Latest Articles Include:
- Change the Equation: Improving science and mathematics education in the US
- Nat Cell Biol 13(8):875 (2011)
Nature Cell Biology | Editorial Change the Equation: Improving science and mathematics education in the US Journal name:Nature Cell BiologyVolume: 13,Page:875Year published:(2011)DOI:doi:10.1038/ncb2318Published online01 August 2011 Change the Equation, a non-profit group of more than 100 corporate organizations, is committed to improving the state of mathematics and science education in the US. Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In 2009, the PISA (Programme for International Student Assessment) test, a standardized assessment developed and administered by the OECD (Organization for Economic Cooperation and Development) to 15-year-olds in participating countries, revealed disappointing academic performances by US students. The results showed that US students lagged significantly behind their peers from participating countries in mathematics and science, with mathematics scores dropping to below average. Students in Shanghai, on the other hand, captured first place in all three categories tested — reading, mathematics and science. Other reports have also noted that an alarming proportion of US students seem to be inadequately prepared for college-level mathematics and science courses. Several indicators suggest that the US is falling behind other developed nations in standards of school education, and a failure to reverse this decline poses a threat to the long-term economic future of the country. Spurred by the current crisis in education, President Obama launched the 'Educate to Innovate' campaign in 2009. This ambitious effort involving partnerships between private companies and non-profit organizations, including foundations and science and engineering societies, has the long-term goal of boosting the performance of US students in science, technology, engineering and mathematics (STEM). In 2010, the corporate community responded to Obama's call for raising STEM literacy by forming Change the Equation (CTEq), a non-profit coalition whose founders include the astronaut Sally Ride and the CEOs of many large corporations. The main aim of CTEq is to enhance STEM education for children, particularly girls and under-represented minorities, and to serve as a long-standing advocate for improved STEM literacy in the country. With this greater purpose in mind, CTEq has three key targets — to improve STEM teaching, to inspire enthusiasm in students for careers in STEM fields and to foster a broad commitment to science education from all sectors of society, including government, educators and business. As an alliance of over 100 companies representing diverse industries such as technology and telecommunications, biotechnology and pharmaceuticals, financial services and consultancy, and entertainment and publishing, CTEq has a combined purse of more than $500 million a! year of financial and in-kind support devoted to this effort. In their first year, a key undertaking has been the development of metrics for evaluating the status of STEM education throughout the US; this 'scorecard' will serve as a basis for assessing future progress in STEM education. In April this year, CTEq released the 'Vital Signs' report on the condition of STEM education in each of the 50 states and in the District of Columbia. The results were compiled from publicly available data, including data from the National Assessment of Education Progress, and highlight several concerns that are widespread across the US. For example, the achievement gap between White students and their Black and Hispanic peers in mathematics and science is significant. There is also an urgent need to provide better training for elementary- and secondary-school mathematics teachers, and more challenging and engaging education in mathematics and science. The results also reveal that although some states have maintained high standards for students in math! ematics and science, many others have dropped the bar, creating a false perception of student ability, and the report makes specific recommendations for improvement. In 2012, CTEq plans to issue a more detailed set of state-wide reports that will represent the most in-depth assessment of the status of STEM literacy to date and which will have the potential to become a crucial tool in evaluating and contrasting progress on a state-by-state basis. Owing to the diverse companies in its network, many of which dominate their individual industries, CTEq has considerable resources, including financial and expert knowledge, within its reach. But the effectiveness of disparate engagement by varied companies is debatable. As part of their efforts to maximize fruitful corporate engagement in improving STEM education, CTEq has taken the important step of developing 'Design Principles for Effective STEM Philanthropy'. These principles serve not only as mechanism to broadly align the efforts of individual members but also to guide effective philanthropic engagement. Nature Publishing Group's mission and strengths in science communication converge with the overall mission of CTEq. As a member of CTEq, Nature Publishing Group has developed the 'Bridge to Science' initiative, which includes six programmes supported by different divisions of Nature Publishing Group. Three programmes from Scientific American — Bring Science Home, 1000 Scientists in 1000 Days, and Citizen Science — are designed to inspire enthusiasm for science amongst parents and children, connect working scientists to teachers and engage the amateur scientist. Bring Science Home, for example, provides a series of quick, easy and fun science projects (making paper, talking through a string phone) to expose 6–12-year-olds to scientific concepts, whereas Citizen Science features diverse projects ranging from predictions of protein structure to understanding declining bee populations, and aims to draw in science hobbyists. The programmes supported by Nature Education, a d! ivision of Nature Publishing Group committed to facilitating global access to science education through innovative technology-driven tools, include Scitable, an open online learning and teaching portal aimed at undergraduates students and faculty in the life sciences, and Bench to Blackboard, a collaboration between Scitable and the Baylor College of Medicine to provide free, online course modules for middle- and high-school biology teachers. Finally, in a joint initiative with Cisco Systems, Nature Publishing Group will be developing a mechanism to assess the returns on national investment in STEM education by OECD countries. The endeavours of CTEq follow in a long tradition of corporate philanthropy in the US and it should be commended for its commitment to tackling the pressing challenges in improving STEM literacy. But philanthropy alone will not suffice. Sustained long-term engagement from both the state and federal governments will be essential in the effort to address the crisis in US education. Additional data - Hunting the elusive oncogene: a stroke of good luck
- Nat Cell Biol 13(8):876 (2011)
Article preview View full access options Nature Cell Biology | Turning Points Hunting the elusive oncogene: a stroke of good luck * Robert A. Weinberg1Journal name:Nature Cell BiologyVolume: 13,Page:876Year published:(2011)DOI:doi:10.1038/ncb2302Published online01 August 2011 Through a succession of happenstances, in 1972 I ended up in the MIT Biology Department faculty, an institution from which I had previously received both my undergraduate and doctoral degrees. I was soon to become an MIT lifer — a proverbial stick-in-the-mud. But this has not involved an enormous sacrifice on my part. MIT was, and is, an exciting place to do science. My return to MIT occurred two years after David Baltimore and Howard Temin had discovered reverse transcriptase, and so I was drawn inexorably into retrovirology. I undertook, in effect, a third postdoctoral stay with Baltimore in the then recently formed MIT Center for Cancer Research, and after two years became a faculty member. In the beginning, my interests were focused on the molecular biology of retrovirus replication, specifically what happens inside an infected cell soon after infection. By 1975, my group discovered that we could trigger a complete viral replication cycle by transfecting murine leukaemia proviral DNA prepared from recently infected cells into NIH3T3 mouse cells. Soon thereafter, this transfection technique, which we had adopted and fine-tuned several years earlier, offered us a unique opportunity to address another question: could we transfect the genomic DNA of cells that had acquired Harvey sarcoma virus (HaSV) proviral DNA in their genome and observe the subsequent transformation of the recipient cells to a neoplastic state? The read-out was, in this case, the formation of foci of transformed cells arising amid the monolayer of NIH3T3 cells that had been exposed to HaSV DNA. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Robert A. Weinberg is at the MIT Ludwig Center for Molecular Oncology, MIT, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Robert A. Weinberg Author Details * Robert A. Weinberg Contact Robert A. Weinberg Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal
- Nat Cell Biol 13(8):877-883 (2011)
Nature Cell Biology | Review The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal * Bin Zhao1, 2 * Karen Tumaneng2 * Kun-Liang Guan2 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:877–883Year published:(2011)DOI:doi:10.1038/ncb2303Published online01 August 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Precise control of organ size is crucial during animal development and regeneration. In Drosophila and mammals, studies over the past decade have uncovered a critical role for the Hippo tumour-suppressor pathway in the regulation of organ size. Dysregulation of this pathway leads to massive overgrowth of tissue. The Hippo signalling pathway is highly conserved and limits organ size by phosphorylating and inhibiting the transcription co-activators YAP and TAZ in mammals and Yki in Drosophila, key regulators of proliferation and apoptosis. The Hippo pathway also has a critical role in the self-renewal and expansion of stem cells and tissue-specific progenitor cells, and has important functions in tissue regeneration. Emerging evidence shows that the Hippo pathway is regulated by cell polarity, cell adhesion and cell junction proteins. In this review we summarize current understanding of the composition and regulation of the Hippo pathway, and discuss how cell polarity and cell! adhesion proteins inform the role of this pathway in organ size control and regeneration. View full text Figures at a glance * Figure 1: The Hippo pathway in Drosophila and mammals. Corresponding proteins in Drosophila () and mammals () are indicated by matching colours. Arrowed or blunted ends indicate activation or inhibition, respectively. Dashed lines indicate unknown mechanisms. * Figure 2: Mechanisms of YAP/TAZ/Yki inhibition by the Hippo pathway. () Phosphorylation-dependent cytoplasmic retention. Phosphorylation of YAP on Ser 127 by Lats1/2 induces 14-3-3 binding and cytoplasmic retention of YAP. The mechanism is conserved in TAZ and Yki. () Phosphorylation-independent cytoplasmic retention. Through WW domain–PPXY motif interactions, Yki binds to Mop, Ex, Hpo and Wts, and YAP/TAZ binds to AMOT family proteins. These interactions physically sequester Yki and YAP/TAZ in the cytoplasm. () Phosphorylation-induced ubiquitylation and degradation. Phosphorylation of YAP on Ser 381 by Lats1/2 primes further phosphorylation of YAP by CK1δ/ε, which induces interaction with SCFβ–TRCP and finally leads to YAP ubiquitylation and degradation. The mechanism is conserved in TAZ. * Figure 3: Mechanisms of the Hippo pathway in regulation of organ size and regeneration. Hexagons denote differentiated cells and circles denote stem/progenitor cells. Blue colour indicates wild-type and yellow colour indicates Hippo-pathway mutant cells. () Hippo pathway inactivation leads to stem/progenitor cell expansion in both cell-autonomous and non-autonomous manners. () Hippo pathway inactivation leads to cell cycle exit defects in some cellular contexts. () Hippo pathway mutants promote proliferation and decrease apoptosis in non-stem/progenitor cells. () Imbalance of Hippo pathway activity in neighbouring cells induces cell competition. Author information * Abstract * Author information Affiliations * Bin Zhao is at the Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China * Bin Zhao, Karen Tumaneng and Kun-Liang Guan are in the Department of Pharmacology and Moores Cancer Center, University of California at San Diego, La Jolla, California 92093-0815, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kun-Liang Guan Author Details * Bin Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Karen Tumaneng Search for this author in: * NPG journals * PubMed * Google Scholar * Kun-Liang Guan Contact Kun-Liang Guan Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The SNXy flavours of endosomal sorting
- Nat Cell Biol 13(8):884-886 (2011)
Article preview View full access options Nature Cell Biology | News and Views The SNXy flavours of endosomal sorting * Ludger Johannes1 * Christian Wunder1 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:884–886Year published:(2011)DOI:doi:10.1038/ncb2300Published online03 July 2011 The retromer complex coordinates retrograde transport of cargo proteins between endosomes and the trans-Golgi network. The sorting nexin SNX3 is required for the retrograde trafficking of Wntless, but not of other retrograde cargo proteins, revealing that the cargo specificity of retromer is provided by the sorting nexins. Figures at a glance * Figure 1: The functional cycle of Wntless depends on retromer. Wnt is lipid modified in the ER and transported to the Golgi, where it binds to Wntless. The Wnt–Wntless complex is transported to the plasma membrane and Wnt is released. Wntless is then internalized and transported back to the trans-Golgi network in a retromer-dependent process. Some Wntless molecules escape recycling and are degraded in late endosomes/lysosomes. * Figure 2: Sorting nexins mediate cargo specificity. Cargo is internalized from the plasma membrane by endocytosis and is delivered to early endosomes, which is depicted to contain early (left) and maturing (right) vacuolar domains. SNX proteins and the retromer VPS subcomplex orchestrate cargo trafficking from early endosomes to different destinations: the SNX-BAR proteins SNX1, 2, 5 and 6 for mannose-6-phosphate receptor (M6R) via tubular structures, SNX3 for Wntless via vesicle budding and SNX27 for β2 adrenergic receptor (β2 AR) on recycling tubules. 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 * Ludger Johannes and Christian Wunder are in the Traffic, Signaling and Delivery Laboratory, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France and the CNRS UMR144, France Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Ludger Johannes or * Christian Wunder Author Details * Ludger Johannes Contact Ludger Johannes Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Wunder Contact Christian Wunder Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - esBAF safeguards Stat3 binding to maintain pluripotency
- Nat Cell Biol 13(8):886-888 (2011)
Article preview View full access options Nature Cell Biology | News and Views esBAF safeguards Stat3 binding to maintain pluripotency * Noa Novershtern1 * Jacob H. Hanna1 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:886–888Year published:(2011)DOI:doi:10.1038/ncb2311Published online01 August 2011 How the unique chromatin configuration of embryonic stem cells (ESCs) integrates inputs from exogenous stimuli to maintain pluripotency remains largely unknown. The ESC-specific ATP-dependent chromatin-remodelling (esBAF) complex maintains the accessibility of the target sites of Stat3, a leukaemia inhibitory factor (LIF) signalling effector, by preventing repressive localized polycomb-mediated trimethylation of Lys 27 of histone 3 (H3K27me3). 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 * Noa Novershtern and Jacob H. Hanna are at The Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Noa Novershtern or * Jacob H. Hanna Author Details * Noa Novershtern Contact Noa Novershtern Search for this author in: * NPG journals * PubMed * Google Scholar * Jacob H. Hanna Contact Jacob H. Hanna Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - FBXW5 controls centrosome number
- Nat Cell Biol 13(8):888-890 (2011)
Article preview View full access options Nature Cell Biology | News and Views FBXW5 controls centrosome number * Julia Pagan1 * Michele Pagano1, 2 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:888–890Year published:(2011)DOI:doi:10.1038/ncb2312Published online01 August 2011 Regulatory mechanisms to prevent centriole overduplication during the cell cycle are not completely understood. In this issue, FBXW5 is shown to control the degradation of the centriole assembly factor HsSAS-6. Moreover, the study proposes that FBXW5 is a substrate of both PLK4 and APC/C, two established regulators of centriole duplication. 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 * Julia Pagan and Michele Pagano are in the Department of Pathology, NYU Cancer Institute, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA * Michele Pagano is at the Howard Hughes Medical Institute, 4000 Jones Bridge Road Chevy Chase, Maryland 20815-6789, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michele Pagano Author Details * Julia Pagan Search for this author in: * NPG journals * PubMed * Google Scholar * Michele Pagano Contact Michele Pagano Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Research highlights
- Nat Cell Biol 13(8):891 (2011)
Article preview View full access options Nature Cell Biology | Research Highlights Research highlights Journal name:Nature Cell BiologyVolume: 13,Page:891Year published:(2011)DOI:doi:10.1038/ncb2317Published online01 August 2011 Extracellular forces lead to increased intracellular tension and matrix remodelling to re-establish force balance. These mechanoreciprocal responses involve the Rho–ROCK (Rho-associated protein kinase) signalling cascade, which regulates actomyosin contractility. Samuel et al. now show how ROCK2 regulates these processes in skin homeostasis and tumorigenesis (Cancer Cell14, 776–791; 2011). Using transgenic mice expressing conditionally active ROCK2, the authors discovered that ROCK2 activation in the skin increases tissue stiffness through collagen deposition and results in epidermal proliferation and skin thickening. Further employment of mouse genetics established that the pro-proliferative effects of ROCK2 depend on the activation of the known mechanoresponsive factor β-catenin and its downstream transcriptional programme. These were linked to a known collagen-initiated signalling cascade downstream of FAK (focal adhesion kinase), involving inactivation of the β-catenin inhibitor GSK3β (glycogen synthase kinase 3β). By inhibiting myosin and ROCK-controlled regulators of actin dynamics, the authors further demonstrated that the cumulative effects of ROCK2 activity on tissue stiffness and β-catenin depend on actomyosin contractility and cellular tension. Finally, using a chemical carcinogenesis protocol that models human squamous cell carcinoma in the mo! use, the authors showed that ROCK2 activation increases papilloma incidence and progression to carcinoma, which is reverted by ROCK inhibition. Thus, ROCK2-induced cellular tension drives tumour growth through its combined effects on tissue stiffness and proliferation. AIZ Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Mitotic internalization of planar cell polarity proteins preserves tissue polarity
- Nat Cell Biol 13(8):893-902 (2011)
