (–) In BFA-treated seedling roots, GNL2 (), when expressed from the GN promoter, co-localizes with the endosomal marker ARF1 () in BFA-compartments; () merged image. (–) PIN1 localization in BFA-treated seedling roots. Loss of polar PIN1 localization in wild-type (WT; ) does not occur in seedlings expressing BFA-resistant GN (GNR; ) or GNL2 from the GN promoter (GN::GNL2R; ). Arrows, polar PIN1 localization; stars, BFA compartments. Scale bars, 5 μm. () Partial rescue (PR) or complete rescue (CR) of the gnom mutant phenotype by expression of GFP- or Myc-tagged BFA-resistant GNL2 from the GN promoter (GN::GNL2R–GFP/Myc). Scale bars, 0.2 cm. (–) Seedlings expressing BFA-resistant GN or GNL2 from the GN promoter (GNR, GN::GNL2R) were treated with BFA. (,) Inhibition of lateral root formation () and gravitropism () in wild-type (WT) but not in BFA-resistant GN (GNR) or GNL2 (GN:: GNL2R) lines. () P values (t-test), *<0.0001, **<0.0001, ***<0.0001. () P values, *<0.0! 001, **<0.0001, ***=0.126596135. () Partial rescue of primary root growth inhibition (wild-type (WT)) in BFA-resistant GN (GNR) or GNL2 (GN:: GNL2R). Numbers of seedlings analysed are in brackets; error bars indicate s.d. P values (t-test), *<0.0001, **<0.0001. (–) Immunolocalization of the Golgi-marker γCOP in BFA-treated seedling roots. Membrane association of γCOP in wild-type () is lost in gnl1 () but restored by BFA-resistant GNL2 in gnl1 mutant background (gnl1 GN::GNL2R; ). Stars, membrane-associated γCOP accumulated around BFA compartments. Scale bars, 5 μm. (–) Wild-type growth (WT; ) is restored in gnl1 dwarfed plants () by expression of GNL2 from the GN or GNL1 promoter (GN::GNL2RGFP gnl1; ; GNL1::GNL2RYFP gnl1; ). Scale bar, 2 cm. * Figure 3: GNL2 mediates polar localization of pectin in pollen grains and pollen tubes. (–) Pectin staining with ruthenium red. Pectin localizes polarly in untreated wild-type pollen tubes (). BFA-treatment interferes with polar pectin localization in wild-type (; arrowhead) but not in BFA-resistant GNL2 pollen tubes (). Pectin localizes polarly in wild-type (WT; ,) and in BFA-resistant Myc-tagged GNL2 (GNL2R–Myc; ) pollen grains in the absence () or presence of BFA (,). Note, polar pectin patches are reduced in size in BFA-treated wild-type pollen grains (). Scale bars, 10 μm. Arrows indicate polar pectin localization. () Quantification of polar pectin localization stained by ruthenium red in wild-type (WT) and BFA-resistant GNL2 (GNL2R–Myc) pollen grains in the absence or presence of BFA. The graph was generated by combining the percentages of three independent experiments. P values (t-test), *=0.01489, **=0.0384. (–) Error bars indicate s.d.; numbers of pollen analysed are indicated in brackets. () Quantification of the polar pectin area in pollen! grains of wild-type (WT) and BFA-resistant GNL2 (GNL2R) in the presence of BFA. P value (t-test), *<0.0001. () Quantification of polar pectin localization in pollen tubes of wild-type (WT) and BFA-resistant GNL2 (GNL2R–Myc) in the absence or presence of BFA. The graph was generated by combining the percentages of three independent experiments. P values (t-test), *=0.081, **=0.0053. * Figure 4: Localized depositions of pectin in the intine layer are dependent on GNL2 action. (–) Electron microscopy of hydrated pollen grains of wild-type (WT; –) and BFA-resistant GNL2 (GNL2R; –) lines in the presence of BFA. N, nucleus. () Higher-magnification image of the area outlined in . (–) Higher-magnification images of the areas outlined in –. Note that depositions (arrowheads) are associated with the bulge of germinating pollen in the BFA-resistant GNL2 line (,). Arrows, secretory vesicles; black asterisks, intine; white asterisks, exine. Scale bars, 5 μm (,,–), 1 μm (,–). (,) Immunofluorescence signal localization of pectin (monoclonal antibody LM18) in wild-type () and GNL2R () pollen grains. Arrowheads point to increased deposition of pectin. Asterisks mark pollen grains. (,) Immunogold localization of pectin (monoclonal antibody LM18) in wild-type pollen grains. (,) Immunogold localization of pectin (monoclonal antibody LM18) in GNL2R pollen grains. Scale bars, 10 μm (,),1 μm (–). * Figure 5: GNL2 is required for polar organization of the pollen tube. Polar localization of Hypo2–YFP (–), the endocytic tracer FM4-64 (–) and the TGN marker RabA4b (–) in pollen tubes. BFA affects polar accumulation of Hypo2, FM4-64 and RabA4b in wild-type (,,), but not in BFA-resistant GNL2 (GNL2R), lines (,,,,,). RabA4b aggregate is still present in BFA-resistant GNL2 lines (, star). (,,,; ) Pollen tube growth. (,,,) Pollen germination in the presence of BFA. Duration of BFA treatment is indicated for each sample (1 h or 3 h). (–) Live-cell imaging of GNL2 and the endocytic tracer FM4-64 in BFA-treated pollen tubes. YFP-tagged GNL2 expressed from the Lat52b promoter (Lat52b::GNL2–YFP; ,) co-localizes with FM4-64 (,) in BFA compartments (,; stars) and also surrounds the BFA compartment (). (, inset) Higher-magnification image of a BFA compartment (left star