Nature Cell Biology | Article Mitotic internalization of planar cell polarity proteins preserves tissue polarity * Danelle Devenport1 * Daniel Oristian1 * Evan Heller1 * Elaine Fuchs1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:893–902Year published:(2011)DOI:doi:10.1038/ncb2284Received11 April 2011Accepted27 May 2011Published online10 July 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 Planar cell polarity (PCP) is the collective polarization of cells along the epithelial plane, a process best understood in the terminally differentiated Drosophila wing. Proliferative tissues such as mammalian skin also show PCP, but the mechanisms that preserve tissue polarity during proliferation are not understood. During mitosis, asymmetrically distributed PCP components risk mislocalization or unequal inheritance, which could have profound consequences for the long-range propagation of polarity. Here, we show that when mouse epidermal basal progenitors divide PCP components are selectively internalized into endosomes, which are inherited equally by daughter cells. Following mitosis, PCP proteins are recycled to the cell surface, where asymmetry is re-established by a process reliant on neighbouring PCP. A cytoplasmic dileucine motif governs mitotic internalization of atypical cadherin Celsr1, which recruits Vang2 and Fzd6 to endosomes. Moreover, embryos transgenic for ! a Celsr1 that cannot mitotically internalize exhibit perturbed hair-follicle angling, a hallmark of defective PCP. This underscores the physiological relevance and importance of this mechanism for regulating polarity during cell division. View full text Figures at a glance * Figure 1: PCP components are selectively internalized in basal epidermal cells undergoing mitosis. Images show planar confocal microscopy sections through the basal layer of E15.5 mouse backskins. Anterior is to the left. () Transgenic embryos mosaically expressing K14–Celsr1–GFP (green). Cell contacts are labelled with E-Cad antibodies (red), and chromatin is labelled with 4,6-diamidino-2-phenylindole (DAPI; blue). Asterisks mark Celsr1–GFP-expressing cells, where Celsr1 accumulates on both the anterior and posterior sides of the plasma membrane. Quantification of fluorescence intensity on anterior and posterior clone borders is on the right (n=65 cells in 26 clones, t-test P=0.8029). () Transgenic embryos mosaically expressing K14–GFP–Vangl2 (green). Backskins are stained with E-Cad antibodies (red) and DAPI (blue). Asterisks indicate cells expressing Vangl2–GFP, which, in contrast to Celsr1, accumulates preferentially on the anterior side of the membrane. Quantification of fluorescence intensity on anterior and posterior clone borders is on the right (n=93c! ells in 22 clones, Mann–Whitney test P<0.0001). (–) Examples of Celsr1 distribution in mitotic basal cells at prophase (), metaphase (), telophase () and cytokinesis (). E15.5 whole-mount backskins were stained with Celsr1 (green) and E-Cad (red) antibodies. DAPI highlights condensed chromosomes of mitotic cells (blue). Note that E-Cad retains its plasma membrane localization during mitosis. () Quantifications of the data in –: the mean percentage of Celsr1 co-localized with E-Cad at the plasma membrane per cell. n=20 cells/cell cycle stage. (,) Fzd6 and Vangl2 (green) internalize and co-localize with Celsr1 (red, ) puncta but not E-Cad (red, ) in mitotic basal cells. White dotted line outlines basal cell in mitosis. () Quantification of co-localization between Vangl2 and Celsr1 or Fzd6 puncta. Whereas 97.5±0.8%of Vangl2 puncta contain Celsr1 (n=25 cells), only 17%±2.9% contain Fzd6 (n=10 cells). Error bars denote s.e.m. Scale bars, 10 μm. * Figure 2: Mitotically internalized PCP components are inherited equally by both daughter cells and redelivered to the plasma membrane in a polarized manner. (,) Celsr1 internalizes in cells dividing both planar () and perpendicular () to the basement membrane. Sagittal sections from E17.5 backskins labelled with Celsr1 (green) and β4 integrin (red) to mark the basement membrane. White dotted line outlines basal cell in cytokinesis. () Quantification of division angles within the plane of the basal epithelium. The orientation of division is not strongly biased towards the anterior–posterior or left–right axis (n=114 planar divisions). () Quantification of number of puncta per anterior or posterior daughter during cytokinesis. Lines connect daughters from the same division. While the total number of puncta varies with the progression of cytokinesis, similar numbers of puncta are observed in daughter cells. For Celsr1, the average difference d=2.6±0.3puncta between daughters; Vangl2 d=1.2±0.2; Fzd6 d=1.5±0.2 (n=25 daughter pairs). A, anterior; P, posterior. (–) Polarized retargeting of Celsr1 to the membrane during cytoki! nesis. Images show planar confocal projections through dividing cells within the basal layer of E15.5 backskins stained with Celsr1 antibodies. Anterior is to the left. (–) Examples of basal cells in cytokinesis extracted from the surrounding epithelia and categorized by orientation of division: ~0°,90°,45°, and perpendicular to the basal lamina (0°=anterior–posterior axis, 90°=left–right axis). Heat maps show Celsr1 intensity (red, high; blue, low). Irrespective of the plane or axis of division, Celsr1 preferentially accumulates on the anterior–posterior sides of daughter cells. Anterior is to the left. () Quantification of Celsr1 intensity during cytokinesis. Polar plots show the intensity of cortically associated Celsr1 in daughter cells during cytokinesis (n=30 cells; see Methods for details). () Celsr1 fluorescence intensity (red, high; blue, low) in Vangl2Lp/Lp embryos mosaically expressing K14–GFP–Vangl2. Vangl2-positive cells are marked with + symbo! ls, whereas Vangl2Lp/Lp cells are marked with − signs. Examp! les of cells dividing in ~0°,90° and 45° orientations are shown. () Quantification of Celsr1 intensity during cytokinesis in Vangl2Lp/Lp mosaic embryos (n=18). Note that Celsr1 enrichment along the anterior–posterior axis during cytokinesis is lost when neighbouring cells are mutant for Vangl2. Scale bars, 10 μm. * Figure 3: On mitosis, PCP components are endocytosed by a clathrin-dependent mechanism and recycled to the plasma membrane. () Time-lapse images of Celsr1–GFP internalization in live mitotic keratinocytes in vitro. The cell on the right (asterisk) is undergoing mitosis. Note that Celsr1 localizes to the cell–cell contact during interphase, but internalizes as the cell enters mitosis. () Time-lapse images of Celsr1–GFP redelivery to sites of intercellular contacts towards the end of mitosis (see Supplementary Movie S1). In and arrowheads point to Celsr1–GFP localization at cell–cell contacts. () Celsr1 co-localizes with Rab11, a marker of recycling endosomes, in mitotic basal cells in vivo. Planar confocal microscopy sections through the basal layer of E15.5 backskin labelled with Celsr1 (red) and Rab11 (green) antibodies. Right, individual channels. Chromatin is labelled with DAPI (blue). White dotted line outlines basal cell in mitosis. () Celsr1–GFP co-localizes appreciably with Rab11, early endosome markers Rab5 and EEA-1, and internalized transferrin, a marker of clathrin-dependen! t endocytosis. Some overlap is observed with caveola marker caveolin. By contrast, Celsr1–GFP is largely independent of Golgi marker GM130, and lysosomal marker lysotracker in mitotic keratinocytes in vitro. Shown is the mean percentage of Celsr1 co-localized with each marker per cell. n=4–10 cells for each marker. Error bars denote s.e.m. (see Supplementary Fig. S2 for images). (,) Celsr1 internalization is increased during mitosis. TIRF microscopy was carried out on live keratinocytes expressing Celsr1ΔN–GFP and DsRed–Clathrin. During interphase Celsr1ΔN–GFP is stably associated at the surface (). In mitosis (arrows), Celsr1ΔN–GFP forms surface puncta, which co-localize with DsRed Clathrin () (see the quantifications in Fig. 5o and Supplementary Movies S3 and S4). Scale bars, 10 μm. * Figure 4: Recruitment of Fzd6 and Vangl2 by Celsr1 to cell contacts during interphase and endocytic vesicles during mitosis. Keratinocytes were transfected with fluorescently tagged constructs as indicated and shifted to high-calcium medium to induce the formation of adherens junctions. Insets show fluorescence channels separately. DAPI staining marks nuclei. (,) Fzd6 in interphase. Fzd6–Cherry (red) is recruited to cell contacts only where Celsr1WT–GFP (green, ) homotypically interacts. (,) Fzd6 in mitosis. Fzd6–Cherry (red) is recruited to endosomes on co-expression of Celsr1WT–GFP (green, ). The cell on the right is entering mitosis. (,) Vangl2 in interphase. Cherry–Vangl2 (red) is recruited to cell contacts where Celsr1WT–GFP (green, ) homotypically interacts. (,) Vangl2 in mitosis. Cherry–Vangl2 (red) is recruited to endosomes by Celsr1WT–GFP (green, ). Scale bar, 10 μm. * Figure 5: Mitotic endocytosis of Celsr1 is mediated by a juxtamembrane dileucine motif. Keratinocytes were transfected with fluorescently tagged constructs, which are shown in schematic form in . Celsr1 contains extracellular cadherin repeats (hexagons), laminin repeats (ovals), epidermal growth factor (EGF) repeats and a HormD domain, followed by a seven-pass transmembrane domain and a 318-amino-acid cytoplasmic tail. (–) Representative examples of mitotic keratinocytes in vitro labelled with acetylated tubulin (spindle, red) and DAPI (nuclei, blue), and transfected with expression vectors encoding full-length wild-type Celsr1–GFP (), Celsr1ΔN–GFP lacking its cadherin, laminin and EGF repeats (), full-length wild-type E-Cad–GFP (), fusion protein between E-Cad extracellular domain and Celsr1's transmembrane and C-terminal tail domains (), fusion between E-Cad's extracellular and transmembrane domains and Celsr1's cytoplasmic tail (), mutation of two juxtamembrane leucines to alanines in the cytoplasmic domain of Celsr1, fused to the extracellula! r and transmembrane domains of E-Cad () and full-length Celsr1LLtoAA–GFP in interphase and mitosis (,). Note that the LLtoAA mutation (amino acids 2,748–2,749) does not affect the localization to cell–cell contacts (). () Fzd6 in interphase. Fzd6–Cherry (red) is recruited to cell contacts only where Celsr1–GFP (green) homotypically interacts and the LLtoAA mutation does not impair this function. () Fzd6 in mitosis. Fzd6–Cherry (red) localizes to the cell cortex in Celsr1LLtoAA–GFP (green)-expressing cells undergoing mitosis (compare with Fig. 3). Insets show fluorescence channels separately. () Vangl2 in interphase. Cherry–Vangl2 (red) is recruited to cell contacts where Celsr1LLtoAA–GFP (green) homotypically interacts. () Vangl2 in mitosis. Cherry–Vangl2 (red) is recruited to the cell cortex in Celsr1LLtoAA–GFP (green)-expressing mitotic keratinocytes. (,) TIRF microscopy was carried out on living keratinocytes expressing Celsr1ΔN-LLtoAA–GFP and Ds! Red–Clathrin. During interphase Celsr1ΔNLLtoAA–GFP is ind! istinguishable from wild type (compare with Fig. 3e). In mitosis Celsr1ΔN-LLtoAA–GFP fails to form puncta and does not co-localize with DsRed–Clathrin (see Supplementary Movies S5 and S6). () Quantification of Celsr1ΔN–GFP and Celsr1ΔN-LLtoAA–GFP surface puncta per cell by TIRF in interphase (n=20cells per construct) and mitosis (n=7 cells per construct). Error bars denote s.e.m. Scale bars, 10 μm. () Table summarizing results presented in – and Supplementary Figs S3–S4. * Figure 6: Mitotic endocytosis of PCP components in basal stem cells is essential for PCP-mediated hair-follicle orientation in the skin. Transgenic founder embryos mosaically expressing wild-type or LLtoAA-mutant Celsr1–GFP (green) in the skin epidermis. Planar confocal microscopy sections through the basal layer of E17.5 whole-mount backskins stained with GFP and E-Cad antibodies and DAPI. Anterior is to the left. () Wild-type Celsr1–GFP showing internalization in a mitotic basal cell. () Wild-type Celsr1–GFP during interphase is asymmetrically localized to the anterior–posterior sides of the plasma membrane. Asterisks mark cells expressing Celsr1–GFP. () LLtoAA-mutant Celsr1–GFP remaining at the plasma membrane of a basal cell undergoing mitosis. () The LLtoAA Celsr1–GFP mutant is asymmetrically localized in basal cells in interphase. Asterisks mark cells expressing Celsr1–GFP. Scale bar, 10 μm. (,) Representative example of hair follicles from E17.5 K14–Celsr1WT–GFP () and K14 Celsr1LLtoAA–GFP () transgenic founders. Planar confocal microscopy sections through hair follicles labell! ed with E-Cad antibodies. Arrows show the direction of follicle growth. Scale bars, 50 μm. Quantifications of hair-follicle angles from transgenic embryos are shown to the right of their corresponding images (wild type, n=396hair follicles, three embryos; LLtoAA, n=386 hair follicles, three embryos). () Quantification of hair-follicle angles observed in K14–Celsr1–GFP transgenics at E15.5 (n=179 wild-type hair follicles, n=146LLtoAA hair follicles) versus E17.5 (n=396 wild-type hair follicles, n=386 LLtoAA hair follicles). (,) Altered polarity surrounding mitotic cells expressing internalization-defective Celsr1. Fluorescence intensity profiles of Celsr1 in planar confocal microscopy sections through the basal layer of wild-type and LLtoAA transgenic backskins (red, high; blue, low). The central cells are in cytokinesis whereas the surrounding neighbours are in interphase. Quantification of Celsr1 intensity at the cell borders of interphase cells directly surrounding! mitotic cells is shown on the right, where the average Celsr1! intensity along a border is plotted against its angle. Levels of Celsr1 were normalized to the minimum (0%) and maximum (100%) border intensities for each image field. Borders were binned into 5° increments and error bars denote s.e.m. within bins. () In K14–Celsr1WT–GFP transgenics, anterior–posterior borders show highest Celsr1 intensity (n=88 borders). () The direction of highest Celsr1 intensity is more randomized in cells neighbouring Celsr1LLtoAA-expressing cells (asterisks; n=219 borders). White dotted lines outline basal cells in cytokinesis.Scale bar, 10 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology & Development, The Rockefeller University, 1230 York Avenue, Box 300, New York, New York 10065, USA * Danelle Devenport, * Daniel Oristian, * Evan Heller & * Elaine Fuchs Contributions E.F. and D.D. designed experiments. D.D. carried out the experiments and analysed their raw data. D.O. carried out injections for generation of transgenic founder animals. E.H. carried out quantitative analyses of image data in Fig. 3. D.D. and E.F. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Elaine Fuchs Author Details * Danelle Devenport Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Oristian Search for this author in: * NPG journals * PubMed * Google Scholar * Evan Heller Search for this author in: * NPG journals * PubMed * Google Scholar * Elaine Fuchs Contact Elaine Fuchs Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (6M) Supplementary Information * Supplementary Movie 2 (14M) Supplementary Information * Supplementary Movie 3 (23M) Supplementary Information * Supplementary Movie 4 (22M) Supplementary Information * Supplementary Movie 5 (10M) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - esBAF facilitates pluripotency by conditioning the genome for LIF/STAT3 signalling and by regulating polycomb function
- Nat Cell Biol 13(8):903-913 (2011)
Nature Cell Biology | Article esBAF facilitates pluripotency by conditioning the genome for LIF/STAT3 signalling and by regulating polycomb function * Lena Ho1, 6 * Erik L. Miller2, 7 * Jehnna L. Ronan3, 7 * Wen Qi Ho1 * Raja Jothi4, 6 * Gerald R. Crabtree5 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:903–913Year published:(2011)DOI:doi:10.1038/ncb2285Received13 December 2010Accepted31 May 2011Published online24 July 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 Signalling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs). This contrasts with most cell types where STAT3 signalling induces differentiation. We find that STAT3 binding across the pluripotent genome is dependent on Brg1, the ATPase subunit of a specialized chromatin remodelling complex (esBAF) found in ESCs. Brg1 is required to establish chromatin accessibility at STAT3 binding targets, preparing these sites to respond to LIF signalling. Brg1 deletion leads to rapid polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg1-activated targets genome wide, including the target genes of the LIF signalling pathway. Hence, one crucial role of Brg1 in ESCs involves its ability to potentiate LIF signalling by opposing PcG. Contrary to expectations, Brg1 also facilitates PcG function at classical PcG targets, including all four Hox loci, reinforcing their repression in ESCs. Therefore,! esBAF does not simply antagonize PcG. Rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency. View full text Figures at a glance * Figure 1: esBAF is dedicated to the LIF/STAT3 signalling pathway. () Western blot showing protein levels of Brg1 and pluripotent markers after 72 h of treatment of Brg1cond ESCs with 4-OHT (Brg1 knockout) or ethanol (Brg1 wild type) vehicle control. Brg1 protein is completely absent only after 48 h of 4-OHT treatment (data not shown). Full-length blots are presented in Supplementary Fig. S9a. () 2D matrix and heat map depicting gene expression changes in Brg1-knockout ESCs and 48 h LIF-starved ESCs, compared with wild-type ESCs for all genes (N=17,030). Axes indicate degree of fold change, from nil (middle of axis) to greater than 1.5-fold (outermost square). Numbers indicate the median fold change of genes in each column or row. The intensity of each square represents the number of genes that fall in that square. () 2D matrix and heat map of direct STAT3 and Brg1 targets (binding sites detected from TSS to transcription end site of the same gene) depicting changes in their expression in Brg1-knockout or LIF-withdrawn ESCs. BR1, bott! om right 1 corner. () STAT3 protein levels in wild-type versus knockout ESCs (72 h 4-OHT; LIFR, LIF receptor) in the presence of LIF (+) or after 18 h of LIF starvation (−). Full-length blots are presented in Supplementary Fig. S9b. () Time course of STAT3 activation in wild-type and Brg1-knockout ESCs. Cells were starved of LIF for 18 h (−), followed by LIF restimuation for the indicated durations. Full-length blots are presented in Supplementary Fig. S9c. * Figure 2: STAT3 binding genome wide is Brg1 dependent. () ChIP assay of STAT3 target regions within BR1 genes in Brg1-knockout and wild-type ESCs. The y axis represents input enrichment over input normalized to a negative control IG3 region. Error bars, s.e.m. of three experiments. See text and Supplementary Information for gene selection criteria. () High-resolution ChIP-seq for total STAT3 levels in wild-type and Brg1-knockout ESCs. Average tag density (yaxis) of each site identified with P<0.01 is plotted against distance in kilobases from the centre of each STAT3-binding sites for wild-type (black) and knockout (red) ESCs. () Top, experimental scheme to generate GFP+Brg1 wild-type and Brg1-knockout ESCs expressing the STAT3ER fusion protein. Bottom, GFP+ cells of the indicated genotype were mixed with GFP− wild-type ESCs at a 1:1 ratio and the GFP ratio of cultures grown in the presence or absence of 4-OHT was measured at each passage by fluorescence-activated cell sorting. Error bars, s.e.m. of three technical replicates.! Results are representative of two independent experiments. () Messenger RNA levels of STAT3/Brg1 co-bound and co-activated targets were measured in wild-type, knockout and knockout;STAT3ERnuc (nuclear) and expressed as a percentage of wild-type levels. Each data point represents a distinct gene from BR1. Error bars, s.e.m. of data points. () ChIP assay for STAT3P-Tyr705 in wild-type, knockout and knockout;STAT3ERnuc ESCs. ChIP levels are measured as the percentage of input, normalized to that of a negative intergenic control IG3, and expressed as a percentage of wild-type levels. * Figure 3: Brg1 is essential to enhance accessibility at STAT3 target genes. () Brg1 dependency correlates with tag density of STAT3 sites. Left, box–whisker plot of ChIP-seq tag numbers of each STAT3 site in Brg1 wild-type ESCs, grouped according to Brg1 dependency. Right, fold change of tag density of each STAT3 site in Brg1-knockout ESCs, compared with wild-type ESCs, grouped according to Brg1 dependency. P values are calculated using a hypergeometric distribution. () Consensus STAT3-binding MOTIFS were calculated by MEME (ref. 45) using STAT3 ChIP-seq data sets from both wild-type and Brg1-knockout ESCs. () DNaseI hypersensitivity assay of Brg1-dependent and Brg1-independent STAT3-binding sites (n=9 each). (See text and Supplementary Information for gene selection criteria.) Error bars, s.e.m. of data for nine sites obtained in two experiments. () DNaseI assay of Brg1-dependent and -independent sites (n=9 each) in wild-type ESCs or Brg1-knockout ESCs. Error bars, s.e.m. of data for nine sites obtained in two experiments. () H3K27me3 ChIP at the! STAT3-binding site of representative Brg1- and LIF- co-activated genes in wild-type and Brg1-knockout ESCs. The y axis represents the ChIP/input ratio for each region, normalized to the ratio at the GAPDH promoter. Error bars, s.e.m. of three independent experiments. * Figure 4: Brg1-deletion leads to genome-wide increased H3K27me3 at Brg1-activated genes and reduced H3K27me3 at Brg1-repressed genes. (,) Average H3K27me3 tag density at TSS of Brg1-repressed () or Brg1-activated () genes (defined as Brg1-bound genes that undergo transcriptional change in Brg1-knockout ESCs) and at the corresponding Brg1-binding regions in wild-type (blue) versus Brg1-knockout (red) ESCs. These genes were grouped according to the fold change in Brg1-knockout ESCs (DR3 represents threefold downregulated, UR3 represents threefold upregulated and so on). The number within parentheses beside each set identifier (top panel) denotes the number of genes within that set. The bottom panel illustrates the average input tag density of UR3 (for ) or DR3 (for ) genes and is representative of other subsets. * Figure 5: Synergistic interaction between Brg1 and PRC2 at Hox genes. () Browser snapshots of average normalized H3K27me3 ChIP-seq tag density in knockout (grey) and wild-type (black) ESCs at the four Hox loci. () Scatter plots of H3K27me3 levels on Hox-containing chromosomes. Each point represents the total number of tags in a particular 0.5 Mb window in Brg1-knockout ESCs (y axis) and the total number of tags in the corresponding 0.5 Mb window in Brg1 wild-type (x axis) ESCs. If a point falls on the diagonal of the plot, there is a similar overall tag number in that window in Brg1-knockout ESCs when compared to wild-type ESCs. The data points corresponding to the window containing Hox genes are labelled. () H3K27me3 ChIP at the TSS of Brg1-repressed Hox genes in wild-type and Brg1-knockout ESCs. The y axis represents the ChIP/input ratio for each region, normalized to the ratio at the GAPDH promoter, which is not H3K27me3-modified in wild-type or knockout ESCs and serves as a negative baseline internal control. Error bars, s.e.m. of thre! e independent experiments. * Figure 6: Increased levels of H3K27me3 at Brg1 and STAT3 co-activated genes in Brg1-knockout ESCs. () Top, high-resolution ChIP-seq for H3K27me3 levels in wild-type (blue) and Brg1-knockout (red) ESCs. STAT3 and Brg1 co-bound target genes were grouped according to their degree of co-activation by Brg1 and STAT3. BR1 represents genes that are highly co-activated and BR49 represents genes that are not co-activated. Graphs, average normalized H3K27me3 tag density across the TSS and over the STAT3 sites of BR1, BR4 and BR49 genes plotted (y axis) against the distance in kilobases (x axis) from the TSS or STAT3 site. The bottom panel depicts average input tag density of BR1 genes, and is representative of all subsets. () UCSC genome browser shots of H3K27me3 profiles at representative BR1 genes in wild-type (black) and knockout (grey) ESCs. () Average H3K27me3 tag density at the TSS of all Brg1 and Oct4 (top, n=70) and Brg1 and Sox2 (bottom, n=13) co-bound co-activated genes in wild-type ESCs (blue) and Brg1-knockout ESCs (red). Oct4 and Sox2 ESCs sites are from ChIP-seq data ! sets from ref. 46. * Figure 7: Opposing activity and localization of esBAF and PRC2 complexes. () 2D matrices depicting the gene expression changes comparing Suz12-knockout ESCs (ref. 34) and 48 h LIF-starved ESCs for all genes (left) and STAT3-bound direct targets (right). () Suz12 ChIP at STAT3-binding sites of BR1 genes in wild-type (black) and Brg1-knockout (grey) ESCs. Error bars, s.e.m. of three biological replicates. The y axis represents the ChIP/input ratio for each region, normalized to the ratio at the GAPDH promoter. () ChIP assay of the PRC2 component Jarid2 at Brg1-dependent STAT3 sites in wild-type versus Brg1-knockout (grey) ESCs. The y axis represents the ChIP/input ratio for each region, normalized to the ratio at the GAPDH promoter. (–) Wild-type ESCs were infected with control (pLKO) or two distinct anti-Suz12 shRNAs (Suz12_1 and Suz12_2) expressing lentiviruses either separately or together (Suz12_1+2). Brg1 deletion was induced with 4-OHT after stable knockdown of Suz12 was achieved. At 72 h post 4-OHT treatment, cells of the indicated geno! type were collected for H3K27me3 ChIP assay (), transcript level () and STAT3P ChIP assay () analysis at Brg1-dependent STAT3-binding sites in BR1. Each point represents a distinct STAT3 target gene or target site. * Figure 8: esBAF both antagonizes and synergizes with PRC2 to promote pluripotency. esBAF antagonizes PRC2 action at LIF target genes preparing them to be activated by phospho-STAT3 entering the nucleus. In contrast, esBAF works with PRC2 to enforce the H3K27me3 repressive mark at all four Hox loci and over many differentiation genes. The levels of pluripotency genes are both repressed and activated by Brg1 (esBAF) as indicated by the blue arrow, a context-dependent function that we have called 'refinement'. Author information * Abstract * Author information * Supplementary information Primary authors * Co-first authors; these authors contributed equally to this work * Lena Ho & * Raja Jothi * Co-second authors; these authors contributed equally to this work * Erik L. Miller & * Jehnna L. Ronan Affiliations * Program in Immunology, Stanford University School of Medicine, Stanford, California 94305, USA * Lena Ho & * Wen Qi Ho * Program in Genetics, Stanford University School of Medicine, Stanford, California 94305, USA * Erik L. Miller * Program in Cancer Biology, Stanford University School of Medicine, Stanford, California 94305, USA * Jehnna L. Ronan * Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health (NIH), 111 T.W. Alexander Drive, Research Triangle Park, North Carolina 27709, USA * Raja Jothi * Howard Hughes Medical Institute and Departments of Developmental Biology and Pathology, Stanford, California 94305, USA * Gerald R. Crabtree Contributions L.H., R.J. and G.R.C. contributed to experimental design, execution and data analysis. R.J. carried out ChIP-seq and all related data analysis. J.L.R. and W.Q.H. contributed to experimental execution. E.L.M. carried out data analysis. L.H. and G.R.C. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Raja Jothi or * Gerald R. Crabtree Author Details * Lena Ho Search for this author in: * NPG journals * PubMed * Google Scholar * Erik L. Miller Search for this author in: * NPG journals * PubMed * Google Scholar * Jehnna L. Ronan Search for this author in: * NPG journals * PubMed * Google Scholar * Wen Qi Ho Search for this author in: * NPG journals * PubMed * Google Scholar * Raja Jothi Contact Raja Jothi Search for this author in: * NPG journals * PubMed * Google Scholar * Gerald R. Crabtree Contact Gerald R. Crabtree Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion
- Nat Cell Biol 13(8):914-923 (2011)
Nature Cell Biology | Article A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion * Martin Harterink1 * Fillip Port2, 4 * Magdalena J. Lorenowicz1, 4 * Ian J. McGough3, 4 * Marie Silhankova1, 5 * Marco C. Betist1 * Jan R. T. van Weering3 * Roy G. H. P. van Heesbeen1 * Teije C. Middelkoop1 * Konrad Basler2 * Peter J. Cullen3, 6 * Hendrik C. Korswagen1, 6 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:914–923Year published:(2011)DOI:doi:10.1038/ncb2281Received23 September 2010Accepted17 May 2011Published online03 July 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 Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1–SNX2 and SNX5–SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 ! is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo. View full text Figures at a glance * Figure 1: SNX3 is required for EGL-20 (Wnt) signalling and MIG-14 (Wls) recycling in C. elegans. () The final positions of the QL.paa and QL.pap cells relative to the invariant positions of the seam cells V1–V6 (n>100). Both snx-1(tm847) and snx-6(tm3790) are viable as single or double mutants and could be propagated as homozygous strains, excluding a contribution of maternally provided protein in our assays. () Expression of the EGL-20 target gene mab-5 in the QL descendants QL.a and QL.p. Cell nuclei are shown by 4,6-diamidino-2-phenylindole (DAPI) staining. The scale bar is 10 μm. () Staining of EGL-20::protein A with rabbit anti-goat-Cy5 (ref. 23) in wild type, vps-35(hu68) and snx-3(tm1595). Expression is visible within the egl-20-expressing cells (solid line) and as a punctate posterior-to-anterior gradient (dotted line). In all images, anterior is to the left and dorsal is up. The scale bar is 10 μm. () Confocal microscopy images of MIG-14::GFP (huSi2) at identical exposure settings in wild type and in snx-1(tm847); snx-6(tm3790), vps-26(tm1523) and snx-3! (tm1595). The scale bar is 10 μm. () Western blot quantification of MIG-14::GFP (huSi2) protein levels. () Confocal images of MIG-14::GFP (huIs71) (green) and LMP-1::mCherry (red) in wild type, vps-35(hu68) and snx-3(tm1595). The arrowheads indicate examples of co-localization between MIG-14::GFP and LMP-1::mCherry. The scale bar is 10 μm. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 2: DSnx3 is required for Wg secretion and Wls recycling in the Drosophila wing imaginal disc. (–) Immunostaining of Wg, Wls and Senseless in wild-type wing disc. (,,–) Expression of Dsnx6 or Dsnx3 RNAi transgenes was induced in the posterior compartment of the wing disc (marked by mCD8–GFP in green) using an hhGal4 driver (see Supplementary Fig. S3a,b for quantification of knockdown efficiency). (,) Immunostaining of Senseless (red). The arrowheads indicate loss of senseless expression in the Dsnx3 RNAi-expressing posterior compartment. () Dsnx3 RNAi was induced in the posterior compartment using hhGal4 or in clones using an actinGal4 driver. The arrowheads indicate notches and loss of sensory bristles. (,) Immunostaining of total Wg (red). The arrowheads indicate Wg accumulation in the Dsnx3 RNAi-expressing posterior compartment. () Immunostaining of extracellular Wg (red). The arrowheads indicate loss of extracellular Wg staining. (,) Immunostaining of Wls (red). The arrowheads indicate loss of Wls in wg-expressing cells in the Dsnx3 RNAi-expressing posterior! compartment. Scale bars, 50 μm. * Figure 3: Co-localization and physical interaction of SNX3 with the cargo-selective subcomplex of the retromer. () SNX3 partially co-localizes with VPS26-positive early endosomes. HeLa cells lentivirally transduced to express GFP–SNX3 (green) were fixed and stained for VPS26, SNX1, EEA1 or LAMP1 (red). Co-localization between GFP–SNX3 and VPS26, SNX1, EEA1, LAMP1, RAB5–mCherry and RAB7–mCherry was quantified as 0.43±0.05, 0.55±0.04, 0.38±0.02, 0.07±0.04, 0.61±0.02 and 0.34±0.02, respectively (Pearson's coefficient, mean±s.d., n=3 with 30 cells per condition; for RAB5 and RAB7, n=20 cells). Scale bar, 11 μm. () At the ultrastructural level, SNX3 and VPS26 localize to common vesicular endosomal profiles. GFP–SNX3 is labelled with 10 nm gold and mCherry–VPS26 with 6 nm gold. The image is representative of that observed from the analysis of five other endosomal vacuoles. Scale bar, 100 nm.() SNX3 interacts with the cargo-selective subcomplex of the retromer. Cell extracts derived from HeLa cells lentivirally transduced with GFP, GFP–SNX3 or both GFP–SNX1 ! and GFP–SNX5 (GFP–SNX1/5) were subjected to a GFP nanotrap. The classical retromer SNX–BARs form heterodimeric complexes, leading to the presence of both endogenous SNX1 and SNX5 in the GFP–SNX1/5 immunoprecipitates3. () 3xFlag–VPS26–VPS29–VPS35–His6 complex (His–VPS) was isolated from BL21 Escherichia coli onto TALON resin and incubated with 2 μM of recombinant SNX3, SNX1 or SNX5 for 2 h at 4 °C. Supernatant (S) and TALON-containing resin (P) were isolated. SNX3 directly associates with His–VPS, as do SNX1 and SNX5 although this is less well pronounced (longer exposures are shown; 2× and 3×). Control: boiled His–VPS resin. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 4: SNX3 co-localizes with Wls and facilitates membrane association of the cargo-selective subcomplex of the retromer. () Co-localization between SNX3–GFP (green) and Wls–mCherry (red) in HeLa cells was quantified as 0.25±0.02 (Pearson's coefficient; mean±s.e.m., n=2 with 23 and 11 cells). The arrowheads show examples of co-localization. Scale bar, 10 μm. (,) Co-localization between Wls–mCherry and endogenous VPS26 (green) in HeLa cells treated with control or SNX3 siRNA was quantified as 0.19±0.02 and 0.08±0.02, respectively (Pearson's coefficient; mean±s.e.m., n=4 with seven to ten cells each). The arrowheads show examples of co-localization. () HeLa cells were transfected with control or SNX3 siRNA and assayed for endogenous Wls, VPS26, SNX3 and tubulin protein levels. () HeLa cells treated with control, SNX3 or RAB7 siRNA were separated into a supernatant (S) fraction containing cytosol and a pellet fraction (P) containing membranes34 and were stained for endogenous VPS26, SNX1 and LAMP1. The amount of VPS26 in the supernatant and pellet fractions was quantified using d! ensitometry and is shown as a percentage of the total. Data are presented as mean±s.e.m. and represent three independent experiments. There was no significant change in SNX1 membrane association on SNX3 knockdown (17.8±3.1% in control versus 22±6.6% in SNX3 knockdown; data are means±s.e.m., n=3, P>0.5 Student's t-test). Knockdown of RAB7 was included as a positive control34. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 5: Wls is contained within SNX3-positive vesicular carriers but is absent from SNX1 retromer-decorated tubular carriers. () RPE-1 cells were transiently co-transfected with pEGFP–SNX1 (green) and Wls–mCherry (red) and cells were subsequently imaged live after a 16 h incubation period. Frames depicting the formation and scission of a GFP–SNX1 tubule from a vesicle positive for both SNX1 and Wls are shown (the arrows indicate the dual-expressing vesicle, whereas the arrowheads indicate the carrier post scission) (see Supplementary Movie S1). Scale bars represent 6 μm. Of the 100 SNX1-decorated tubulating endosomes that were analysed, 22 were positive for Wls; 18/22 tubules emanating from these endosomes were negative for Wls, whereas 4/22 were weakly positive. Quantification of Wls–mCherry and GFP–SNX1 levels in an endosome and corresponding tubule is shown in Supplementary Fig. S4d. () Further examples of SNX1 retromer tubules negative for Wls. (1) An example of a SNX1 retromer-positive endosome and tubule both of which are negative for Wls. (2,3) Further examples of SNX1-labelle! d endosomes positive for Wls, but with tubules negative for Wls. Scale bars represent 6 μm. () RPE-1 cells were transiently co-transfected with pEGFP–SNX3 (green) and Wls–mCherry (red) and cells were subsequently imaged live after a 16 h incubation period. Frames depicting the formation and scission of GFP–SNX3-labelled buds from vesicles positive for both SNX3 and Wls are shown. Note the 1 s delay between acquisitions for a given image pair. The arrows and arrowheads show two examples of buds positive for both Wls and SNX3 (see Supplementary Movie S2). Scale bars represent 6 μm. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Fillip Port, * Magdalena J. Lorenowicz & * Ian J. McGough Affiliations * Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands * Martin Harterink, * Magdalena J. Lorenowicz, * Marie Silhankova, * Marco C. Betist, * Roy G. H. P. van Heesbeen, * Teije C. Middelkoop & * Hendrik C. Korswagen * Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland * Fillip Port & * Konrad Basler * Henry Wellcome Integrated Signaling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK * Ian J. McGough, * Jan R. T. van Weering & * Peter J. Cullen * Present address: Department of Cell Biology, Faculty of Science, Charles University Prague, Vinicna 7, 128 00 Prague 2, Czech Republic * Marie Silhankova * Joint senior authors * Peter J. Cullen & * Hendrik C. Korswagen Contributions M.H., M.S., T.C.M., M.C.B., R.G.H.P.H. and H.C.K. designed and carried out the C. elegans experiments, F.P. and K.B. designed and carried out the Drosophila experiments, M.J.L., I.J.M., J.R.T.W., H.C.K. and P.J.C. designed and carried out the cell biological analysis of SNX3 function in tissue culture cells and M.H., K.B., P.J.C. and H.C.K. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Peter J. Cullen or * Hendrik C. Korswagen Author Details * Martin Harterink Search for this author in: * NPG journals * PubMed * Google Scholar * Fillip Port Search for this author in: * NPG journals * PubMed * Google Scholar * Magdalena J. Lorenowicz Search for this author in: * NPG journals * PubMed * Google Scholar * Ian J. McGough Search for this author in: * NPG journals * PubMed * Google Scholar * Marie Silhankova Search for this author in: * NPG journals * PubMed * Google Scholar * Marco C. Betist Search for this author in: * NPG journals * PubMed * Google Scholar * Jan R. T. van Weering Search for this author in: * NPG journals * PubMed * Google Scholar * Roy G. H. P. van Heesbeen Search for this author in: * NPG journals * PubMed * Google Scholar * Teije C. Middelkoop Search for this author in: * NPG journals * PubMed * Google Scholar * Konrad Basler Search for this author in: * NPG journals * PubMed * Google Scholar * 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 Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Table 1 (50K) Supplementary Information Movies * Supplementary Movie 1 (150K) Supplementary Information * Supplementary Movie 2 (120K) Supplementary Information PDF files * Supplementary Information (12M) Supplementary Information Additional data - Cdk1-phosphorylated CUEDC2 promotes spindle checkpoint inactivation and chromosomal instability
- Nat Cell Biol 13(8):924-933 (2011)
Nature Cell Biology | Article Cdk1-phosphorylated CUEDC2 promotes spindle checkpoint inactivation and chromosomal instability * Yan-Fei Gao1, 2 * Teng Li1, 2 * Yan Chang1, 2 * Yu-Bo Wang1 * Wei-Na Zhang1 * Wei-Hua Li1 * Kun He1 * Rui Mu1 * Cheng Zhen1 * Jiang-Hong Man1 * Xin Pan1 * Tao Li1 * Liang Chen1 * Ming Yu1 * Bing Liang1 * Yuan Chen1 * Qing Xia1 * Tao Zhou1 * Wei-Li Gong1 * Ai-Ling Li1 * Hui-Yan Li1 * Xue-Min Zhang1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:924–933Year published:(2011)DOI:doi:10.1038/ncb2287Received04 April 2011Accepted01 June 2011Published online10 July 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 Aneuploidy and chromosomal instability are major characteristics of human cancer. These abnormalities can result from defects in the spindle assembly checkpoint (SAC), which is a surveillance mechanism for accurate chromosome segregation through restraint of the activity of the anaphase-promoting complex/cyclosome (APC/C). Here, we show that a CUE-domain-containing protein, CUEDC2, is a cell-cycle regulator that promotes spindle checkpoint inactivation and releases APC/C from checkpoint inhibition. CUEDC2 is phosphorylated by Cdk1 during mitosis. Depletion of CUEDC2 causes a checkpoint-dependent delay of the metaphase–anaphase transition. Phosphorylated CUEDC2 binds to Cdc20, an activator of APC/C, and promotes the release of Mad2 from APC/C–Cdc20 and subsequent APC/C activation. CUEDC2 overexpression causes earlier activation of APC/C, leading to chromosome missegregation and aneuploidy. Interestingly, CUEDC2 is highly expressed in many types of tumours. These results s! uggest that CUEDC2 is a key regulator of mitosis progression, and that CUEDC2 dysregulation might contribute to tumour development by causing chromosomal instability. View full text Figures at a glance * Figure 1: The Cdk1-dependent phosphorylation of CUEDC2 is required for the metaphase–anaphase transition. () Immunoblot analysis of CUEDC2 in HeLa cells synchronized at the indicated cell-cycle phases as described in Methods. () Asynchronous (Asy.), nocodazole-arrested (Noc.) and taxol-arrested extracts from HeLa cells were treated with/without λ-PPase, followed by immunoblotting with antibodies against CUEDC2, phosphorylated CUEDC2 (P-CUEDC2) and α-tubulin. () Extracts from asynchronous or nocodazole-treated HeLa cells (shake-off) ectopically expressing Flag–CUEDC2 wild type or its phosphomutants were immunoblotted (IB) with an anti-Flag antibody. () HeLa cells were synchronized by a double thymidine treatment and then released into nocodazole. Cell extracts were prepared at the times indicated after nocodazole treatment. Immunoblot analysis was carried out using the indicated antibodies. () In vitro Cdk1–Cyclin B1 kinase assay. Top, active Cdk1–Cyclin B1 was incubated with recombinant proteins as indicated in the presence of [γ-32P]-ATP; bottom, the amounts of recombi! nant proteins from the same reaction were analysed by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie staining. () Selected frames from time-lapse movies of representative HeLa/GFP–H2B cells transfected with control or CUEDC2 siRNA. The time on the images is in minutes. NEB, nuclear envelope breakdown; M, metaphase; A, anaphase. Scale bar, 10 μm. () A box-and-whisker plot showing the duration from NEB to anaphase onset in HeLa/GFP–H2B cells with CUEDC2 knockdown (n=135cells) or control knockdown (n=135 cells). Open circles represent individual cells; filled circles represent statistical outliers excluded from the analyses. () The lengths of prometaphase and metaphase in control (n=135cells) and CUEDC2-knockdown cells (n=135 cells) described as in were analysed. Data are shown as mean±s.e.m. () Complementation of RFP–CUEDC2 in knockdown HeLa/GFP–H2B cells rescues anaphase onset. The CUEDC2-knockdown cells were transfected with CUEDC2 siRNA-resistant! expression construct including RFP–CUEDC2 wild type (wild t! ype) or its mutant RFP–CUEDC2S110A. The data show the percentage of RFP-positive cells taking longer than 2 h for metaphase. Data are representative of two independent experiments. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 2: The knockdown of CUEDC2 did not affect mitotic spindle, BubR1 and Mad2 signals at kinotechores. () Quantification of metaphase cells with multipolar spindles in control (n=645cells) and CUEDC2-knockdown (n=669 cells) cells. Data are shown as mean±s.d. and are representative of three independent experiments. () Representative images of control or CUEDC2-knockdown cells in metaphase. The cells were stained with 4,6-diamidino-2-phenylindole (DAPI; for DNA; blue), anti-α-tubulin (microtubules; green) and CREST antisera (kinetochores; red). Insets, a single z slice of the boxed regions. Scale bar, 10 μm. () Average inter-kinetochore distance from control siRNA and CUEDC2 siRNA cells treated as in . The quantification was based on the location of CREST staining in HeLa cells. Data are shown as mean±s.d. and n=160 kinetochore pairs from 18 cells. () Quantification of Mad2 and BubR1 levels on prometaphase and metaphase kinetochores. HeLa cells were transfected with control or CUEDC2 siRNA, stained with anti-Mad2 or anti-BubR1 antibody and visualized using an inverted flu! orescence microscope. Data are shown as mean±s.d. and n=160 kinetochores from 15 cells. () HeLa/GFP–H2B cells were co-transfected with control siRNA or CUEDC2 siRNA together with RFP–Mad2, and synchronized by a thymidine block. After 8 h following release, the cells through mitosis were imaged by fluorescence time-lapse microscopy. Data are shown as mean±s.e.m. (n=30 cells). () Representative images of control and CUEDC2-knockdown cells in mitosis, as described in . Scale bar, 10 μm. * Figure 3: CUEDC2 regulates APC/C-mediated ubiquitylation and degradation depending on SAC. () A box-and-whisker plot showing the duration from NEB to anaphase onset in HeLa/GFP–H2B cells with CUEDC2 knockdown or with Mad2 co-knockdown. Open circles represent individual cells; filled circles represent statistical outliers excluded from the analyses (n=155 cells). () HeLa cells were transfected with control siRNA or CUEDC2 siRNA, and synchronized at prometaphase by a thymidine–nocodazole (Noc.) arrest. Mitotic cells were collected by shake-off, released into fresh medium and then collected at the indicated times after release. Levels of cyclin B1, securin, Cdc27, CUEDC2 and heat-shock protein 70 (Hsp70) were determined by western blot analysis. () HeLa cells were transfected as described in , and synchronized at prometaphase by a taxol arrest. Mitotic cells were collected by shake-off, and then collected at the indicated times after adding ZM447439. Levels of cyclin B1, securin, CUEDC2 and Hsp70 were determined by western blot analysis. () In vitro degradation a! ssays. Control or CUEDC2-knockdown extracts from nocodazole-arrested HeLa cells were supplemented with an energy-regenerating system and reactions were initiated by the addition of 35S-labelled cyclin B1 or securin proteins. Samples were then taken at the indicated times and analysed by SDS–PAGE and autoradiography. () In vitro ubiquitylation assay. The assay was carried out as described in , except replacing substrate with geminin. () Quantification of geminin ubiquitylation. Geminin–ubiquitin conjugates from experiment in were quantified. The relative intensity was obtained by converting the intensity at 70 min from control siRNA cells to unity. () HeLa cells were transfected as described in , and then were synchronized by double-thymidine block and cells collected at different times after release into nocodazole. Levels of cyclin A, Cdc27, CUEDC2 and Hsp70 were determined by western blot analysis. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 4: The interaction of CUEDC2 with Cdc20 depends on the phosphorylation at Ser 110, but not the CUE domain. () HeLa cells were synchronized at prometaphase by a thymidine–nocodazole (Noc.) arrest and collected by shake-off. Cell lysates were immunoprecipitated (IP) with an anti-CUEDC2 antibody or normal mouse IgG and then the immunoprecipitates were analysed by immunoblotting. () HeLa cells were synchronized at prometaphase as described in and released into fresh medium. Cell samples taken at the indicated times were immunoprecipitated with anti-CUEDC2 antibody. The immunoprecipitates were analysed by immunoblotting. () 293T cells were co-transfected with vectors expressing Myc-tagged CUEDC2 and GFP-tagged Cdc20 or Flag-tagged Mad2, and treated with nocodazole. Cell lysates were subjected to immunoprecipitation with an anti-Myc antibody. The immunoprecipitates and cell lysates were analysed by immunoblot (IB). () M2-bead-bound phosphorylated Flag–CUEDC2 proteins were prepared as described in Methods, and then incubated with in vitro-translated 35S-labelled Cdc20 or Mad2 protei! ns as indicated. The samples were assayed for Cdc20 and Mad2 by autoradiography (top panel) and blotted with anti-Flag (bottom panel). () Immunoassay of 293T cells co-transfected with vectors expressing Flag-tagged CUEDC2 and GFP-tagged Cdc20 or two deletion mutants of Cdc20; cells were treated with nocodazole and lysates were immunoprecipitated with anti-Flag and whole-cell lysates were analysed by immunoblotting with anti-Flag and anti-GFP. () Immunoassay of 293T cells co-transfected with vectors expressing GFP-tagged Cdc20 and Flag-tagged CUEDC2 or two deletion mutants of CUEDC2; cells were treated with nocodazole, lysates were immunoprecipitated with anti-Flag M2 beads and immunoprecipitates were probed with an anti-GFP or anti-Flag antibody. () Immunoassay of 293T cells co-transfected with vectors expressing GFP-tagged Cdc20 and Flag-tagged CUEDC2 or various CUEDC2 mutants; cells were treated with nocodazole and lysates were immunoprecipitated with anti-Flag M2 beads a! nd immunoprecipitates were probed with an anti-GFP or anti-Fla! g antibody. () In vitro binding assay of CUEDC2 and Cdc20. M2-bead-bound phosphorylated Flag–CUEDC2 or various mutant proteins were prepared as described in , and then incubated with 35S-labelled Cdc20 as indicated. The samples were assayed for Cdc20 by autoradiography and blotted with anti-Flag. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 5: CUEDC2 is required for the disassociation of Mad2 from APC/C–Cdc20 complex. () HeLa cells were treated with the indicated siRNAs before nocodazole (Noc.) arrest. Cdc20 was immunoprecipitated (IP) at the indicated times after nocodazole washout. The amounts of co-precipitated Mad2 were visualized by western blotting (upper panel). Whole-cell lysates were analysed by immunoblotting with indicated antibodies (lower panel). () HeLa cells were treated with the indicated siRNAs before taxol arrest. For the last 2 h before shake-off, MG132 was added. Then taxol-arrested cells were shaken off and treated with ZM447439 for the indicated times. Cdc20 was immunoprecipitated, and co-precipitated Mad2 was detected as described in . () Immunoassay of 293T cells transfected with increasing amounts of Flag-tagged CUEDC2 or its various mutants as indicated and synchronized at prometaphase by thymidine–nocodazole; Cdc20 in lysates was immunoprecipitated with anti-Cdc20, followed by immunoblot analysis of immunoprecipitates. () HeLa cells were transfected with CUE! DC2 siRNA as described in , and synchronized at prometaphase by thymidine–nocodazole. Nocodazole-arrested extracts were incubated with purified Flag–CUEDC2 or its mutant proteins along with an energy-regenerating system for 60 min in vitro and then immunoprecipitated with an anti-Cdc20 antibody. Co-immunoprecipitated Mad2 was detected by western blot. () In vitro ubiquitylation assay. HeLa cells were treated and synchronized as described in . Nocodazole-arrested extracts were incubated with in vitro-translated 35S-labelled geminin and purified Flag–CUEDC2 or its mutant proteins along with an energy-regenerating system at 30 °C for indicated times. Samples were detected by SDS–PAGE and autoradiography. () In vitro degradation assays. Mock- or CUEDC2-immunodepleted extracts from nocodazole-arrested HeLa cells were supplemented with an energy-regenerating system and purified Flag–CUEDC2 or its mutant proteins. The reactions were initiated by the addition of 35S-! labelled cyclin B1 proteins. Samples were then taken at the in! dicated times and analysed by SDS–PAGE and autoradiography. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 6: Overexpression of CUEDC2 leads to chromosome missegregation and aneuploidy. () CUEDC2 overrides the spindle checkpoint in taxol-arrested cells. MCF10A cells were treated with taxol. After 24 h, the mitotic index and multilobed nucleus index were determined. Error bars, s.e.m. Data are representative of three independent experiments. () CUEDC2 overrides the spindle checkpoint in taxol-arrested cells. HeLa cells were transfected with wild-type CUEDC2 or CUEDC2S110A, and then were synchronized with thymidine treatment. After 8 h release from thymidine, the cells were treated with taxol and imaged by fluorescence time-lapse and differential interference contrast microscopy at 10 min intervals for 30 h. Data are shown as mean±s.e.m. (n=73 cells). () Analysis of chromosome-segregation defects in MCF10A cells expressing wild-type CUEDC2 or CUEDC2S110A. Error bars, s.e.m. Data are representative of three independent experiments. Bottom, representative images of normal and lagging chromosomes in MCF10A cells. Scale bar, 10 μm. () The proportion of ! cells with a karyotype deviating from the modal chromosome number was determined within 30 generations in MCF10A, MCF10A/wild-type CUEDC2 or MCF10A/CUEDC2S110A cells (n=328, 355 and 339 cells (MCF10A, MCF10A/wild-type CUEDC2 and MCF10A/CUEDC2S110A, respectively). Data are shown as mean±s.d. and are representative of three independent experiments. () Individual chromosome numbers from metaphase spreads of cultured MCF10A cells stably expressing CUEDC2 wild type or CUEDC2S110A were analysed within 30 generations (n≥100). () The tissue array of kidney, ovarian and brain tumours was carried out by immunohistochemistry with anti-CUEDC2 antibody. CUEDC2 expression was plotted using the score described in the 'Methods' section. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Yan-Fei Gao, * Teng Li & * Yan Chang Affiliations * Institute of Basic Medical Sciences, National Center of Biomedical Analysis, 27 Tai-Ping Road, Beijing 100850, China * Yan-Fei Gao, * Teng Li, * Yan Chang, * Yu-Bo Wang, * Wei-Na Zhang, * Wei-Hua Li, * Kun He, * Rui Mu, * Cheng Zhen, * Jiang-Hong Man, * Xin Pan, * Tao Li, * Liang Chen, * Ming Yu, * Bing Liang, * Yuan Chen, * Qing Xia, * Tao Zhou, * Wei-Li Gong, * Ai-Ling Li, * Hui-Yan Li & * Xue-Min Zhang Contributions X-M.Z. and H-Y.L. supervised the project; Y-F.G., Teng L. and Yan C. designed and carried out most of the experiments; Y-F.G. and Y-B.W. contributed to chromosome spread analysis; W-H.L. and K.H. analysed the phosphorylation modification with mass spectrometry; W-N.Z. and R.M. carried out immunohistochemistry analysis; Y-B.W., Tao L. and C.Z. contributed to the preparation of complementary DNA vector constructs; M.Y., Yuan C. and R.M. prepared the CUEDC2 antibody; W-L.G., B.L. and L.C. developed stable cell lines; J-H.M., Q.X. and X.P. carried out the statistics; T.Z., A-L.L., X-M.Z. and H-Y.L. analysed the data; Y-F.G., Teng L., Yan C., H-Y.L. and X-M.Z. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Hui-Yan Li or * Xue-Min Zhang Author Details * Yan-Fei Gao Search for this author in: * NPG journals * PubMed * Google Scholar * Teng Li Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Chang Search for this author in: * NPG journals * PubMed * Google Scholar * Yu-Bo Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Wei-Na Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Wei-Hua Li Search for this author in: * NPG journals * PubMed * Google Scholar * Kun He Search for this author in: * NPG journals * PubMed * Google Scholar * Rui Mu Search for this author in: * NPG journals * PubMed * Google Scholar * Cheng Zhen Search for this author in: * NPG journals * PubMed * Google Scholar * Jiang-Hong Man Search for this author in: * NPG journals * PubMed * Google Scholar * Xin Pan Search for this author in: * NPG journals * PubMed * Google Scholar * Tao Li Search for this author in: * NPG journals * PubMed * Google Scholar * Liang Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Ming Yu Search for this author in: * NPG journals * PubMed * Google Scholar * Bing Liang Search for this author in: * NPG journals * PubMed * Google Scholar * Yuan Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Qing Xia Search for this author in: * NPG journals * PubMed * Google Scholar * Tao Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Wei-Li Gong Search for this author in: * NPG journals * PubMed * Google Scholar * Ai-Ling Li Search for this author in: * NPG journals * PubMed * Google Scholar * Hui-Yan Li Contact Hui-Yan Li Search for this author in: * NPG journals * PubMed * Google Scholar * Xue-Min Zhang Contact Xue-Min Zhang Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (500K) Supplementary Information * Supplementary Movie 2 (1M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway
- Nat Cell Biol 13(8):934-943 (2011)
Nature Cell Biology | Article N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway * Eva M. Kovacs1 * Suzie Verma1 * Radiya G. Ali1, 2 * Aparna Ratheesh1 * Nicholas A. Hamilton3 * Anna Akhmanova4 * Alpha S. Yap1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:934–943Year published:(2011)DOI:doi:10.1038/ncb2290Received01 December 2010Accepted03 June 2011Published online24 July 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 N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WAS! P WH1 domain. We conclude that N-WASP–WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity. View full text Figures at a glance * Figure 1: The epithelial junctional actin cytoskeleton contains two pools of actin filaments that are predominantly nucleated at the membrane. () Left, confluent Caco-2 cells immunostained for E-cadherin (red) and F-actin (blue). Representative confocal micrographs were taken at the zonula adherens. Lower-magnification images are shown in Supplementary Fig. S1a. Right, E-cadherin and F-actin at the zonula adherens were quantified by line-scan analysis of fluorescence intensity. Data represent mean±s.e.m. pooled from three individual experiments (n=8). The outlined area indicates the region used for line-scan analysis. () Measurement of junctional barbed ends and F-actin. Left, confluent Caco-2 cells were semi-permeabilized with saponin and incubated with Alexa594–G-actin (red), then fixed and stained for F-actin (phalloidin, blue). Representative confocal micrographs were taken at the zonula adherens and show apical actin rings with barbed ends localized at the membrane between the actin rings. Lower-magnification images are shown in Supplementary Fig. S1b. Right, accumulation of F-actin and barbed ends at the z! onula adherens was quantified by line-scan analysis of fluorescence intensity. Data represent mean±s.e.m. of data pooled from three individual experiments (F-actin, n=22; barbed ends, n=15). The outlined area indicates the region used for line-scan analysis. () Spatiotemporal pattern of junctional actin turnover in Caco-2 cells examined by FRAP analysis of transiently expressed GFP–actin. () Kymograph analysis of the FRAP data was carried out using line scans perpendicular to the junctional GFP–actin (in the region outlined in ). A representative kymograph is shown and a complete movie is provided in Supplementary Movie S1. (,) Effect of Arp2/3 inhibition on apical actin rings and barbed end generation. Caco-2 cells were treated with either an Arp2/3 inhibitor (CK-869; ) or a control drug (CK-312; ). F-actin and barbed ends were then visualized with phalloidin and Alexa594–G-actin, respectively, and quantified by line-scan analysis. Data represent mean±s.e.m. of dat! a pooled from three individual experiments (, n=42; , n=45). R! epresentative images are shown in Supplementary Fig. S1e,f. Scale bars: , 10 μm; , 6.5 μm; , 1 μm (left panel) and 1.5 μm (right panel); , 0.75 μm. * Figure 2: N-WASP regulates the junctional actin cytoskeleton without affecting filament nucleation. () Confluent Caco-2 cells were fixed and immunostained for E-cadherin (red) and N-WASP (blue). Representative confocal images were taken at the zonula adherens. () Monolayers of Caco-2 cells stained with N-WASP (n=31) and E-cadherin (n=30), or N-WASP (n=25) and actin (n=27), were analysed by line-scan analysis of fluorescence intensity at the contacts. Data represent mean±s.e.m. of data pooled from three individual experiments. () Immunoprecipitates (IP) of E-cadherin or N-WASP were immunoblotted for E-cadherin and N-WASP. Rabbit IgG and mouse IgG are negative immunoprecipitation controls. Further representative full blots are shown in Supplementary Fig. S2g. () Junctional N-WASP in monolayers of control Caco-2 cells or Caco-2 cells transduced with an shRNA directed against CDH1. Data represent mean±s.e.m. pooled from three individual experiments (n=10;***P<0.0001; Student's t-test). () Junctional N-WASP in monolayers of Caco-2 cells incubated with either a control antib! ody (mouse IgG) or an E-cadherin-blocking antibody (SHE78-7). Data represent mean±s.e.m. pooled from three individual experiments (n=24;***P<0.0001; Student's t-test). () Lysates from Caco-2 cells transfected with an empty vector control or an shRNA directed against N-WASP (N-WASP-knockdown cells) were immunoblotted for N-WASP and GFP. Full blots are shown in Supplementary Fig. S6. () N-WASP-knockdown cells were stained for barbed ends and F-actin, imaged at the level of the zonula adherens (micrographs) and quantified by line-scan analysis of fluorescence intensity (graph). Data represent mean±s.e.m. pooled from three individual experiments (F-actin, n=22; barbed ends, n=15). Lower-magnification images are shown in Supplementary Fig. S2a. (–) Control and N-WASP-knockdown (KD) cells were further analysed at the zonula adherens for total F-actin (phalloidin) content (), total barbed ends () and apical F-actin rings (). Data represent mean±s.e.m. of data pooled from th! ree individual experiments (, n=23, *P<0.01; , n=23, not signi! ficant; , n=26, ***P=0.0004; Student's t-test). Scale bars: , 30 μm; , 5 μm. * Figure 3: N-WASP regulates junctional filament stability and organization by a VCA-independent, WH1-dependent mechanism. (–) Effects on junctional F-actin and actin nucleation. Caco-2 cells were infected with N-WASP shRNA lentivirus (KD) that co-expressed RNAi-resistant full-length N-WASP (FL, ), N-WASPΔVCA () or N-WASPΔWH1 (). Micrographs, representative confocal images of barbed ends at the zonula adherens identified with Alexa594–G-actin (red) and of F-actin (phalloidin, blue); lower-magnification images are shown in Supplementary Fig. S2b–d. Graphs, F-actin and barbed ends were quantified by line-scan analysis of fluorescence intensity. Data represent mean±s.e.m. of data pooled from three individual experiments (, n=47; , n=50; , n=47). (,) Line scans from – were further analysed to extract total F-actin content at the contacts (*P<0.01; Student's t-test; ) and total barbed ends at the contacts (not significant; Student's t-test; ); data represent mean±s.e.m. () Apical F-actin rings at the zonula adherens. Data represent mean±s.e.m. of data pooled from three individual exp! eriments (FL, n=47; KD, n=47; ΔVCA, n=50; ΔWH1, n=47, ***P<0.0001; Student's t-test). Scale bars: 5 μm. * Figure 4: The N-WASP WH1 domain regulates junctional filament stability and dynamic spatiotemporal organization of F-actin. Actin filament dynamics and organization were analysed in N-WASP-knockdown (KD) cells and knockdown cells expressing RNAi-resistant full-length N-WASP (FL), N-WASPΔVCA or N-WASPΔWH1. () Junctional actin filament stability was measured by transient expression of mRFP–PA-GFP–actin. Transgene expression at the junctions was identified by visualizing mRFP, then PA-GFP was activated and fluorescence signal decay monitored. Average decay of fluorescence intensity is shown (n=8). Individual curves for each of the mutants are shown in Supplementary Fig. S2e. () Turnover of junctional actin was measured by FRAP of transiently expressed GFP–actin. Average recovery of fluorescence intensity is shown (n=10). Individual curves for each of the mutants are shown in Supplementary Fig. S2f. (–) Spatiotemporal patterns of junctional GFP–actin recovery following photobleaching in N-WASP-knockdown cells () and knockdown cells expressing full-length N-WASP (), N-WASPΔVCA () and N-WA! SPΔWH1 (). Representative kymographs (left), and surface intensity plots (right) derived from the kymographs, are shown. Representative frames from the movies for each cell line are shown in Supplementary Fig. S3a. Complete movies are shown in Supplementary Movies S2–5. Scale bars: 0.5 μm. () Lateral pattern of recovery of the GFP–actin at contacts was quantified from kymographs by measuring the angle between the initial point of actin recovery and the final position in N-WASP-knockdown cells (n=6) and knockdown cells reconstituted with full-length N-WASP (n=4), N-WASPΔVCA (ΔVCA, n=8) or N-WASPΔWH1 (ΔWH1, n=6). The angle used for quantification is indicated in . (Data are means±s.e.m. of data pooled from four individual experiments; ***P<0.0001; Student's t-test.) * Figure 5: N-WASP recruits WIRE to regulate the junctional actin cytoskeleton. () Transgenes in N-WASP-knockdown (KD) cells expressing GFP alone or GFP-tagged full-length N-WASP (FL), N-WASPΔVCA or N-WASPΔWH1 isolated by GFP-Trap then immunoblotted for WIRE or GFP. () E-cadherin or WIRE immunoprecipitates (IP) from control or N-WASP-knockdown cells immunoblotted for WIRE, E-cadherin and N-WASP. Full blots are shown in Supplementary Fig. S6. () Confocal images of E-cadherin and WIRE localization at the zonula adherens of confluent Caco-2 cells. Lower-magnification images are in Supplementary Fig. S4a. () Junctional WIRE in confluent N-WASP-knockdown cells or N-WASP-knockdown cells reconstituted with full-length N-WASP. Data represent mean±s.e.m. of data pooled from three individual experiments (FL, n=35; KD, n=43, ***P<0.0001; Student's t-test). (–) WIRE knockdown replicates the N-WASP-knockdown actin phenotype. Control siRNA cells and WIRE siRNA cells stained for WIRE (red) and phalloidin (blue) (). (Lower-magnification images are in Supplementa! ry Fig. S5a,b.) In control cells, WIRE localizes to the zonula adherens between the apical actin rings as assessed by line-scan analysis (). (,) Line-scan analysis of F-actin and barbed ends at the zonula adherens in control siRNA cells () and WIRE-knockdown cells (). Data represent mean±s.e.m. pooled from three individual experiments (n=27–37). (,) Data from and were further analysed for total F-actin content at the zonula adherens (*P<0.01, Student's t-test; ) and total barbed ends (not significant; ). Data represent mean±s.e.m. pooled from three individual experiments (n=27–37, Student's t-test). () Apical actin rings at contacts in control or WIRE siRNA cells. Data represent mean±s.e.m. (n=27–37, ***P<0.0001; Student's t-test). Data in – represent mean ± s.e.m. pooled from three individual experiments (controls, n=27; WIRE knockdown, n=37). (–) Full-length WIRE and WIREΔV can rescue the actin phenotype. Control siRNA and WIRE siRNA Caco-2 cells were! subsequently transfected with empty vector (), full-length WI! RE (), a WIRE mutant lacking the G-actin binding domain (WIREΔV; ) or a WIRE mutant lacking the F-actin binding region (WIREΔBS; ). Monolayers were stained for transgene expression and phalloidin. F-actin phenotype was determined by line-scan analysis of fluorescence intensity. Data represent mean±s.e.m. of data pooled from four individual experiments (, control, n=33, KD, n=31; , control, n=32, KD, n=28; , control, n=26, KD, n=29; , n=27). Representative high-magnification images of F-actin phenotypes and transgene localization are shown in Supplementary Fig. S5c. Scale bars: and , 5 μm. * Figure 6: Junctional biogenesis is perturbed by N-WASP or WIRE knockdown. (–) Monolayers of N-WASP-knockdown (KD) cells reconstituted with full-length N-WASP (FL) cells (), N-WASP-knockdown cells (), Caco-2 cells transfected with a control siRNA () or Caco-2 cells transfected with siRNAs directed against WIRE () were immunostained for N-WASP (,) or WIRE (,) and E-cadherin. Representative images are shown at the region of the zonula adherens. () Effect of N-WASP knockdown on linear integrity of E-cadherin staining at the zonula adherens. Data are mean±s.e.m. (FL, n=251; KD, n=117, P<0.0001; *** Student's t-test). () Effect of WIRE knockdown on linear integrity of E-cadherin staining at the zonula adherens. Data are mean±s.e.m. (control, n=213; WIRE knockdown, n=175, P<0.0001; *** Student's t-test). (,) Effect of N-WASP () or WIRE () RNAi on the reassembly of E-cadherin cell–cell contacts. The length of E-cadherin contacts (pixels) was measured in untreated control monolayers (UT) or at various times after extracellular calcium was restore! d to calcium-depleted monolayers. Data represent mean±s.e.m. (, n=165; , n=74, ***P<0.0001; Student's t-test). Scale bars: 20 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia * Eva M. Kovacs, * Suzie Verma, * Radiya G. Ali, * Aparna Ratheesh & * Alpha S. Yap * School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia * Radiya G. Ali * Division of Genomics and Computational Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia * Nicholas A. Hamilton * Cell Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands * Anna Akhmanova Contributions E.M.K., R.G.A. and A.S.Y. conceived the project and designed experiments; E.M.K., S.V. and R.G.A. carried out experiments; A.R. developed reagents; A.A. carried out protein partner analysis; E.M.K., N.A.H. and A.S.Y. analysed the data; E.M.K. and A.S.Y. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alpha S. Yap Author Details * Eva M. Kovacs Search for this author in: * NPG journals * PubMed * Google Scholar * Suzie Verma Search for this author in: * NPG journals * PubMed * Google Scholar * Radiya G. Ali Search for this author in: * NPG journals * PubMed * Google Scholar * Aparna Ratheesh Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas A. Hamilton Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Akhmanova Search for this author in: * NPG journals * PubMed * Google Scholar * Alpha S. Yap Contact Alpha S. Yap Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (700K) Supplementary Information * Supplementary Movie 2 (700K) Supplementary Information * Supplementary Movie 3 (1M) Supplementary Information * Supplementary Movie 4 (1400K) Supplementary Information * Supplementary Movie 5 (700K) Supplementary Information * Supplementary Movie 6 (1M) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Variegated gene expression caused by cell-specific long-range DNA interactions