in ). Scale bars, 5 μm. Author information * Author information * Supplementary information Affiliations * ZMBP, Entwicklungsgenetik, Universität Tübingen, Auf der Morgenstelle 3, 72076 Tübingen, Germany * Sandra Richter, * Lena M. Müller, * Ulrike Mayer, * Nozomi Takada, * Anne Vieten, * Niko Geldner & * Gerd Jürgens * ZMBP, Mikroskopie, Universität Tübingen, Auf der Morgenstelle 5, 72076 Tübingen, Germany * York-Dieter Stierhof & * Ulrike Mayer * Cell Biology, Department of Biology, University of Erlangen-Nürnberg, 91058 Erlangen, Germany * Benedikt Kost * Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829 Köln, Germany * Csaba Koncz * Present addresses: Institute of Plant Biology, University of Zürich, 8008 Zürich, Switzerland (L.M.M.); DBMV, Université de Lausanne, UNIL-Sorge, 1015 Lausanne, Switzerland (N.G.) * Lena M. Müller & * Niko Geldner Contributions S.R. and G.J. planned the experiments. S.R. carried out most of the experiments. L.M.M. did the analysis of pollen germination. N.T. cloned some GNL2 constructs. Y.D.S. and U.M. carried out electron microscopy and assisted in light microscopy. C.K. isolated the gnl2 mutant and A.V. did initial work on gnl2. B.K. cloned the Hypo2 construct. N.G. initiated the project. All authors analysed and discussed the data; S.R. and G.J. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gerd Jürgens Author Details * Sandra Richter Search for this author in: * NPG journals * PubMed * Google Scholar * Lena M. Müller Search for this author in: * NPG journals * PubMed * Google Scholar * York-Dieter Stierhof Search for this author in: * NPG journals * PubMed * Google Scholar * Ulrike Mayer Search for this author in: * NPG journals * PubMed * Google Scholar * Nozomi Takada Search for this author in: * NPG journals * PubMed * Google Scholar * Benedikt Kost Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Vieten Search for this author in: * NPG journals * PubMed * Google Scholar * Niko Geldner Search for this author in: * NPG journals * PubMed * Google Scholar * Csaba Koncz Search for this author in: * NPG journals * PubMed * Google Scholar * Gerd Jürgens Contact Gerd Jürgens Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Movies * Supplementary Movie 1 (1200K) Supplementary Information Excel files * Supplementary Table 1 (19K) Supplementary Information * Supplementary Table 2 (57K) Supplementary Information * Supplementary Table 3 (34K) Supplementary Information Additional data - The adaptor protein CRK is a pro-apoptotic transducer of endoplasmic reticulum stress
- Nat Cell Biol 14(1):87-92 (2012)
Nature Cell Biology | Letter The adaptor protein CRK is a pro-apoptotic transducer of endoplasmic reticulum stress * Kathryn Austgen1 * Emily T. Johnson1 * Tae-Ju Park2 * Tom Curran2 * Scott A. Oakes1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:87–92Year published:(2012)DOI:doi:10.1038/ncb2395Received16 March 2011Accepted07 November 2011Published online18 December 2011 Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Excessive demands on the protein-folding capacity of the endoplasmic reticulum (ER) cause irremediable ER stress and contribute to cell loss in a number of cell degenerative diseases, including type 2 diabetes and neurodegeneration1, 2. The signals communicating catastrophic ER damage to the mitochondrial apoptotic machinery remain poorly understood3, 4, 5, 6. We used a biochemical approach to purify a cytosolic activity induced by ER stress that causes release of cytochrome c from isolated mitochondria. We discovered that the principal component of the purified pro-apoptotic activity is the proto-oncoprotein CRK (CT10-regulated kinase), an adaptor protein with no known catalytic activity7. Crk−/− cells are strongly resistant to ER-stress-induced apoptosis. Moreover, CRK is cleaved in response to ER stress to generate an amino-terminal Mr~14K fragment with greatly enhanced cytotoxic potential. We identified a putative BH3 (BCL2 homology 3) domain within this N-terminal C! RK fragment, which sensitizes isolated mitochondria to cytochrome c release and when mutated significantly reduces the apoptotic activity of CRK in vivo. Together these results identify CRK as a pro-apoptotic protein that signals irremediable ER stress to the mitochondrial execution machinery. View full text Figures at a glance * Figure 1: Biochemical purification of ER stress apoptotic activity identifies CRK. () Induction of cytochrome c release from isolated Jurkat mitochondria by cytosolic extracts (S100) from untreated (UNT) and 24-h-BFA (2.5 μg ml−1)-treated Bax−/−Bak−/− MEFs. n=3, error bars represent s.d. () Fast protein liquid chromatography purification scheme for CcRA present in BFA Bax−/−Bak−/− S100. Active fractions from each purification step are indicated. () CcRA assay of the fractions from the final step of the purification (Mono Q ion exchange gradient). FT, flow through. n=3, error bars represent s.d. () Diagram of CRK isoforms, domains and amino-acid sequence. The asterisk indicates the stop codon. * Figure 2: Crk−/− MEFs are significantly resistant to ER-stress-induced apoptosis. (,) 18-h-BFA (2.5 μg ml−1)-treated Crk−/− MEF S100 contains significantly less CcRA when compared with 18-h-BFA (2.5 μg ml−1)-treated wild-type (WT) MEF S100. UNT, untreated. n=3, error bars represent s.d. () Crk−/− MEFs are visually resistant (phase contrast) to ER-stress-induced apoptosis (BFA 2.5 μg ml−1). Scale bar, 100 μm. () Crk−/− MEFs are strongly resistant to BFA- and tunicamycin (TUN)-induced apoptosis, but equally sensitive to staurosporine (STS), in comparison with wild-type MEFs. n=3, error bars represent s.d. NS, not significant. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 3: CRKI or CRKII restores sensitivity of Crk−/− MEFs to ER-stress-induced apoptosis. (,) Transient expression of CRKI or CRKII sensitizes Crk−/− MEFs to BFA-induced apoptosis. pmx, empty vector. n=3, error bars represent s.d. (–) Stable CRKII expression in Crk−/− MEFs rescues sensitivity to 24-h-BFA- and 18-h-tunicamycin (TUN)-induced apoptosis, but does not change sensitivity to staurosporine (STS)-induced apoptosis. UNT, untreated; NS, not significant. n=3, error bars represent s.d. Scale bar, 100 μm. (,) Stable overexpression of CRKII in wild-type (WT) MEFs further increases sensitivity to 18-h-BFA-induced apoptosis. n=3, error bars represent s.d. (,) Transient overexpression of CRKI sensitizes wild-type MEFs to 18-h-BFA (1.25 μg ml−1)-induced apoptosis. n=3, error bars represent s.d. The asterisk in indicates endogenous CRKII. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 4: CRK is proteolytically cleaved into an apoptotic signal following irremediable ER stress. () Following 24 h BFA (2.5 μg ml−1) treatment of Bax−/−Bak−/− MEFs, full-length CRKII is depleted in the cytosol and at the ER. CRKII-specific fragments (*) appear in the cytosol, ER and mitochondria (MITO). UNT, untreated. () Transiently expressed CRKI is also cleaved following 24 h BFA (2.5 μg ml−1) treatment in Crk−/− MEFs. *, CRKI-specific fragment. () Loss of full-length, endogenous CRKI and CRKII observed following 18 h BFA treatment of wild-type (WT) and Bax−/−Bak−/− MEFs. () Following ER stress, CRK is cleaved at Asp 110. Mutation of this site (D110A) in CRKII prevents cleavage following 24 h 2.5 μg ml−1 BFA treatment in stably reconstituted Crk−/−MEFs. *, nonspecific band. (,) CRKIID110A is not able to rescue Crk−/− MEF sensitivity to ER-stress-induced apoptosis induced by 24 h 2.5 μg ml−1 BFA treatment, in contrast to Crk−/− MEFs stably expressing wild-type CRKII. n=3, error bars represent s.d. (! ) Diagram of the CRK (1–110 amino acids) cleavage fragment (NF110) produced following ER stress. (,) Transient expression of NF110 induces apoptosis independently of ER stress. n=3, error bars represent s.d. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 5: CRKII contains a putative BH3 domain and triggers BAX/BAK-dependent apoptosis. (,) CRKII and empty vector (pmx) were transiently overexpressed in wild-type (WT) and Bax−/−Bak−/− MEFs using retroviral infection. At 24 h after retroviral infection, cells were treated with BFA (2.5 μg ml−1) for an additional 24 h and analysed for Annexin-V expression by flow cytometry. n=3, error bars represent s.d. (,) CRKI and empty vector (pmx) were transiently overexpressed in wild-type and Bax−/−Bak−/− MEFs. At 24 h after transfection, cells were treated for an additional 18 h with BFA (2.5 μg ml−1) and analysed for Annexin-V expression by flow cytometry. n=3, error bars represent s.d. () The sequences of the putative BH3-only domain of CRK and the 'BH3 domain' point mutation D91A are aligned against BH3 domains of several known BH3-only proteins. () HEK293 cells were transiently transfected for 24 h with Flag-tagged CRKII or untagged CRKII, and were then treated for 14 h with BFA (1.25 μg ml−1). Lysates were incub! ated with Flag-specific agarose beads. Beads were immunoblotted for endogenous BCL-XL. IP, immunoprecipitate. () Cytochrome c release from isolated Jurkat mitochondria incubated with decreasing doses of tBID and CRK BH3 domain peptide (CRKp). DMSO, dimethylsulphoxide. n=3, error bars represent s.d. (,) Stable reconstitution of CRKIID91A into Crk−/− MEFs is significantly less effective at restoring ER-stress-induced apoptosis (24 h BFA treatment) when compared with expression of wild-type CRKII. UNT, untreated. n=3, error bars represent s.d. Uncropped images of blots are shown in Supplementary Fig. S6. Author information * Author information * Supplementary information Affiliations * Department of Pathology, University of California-San Francisco, 513 Parnassus Avenue, HSW-517, Box 0511, San Francisco, California 94143-0511, USA * Kathryn Austgen, * Emily T. Johnson & * Scott A. Oakes * Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania 19104, USA * Tae-Ju Park & * Tom Curran Contributions K.A. and E.T.J. designed and carried out experiments and contributed to the manuscript. T-J.P. and T.C. contributed reagents and data interpretation. S.A.O. designed the study and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Scott A. Oakes Author Details * Kathryn Austgen Search for this author in: * NPG journals * PubMed * Google Scholar * Emily T. Johnson Search for this author in: * NPG journals * PubMed * Google Scholar * Tae-Ju Park Search for this author in: * NPG journals * PubMed * Google Scholar * Tom Curran Search for this author in: * NPG journals * PubMed * Google Scholar * Scott A. Oakes Contact Scott A. Oakes Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (2500K) Supplementary Information Additional data - Defining human ERAD networks through an integrative mapping strategy
- Nat Cell Biol 14(1):93-105 (2012)
Nature Cell Biology | Resource Defining human ERAD networks through an integrative mapping strategy * John C. Christianson1, 2, 5 * James A. Olzmann1, 5 * Thomas A. Shaler3 * Mathew E. Sowa4 * Eric J. Bennett4, 6 * Caleb M. Richter1 * Ryan E. Tyler1 * Ethan J. Greenblatt1 * J. Wade Harper4 * Ron R. Kopito1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:93–105Year published:(2012)DOI:doi:10.1038/ncb2383Received18 April 2011Accepted21 October 2011Published online27 November 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * pdf options * download pdf * view interactive pdfpowered by ReadCube * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organized into a functional network that coordinates recognition, ubiquitylation and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organization of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics and the transcriptional response to ER stress. This analysis supports an adaptive organization for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD. View full text Figures at a glance * Figure 1: Hierarchical cluster analysis of CompPASS-identified high-confidence candidate interaction proteins (HCIPs). Hierarchical clustering of HCIPs for interactions present in digitonin (left) and Triton X-100 (right). Prominent HCIP clusters identified in digitonin (1–8D) and Triton X-100 (1–3, 5 and 8T) were manually selected and are highlighted below. The colour of the square indicates the WDN-score. * Figure 2: The INfERAD. Interaction network for ERAD isolated in digitonin and Triton X-100 represented by baits (squares) and their HCIPs (circles). Unidirectional (dashed, single arrow) and reciprocal (solid black, double arrows) interactions are shown. Each bait protein is rendered in a unique colour and line colour reflects the bait protein used to identify the interaction with the HCIP. Dotted lines marked with a circle indicate interactions detected in both digitonin and Triton X-100, and long dashed lines represent those found only in Triton X-100. The inset table lists the determined constituents of the mammalian ER membrane complex (mEMC), their size (in amino acids, aa), cellular localization (IMP, integral membrane protein; Cyto., cytosolic), and corresponding yeast orthologues (SC) and ID in the Saccharomyces Genome Database (SGD). For clarity, a selection of additional digitonin HCIPs not included in the map is shown on the bottom, with a circle's colour corresponding to the bait for! which the HCIP was observed and asterisks denoting an HCIP also detected in Triton X-100. * Figure 3: shRNA-mediated refinement of ERAD E3 ligase subnetworks. (–) S-tagged ERAD baits were transiently co-expressed with the indicated shRNAs in HEK293 cells. All complexes were affinity purified (AP) in 1% digitonin and analysed by immunoblotting. S-prot, S-protein; FF, firefly. () XTP3-B–S expression, probe for Hrd1 and SEL1L simultaneously. () Co-expression of myc–UBE2J1 and XTP3-B–S, probe for myc and SEL1L; () XTP3-B–S expression, probe for FAM8A1 and SEL1L. () S–OS-9 expression, probe for Hrd1 and SEL1L simultaneously. () Incorporation of refinements (–) to the Hrd1 complex. () S–FAM8A1 expression, probe for Hrd1 and SEL1L. () Co-expression of myc–UBE2J1 and Hrd1–S, probe for myc and SEL1L. () Co-expression of myc–UBE2J1 and S–SEL1L, probe for myc and Hrd1. () AUP1–S expression, probe for Hrd1 and SEL1L. () Co-expression of myc–UBE2G2 and AUP1–S, probe for myc and Hrd1. () Refined interaction map for Hrd1 complex. () UBAC2–S expression with UBXD8 knockdown, probe for gp78. () UBAC2–S expression w! ith gp78 knockdown, probe for UBXD8. () UBXD8–S expression with gp78 knockdown, probe for UBAC2. () UBXD8–S expression with UBAC2 knockdown, probe for Derlin-2, UBAC2 and gp78. () UBXD8–S expression with Derlin-2 knockdown, probe for gp78. () Refined interaction map for the gp78 complex. Uncropped images of blots are shown in Supplementary Fig. S12. * Figure 4: Functional genomic screen to identify essential substrate-specific ERAD components. () Localization and topology of GFP reporters (TTRD18G–GFP, A1ATNHK–GFP, A1ATNHK-QQQ–GFP, GFP–GluR1, GFP–CFTRΔF508 and GFPu) and GFP. () Time course of relative mean GFP fluorescence intensity levels for each ERAD reporter cell line treated with MG132 (10 μM). Cyto., cytosolic. () Heat maps reflecting the normalized fold change in mean GFP fluorescence intensity of ERAD