- Nat Cell Biol 13(8):944-951 (2011)
Nature Cell Biology | Article Variegated gene expression caused by cell-specific long-range DNA interactions * Daan Noordermeer1, 4, 5 * Elzo de Wit1, 5 * Petra Klous1, 5 * Harmen van de Werken1 * Marieke Simonis1 * Melissa Lopez-Jones2 * Bert Eussen3 * Annelies de Klein3 * Robert H. Singer2 * Wouter de Laat1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:944–951Year published:(2011)DOI:doi:10.1038/ncb2278Received12 September 2010Accepted09 May 2011Published online26 June 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mammalian genomes contain numerous regulatory DNA sites with unknown target genes. We used mice with an extra β-globin locus control region (LCR) to investigate how a regulator searches the genome for target genes. We find that the LCR samples a restricted nuclear subvolume, wherein it preferentially contacts genes controlled by shared transcription factors. No contacted gene is detectably upregulated except for endogenous β-globin genes located on another chromosome. This demonstrates genetically that mammalian trans activation is possible, but suggests that it will be rare. Trans activation occurs not pan-cellularly, but in 'jackpot' cells enriched for the interchromosomal interaction. Therefore, cell-specific long-range DNA contacts can cause variegated expression. View full text Figures at a glance * Figure 1: An ectopic LCR does not activate a natural target gene on the homologous chromosome. () Schematic representation of the endogenous mouse and human β-globin loci. Below each globin gene, gene activity in (transgenic) mice is indicated. () Targeting strategy for the insertion of the human β-globin LCR and a human Aγ-globin–green fluorescent protein (GFP) reporter gene into the 8C3–C4 locus on mouse chromosome eight. () RT–qPCR of Aγ-globin transcript levels, normalized to Hprt1 transcript levels. Data are from at least two independent samples. () Representative examples of DNA FISH showing co-localized and separate 8C3–C4 alleles. DAPI, 4,6-diamidino-2-phenylindole. Scale bar: 2 μm. () Co-localization frequencies of 8C3–C4 alleles. Significance levels are indicated above the graph (G-test). * Figure 2: Contacts of 8C3–C4 with and without the LCR are similar. () Intrachromosomal DNA interactions of 8C3–C4 with (top) and without (bottom) an integrated β-globin LCR are essentially similar, as determined by 4C analysed with a running-mean analysis of microarray data (average probe spacing: 7 kb). () Intrachromosomal DNA interactions of 8C3–C4 with (top) and without (bottom) an integrated β-globin LCR are essentially similar, as determined by 4C analysed with domainograms that visualize probability scores (P-values indicated with colour codes) for the clustering of positive 4C signals over windows ranging in size from 1 to 200 probes. () Interchromosomal 4C data for two chromosomes (7 and 11), analysed as described above. () Validation of 4C results by cryo-FISH; examples of results. Scale bar: 2 μm. () Interaction frequencies with a series of genomic regions, measured by cryo-FISH in wild-type and LCR transgenic fetal livers. The number of cells analysed (n) is indicated. Colour codes indicate the significance of the 4C s! ignal (probe clustering), with green referring to P<0.01 and red referring to P≥0.01. * Figure 3: Within a predetermined genomic environment the ectopic LCR shows preferential interactions with specific genes. () 4C results (running median over sliding windows of 21 probes) for 8C3–C4 with (blue) and without (red) the integrated LCR, at the endogenous β-globin locus on chromosome 7 (left) and the α-globin locus on chromosome 11 (right). () Probes were binned according to increasing difference in (LCR − wild-type) 4C signal and characterized depending on their location relative to highly expressed and GATA-1- and EKLF-regulated genes. The yellow dashed line represents the expected frequency on the basis of all probes. () Venn diagram showing the number of, and overlap between, probes captured more frequently in the integrated LCR 4C experiment for each category of genes analysed in the population marked with an asterisk in . * Figure 4: The ectopic LCR on chromosome 8 enhances the expression of the endogenous βh1 gene on chromosome 7. () Affymetrix gene-expression data for all probe-sets analysing βh1 transcripts (n=3). () RT–qPCR comparison of βh1 gene expression between multiple wild-type and homozygous LCR-AS littermates, normalized to Hprt1 transcript levels and to own wild-type littermates. () RT–qPCR analysis showing that the insertion at 8C3–C4 of a neomycin selection cassette instead of the hLCR does not lead to upregulation of the βh1 gene. Data from two independent samples. () RT–qPCR analysis of expression of β- and α-globin genes in wild-type and homozygous LCR littermates. Error bars: standard error on the basis of 3 littermates (n). * Figure 5: Increased βh1 mRNA levels in cells showing interchromosomal LCR–βh1 interactions. () RNA FISH on E14.5 fetal liver cells, with one cell showing strongly increased βh1 mRNA levels in the cytoplasm ('jackpot cell'). Scale bars: 2 μm. () Enlargement of the 'jackpot cell', revealing an interchromosomal interaction between the endogenous β-globin locus on chromosome 7 and the ectopic LCR on chromosome 8. (I–IV) Probes from one focal plane are shown separately and merged. (V) Z stack showing all RNA signals for βmaj and 8C3–C4. () Quantification of RNA FISH. Determining interchromosomal interaction frequencies between the endogenous β-globin locus and 8C3–C4 without (wild type) and with an integrated LCR (hLCR-AS), in all red blood cells and in 'βh1 jackpot cells'. The number of cells analysed (n) is indicated. NS: no significant difference. * Figure 6: Increased βh1 and β-major mRNA levels in cells showing interchromosomal LCR–βh1 interactions. (,) Automated RNA-FISH image-analysis (see Methods) results, showing that cells in which the ectopic LCR interacts in trans with the endogenous β-globin locus more often have high βh1 () or β-major () transcript levels than cells that have the loci apart. () Cells in which the ectopic LCR interacts in trans with the endogenous α-globin locus do not differ in their levels of mRNA for α-globin from cells without this interchromosomal interaction. The probability score for the difference in distributions for interacting and non-interacting cells is calculated by a one-sided Kolmogorov–Smirnov test. () SEV: variegated expression among otherwise identical cells caused by cell-specific long-range DNA interactions (intra- or interchromosomal) that are relatively stable during interphase. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24614 * GSE5891 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Daan Noordermeer, * Elzo de Wit & * Petra Klous Affiliations * Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands * Daan Noordermeer, * Elzo de Wit, * Petra Klous, * Harmen van de Werken, * Marieke Simonis & * Wouter de Laat * Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA * Melissa Lopez-Jones & * Robert H. Singer * Department of Clinical Genetics, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam, The Netherlands * Bert Eussen & * Annelies de Klein * Present address: Laboratory of Developmental Genomics, School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland * Daan Noordermeer Contributions D.N. and W.d.L. designed the experiments, analysed the data and, with help from E.d.W., wrote the manuscript. D.N. and P.K. carried out experiments. E.d.W. analysed 4C data and developed the automated FISH image analysis. H.v.d.W. analysed 4C and microarray expression data. M.S. carried out 4C experiments. M.L-J. and R.H.S. designed and synthesized RNA-FISH probes. B.E. and A.d.K. helped with the FISH experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Wouter de Laat Author Details * Daan Noordermeer Search for this author in: * NPG journals * PubMed * Google Scholar * Elzo de Wit Search for this author in: * NPG journals * PubMed * Google Scholar * Petra Klous Search for this author in: * NPG journals * PubMed * Google Scholar * Harmen van de Werken Search for this author in: * NPG journals * PubMed * Google Scholar * Marieke Simonis Search for this author in: * NPG journals * PubMed * Google Scholar * Melissa Lopez-Jones Search for this author in: * NPG journals * PubMed * Google Scholar * Bert Eussen Search for this author in: * NPG journals * PubMed * Google Scholar * Annelies de Klein Search for this author in: * NPG journals * PubMed * Google Scholar * Robert H. Singer Search for this author in: * NPG journals * PubMed * Google Scholar * Wouter de Laat Contact Wouter de Laat Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information Excel files * Supplementary Information (126K) Supplementary Table 1 PDF files * Supplementary Information (3600K) Supplementary Information Additional data - Cytoskeletal polarity mediates localized induction of the heart progenitor lineage
- Nat Cell Biol 13(8):952-957 (2011)
Nature Cell Biology | Letter Cytoskeletal polarity mediates localized induction of the heart progenitor lineage * James Cooley1 * Stacia Whitaker1 * Sarah Sweeney1, 2 * Scott Fraser2 * Brad Davidson1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:952–957Year published:(2011)DOI:doi:10.1038/ncb2291Received03 June 2010Accepted06 June 2011Published online24 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cells must make appropriate fate decisions within a complex and dynamic environment1. In vitro studies indicate that the cytoskeleton acts as an integrative platform for this environmental input2. External signals regulate cytoskeletal dynamics and the cytoskeleton reciprocally modulates signal transduction3, 4. However, in vivo studies linking cytoskeleton/signalling interactions to embryonic cell fate specification remain limited5, 6, 7. Here we show that the cytoskeleton modulates heart progenitor cell fate. Our studies focus on differential induction of heart fate in the basal chordate Ciona intestinalis. We have found that differential induction does not simply reflect differential exposure to the inductive signal. Instead, pre-cardiac cells employ polarized, invasive protrusions to localize their response to an ungraded signal. Through targeted manipulation of the cytoskeletal regulator CDC42, we are able to depolarize protrusive activity and generate uniform heart pro! genitor fate specification. Furthermore, we are able to restore differential induction by repolarizing protrusive activity. These findings illustrate how bi-directional interactions between intercellular signalling and the cytoskeleton can influence embryonic development. In particular, these studies highlight the potential for dynamic cytoskeletal changes to refine cell fate specification in response to crude signal gradients. View full text Figures at a glance * Figure 1: Nature and timing of heart progenitor lineage induction. (–) Top, ventral and lateral optical sections of FGF9in situ hybridizations (m, mesenchyme; tm, tail muscle; scale bars, 30 μm). () Top, in vivo dpERK antibody staining indicates differential MAPK activation in the smaller heart progenitor cells (arrowheads) shortly after founder cell division; lateral view; scale bar, 10 μm. (–) Bottom, illustrations of the spatial relationship between founder cells (B8.9 and 8.10) and FGF9-expressing cells (hp, heart progenitor; atm, larger sister cell lineage; m, mesenchyme; GPI-GFP refers to a membrane-anchored GFP fusion, glycosylphosphatidylinositol-GFP driven by the Mesp enhancer). (–) Representative founder cell clone pairs resulting from staged dissociations; scale bars, 5 μm. () Percentage of induction (FoxF–RFP-positive cells) produced by founder cells isolated at discrete stages; n=1,117 for stage 10–12, n=658 for stage 13, n=651 for stage 14 and n=693 for stage 15. Embryos are shown anterior to the left in the! se and all subsequent figures. Note that in these and subsequent figures founder cells are labelled by Mesp-ensGFP (a fusion of the microtubule binding protein ensconsin with multiple copies of GFP). See Methods for further details. * Figure 2: Localized protrusive activity correlates with localized induction. (–) Top, ventral projections of membrane-anchored-GFP (GPI–GFP)-labelled founder lineage cells, stage 14–15. Bottom left, lateral optical sections through corresponding stacks at the position indicated in - by the white line. Bottom right, diagrams illustrating invasion of underlying ventral epidermis. (–) Representative, lateral optical sections through staged B8.9 founder cells (,) and their progeny (); green represents utrophin–GFP and red represents pTyr for both images and accompanying schematics. Asterisks indicate the anterior-ventral position at which heart progenitor cells (hp) consistently emerge; atm, anterior tail muscle lineage. () Schematic of a dividing founder cell (after Fig. 2e) illustrating the method for comparing pTyr levels between the anterior-ventral (AV) and posterior-ventral (PV) membranes. () Quantitative analysis of membrane pTyr ratios (AV versus PV). Note that significant pTyr polarization (asterisk) was first observed at stage 14 (sta! ge 12 P=0.64, n=92; stage 13 P=0.21, n=89; stage 14 P=9.3×10−12, n=89) and that this polarization was dependent on FGF signalling (stage 14 Mesp–FGFRdn P=0.103, n=57). Scale bars, 10 μm. * Figure 3: Localized CDC42 activity is required for differential induction. () Lateral optical section of a stage 14 GPI–GFP-labelled founder cell illustrating the anterior-ventral (AV) and posterior-ventral (PV) regions used to generate FRET data. () FRET data, n=30 for wild-type sensor and n=26 for T17N; the inactive T17N probe serves as a negative control. (–) Representative results from induction assays, fluorescent reporters and transgenic backgrounds as indicated above and to the left respectively. In , the red channel in the merged image was amplified to better visualize the embryo. (,) Quantitative data showing the percentage of transgenic embryos exhibiting loss of localized induction (n=375 for Cdc42, n=466 for Q61L, n=461 for F28L, n=240 for F28L ΔRho and n=250 for ΔRho; ) and loss of polarized CDC42–GFP enrichment along the heart progenitor/ventral membrane (n=31 for Cdc42, n=31 for Q61L, n=25 for F28L and n=30 for F28L ΔRho; ). () Lateral projection of dividing founder cell exhibiting enrichment of CDC42–GFP (green) along! the presumptive heart progenitor membrane (asterisk). Scale bars, 10μm, except for , main panel, 20μm. * Figure 4: Cytoskeletal polarity directs differential induction. (–) Representative results from induction assays, reporters and transgenic backgrounds as indicated above and to the left respectively. In merged images the red channel has been amplified to better visualize the whole embryo. Scale bars, 20 μm (columns 1–3); 40 μm (column 4). () pTyr ratio comparing AV with PV membranes; n=25 for Cdc42, n=29 for WaspΔVCA, n=28 for Q61L and n=33 for Q61L+WaspΔVCA; asterisks indicate a significant difference (P<0.005) in the AV versus PV measurements for that sample set. () Quantitative data for induction assays showing the percentage of transgenic embryos with expanded induction; n=374 for Q61L, n=266 for Q61L+WaspΔVCA, n=178 for Q61L+Wasp and n=307 for Q61L+Par6-CRIB; Q61L versus Q61L+Wasp, P=0.53; Q61L versus Q61L+Par6-CRIB, P=0.81. () Four-step model for differential specification of the heart progenitor lineage. (1) Ungraded exposure to growth factor leads to uniform receptor occupancy. (2) Receptor activation is enriched alo! ng the ventral membrane in association with enhanced protrusive activity. (3) As founder cells enter mitosis, localized invasive protrusions facilitate restriction of receptor activation to the ventral-anterior membrane. (4) Following division, MAPK pathway activation (nuclear dpERK) is restricted to the ventral daughter, leading to differential expression of heart progenitor genes. Polarization of a non-graded inductive signal or amplification of a weakly graded signal may involve: reciprocal feedback between RTK signalling, adhesion, GTPase regulation and actin dynamics4; restricted contact with epidermal cells or matrix as founder cells round up during mitosis; or intrinsic polarization of microtubule dynamics, GTPase activity or endosome trafficking associated with asymmetric spindle positioning32, 37. Author information * Author information * Supplementary information Affiliations * Department of Molecular and Cellular Biology, Molecular Cardiovascular Research Program, University of Arizona, Arizona 85724, USA * James Cooley, * Stacia Whitaker, * Sarah Sweeney & * Brad Davidson * Department of Biology, Imaging Center, Beckman Institute, California Institute of Technology, California 91125, USA * Sarah Sweeney & * Scott Fraser Contributions J.C., S.W. and B.D. designed the project and carried out most of the experiments including data analysis. S.F. provided material and technical support for live-cell imaging carried out by S.S., J.C. and B.D. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Brad Davidson Author Details * James Cooley Search for this author in: * NPG journals * PubMed * Google Scholar * Stacia Whitaker Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah Sweeney Search for this author in: * NPG journals * PubMed * Google Scholar * Scott Fraser Search for this author in: * NPG journals * PubMed * Google Scholar * Brad Davidson Contact Brad Davidson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (400K) Supplementary Information * Supplementary Movie 2 (800K) Supplementary Information * Supplementary Movie 3 (500K) Supplementary Information * Supplementary Movie 4 (3M) Supplementary Information * Supplementary Movie 5 (5M) Supplementary Information * Supplementary Movie 6 (7M) Supplementary Information PDF files * Supplementary Information (1300K) Supplementary Information Additional data - Mir193b–365 is essential for brown fat differentiation
- Nat Cell Biol 13(8):958-965 (2011)
Nature Cell Biology | Letter Mir193b–365 is essential for brown fat differentiation * Lei Sun1, 7 * Huangming Xie1, 2, 7, 8 * Marcelo A. Mori3 * Ryan Alexander1, 4 * Bingbing Yuan1 * Shilpa M. Hattangadi1, 5 * Qingqing Liu1 * C. Ronald Kahn3 * Harvey F. Lodish1, 4, 6 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:958–965Year published:(2011)DOI:doi:10.1038/ncb2286Received24 March 2011Accepted27 May 2011Published online10 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mammals have two principal types of fat. White adipose tissue primarily serves to store extra energy as triglycerides, whereas brown adipose tissue is specialized to burn lipids for heat generation and energy expenditure as a defence against cold and obesity1, 2. Recent studies have demonstrated that brown adipocytes arise in vivo from a Myf5-positive, myoblastic progenitor by the action of Prdm16 (PR domain containing 16). Here, we identified a brown-fat-enriched miRNA cluster, MiR-193b–365, as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes markedly impaired brown adipocyte adipogenesis by enhancing Runx1t1 (runt-related transcription factor 1; translocated to, 1) expression, whereas myogenic markers were significantly induced. Forced expression of Mir193b and/or Mir365 in C2C12 myoblasts blocked the entire programme of myogenesis, and, in adipogenic conditions, miR-193b induced myoblasts to differentiate into bro! wn adipocytes. Mir193b–365 was upregulated by Prdm16 partially through Pparα. Our results demonstrate that Mir193b–365 serves as an essential regulator for brown fat differentiation, in part by repressing myogenesis. View full text Figures at a glance * Figure 1: Mir193b–365 is enriched in BAT. () Heat map showing the expression of miRNAs that are enriched in BAT when compared with epididymal WAT and skeletal muscle. Red denotes higher and green denotes lower relative to the mean of the six samples for each miRNA. (P<0.1, ANOVA—analysis of variance.) () rtPCR analysis of miR-193b, miR-365 and a control miRNA, miR-223, expression levels in BAT relative to other adult mouse tissues. n=3. () rtPCR analysis of miR-193b and miR-365 expression levels during adipogenesis of primary brown adipocyte cultures. n=3. Means±s.e.m. * Figure 2: Mir193b–365 is required for brown adipocyte adipogenesis. () SVF cells from brown fat were transfected with LNA miRNA inhibitors (100 nM) one day before differentiation. RNAs were collected at day 4. rtPCR was used to examine the expression of these miRNAs. n=3. NC, negative control. () mRNAs from cultured primary brown adipocytes (day 4) transfected with each inhibitor or control inhibitor were analysed by microarray analysis. On the x axis is the relative expression of each gene calculated as a log2 ratio (x axis) between its intensity in the miRNA-inhibited sample and its intensity in the control-inhibitor sample. The cumulative fraction (y axis) was plotted as a function of the relative expression (x axis). 'miRNA targets' (red line) represents the population of genes containing miRNA-binding sites predicted by TargetScan, and 'Control' (black line) represents all genes lacking binding sites for the miRNA. The 'targets' curve shifts to the right with a P value <0.05 as determined by the one-sided Kolmogorov–Smir! nov test, indicating a trend of upregulation of predicted targets in response to transfection of the miRNA inhibitor. () Oil red O staining was used to determine the accumulation of lipid droplets in brown adipocytes (day 4). () SVF cells from brown fat were co-transfected with LNA miRNA inhibitors (100 nM) and miRNA mimics (16.7 nM mimic-Mir193a, 16.7 nM mimic-Mir193b and 16.7 nM mimic-Mir365, or 50 nM control, ctl, mimic) one day before differentiation. Four days after differentiation, oil red O staining was used to determine lipid-droplet content. (,) rtPCR analysis of the expression of adipogenesis markers () and brown fat markers (). n=3. () Western blot to examine the expression of Ucp1. Ucp1, uncoupling protein 1; Gapdh, glyceraldehyde 3-phosphate dehydrogenase. () Effect of miR-193 knockdown on expression of Runx1t1. rtPCR was carried out to examine the expression of Runx1t1. Runx1t1-P1 and Runx1t1-P2 represent two sets of PCR primers. n=3. () Luciferase r! eporter assay to examine the interactions between miR-193b and! the predicted target site in the Runx1t1 3′UTR. Plasmids with the Runx1t1 3′UTRs or mutated UTRs were co-transfected with miR-193b mimic or a control mimic into 293T cells. Renilla luciferase activity was measured by a Dual-Glo luciferase assay system and normalized to internal control firefly luciferase activity. n=6. *P<0.05, Student's t-test; means±s.e.m. Uncropped images of blots are shown in Supplementary Fig. S2. * Figure 3: Ectopic expression of miR-193b and/or miR-365 inhibits C2C12 myogenic differentiation. () SVF cells from brown fat were transfected with LNA miRNA inhibitors (100 nM) one day before differentiation. At day 4, RNAs were extracted and rtPCR was carried out to detect the expression of myogenic markers. n=3. () Representative micrographs of cells (day 6 in 2% horse serum) differentiated from C2C12 myoblasts expressing retroviral miR-193b and/or miR-365, or control. GFP (green fluorescent protein) was expressed under control of an internal ribosome entry site downstream of the miRNA to visualize transfected cells. () Western blot with triplicate biological repeats to examine the expression of myosin on ectopic expression of miR-193b and/or miR-365. () rtPCR analysis for expression of muscle markers. n=3. (,) rtPCR () and western () analysis for predicted miR-193b targets, Cdon and Igfbp5, in C2C12 cells expressing miR-193b or a control vector. n=3. () SVF cells from brown fat were transfected with LNA–miR-193a and/or LNA–miR-193b, and differentiated for 4 day! s. rtPCR was carried out to examine the expression of Cdon and Igfbp5. n=6. () Luciferase reporter assay to examine the interactions between miR-193b and the predicted target site in the Cdon and Igfbp5 3′UTRs. Plasmids with the Igfbp5 or Cdon 3′UTRs or mutated UTRs were co-transfected with miR-193b mimic or a control mimic into 293T cells. Renilla luciferase activity was measured by a Dual-Glo luciferase assay system, normalized to internal control firefly luciferase activity. n=6. *P<0.05, Student's t-test; means±s.e.m. Uncropped images of blots are shown in Supplementary Fig. S4. * Figure 4: Ectopic expression of miR-193b induces C2C12 to form brown adipocytes under adipogenic differentiation conditions. () C2C12 cells ectopically expressing miR-193b or control were exposed to pro-adipogenic conditions (described in Methods) for 5 days. Oil red O staining was used to assess lipid accumulation in cells. Representative micrographs of these cells are depicted (bottom row). () Western blot for myogenic markers Pax3 and MyoD and brown fat marker Ucp1. n=3. () rtPCR analysis for expression of common adipogenesis markers (upper panel) and brown-fat-selective markers (lower panel). n=3. () C2C12 cells expressing miR-193b (day 6) were stimulated with 500 μM dibutyryl cAMP for 4 h, and the expression of thermogenic markers, Pgc-1αand Ucp1, was examined by rtPCR. n=3. () The metabolic profile of C2C12 cells expressing miR-193b (day 6) was assessed using a Seahorse XF24 extracellular flux analyser. A representative curve of the OCRs of control and miR-193b-expressing cells in their basal states and on treatment with drugs used to dissect the multiple components of the respiration ! process is plotted in the top left panel. The parameters analysed are represented by different colours in the upper panel and quantitated in other panels. In vitro differentiated primary brown adipocytes (BAT) were used as a reference. n=8. *P<0.05, Student's t-test; means±s.e.m. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 5: Mir193b–365 is regulated by Prdm16. () Subcutaneous white preadipocytes (left) and C2C12 myoblasts (right) were infected by retrovirus expressing Prdm16 two days before differentiation. Five days after differentiation, rtPCR was carried out to examine the expression of miR-193b, miR-365 and as a control miR-223. n=3. () Primary brown preadipocytes were infected by retrovirus expressing shRNA (short hairpin RNA) targeting Prdm16 two days before differentiation. By day 5 of differentiation, rtPCR was used to examine the mRNA level of Prdm16 (left) and of miR-193b, miR-365 and the control miR-223 (right). n=3. () rtPCR analysis for Pparα in primary brown adipocytes retrovirally expressing shRNA for Prdm16. () rtPCR analysis of miR-193b, miR-365, Prdm16 and Pparα expression levels during adipogenesis of primary brown adipocyte cultures. n=3. () ChIP analysis for the interaction between Pparα and the promoter region of Mir193b–365. Immortalized brown adipocyte cultures (day 5) were fixed and sheared by ultraso! nication. Immunoprecipitation was carried out with anti-Pparα and control IgG. Recovered DNA was amplified by three pairs (P1, P2 and P3) of primers designed to span the 1 kb promoter region. Input samples are in the upper panel and immunoprecipitated ones in the lower. () Primary brown preadipocytes were transfected with siRNA targeting Pparα, and differentiated for three days. rtPCR was carried out to examine the expression of Pparα and of miR-193b and miR-365. Pparγ and miR-223 were used as controls. n=3. NC, negative control. () rtPCR was carried out to examine the expression of miRNAs in brown adipose tissue isolated from Pparα knockout mice (8 weeks old, male). Age-matched wild-type mice were used as controls. n=6. *P<0.05, Student's t-test; means±s.e.m. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Lei Sun & * Huangming Xie Affiliations * Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA * Lei Sun, * Huangming Xie, * Ryan Alexander, * Bingbing Yuan, * Shilpa M. Hattangadi, * Qingqing Liu & * Harvey F. Lodish * Computation and Systems Biology, Singapore-MIT Alliance, National University of Singapore, 4 Engineering Drive 3, Singapore 117576, Singapore * Huangming Xie * Section on Integrative Physiology and Metabolism, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA * Marcelo A. Mori & * C. Ronald Kahn * Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Ryan Alexander & * Harvey F. Lodish * Department of Hematology, Boston Children's Hospital, Boston, Massachusetts 02115, USA * Shilpa M. Hattangadi * Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Harvey F. Lodish * Present address: Division of Newborn Medicine, Department of Medicine, Children's Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115, USA * Huangming Xie Contributions L.S., H.X. and H.F.L. conceived the project and designed the experiments. L.S., H.X., M.A.M., R.A., B.Y., S.M.H. and Q.L. carried out the experiments. All authors analysed data. L.S., H.X., M.A.M. and H.F.L. wrote the manuscript. C.R.K. and H.F.L. supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Harvey F. Lodish Author Details * Lei Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Huangming Xie Search for this author in: * NPG journals * PubMed * Google Scholar * Marcelo A. Mori Search for this author in: * NPG journals * PubMed * Google Scholar * Ryan Alexander Search for this author in: * NPG journals * PubMed * Google Scholar * Bingbing Yuan Search for this author in: * NPG journals * PubMed * Google Scholar * Shilpa M. Hattangadi Search for this author in: * NPG journals * PubMed * Google Scholar * Qingqing Liu Search for this author in: * NPG journals * PubMed * Google Scholar * C. Ronald Kahn Search for this author in: * NPG journals * PubMed * Google Scholar * Harvey F. Lodish Contact Harvey F. Lodish Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1200K) Supplementary Information Additional data - Cleavage of cohesin rings coordinates the separation of centrioles and chromatids
- Nat Cell Biol 13(8):966-972 (2011)
Nature Cell Biology | Letter Cleavage of cohesin rings coordinates the separation of centrioles and chromatids * Laura Schöckel1 * Martin Möckel1 * Bernd Mayer1 * Dominik Boos2 * Olaf Stemmann1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:966–972Year published:(2011)DOI:doi:10.1038/ncb2280Received14 October 2010Accepted13 May 2011Published online10 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cohesin pairs sister chromatids by forming a tripartite Scc1–Smc1–Smc3 ring around them1, 2. In mitosis, cohesin is removed from chromosome arms by the phosphorylation- dependent prophase pathway3. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1–PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4, 5, 6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase7. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic8, 9. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 i! s replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex. View full text Figures at a glance * Figure 1: Centriole disengagement is promoted by separase and inhibited by non-cleavable Scc1. () Characterization of recombinant, securin-less human separases. Wild-type (WT), protease-dead (PD), Cdk1-binding-deficient, phosphorylation-site mutant (SA; S1126A) and doubly mutant (SA+PD; S1126A+protease-dead) separases were characterized by Coomassie staining (CBB) and for their ability to cleave 35S-labelled wild-type (WT) Scc1 or a non-cleavable (NC) mutant. Scc1 fragments are labelled by arrowheads. () Xenopus laevis separase was blocked by addition of securinΔN or cyclin B1ΔN to CSF extract (see Supplementary Fig. S1b). The extract was supplemented with engaged centrioles and securin-free human separase (see ) and released. Finally, centrosomes were re-isolated, immunostained for centrin 2 and C-NAP1 and analysed by immunofluorescence microscopy. Scale bars =0.5 μm. Centriole disengagement specifically induced by the proteolytic activity of separase was calculated by subtraction of the corresponding background, that is, centriole disengagement in the presence ! of protease-dead or SA+PD separase (20–40%). Shown are averages (grey bars) of three or four independent experiments (circles). More than 60 centrosomes per sample were counted. Note that depending on arrest efficiency and individual preparation a subset of centrosomes appeared disengaged even in negative controls. Thus, values within each colour-coded group were normalized to an average background of the corresponding negative control in all further quantifications. () Whole-cell extracts (WCE, upper panels) and isolated chromatin (lower panels) from thymidine/nocodazole-synchronized S1126A cells expressing GFP-tagged wild-type or non-cleavable Scc1 were characterized by western blot or CBB. Lanes in the lower panels, although not directly juxtaposed, are from the same gel. () Separase-induced chromosome splitting was determined from spreads by background subtraction (5–10%), that is, percentage of cells with one-chromatid chromosomes in the absence of Tet (negative co! ntrols). Shown are averages (bars) of two independent experime! nts (circles). 100 cells for each were counted. () Centriole disengagement is induced by activation of separase in prometaphase and is blocked by non-cleavable Scc1. Centrosomes were spun from lysates of siRNA- and nocodazole-treated S1126A cells directly onto coverslips and analysed by immunofluorescence microscopy. Separase-induced centriole disengagement was determined by background subtraction (25%), that is, centriole disengagement in the absence of Tet (negative controls). Bars, averages; circles, two independent experiments (150 centrosomes per column). Uncropped images of blots are shown in Supplementary Fig. S4. * Figure 2: Cleavage of an engineered Scc1 triggers centriole disengagement in vitro. () The first separase cleavage site in Scc1 was replaced by one for HRV 3C protease (schematic representation). 35S-labelled wild-type and HRV-Scc1 were incubated with separase (Sep.) or HRV protease and analysed by SDS–polyacrylamide gel electrophoresis autoradiography. Main Scc1 cleavage fragments are labelled by arrowheads. Note that HRV-Scc1 can still be cleaved by separase at the second cleavage site. () HRV-Scc1 interacts with other cohesin subunits as judged by immunoprecipitation–western blot analysis of transiently transfected Hek293T cells. GFP-expressing cells served as a negative control (−). () Episomally encoded wild-type and HRV-Scc1 are inducibly and equally expressed in Hek293T cells as judged by immunoblotting. () Transfection of a 3′UTR-directed siRNA results in depletion of endogenous and increase of recombinant Scc1 as exemplified by western blot analysis of the stable HRV-Scc1 cell line. Notably, the knockdown reproducibly resulted in greater le! vels of the recombinant Scc1, indicating that cells try to keep a constant total Scc1 concentration. () Episomal cell lines expressing wild-type or HRV-Scc1 were transfected with SCC1 siRNA or mock treated as indicated. Four days later, cells were treated for 12 h with nocodazole and then collected. Lysates were combined with bacterially expressed HRV protease before spinning of centrosomes onto coverslips. Given are the percentages of centriole disengagement after subtraction of the background (wild-type Scc1 samples; −siRNA: 18–26%, +siRNA: 14–17%= negative controls). Experiments were normalized to wild-type Scc1 with or without siRNA (blue and orange circles, respectively). Shown are averages (grey bars) of three or four independent experiments. Between 30 and 200 centrosomes per sample were counted. () Western blot analysis of HRV protease-treated lysates (see ) revealed specific cleavage of HRV-Scc1 (arrowhead). Note that the shown lanes, although not directly ! juxtaposed, nevertheless originate from the same gel. Uncroppe! d images of blots are shown in Supplementary Fig. S4. * Figure 3: Cleavage of HRV-Scc1 triggers centriole disengagement in vivo. () Expression of HRV protease triggered centriole disengagement in Hek293T cells, in which endogenous Scc1 had been replaced by HRV-Scc1. Episomal cell lines were induced with Tet and transfected with SCC1 siRNA and expression plasmids as indicated at the top. In parallel (see time line at the top), cells were thymidine/nocodazole synchronized before centrosome isolation and immunofluorescence microscopy. Given are the percentages of centriole disengagement after subtraction of the background (23–39%), that is, disengagement in the corresponding negative controls (−Tet or GFP instead of HRV protease, colour coded). Averages (grey bars) of three independent experiments (circles) are shown, representing at least 370 centrosomes per column. () Tet-induced episomal cell lines were transfected with SCC1 siRNA. Three days later, cells were additionally transfected with HRV protease- and securinΔN expression plasmids in nocodazole-containing medium (see the time line at the to! p). After release (Rel.) into G1 phase, cells were fixed and stained for centrin 2, C-NAP1 and DNA. Insets from the top images (scale bars =1 μm) are shown magnified below (scale bars =250 nm). Between 100 and 400 centrosomes per sample were counted. Shown are averages (grey bars) of two independent experiments (dots). The background (46.5% disengaged centrioles in wild-type Scc1 samples = negative controls) was subtracted to quantify HRV protease-induced centriole disengagement in HRV-Scc1-expressing cells. * Figure 4: Ectopic opening of the cohesin ring within Smc3 triggers centriole disengagement. () TEV protease-cleavable human Smc3 (TEV-Smc3) was generated by integration of three consecutive sites (TEV3) into two opposing positions of minimal coiled-coil probabilities within the Smc3 arm (schematic representation). Treatment of 35S-labelled TEV-Smc3 but not wild-type Smc3 with TEV protease resulted in three fragments, marked N, M and C for N-terminal, middle and carboxy-terminal, respectively, as judged by SDS–polyacrylamide gel electrophoresis autoradiography. () TEV-Smc3 retains competence to interact with other cohesin subunits as judged by immunoprecipitation–western blot analysis of transiently transfected Hek293T cells. GFP-expressing cells served as negative control (−). () Hek293T cells were co-transfected with wild-type or TEV-Smc3 expression plasmids and SMC3 or GL2 siRNA, as indicated. Two days later and 12 h after nocodazole addition, lysates were prepared, incubated with TEV protease and analysed by western blot and immunofluorescence microscopy! of pelleted centrosomes. Endogenous Smc3 and cleavage fragments of TEV-Smc3 are labelled by a black arrow and grey arrowheads, respectively. Percentages of centriole disengagement are indicated at the bottom. The background (21 and 25% disengaged centrioles in wild-type Smc3 with and without siRNA, respectively = negative controls) was subtracted from the corresponding TEV-Smc3 samples to quantify TEV protease-induced centriole disengagement (colour coding). 200 centrosomes for each were counted. n.d., not determined. () Hek293T cells were transfected with expression plasmids encoding wild-type or HRV-Scc1 or wild-type or TEV-Smc3 as indicated. In the case of Smc3, a corresponding siRNA was co-transfected, which targets the 3′UTR of the endogenous mRNA. Following thymidine/nocodazole synchronization, centrosomes were isolated and incubated either alone or together with TEV or HRV protease in vitro. Finally, centrosomes were analysed by immunofluorescence microscopy. Give! n are averages (grey bars) of centriole disengagement from thr! ee independent experiments. Colour coding of circles identifies the corresponding background (27–30%) used for normalization. Between 400 and 700 centrosomes were counted in each sample. Uncropped images of blots are shown in Supplementary Fig. S4. * Figure 5: The prophase pathway promotes centriole disengagement. () Wapl depletion partially rescues premature sister chromatid separation in nocodazole-treated S1126A cells as judged by chromosome spreading. Shown are averages of three independent experiments representing a total of 160–200 cells per column. () Wapl depletion partially rescues premature centriole disengagement. Centrosomes from WAPL siRNA-treated S1126A cells were isolated and processed for immunofluorescence microscopy. Separase-induced centriole disengagement was determined as described in Fig. 1e. Shown are averages (grey bars) of three independent experiments (circles) representing a total of 400 centrosomes per column. Colour coding of circles identifies the corresponding background used for normalization (15–27% for GL2 siRNA without Tet and 21–28% for WAPL siRNA without Tet). () Cleavage of HRV-Scc1 by HRV protease is unaffected by Plk1. 35S-labelled wild-type or HRV-Scc1 was incubated with active separase or HRV protease in the presence or absence of Plk1. ! Cleavage reactions were resolved by SDS–polyacrylamide gel electrophoresis and analysed by autoradiography. () Plk1 stimulates separase-dependent centriole disengagement. Wild-type or HRV-Scc1-containing centrosomes were incubated in CSF extract in the presence or absence of active separase (Sep.), HRV protease or BI2536 (200 nM), as indicated, re-isolated and imaged by immunofluorescence microscopy. For each condition, 100–200 centrosomes were analysed. The percentage of centriole disengagement in the absence of protease and Plk1 inhibitor (20 and 24% for wild-type and HRV-Scc1, respectively) was used for background subtraction. This correction also explains the slightly negative values in the absence of Plk1- and protease function. The slight inhibitory effect of BI2536 on HRV samples indicates that disengagement of a small fraction of G2 centrosomes is aided by the prophase pathway owing to limiting protease activity. () The dual use of cohesin ensures coordination o! f the chromosome- and the centrosome cycle. The model proposes! that centrioles (shades of grey), in a similar way to sister chromatids (light blue), are held together by two pools of cohesin (red), that is, a prophase-responsive fraction and one protected by shugoshin 1 (dark blue). Consequently, centriole disengagement and sister-chromatid separation occur in highly similar fashions, with separase removing the last of cohesin by proteolytic cleavage of Scc1. Author information * Author information * Supplementary information Affiliations * University of Bayreuth, 95440 Bayreuth, Germany * Laura Schöckel, * Martin Möckel, * Bernd Mayer & * Olaf Stemmann * Cancer Research UK, Clare Hall Laboratories, EN6 3LD Hertfordshire, UK * Dominik Boos Contributions L.S. carried out all experiments, M.M. created the episomal cell lines, helped with cloning of TEV-Smc3 and independently reproduced many experiments, B.M. produced Plk1, designed the experiment shown in Fig. 3b and independently confirmed several experiments, D.B. started the project and O.S. designed the research and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Olaf Stemmann Author Details * Laura Schöckel Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Möckel Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd Mayer Search for this author in: * NPG journals * PubMed * Google Scholar * Dominik Boos Search for this author in: * NPG journals * PubMed * Google Scholar * Olaf Stemmann Contact Olaf Stemmann Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (400K) Supplementary Information Additional data - A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells
- Nat Cell Biol 13(8):973-980 (2011)