reporter lines transfected with wild-type or dominant-negative VCP (wild-type or H317A, top panel) and time course of treatment with kifunensine (30 μM, bottom panel). Fold change in mean GFP fluorescence intensity was normalized to the levels measured for each reporter at the 3 h time point of MG132 treatment, and thus a degradation score of 3 is equivalent to the impairment induced by 3 h MG132 treatment. () Target composition of the shRNA library. () Overview of the functional genomic screen. () Hierarchically clustered heat map of the normalized fold change in mean GFP fluorescence in! tensity of ERAD reporter lines in response to shRNA-mediated knockdown of ERAD components. The normalization and colour scale are the same as in . FF, firefly. () Functional data from the heat map shown in were mapped onto the refined Hrd1 physical interaction network (Fig. 3k) to provide an integrated snapshot of substrate-specific functional requirements for Hrd1 network components. Uncropped images of blots are shown in Supplementary Fig. S12. * Figure 5: Coordinated ER stress response of ERAD genes. qRT–PCR results for validated and suspected ERAD components following treatment of HEK293 cells with tunicamycin (10 μg ml−1, 6 h). Data are presented as fold induction (log2) normalized to β-actin. Tunicamycin-induced expression changes in ERAD genes plotted as groups according to: () fold induction of gene expression represented by functional category, and () fold induction of gene expression from mapped onto the ERAD interactome from Fig. 2. Additional genes of interest are presented alongside the induction map. * Figure 6: Characterization of the Hrd1-binding partner FAM8A1. () Domain structure and interaction network of FAM8A1. aa, amino acids. () Immunoprecipitation (IP) with anti-FAM8A1 from HEK293 digitonin-soluble lysates was analysed by immunoblotting with the indicated antibodies. () Consensus TOPCONS prediction of FAM8A1 membrane orientation (http://topcons.cbr.su.se). The reliability index indicates the likelihood for consensus prediction at each position using a sliding 21 amino-acid window. Cyto., cytosolic; TMD, transmembrane domain. () HEK293 membrane fractions incubated with 1 M NaCl, 0.1 M Na2CO3 at pH 12 or 1% SDS. Following 100,000g centrifugation, equal volumes of soluble (S) and pellet (P) fractions were analysed by western blotting with anti-FAM8A1. () HeLa cells expressing S–FAM8A1 or Hrd1–S were permeabilized with digitonin or Triton X-100 to allow antibody access to cytosolic epitopes or cytosolic and luminal epitopes, respectively, immunostained and analysed by fluorescence microscopy. Scale bar, 10 μm. () Hr! d1–S-expressing HEK293 cell lysates separated on a continuous 10–40% sucrose gradient. S-tagged Hrd1 protein complexes were affinity purified from each 1 ml fraction (fractions 1–12) or from 150 mg whole-cell lysate (10% AP), and analysed by western blotting for Hrd1 (S-tag), SEL1L and FAM8A1. () Heat map representing the normalized change in mean GFP fluorescence intensity (20,000 cells, n=3) of the indicated ERAD reporter cell lines following transfection with the indicated Hrd1, SEL1L and FAM8A1 plasmids. DFP indicates dead fluorescent protein, a non-fluorescent GFP variant. Data are represented as a normalized heat map as in Fig. 4c. Uncropped images of blots are shown in Supplementary Fig. S12. * Figure 7: Characterization of UBAC2, a ubiquitin-binding ERAD component. () Predicted domain structure and interaction network of UBAC2. aa, amino acids. () Immunoprecipitation (IP) with anti-UBXD8 from HEK293 digitonin-soluble lysates was analysed by western blotting with the indicated antibodies. () Analysis of multiple UBAC2-targeting shRNAs on the Hrd1 substrate TTRD18G–GFP by flow cytometry. FF, firefly. () Sequence alignment of the predicted UBA domains from UBAC2 (304–344) and UBXD8 (8–53) with characterized human and yeast UBA domains. () HeLa cells expressing C-terminally S-tagged UBAC2 or gp78 were permeabilized, immunostained and analysed by fluorescence microscopy as in Fig. 6e. Scale bar, 10 μm. () Recombinantly expressed UBA domains of hPlic2, UBXD8 and UBAC2 were coupled to Affi-Gel and incubated with HEK293 cell lysates (±10 μM MG132, 6 h). Samples were separated by SDS–PAGE, and ubiquitin binding was determined by immunoblotting with anti-ubiquitin. Uncropped images of blots are shown in Supplementary Fig. S12. * Figure 8: Functional integration of mammalian ERAD networks. The schematic model of the ERAD protein interaction network is topologically organized with respect to the ER membrane and arranged as an array of six colour-coded functional modules. Individual components from this study (baits or HCIPs) are indicated as nodes with reported components (black) and previously unknown components (red). Similarly, reported interactions confirmed in this study (black lines) and previously unknown interactions (red lines) are shown. Symbols for protein–protein interactions, UPR induction and functional requirements are indicated in the legend. Inter-module interactions represented terminate either at the specific node within a module that establishes the link with the module periphery or at the module itself (where there are interactions with multiple components and that module is a single complex; for example, the mEMC or proteasome). Asterisks indicate components that were identified by proteomics, but exhibited a subthreshold CompPASS score ! (WDN-score<1.0). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * John C. Christianson & * James A. Olzmann Affiliations * Department of Biology & Bio-X Program, Stanford University, Lorry Lokey Building, 337 Campus Drive, Stanford, California 94305, USA * John C. Christianson, * James A. Olzmann, * Caleb M. Richter, * Ryan E. Tyler, * Ethan J. Greenblatt & * Ron R. Kopito * Ludwig Institute for Cancer Research, University of Oxford, ORCRB, Headington, Oxford OX3 7DQ, UK * John C. Christianson * SRI International, Menlo Park, California 94025, USA * Thomas A. Shaler * Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA * Mathew E. Sowa, * Eric J. Bennett & * J. Wade Harper * Present address: Division of Biological Sciences, UC San Diego, La Jolla, California 92093, USA * Eric J. Bennett Contributions The manuscript was written collectively by J.C.C., J.A.O. and R.R.K. Experiments and data analysis were carried out by J.A.O. and J.C.C. with assistance from C.M.R. R.E.T. and E.J.G. LC–MS/MS analysis was carried out by T.A.S. CompPASS analysis was carried out by M.E.S. and E.J.B with support from J.W.H. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ron R. Kopito Author Details * John C. Christianson Search for this author in: * NPG journals * PubMed * Google Scholar * James A. Olzmann Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas A. Shaler Search for this author in: * NPG journals * PubMed * Google Scholar * Mathew E. Sowa Search for this author in: * NPG journals * PubMed * Google Scholar * Eric J. Bennett Search for this author in: * NPG journals * PubMed * Google Scholar * Caleb M. Richter Search for this author in: * NPG journals * PubMed * Google Scholar * Ryan E. Tyler Search for this author in: * NPG journals * PubMed * Google Scholar * Ethan J. Greenblatt Search for this author in: * NPG journals * PubMed * Google Scholar * J. Wade Harper Search for this author in: * NPG journals * PubMed * Google Scholar * Ron R. Kopito Contact Ron R. Kopito Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (5400K) Supplementary Information Excel files * Supplementary Tables 1–10 (3200K) Supplementary Information Additional data - Single-molecule transcript counting of stem-cell markers in the mouse intestine
- Nat Cell Biol 14(1):106-114 (2012)
Nature Cell Biology | Technical Report Single-molecule transcript counting of stem-cell markers in the mouse intestine * Shalev Itzkovitz1, 2 * Anna Lyubimova1, 2, 3 * Irene C. Blat2, 4 * Mindy Maynard4 * Johan van Es3 * Jacqueline Lees2, 4 * Tyler Jacks2, 4, 5 * Hans Clevers3 * Alexander van Oudenaarden1, 2, 3, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 14,Pages:106–114Year published:(2012)DOI:doi:10.1038/ncb2384Received19 May 2011Accepted21 October 2011Published online27 November 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Determining the molecular identities of adult stem cells requires technologies for sensitive transcript detection in tissues. In mouse intestinal crypts, lineage-tracing studies indicated that different genes uniquely mark spatially distinct stem-cell populations, residing either at crypt bases or at position +4, but a detailed analysis of their spatial co-expression has not been feasible. Here we apply three-colour single-molecule fluorescent in situ hybridization to study a comprehensive panel of intestinal stem-cell markers during homeostasis, ageing and regeneration. We find that the expression of all markers overlaps at crypt-base cells. This co-expression includes Lgr5, Bmi1 and mTert, genes previously suggested to mark distinct stem cells. Strikingly, Dcamkl1 tuft cells, distributed throughout the crypt axis, co-express Lgr5 and other stem-cell markers that are otherwise confined to crypt bases. We also detect significant changes in the expression of some of the marke! rs following irradiation, indicating their potential role in the regeneration process. Our approach can enable the sensitive detection of putative stem cells in other tissues and in tumours, guiding complementary functional studies to evaluate their stem-cell properties. View full text Figures at a glance * Figure 1: Three-colour single-molecule FISH of intestinal stem-cell markers. () Small-intestinal fixed tissue sections were simultaneously hybridized with three differentially labelled probe libraries (here Lgr5–TMR (tetramethylrhodamine, green), Bmi1–cy5 (red) and Prominin-1–Alexa594 (blue)). Single transcripts appear as diffraction-limited spots under a fluorescent microscope. Fluorescein isothiocyanate (FITC)–E-cadherin antibody labels cell membranes. Magnification of a representative area highlighted in red is