Nature Cell Biology | Letter A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells * Sungjin Park1 * Amy L. Szumlanski1 * Fangwei Gu1 * Feng Guo1 * Erik Nielsen1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:973–980Year published:(2011)DOI:doi:10.1038/ncb2294Received18 October 2010Accepted07 June 2011Published online17 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material1. In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae1. We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP–CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (14)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells. View full text Figures at a glance * Figure 1: Localization and role of cell-wall polysaccharides in root-hair cells. (,), The presence and distribution of cellulose or cellulose-like polysaccharides were visualized in seven-day-old wild-type (Col-0) A. thaliana seedlings stained with S4B () or CBM3a (). Medial confocal sections were collected using a 40× lens with bright-field optics (upper panels) or spinning-disc confocal microscopy and appropriate excitation and emission filters (lower panels). Immunodetection of root-hair-bound CBM3a was specific, as treatment of root hairs with rabbit anti-His–FITC antibodies alone gave no signal (, right panels). Scale bars, 10 μm. () Seven-day-old wild-type A. thaliana seedlings were transferred to a perfusion chamber for bright-field microscopy (left panel). Normal root-hair expansion was observed with a ×10 lens for 10 min while seedlings were grown in 0.25× Murashige and Skoog (MS) medium (middle panel). After 10 min of equilibration, perfusion of the chamber was changed to medium containing 20 μM DCB and the effect on growing root! hairs was observed for another 30 min (right panel). Root hairs that popped are indicated by arrowheads, whereas root hairs that formed bulges are indicated by asterisks (scale bars, 10 μm). (,) Quantification of DCB () and cell-wall-degrading enzyme () effects. Growing root hairs from seven-day-old seedlings were treated with 0.25× MS containing the indicated DCB concentrations (), or were treated with the indicated cell-wall-degrading enzymes (XGase, xyloglucanase; PLase, pectate lyase) at 5 U ml−1 either in 0.25× MS or 0.25×MS+0.8 mg ml−1 carboxymethyl (CM)-cellulose (), and growth of root hairs was assessed after 30 min. Data show the averages of 3 seedlings per treatment, counting >20 hairs per seedling; error bars, ±s.d. * Figure 2: Localization of fluorescent CESA and CSLD3 fusion proteins in growing root-hair cells. () Medial confocal sections were collected from growing root-hair cells of seven-day-old A. thaliana seedlings stably expressing eYFP–CESA3 (left), eGFP–CESA6 (middle) and eYFP–CSLD3 (right) using a ×40 lens with bright-field optics (upper panels) or spinning-disc confocal microscopy (lower panels; scale bars, 10 μm). Insets, expanded images to show details of subcellular localization in root-hair tips. Subcellular distribution of fluorescent CESA and CSLD3 fusions in non-hair-forming epidermal cells. () x–y (upper panels) and x–z (lower panels) optical sections (designated graphically on the left) of root epidermal cells from seedlings stably expressing eYFP–CSLD3, eGFP–CESA3 and eYFP–CESA6 were collected using a ×40 lens and spinning-disc confocal microscopy (scale bars, 20 μm). () Schematic representation of characterized CSLD3 mutations kjk-2 (resulting in a premature stop codon; Q121X), kjk-3 (resulting in an amino-acid substitution; E829K) and a! new mutation, kjk-4 (resulting in a premature stop codon; W978X). () Representative bright-field microscopy images were collected from seven-day-old wild-type or CSLD3 mutant (kjk-2, kjk-3, kjk-4) seedlings using a ×10 lens on a Nikon E600 compound microscope (scale bars, 50 μm). (,) Medial confocal sections were collected using a ×40 lens on a spinning-disc confocal microscope from growing root-hair cells of wild-type A. thaliana seedlings stably expressing eYFP fused to kjk-3 () or kjk-4 () mutant sequences (scale bars, 10 μm). * Figure 3: Surface accessibility of CESA and CSLD3 proteins in A. thaliana roots and effect of actin depolymerization on eYFP–CSLD3 localization. (,) Total membranes were isolated and detergent solubilized from root tissues of two-week-old A. thaliana seedlings expressing BRI1–GFP, eYFP–CESA6, eYFP–CSLD3 or eYFP–RabA4b that had been previously treated with the membrane-impermeable sulpho-NHS-SS-biotin modifying reagent. Immunoblotting with anti-GFP antibodies was carried out on equivalent fractions of either total membrane proteins () or surface-exposed, biotinylated proteins selectively purified and eluted after Neutravidin affinity column purification (). WTCO, wild-type Columbia. () Seedlings expressing eYFP–CSLD3 were grown in liquid medium and transferred to a perfusion chamber for spinning-disc confocal microscopy using a ×40 lens. Normal root-hair elongation was observed for 15 min (a thin white line denotes the relative position of the root-hair tip at the beginning of the time-course analysis). Within 3 min of treatment with 200 nM latrunculin B (LB; 15 min) accumulation of internal eYFP–! CSLD3 compartments in the root-hair tip was rapidly lost (18 min). Loss of eYFP–CSLD3 from the tip-polarized plasma-membrane domain was much slower and polarized eYFP–CSLD3 was still detected in an apical plasma-membrane domain up to 10 min after latrunculin B treatment (18–24 min). This effect was reversible: washout of latrunculin B (30 min) resulted in reorganization of tip-localized eYFP–CSLD3 after a short lag (33 min) and resumption of root-hair elongation (scale bars, 5 μm). (,) Quantification of latrunculin B effects on eYFP–CSLD3 subcellular localization and root-hair growth in two independent root hairs (black triangles and grey squares). Overall root-hair elongation on treatment with latrunculin B was measured (), and compared with relative eYFP–CLSD3 fluorescence () expressed as a percentage of fluorescence detected at time zero. Fluorescence was measured for eYFP–CSLD3 in the vesicle-rich zone (dashed lines), or for eYFP–CSLD3 prese! nt in apical membranes (solid lines). The time period in which! latrunculin B was present in the perfusion medium is represented by the shaded area. * Figure 4: Effect of cellulose synthase inhibitors on root-hair growth. DCB effect on eYFP–CSLD3 localization. () Seedlings expressing eYFP–CSLD3 were grown in liquid medium and transferred to a perfusion chamber for spinning-disc confocal microscopy with a ×40 lens. Growing root hairs were selected (left panel) and elongation was observed for 15 min, at which point 25 μM DCB was added (middle panel). Over time, root hairs showed increasingly large bulges, which were associated with an increase in the size of the eYFP–CSLD3 polarized plasma-membrane domain (right panel; scale bars, 10 μm). () Effect of isoxaben on root elongation in light-grown A. thaliana seedlings. Wild-type seedlings were germinated and grown vertically at 22 °C and in 16 h light/day for six days on 0.25× MS plates containing no additions, or containing 40 pM, 200 pM, 1 nM or 5 nM isoxaben. Representative images of seedlings were collected using a dissecting scope with a ×1 lens (scale bar, 5 mm). () Overall root length was quantified for seedlings grown at various isoxaben concentrat! ions (n=10 seedlings). () Representative images of isoxaben effect on root-hair growth in light-grown A. thaliana seedlings (scale bar, 200 μm). () Average lengths of 10 root hairs were measured from seedlings grown at various isoxaben concentrations (n=7 seedlings; error bars, ±s.d.). * Figure 5: The cellulose-synthase inhibitor CGA 325′615 stimulates recruitment of A. thaliana CESA proteins and CSLD3 into cell-wall extracts. () Root tissue from two-week-old seedlings stably expressing eYFP–CESA6, eGFP–CESA3 or eYFP–CSLD3 was treated with 25 μM CGA or DMSO (control). Equivalent DMSO- and CGA-treated samples from either total soluble protein (left panel) or extracted cell-wall fractions (right panel) were immobilized on nitrocellulose using a dot-blotting apparatus. The presence of eYFP–CESA6 (CESA6), eGFP–CESA3 (CESA3) or eYFP–CLSD3 (CSLD3) in these fractions was detected by immunoblotting with antibodies specific for GFP. The presence or absence of RabA4b and PIP2B in these fractions (from eYFP–CESA6-expressing plants) was detected by immunoblotting with an affinity-purified antibody specific for RabA4b or PIP2B. () Schematic representations of CSLD3 (green), CESA6 (yellow) and the CSLD3::A6CD chimaera (D3 portions in green, A6 portions in yellow). Zinc-finger domains are represented as blue diamonds, and predicted trans membrane domains are represented as black bars. () Subcell! ular localization of eYFP–CSLD3::A6CD chimaeras in the growing root-hair cells of a rescued kjk-2 mutant seedling (scale bar, 10 μm). () Subcellular localization of eYFP–CSLD3 and eYFP–CSLD3::A6CD, indicating the presence of this fluorescent chimaera in membranes at the periphery of non-hair-forming epidermal cells (scale bars, 20 μm). Author information * Author information * Supplementary information Affiliations * Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA * Sungjin Park, * Amy L. Szumlanski, * Fangwei Gu, * Feng Guo & * Erik Nielsen Contributions S.P. carried out most experiments. A.L.S., F. Gu, F. Guo and E.N. all contributed to experimental work and data analysis in this manuscript. E.N. was also responsible for project planning. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Erik Nielsen Author Details * Sungjin Park Search for this author in: * NPG journals * PubMed * Google Scholar * Amy L. Szumlanski Search for this author in: * NPG journals * PubMed * Google Scholar * Fangwei Gu Search for this author in: * NPG journals * PubMed * Google Scholar * Feng Guo Search for this author in: * NPG journals * PubMed * Google Scholar * Erik Nielsen Contact Erik Nielsen Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (8M) Supplementary Information * Supplementary Movie 2a (8M) Supplementary Information * Supplementary Movie 2b (8M) Supplementary Information * Supplementary Movie 2c (8M) Supplementary Information * Supplementary Movie 3 (7M) Supplementary Information * Supplementary Movie 4 (1M) Supplementary Information * Supplementary Movie 5 (2M) Supplementary Information * Supplementary Movie 6 (7M) Supplementary Information * Supplementary Movie 7 (6M) Supplementary Information * Supplementary Movie 8a (1M) Supplementary Information * Supplementary Movie 8b (1M) Supplementary Information * Supplementary Movie 8c (1M) Supplementary Information * Supplementary Movie 8d (2M) Supplementary Information * Supplementary Movie 9 (3M) Supplementary Information * Supplementary Movie 10 (2M) Supplementary Information * Supplementary Movie 11 (3M) Supplementary Information * Supplementary Movie 12 (2M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis
- Nat Cell Biol 13(8):981-988 (2011)
Nature Cell Biology | Letter Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis * Daphné Dambournet1, 2 * Mickael Machicoane1, 2 * Laurent Chesneau1, 2 * Martin Sachse3 * Murielle Rocancourt1, 2 * Ahmed El Marjou4, 5 * Etienne Formstecher6 * Rémi Salomon7 * Bruno Goud4, 5 * Arnaud Echard1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:981–988Year published:(2011)DOI:doi:10.1038/ncb2279Received29 November 2010Accepted09 May 2011Published online26 June 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Abscission is the least understood step of cytokinesis. It consists of the final cut of the intercellular bridge connecting the sister cells at the end of mitosis, and is thought to involve membrane trafficking as well as lipid and cytoskeleton remodelling1, 2, 3, 4, 5, 6. We previously identified the Rab35 GTPase as a regulator of a fast recycling endocytic pathway that is essential for post-furrowing cytokinesis stages7. Here, we report that the phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) 5-phosphatase OCRL, which is mutated in Lowe syndrome patients8, 9, is an effector of the Rab35 GTPase in cytokinesis abscission. GTP-bound (active) Rab35 directly interacts with OCRL and controls its localization at the intercellular bridge. Depletion of Rab35 or OCRL inhibits cytokinesis abscission and is associated with local abnormal PtdIns(4,5)P2 and F-actin accumulation in the intercellular bridge. These division defects are also found in cell lines derived from Lowe patie! nts and can be corrected by the addition of low doses of F-actin depolymerization drugs. Our data demonstrate that PtdIns(4,5)P2 hydrolysis is important for normal cytokinesis abscission to locally remodel the F-actin cytoskeleton in the intercellular bridge. They also reveal an unexpected role for the phosphatase OCRL in cell division and shed new light on the pleiotropic phenotypes associated with Lowe disease. View full text Figures at a glance * Figure 1: OCRL directly interacts with GTP-bound Rab35. () The Saccharomyces cerevisiae strain L40 was transformed with plasmids encoding GAD fused to full-length OCRL-b (left) or GAD alone (control; right) to examine interactions with LexA fused to wild-type Rab35, Rab35Q67L (GTP-bound mutant), Rab35S22N (GDP-bound mutant) and Rab11Q70L (GTP-bound mutant). Growth on medium without His (−His) indicates an interaction with the indicated proteins. Part of the ASH domain (amino acids 540–676) was sufficient to interact specifically with GTP-bound Rab35 in a yeast two-hybrid assay (data not shown). () Recombinant purified GST-tagged wild-type Rab35 or Rab11 proteins were loaded with either GDP or GTP-γS. After incubation with HeLa cell extracts, endogenous OCRL bound to either GST or GST–Rab beads was detected by western blot (WB) analysis using an anti-OCRL antibody. Input represents 5% load of the total cell extracts used in all conditions. () HeLa cells were co-transfected with plasmids encoding GFP–OCRL-b and either Myc-! tagged wild-type Rab35, Rab35S22N or Rab35Q67L. Rab35 was immunoprecipitated (IP) with anti-Myc antibodies, and co-immunoprecipitated OCRL was revealed by anti-GFP antibodies (middle). The amount of GFP–OCRL (1% lysate, top) and immunoprecipitated Rab35 (2%, bottom) is shown in all conditions. () Recombinant GST or GST–OCRL-b (amino acid 540–893) immobilized on glutathione beads was incubated with recombinant 6×His-tagged wild-type Rab35, Rab6 or Rab11 loaded with either GDP or GTP-γS (1% inputs are shown on the left). The amount of each Rab directly bound to GST (top western blot) or GST–OCRL-b (bottom western blot) was detected with anti-6×His antibodies. 1% bead inputs are shown below each western blot. () The same experiment as in , except that recombinant 6×His-tagged GTP-bound mutant proteins (Q/L) of the indicated Rab were used. () 96-well plates were coated with recombinant GST–OCRL-b (amino acids 540–893) and incubated with increasing amounts of rec! ombinant 6×His-tagged GTP-bound Rab mutant proteins, as indic! ated (solid-phase assay). After washes, Rab proteins bound to OCRL were detected (arbitrary units) using anti-6×His antibodies and chromogenic substrate (mean±s.e.m., n=3 experiments). No binding to GST alone was detected (data not shown). Uncropped images of blots are shown in Supplementary Fig. S8a–d. * Figure 2: Rab35 activation controls the accumulation of an OCRL pool in the cytokinesis intercellular bridge. () HeLa cells in cytokinesis were stained for endogenous OCRL (green) and the Golgi marker GM130 (red). Right, higher-magnification images. Blue, DAPI (4,6-diamidino-2-phenylindole) staining. G, distal Golgi; g, proximal Golgi that is being reformed during cytokinesis (see ref. 6). Arrow, intercellular bridge. Scale bar, 10 μm. () OCRL and Rab35 co-localize at the intercellular bridge. HeLa cells were co-transfected with GFP-tagged wild-type Rab35 and mCherryFP–OCRL-b. Top left: OCRL channel. Top right: Rab35 channel. Bottom right: merge channels (Rab35 in green and OCRL in red). These images correspond to a snapshot of a time-lapse movie acquired with a spinning-disc confocal microscope. Tracks (temporal projection) of individual OCRL vesicles trafficking to (no. 1,2,4,5,7,8) or from (no. 3,6) the intercellular bridge are indicated in the bottom left panel. Scale bar, 5 μm. () HeLa cells were co-transfected with plasmids encoding GFP–OCRL-b and Myc-tagged wild-typ! e Rab35. GFP-positive cells with late cytokinesis bridges (arrowhead, top panel, scale bar, 10 μm) were identified and fixed. Cells were then processed for double immuno-electron microscopy localization of Rab35 (10 nm gold particles) and OCRL (15 nm gold particles; middle panel). The regions indicated with arrows are presented at a higher magnification (bottom panels). Scale bar, 500 nm. () Rab35 is required for proper localization of OCRL at the intercellular bridge. HeLa cells were transfected with control or Rab35-specific siRNA and stained for Aurora B kinase to identify intercellular bridges (red) and for endogenous OCRL (green). Top left: The western blot shows the efficiency of Rab35 RNAi (depletion by 70%–85% of endogenous levels), using the β-tubulin signal as a loading control. Top right and bottom right panels: Higher-magnification images of the bridge regions for each siRNA. Bottom left: Quantification of the OCRL intensity at the intercellular brid! ge in control and Rab35-depleted cells is presented (mean±s.e! .m., n=30 cells analysed per experiment, four independent experiments). g, proximal Golgi. Blue, DAPI staining. Scale bar, 10 μm. () HeLa cells were transfected with either control (empty vector), active Rab35Q67L or dominant-negative Rab35S22N mutants. OCRL staining (right panels) and quantification in intercellular bridges (left panel) were carried out as in (mean±s.e.m., n=30 cells analysed per experiment, 3–6 independent experiments). Blue, DAPI staining. Scale bar, 1 μm. ** P<6×10−3 and *** P<3×10−5 (Student's t-test). Uncropped images of blots are shown in Supplementary Fig. S8e. * Figure 3: Cytokinesis abscission defects in cells depleted for Rab35 or OCRL, and in Lowe syndrome patient cells. () Snapshots of time-lapse phase-contrast microscopy movies of HeLa cells transfected with either control, OCRL- or Rab35-specific siRNAs. Insets, high-magnification images of the intercellular bridges (black arrowheads) before and after abscission. () Distribution of the abscission times in the cell populations described in , as indicated. The distributions after OCRL and Rab35 depletion are different from the control, with P<10−5 (Kolmogorov–Smirnov test). () Snapshot of OCRL-depleted cells in which cytokinesis abscission failed at two successive cell divisions (see also Supplementary Fig. S3c). Note the presence of a chain of four cells connected by the old intercellular bridge (arrowhead) and by two new intercellular bridges (arrows). () Left, mean abscission times were measured on time-lapse movies in HeLa cells transfected first with control or OCRL siRNAs, then with either GFP alone, wild-type GFP–OCRL-b or the GFP–OCRL-bH524R catalytically inactive mutant (me! an±s.e.m., n=40–70 cells, three independent experiments). n.s.=not significant, *** P<8×10−5 (Student's t-test). Right, western blot analysis showing that the amounts of wild-type and mutant GFP–OCRL expressed are comparable (loading control, PLC-γ1). () The recombinant phosphatase domain (amino acids 202–618) of either wild-type, G421E or H524R OCRL fused to GST was purified and the in vitro phosphatase activity towards dC4-PtdIns(4,5)P2 was measured (mean±s.e.m., representative triplicate experiment). Inset, amount of inactive OCRLG421E expressed in the Lowe patient cells analysed in this study (western blot loading control, β-tubulin). () Renal epithelial cells from a patient expressing the OCRLG421E mutant were transfected with GFP alone or wild-type GFP–OCRL-b and abscission times were measured as in . For comparison, renal epithelial cells from a donor not mutated in OCRL (normal cells) were analysed after transfection of GFP alone (mean±s.e.m., n=60! –80 cells, three independent experiments). *** P<2×10−3 (! Student's t-test). () Distribution of the abscission times in the different cell populations described in . Distributions in Lowe cells transfected with GFP versus GFP–OCRL wild type are different, with P<10−5 (Kolmogorov–Smirnov test). Uncropped images of blots are shown in Supplementary Fig. S8g,h. * Figure 4: Abnormal local accumulation of PtdIns(4,5)P2 and F-actin at the intercellular bridges in cells depleted for OCRL or Rab35 and in Lowe syndrome patient cells. () Abnormal accumulation of PtdIns(4,5)P2 in HeLa cells transfected with OCRL or Rab35 siRNAs. Left, cells were stained with an anti-PtdIns(4,5)P2 antibody (green) and for α-tubulin (red). Blue, DAPI staining. Scale bars, 10 μm. Right, quantification of the mean intensity of the PtdIns(4,5)P2 staining in each condition (mean±s.e.m., n=30 cells analysed per experiment, four independent experiments). *** P<3×10−4 (Student's t-test). For all panels, representative images of cells in late cytokinesis (top images) and higher-magnification images of the indicated intercellular bridge regions (bottom images). () Abnormal accumulation of PtdIns(4,5)P2 in Lowe patient renal cells, which expressed the inactive OCRLG421E mutant. Stainings and quantifications as described in . Mean±s.e.m., n=30 cells analysed per experiment, four independent experiments. *** P=3×10−3 (Student's t-test). () Abnormal accumulation of F-actin in HeLa cells transfected with OCRL or Rab35 siRN! As. Left, cells were stained with the fluorescently labelled phalloidin (green) and for Aurora B kinase (red). Blue, DAPI staining. Scale bars, 10 μm. Right, quantification of the mean intensity of the phalloidin staining in each condition (mean±s.e.m., n=30 cells analysed per experiment, four independent experiments). ** P=9×10−3 and *** P=5×10−3 (Student's t-test). () Abnormal accumulation of F-actin in Lowe patient renal cells, which expressed the inactive OCRLG421E mutant. Stainings and quantifications were carried out as described in . Mean±s.e.m., n=30 cells analysed per experiment, four independent experiments. *** P=5×10−3 (Student's t-test). * Figure 5: Abscission defects observed in cells depleted for OCRL or Rab35 and in Lowe syndrome patient cells are suppressed by low doses of F-actin depolymerizing drugs. () Abnormal F-actin accumulation in HeLa cells depleted for OCRL or Rab35 (left) and in Lowe patient cells (right) is suppressed by LatA treatment. HeLa cells transfected with control, OCRL or Rab35 siRNAs were treated with either dimethylsulphoxide (DMSO) or DMSO+4 nM LatA, as indicated. Renal cells from a donor not mutated in OCRL (normal cells) and from the Lowe patient were treated with either DMSO or DMSO+1 nM LatA, as indicated. F-actin intensities at the intercellular bridges were quantified as described in Fig. 4c,d (mean±s.e.m., n=90–150 cells, 3–5 independent experiments). () HeLa cells depleted for OCRL or Rab35 were treated with either DMSO or DMSO+LatA as described in . Mean abscission times in each condition were measured by video microscopy (mean±s.e.m., n=60–77 cells, three independent experiments). () Normal and Lowe patient cells were treated with either DMSO or DMSO+LatA as described in and abscission times were measured as described in (mean±! s.e.m., n=59–80 cells, three independent experiments). () Low doses of LatA are sufficient to restore normal distributions of abscission times in HeLa cells depleted for OCRL or Rab35. The cells were treated with either DMSO or DMSO+LatA as described in . Distributions were plotted as described in Fig. 3b. () Low doses of LatA are sufficient to restore normal distributions of abscission times in Lowe patient cells. Normal and Lowe patient cells were treated with either DMSO or DMSO+LatA as described in . Distributions were plotted as described in Fig. 3g. Whereas the distributions between red, blue and light green curves to their respective controls were different, with P<1.5×10−2–10−5 (Kolmogorov–Smirnov test), the distributions between black, grey, cyan and yellow curves () or between black, grey and dark green curves () were not significantly different. n.s.