shown below. Images are maximal projections of stacks of 20 optical sections spaced 0.3 μm apart. () Segmented crypt with transcripts for Lgr5 (green triangles), Bmi1 (red diamonds) and Prominin-1 (blue circles). Dots and cell borders are based on ten optical sections from . () Area highlighted in showing the simultaneous detection of transcripts for Lgr5 (green), Bmi1 (red) and Prominin-1 (blue). Dashed outlines denote cell borders. Scale bar, 5 μm. * Figure 2: Single-molecule FISH correlates with reporter expression in transgenic mice but provides a much broader sampling. () Expression analysis in the Lgr5–eGFP transgenic mice. Shown are two crypts, one positive for the transgene expression (right) and one negative (left). The grey scale reflects the GFP measurements. Green dots are automatically detected Lgr5 endogenous transcripts, red dots are eGFP transcripts and dashed lines mark cell borders, on the basis of immunofluorescence with FITC–E-cadherin. Arrows point to cells with high GFP fluorescence. Unlike the transgene, which was expressed once every ten crypts, the endogenous transcripts were detected in each and every crypt. () Endogenous Lgr5 transcript levels are highly correlated with eGFP transcript levels in the crypts in which the transgene is active (Spearman correlation R=0.68,P<10−68). Analysis on the basis of simultaneous single-molecule FISH with probe libraries for Lgr5 and eGFP. (,) Hybridization with single-molecule FISH libraries yields highly localized and specific expression patterns. () Intestinal crypt hybridiz! ed with probes for Paneth-cell marker Lysozyme (red), goblet-cell marker Gob5 (green) and stem-cell marker Lgr5 (yellow). () Intestinal crypt hybridized with the proliferation marker Ki67 (green) and with the enterocyte marker Creb3l3 (red). The image was filtered with a Laplacian of Gaussian filter (Methods). The sharp decline in expression at the crypt–villus border demonstrates that rates of transcript degradation are faster than cell migration rates in intestinal crypts. Dashed lines mark cell borders. Scale bars, 5 μm. * Figure 3: Spatial expression profiles of stem-cell markers are broad and overlap at CBC cells. () Spatial expression profiles of stem-cell markers are invariant between crypts and to ageing. Rows are different crypts; columns are crypt positions, position 0 is the crypt apex. All crypts above the white horizontal lines are from a 4-month-old mouse (marked with grey vertical bars); all crypts below the white lines are from an 11-month-old mouse (marked with black vertical bars). () Bmi1 and Lgr5 are extensively co-expressed in a non-correlated manner (R=−0.025,P=0.9). Dots represent pooled single cells from crypts of a wild-type 4-month-old mouse. Coordinates are the transcript concentrations divided by the average concentration within the crypt from which the cell was sampled (cells with no transcripts were assigned the lowest non-zero concentration detected). Dot colours correspond to position along the crypt axis. 76% of Lgr5-positive cells contain Bmi1 transcripts (1,073/1,417) whereas 48% of Bmi1-positive cells contain Lgr5 transcripts (1,073/2,221). () mTert an! d Lgr5 are co-expressed in CBC cells (R=0.13,P=0.002). (,) Lgr5 (green dots) and Bmi1 (red dots, ) as well as mTert (red dots, ) are co-expressed in crypt-base cells. Dashed lines mark cell borders. Images are maximal projections of 15 optical sections spaced 0.3 μm apart, filtered with a Laplacian of Gaussian filter (Methods). Scale bars, 5 μm. * Figure 4: Single-cell correlations of stem-cell markers are validated with mutants for key regulator genes. () Musashi-1 (Msi1) and Ascl2 are highly correlated at the single-cell level (Spearman correlation R=0.7,P<10−16). () Bmi1 and Ascl2 are not significantly correlated (R=−0.05,P=0.74). Dots in , represent pooled single cells from crypts of a 4-month-old mouse. Coordinates are the transcript concentrations divided by the average concentration within the crypt from which the cell was sampled (cells with no transcripts were assigned the lowest non-zero concentration detected). Dot colours correspond to position along the crypt axis. () Bmi1 transcripts are detected in a wild-type mouse (left) but not in a Bmi1 homozygous knockout mouse (right). () Ascl2 transcripts are significantly reduced in an Ah-Cre/Ascl2floxed/floxed mouse 5 days after βNF induction (right) when compared with non-induced controls (left). Scale bars, 5 μm. () Deletion of Bmi1 significantly reduces the expression of Cd44 and Bmi1. Shown are the distributions of the mean transcript concentrations per c! rypt cell for the wild-type (WT, Bmi1+/+) and the mutant (KO, Bmi1−/−) mice, where horizontal red lines are median concentrations and boxes delimit the 25–75 percentiles. *P<0.02, **P<0.001. () As in , distributions of mean transcript concentrations per crypt cell for non-induced (WT, Ascl2+/+) and induced (KO, Ascl2−/−) mice. () Reduction in expression of stem-cell markers in mice mutant for Bmi1 and Ascl2 is correlated with the Spearman correlation coefficients of these markers with either Bmi1 or Ascl2 respectively in the wild-type (R=0.76,P=0.0045). Expression reduction for each gene is the difference in median transcript concentration between the wild type and the mutant, divided by the wild-type median levels. () Correlation map of stem-cell markers with Bmi1 and Lgr5. Axes are the Spearman correlations between single-cell transcript concentration in either Lgr5 (x axis) or Bmi1 (y axis). Red denotes significant correlation with Lgr5, blue denotes significan! t correlation with Bmi1 and green with both. () Pairwise corre! lations are highly reproducible between a 4-month-old mouse and an 11-month-old mouse (R=0.88). * Figure 5: Dcamkl1 marks tuft cells occurring throughout the crypt axis that significantly co-express stem-cell markers otherwise confined to crypt bottoms. () Example of significant co-expression of Lgr5 (green dots) in a cell with high Dcamkl1 transcript levels (red dots). () Example of a Dcamkl1 cell with no Lgr5 expression. Images in , are maximal projections of 15 optical sections spaced 0.3 μm apart, filtered with a Laplacian of Gaussian filter (Methods). Blue dots are Cox1 transcripts, dashed lines mark cell borders and arrows mark the tuft cells. Scale bars, 5 μm. () Expression signature for Dcamkl1 cells. Shown are the median ratios of transcripts for different genes between Dcamkl1high cells (cells with more than five transcripts) and the average levels in their immediate two neighbouring cells (above and below). Red bars are ratios that are significant relative to permuted crypts (see Methods), using a false discovery rate of 10%. () Passive-migration model for the elevated transcript levels of Lgr5 in Dcamkl1 cells. Green dots represent transcripts of Lgr5 (or other stem-cell markers co-expressed in Dcamkl1 cel! ls) in the progenies of two different cells migrating away from the stem-cell zone at the crypt bottoms—a Dcamkl1-positive cell (red) and Dcamkl1-negative cell (blue). t1 and t2 indicate successive times. If Dcamkl1-positive cells do not divide and migrate more rapidly than other cells, the spatial decay rate of the stem-cell marker transcripts such as Lgr5 will be lower. () Lgr5 transcripts in Dcamkl1high cells (black dots) decay more slowly with crypt position than Lgr5 transcripts in Dcamkl1-negative cells (grey dots). Transcripts of Olfm4 in Dcamkl1high cells (black) exhibit the same decay rate as in Dcamkl1-negative cells (grey). Lines are exponential fits. () Transcript decay of Lgr5 and Musashi-1 is less than half as fast in Dcamkl1high cells when compared with Dcamkl1-negative cells, but comparable for Cd44 and Olfm4. * Figure 6: 12 Gy whole-body irradiation results in significant changes in the levels and range of stem-cell markers. (,) Average spatial expression profiles for Cd44 () and Olfm4 () in a non-irradiated control mouse (black) and in mice irradiated with 12 Gy and killed after 48 h (red) and 7 days (blue). () Transcript levels of Musashi-1 (green dots) and Ascl2 (red dots) are significantly increased 48 h after whole-body 12 Gy irradiation. Images are maximal projections of six optical sections spaced 0.3 μm apart. Scale bar, 10 μm. () The medians of the spatial expression profiles significantly increase for most stem-cell markers following gamma irradiation. Shown are the distributions among different crypts of the median cell positions of the spatial expression profiles. Horizontal red lines are the medians of the distributions and boxes delimit the 25–75 percentiles. *P<0.03,**P<0.001. () Mean transcript concentration per crypt cell significantly increases for some stem-cell markers (Musashi-1, Ascl2, Cd44, Olfm4) but not for others (Lgr5, Bmi1). Horizontal red lines are th! e medians of the distributions and boxes delimit the 25–75 percentiles. *P<0.01,**P<0.001. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Shalev Itzkovitz, * Anna Lyubimova & * Alexander van Oudenaarden * Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Shalev Itzkovitz, * Anna Lyubimova, * Irene C. Blat, * Jacqueline Lees, * Tyler Jacks & * Alexander van Oudenaarden * Hubrecht Institute–KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, Netherlands * Anna Lyubimova, * Johan van Es, * Hans Clevers & * Alexander van Oudenaarden * Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Irene C. Blat, * Mindy Maynard, * Jacqueline Lees, * Tyler Jacks & * Alexander van Oudenaarden * Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Tyler Jacks Contributions S.I. and A.v.O. conceived the project. S.I., I.C.B. and A.L. carried out most of the experiments. S.I. analysed the data. M.M., J.L., J.v.E., T.J. and H.C. provided mice and assisted with experiments. S.I., H.C. and A.v.O. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alexander van Oudenaarden Author Details * Shalev Itzkovitz Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Lyubimova Search for this author in: * NPG journals * PubMed * Google Scholar * Irene C. 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