=not significant. *** P<10−3 and ** P<2×10−2 (Student's t-test). Author information * Author information * Supplementary information Affiliations * Institut Pasteur, Membrane Traffic and Cell Division Lab. 25–28 rue du Dr Roux, 75724 Paris cedex 15, France * Daphné Dambournet, * Mickael Machicoane, * Laurent Chesneau, * Murielle Rocancourt & * Arnaud Echard * CNRS URA2582, France * Daphné Dambournet, * Mickael Machicoane, * Laurent Chesneau, * Murielle Rocancourt & * Arnaud Echard * Institut Pasteur, Imagopole, Plate-forme de microscopie ultrastructurale, 25–28 rue du Dr Roux, 75724 Paris cedex 15, France * Martin Sachse * Institut Curie, Molecular Mechanisms of Intracellular Transport Lab. 25 rue d'Ulm, 75005 Paris, France * Ahmed El Marjou & * Bruno Goud * CNRS UMR144, France * Ahmed El Marjou & * Bruno Goud * Hybrigenics SA, 3–5 impasse Reille, 75014 Paris, France * Etienne Formstecher * AP-HP Hôpital Necker, Service de Néphrologie Pédiatrique, Inserm U983, Paris, France * Rémi Salomon Contributions D.D., M.M., L.C., M.S. and A.E. designed and analysed the experiments; D.D., M.M., L.C., M.S., M.R., A.E.M., E.F. and A.E. did the experimental work; R.S. and B.G. provided reagents; D.D., M.M., L.C., M.S., B.G. and A.E. wrote or edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Arnaud Echard Author Details * Daphné Dambournet Search for this author in: * NPG journals * PubMed * Google Scholar * Mickael Machicoane Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent Chesneau Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Sachse Search for this author in: * NPG journals * PubMed * Google Scholar * Murielle Rocancourt Search for this author in: * NPG journals * PubMed * Google Scholar * Ahmed El Marjou Search for this author in: * NPG journals * PubMed * Google Scholar * Etienne Formstecher Search for this author in: * NPG journals * PubMed * Google Scholar * Rémi Salomon Search for this author in: * NPG journals * PubMed * Google Scholar * Bruno Goud Search for this author in: * NPG journals * PubMed * Google Scholar * Arnaud Echard Contact Arnaud Echard Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (900K) Supplementary Information Additional data - Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons
- Nat Cell Biol 13(8):989-995 (2011)
Nature Cell Biology | Letter Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons * Elaine M. Pinheiro1 * Zhigang Xie2 * Amy L. Norovich1 * Marina Vidaki1 * Li-Huei Tsai3, 4 * Frank B. Gertler1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:989–995Year published:(2011)DOI:doi:10.1038/ncb2292Received18 October 2010Accepted06 June 2011Published online24 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg During corticogenesis, pyramidal neurons (~80% of cortical neurons) arise from the ventricular zone, pass through a multipolar stage to become bipolar and attach to radial glia1, 2, and then migrate to their proper position within the cortex1, 3. As pyramidal neurons migrate radially, they remain attached to their glial substrate as they pass through the subventricular and intermediate zones, regions rich in tangentially migrating interneurons and axon fibre tracts. We examined the role of lamellipodin (Lpd), a homologue of a key regulator of neuronal migration and polarization in Caenorhabditis elegans, in corticogenesis. Lpd depletion caused bipolar pyramidal neurons to adopt a tangential, rather than radial-glial, migration mode without affecting cell fate. Mechanistically, Lpd depletion reduced the activity of SRF, a transcription factor regulated by changes in the ratio of polymerized to unpolymerized actin. Therefore, Lpd depletion exposes a role for SRF in directing p! yramidal neurons to select a radial migration pathway along glia rather than a tangential migration mode. View full text Figures at a glance * Figure 1: Lpd silencing impairs neuronal positioning. (–) Mouse embryos were electroporated in utero at E14.5 and collected at E18.5 (–) or P3 (). () Quantification of cell distribution in cortical sections co-electroporated with mCherry and either Lpd shRNA, control shRNA or Lpd shRNA+Lpd* (Lpd rescue construct; **P<0.01, ***P<0.001, one-way ANOVA). () Rescue of the shRNA-mediated phenotype with an RNAi-resistant Lpd construct (Lpd*) expressed under the NeuroD1 promoter (*P<0.05, **P<0.01, Student's t-test). () Images of a sequential electroporation of an embryo at E13.5 with Lpd shRNA plus Venus and subsequently at E14.5 with mCherry. () Distribution of cells at P3 (postnatal day 3) after electroporation at E14.5 (*P<0.05, **P<0.01, ***P<0.001, Student's t-test). Scale bars, 50 μm (,,). Bar graphs are plotted as mean±s.e.m. The number of brains per condition (n) is indicated in the figure. * Figure 2: Suppression of Lpd increases the number of tangentially oriented bipolar pyramidal neurons in the intermediate/subventricular zone. (–) Mouse embryos were electroporated in utero at E14.5 and collected at E18.5. () Percentage of multipolar versus bipolar cells in the subventricular and lower intermediate zones of brains electroporated with either control or Lpd shRNA. () Orientation of control (orange arrowhead) and Lpd shRNA (blue arrowhead) bipolar cells that co-express Venus in the subventricular/intermediate zone. The graph represents the distribution of bipolar cells based on the angle of their leading process with respect to the pial surface (90°) or ventricular surface (−90°). Cells were counterstained with Hoechst 33258 dye to visualize nuclei (blue). () Percentage of Lpd shRNA or control bipolar cells that are tangentially oriented in the intermediate/subventricular zone (leading process angles between 30° and −30°). The average percentage per brain was calculated and the analysis was based on 3 brains for Lpd shRNA (analysing 173 total cells among all 3 brains) and 3 brains for contro! l (analysing 111 total cells among all 3 brains. () Image of a tangentially oriented bipolar cell expressing Lpd shRNA identified by mCherry expression (red) in brain sections immunostained with axon fibre tract marker Neurofilament (green). () Tangential cells expressing Lpd shRNA do not align with radial glial fibres. The angle of the leading process of the tangential (as determined in ) Lpd-shRNA-expressing cells was measured with respect to the radial glial fibres (identified by the expression of Nestin) and compared with that of bipolar control cells. Scale bars, 5 μm (, and ). Bar graphs are plotted as mean±s.e.m. (**P<0.01, ***P<0.001, Student's t-test, n=20 cells (from 3 brains per condition). * Figure 3: Lpd-depleted bipolar pyramidal neurons migrate tangentially within the intermediate/subventricular zones but do not exhibit a change in cell fate. () E18.5 cortical sections electroporated at E14.5 with Lpd shRNA and Venus. Cells were counterstained with Hoechst 33258 dye to visualize nuclei (blue). An enlargement of the outlined region is shown below. The distance between the cell body of individual tangential cells and the border of the electroporated region is indicated (arrows). () Time-lapse sequence of a tangentially oriented bipolar cell migrating in the intermediate/subventricular zone (arrowhead). () Image of a tangentially oriented bipolar cell, co-electroporated with Lpd shRNA and Venus, that expressed the neuronal marker, Cux1 (red). Nuclei were visualized with Hoechst 33342 dye. (,) Cortical neurons expressing Lpd shRNA and Venus (arrowheads) lack detectable GABA expression (red; ) and CXCR4 (red; ) expressed by migrating interneurons (arrow). () Orientation of the leading process of Lpd-depleted tangentially oriented bipolar cells in the medial, mediolateral and lateral regions of the cortex. GE, ganglion! ic eminence. Brains were electroporated at E11.5 and collected 37 h later n=3. Bar graphs are plotted as mean±s.e.m. Scale bars, 50 μm (), 40 μm () 5 μm (), 10 μm () and 10 μm (). * Figure 4: Lpd affects the orientation of cortical bipolar cells through an SRF/MAL-dependent pathway. (–) Mouse embryos were electroporated in utero at E14.5 and collected at E18.5. () Expression levels of rhodamine–phalloidin (red) in control and Lpd shRNA bipolar cells that co-express Venus in the subventricular/intermediate zone. Nuclei were visualized with Hoechst 33258 dye (blue). Scale bar, 5 μm. Quantification of F-actin levels in Lpd-knockdown tangentially oriented and control bipolar neurons as measured by relative phalloidin fluorescence intensity normalized to background fluorescence intensity (***P<0.001; cells from 3 control and 3 Lpd-shRNA brains were included for quantification). () In utero luciferase reporter assay. Tissue was from brains that were co-electroporated with SRF reporter 3D.ALuc and pRL–TK plasmids along with either Lpd shRNA, control shRNA or control (empty NeuroD1 vector), DN-SRF or R62D expression vectors and subsequently analysed for luciferase activity. Lpd knockdown significantly decreased the SRF activity level, represented by th! e relative firefly luciferase activity normalized to Renilla luciferase activity (***P<0.001, Student's t-test; *P<0.05, **P<0.01, one-way ANOVA). (,) Percentage of control and SRF knockdown (*P<0.05, Student's t-test, n=3 brains per condition; ) or control, DN-SRF and R62D bipolar cells () that are tangentially oriented (leading process angles between 30° and −30°) in the intermediate/subventricular zone (***P<0.001, one-way ANOVA). The number of brains (n) per condition is indicated within each bar. * Figure 5: Expression of MAL G-actin binding motifs (RPEL) rescues the Lpd-knockdown orientation and positioning defects of bipolar pyramidal neurons. Mouse embryos were electroporated in utero at E14.5 and collected at E18.5. () Schematic representation of SRF/MAL activity on RPEL–NLS (ref. 23) rescue of the Lpd-knockdown phenotype. () Percentage of bipolar tangentially oriented cells in the intermediate/subventricular zone of samples co-electroporated with Lpd-knockdown vector and RPEL–NLS or RPEL(*)–NLS (which contains mutations in the RPEL motif that disrupt G-actin binding; **P<0.01, Student's t-test, n=3 brains per condition). Bar graphs are plotted as mean±s.e.m. () Quantification of cell distribution in cortical sections co-electroporated with Venus, Lpd shRNA and either RPEL–NLS or RPEL(*)–NLS (*P<0.05, **P<0.01, ***P<0.001, Student's t-test, n=3 brains per condition). Scale bar, 50 μm. Bar graphs are plotted as mean±s.e.m. Author information * Author information * Supplementary information Affiliations * Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Elaine M. Pinheiro, * Amy L. Norovich, * Marina Vidaki & * Frank B. Gertler * Department of Neurosurgery, Boston University School of Medicine, Boston, Massachusetts 02118, USA * Zhigang Xie * Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA * Li-Huei Tsai * Howard Hughes Medical Institute, Cambridge, Massachusetts 02139, USA * Li-Huei Tsai Contributions E.M.P. designed experiments, analysed data and wrote the paper. Z.X. designed and carried out experiments. A.L.N. and M.V. carried out experiments. L-H.T. provided advice and commented on the manuscript. E.M.P., Z.X. and F.B.G. discussed the results and implications and commented on the manuscript at all stages. F.B.G. designed experiments, gave technical support and conceptual advice and revised the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Elaine M. Pinheiro or * Amy L. Norovich or * Frank B. Gertler Author Details * Elaine M. Pinheiro Search for this author in: * NPG journals * PubMed * Google Scholar * Zhigang Xie Search for this author in: * NPG journals * PubMed * Google Scholar * Amy L. Norovich Search for this author in: * NPG journals * PubMed * Google Scholar * Marina Vidaki Search for this author in: * NPG journals * PubMed * Google Scholar * Li-Huei Tsai Search for this author in: * NPG journals * PubMed * Google Scholar * Frank B. Gertler Contact Frank B. Gertler Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1a (3M) Supplementary Information * Supplementary Movie 1b (3M) Supplementary Information PDF files * Supplementary Information (500K) Supplementary Information Additional data - COPI acts in both vesicular and tubular transport
- Nat Cell Biol 13(8):996-1003 (2011)
Nature Cell Biology | Letter COPI acts in both vesicular and tubular transport * Jia-Shu Yang1 * Carmen Valente2, 3 * Roman S. Polishchuk2 * Gabriele Turacchio3 * Emilie Layre1 * D. Branch Moody1 * Christina C. Leslie4 * Michael H. Gelb5 * William J. Brown6 * Daniela Corda3 * Alberto Luini2, 3 * Victor W. Hsu1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:996–1003Year published:(2011)DOI:doi:10.1038/ncb2273Received26 November 2010Accepted04 May 2011Published online03 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Intracellular transport occurs through two general types of carrier, either vesicles1, 2 or tubules3, 4. Coat proteins act as the core machinery that initiates vesicle formation1, 2, but the counterpart that initiates tubule formation has been unclear. Here, we find that the coat protein I (COPI) complex initially drives the formation of Golgi buds. Subsequently, a set of opposing lipid enzymatic activities determines whether these buds become vesicles or tubules. Lysophosphatidic acid acyltransferase-γ (LPAATγ) promotes COPI vesicle fission for retrograde vesicular transport. In contrast, cytosolic phospholipase A2-α (cPLA2α) inhibits this fission event to induce COPI tubules, which act in anterograde intra-Golgi transport and Golgi ribbon formation. These findings not only advance a molecular understanding of how COPI vesicle fission is achieved, but also provide insight into how COPI acts in intra-Golgi transport and reveal an unexpected mechanistic relationship betwe! en vesicular and tubular transport. View full text Figures at a glance * Figure 1: LPAAT activity promotes COPI vesicle formation and inhibits tubule formation from Golgi membrane. () Examination of Golgi membrane by electron microscopy on incubation with CI-976 in the COPI reconstitution system; scale bar, 50 nm. () Quantification of Golgi tubules on the addition of CI-976 to the reconstitution system. Golgi tubules within particular lengths, as indicated, were quantified, and then grouped and expressed as a fraction of the total. The mean from three experiments with standard error is shown. () COPI-dependent retrograde transport, as tracked by the redistribution of VSVG–KDELR from the Golgi to the endoplasmic reticulum (ER), requires the catalytic activity of LPAATγ. HeLa cells were left untreated (None), or were treated with siRNA against AGPAT3 or against an irrelevant sequence (encoded by GFP), and also transfected with VSVG–KDELR. For rescue experiments, cells were also transfected with an siRNA-resistant form of wild-type (WT) LPAATγ or a catalytic-dead point mutant (H96A). The endoplasmic reticulum pattern for the chimaeric KDELR was th! en quantified. The mean from three experiments with standard error is shown. () An anti-LPAATγ antibody inhibits COPI vesicle formation. Antibodies as indicated were incubated with Golgi membrane, and then added to the COPI reconstitution system. The release of β-COP from Golgi membranes after the second-stage incubation was then quantified. The mean from three experiments with standard error is shown. * Figure 2: PLA2 activity inhibits COPI vesicle formation and promotes tubule formation. () Inhibition of retrograde COPI transport requires the catalytic activity of cPLA2α. HeLa cells were transfected with wild-type or the catalytic-dead point mutant (S228A) of cPLA2α, and then quantified for an endoplasmic reticulum (ER) pattern of VSVG–KDELR. The mean from three experiments with standard error is shown. () Inhibition of COPI vesicle formation by cPLA2α. The reconstitution system was carried out, with the second-stage incubation containing components as indicated. BARS was added in all conditions. After centrifugation, β-COP levels in the pellet and supernatant fractions were quantified, and then calculated for the fractional release. The mean from three experiments with standard error is shown. () Binding of recombinant cPLA2α to Golgi membrane requires calcium. After incubation of the recombinant protein with Golgi membrane in conditions as indicated, centrifugation was used to pellet Golgi membrane followed by immunoblotting for cPLA2α. A full scan! of the gel is shown in Supplementary Fig. S6. P, pellet; S, supernatant. () Inhibition of COPI vesicle formation by cPLA2α requires calcium. The reconstitution system was carried out, with the second-stage incubation containing components as indicated. BARS was added in all conditions. The mean from three experiments with standard error is shown. () An anti-cPLA2α antibody added to Golgi membrane promotes COPI vesicle formation. The reconstitution system was carried out using Golgi membrane washed with 0.5 M KCl. ARFGAP1 was added at a reduced level, so that potential enhancement in COPI vesicle formation above the basal level could be more readily detected. The fractional release of β-COP from Golgi membrane after the second-stage incubation was normalized to the control (no antibody added). The mean from three experiments with standard error is shown. () Quantification of Golgi tubules seen on adding recombinant cPLA2α to the reconstitution system. The reconstituti! on system was carried out, with the second-stage incubation co! ntaining ARFGAP1, BARS and recombinant cPLA2α. Subsequently, Golgi tubules within particular lengths, as indicated, were quantified, and then grouped and expressed as a fraction of the total. The mean from three experiments with standard error is shown. * Figure 3: The relative roles of COPI and lipid enzymes in vesicle versus tubule formation. () Adding CI-976 decreases the level of phosphatidic acid on Golgi membrane. Golgi membrane was incubated with or without CI-976, followed by lipid extraction and comparative LC–MS analysis. Blank indicates injection with solvent. () Adding cPLA2α decreases the level of phosphatidic acid on Golgi membrane. Golgi membrane was incubated with or without recombinant cPLA2α, followed by lipid extraction and comparative LC–MS analysis. Blank indicates injection with solvent. () The anti-coatomer antibody inhibits COPI vesicle formation. The reconstitution system was carried out with the anti-coatomer antibody (CM1A10) also added to the incubation. Vesicle formation was assessed by the release of β-COP from Golgi membrane after the second-stage incubation. A full scan of the gel is shown in Supplementary Fig. S6. P, pellet; S, supernatant. () Bud formation from Golgi membrane is inhibited by an anti-coatomer antibody. The antibody was added to the reconstitution system follo! wed by examination of Golgi membrane using electron microscopy. Representative images are shown; scale bar, 50 nm. () Tubule formation is inhibited by the anti-coatomer antibody. The reconstitution system was carried out with additional components as indicated. The number of tubules (>100 nm in length) was then quantified by electron microscopy, and expressed as a percentage of all protrusions seen on Golgi membrane. The mean from three experiments with standard error is shown. () Summary of how key determinants act in COPI vesicle versus tubule formation. The COPI complex initially drives the formation of buds from Golgi membrane. Subsequently, cPLA2α activity promotes elongation of these buds to become tubules. In contrast, LPAATγ activity commits these buds towards vesicle formation by promoting the early stage of fission, which initiates the constriction of the bud neck, followed by PLD2 activity that completes vesicle fission. * Figure 4: Characterizing how COPI acts in Golgi structure and transport. () Microinjection of an anti-coatomer antibody transforms the Golgi from ribbons to dispersed punctate structures. HeLa cells were microinjected with the anti-coatomer antibody or with vehicle as a control, followed by examination using immunofluorescence microscopy to assess the distribution of a Golgi marker (TGN46); scale bar, 5 μm. () Microinjection of the anti-coatomer antibody transforms Golgi ribbons to mini-stacks. Electron microscopy was then carried out after microinjection; scale bar, 400 nm. In control cells, the Golgi exhibits an extended ribbon structure (highlighted by the row of red arrows). In cells with coatomer inhibited, the Golgi is converted into 'mini-stacks' (outlined by red ovals). () Microinjection of the anti-coatomer antibody reduces the level of Golgi vesicles. HeLa cells were microinjected with the anti-coatomer antibody or with vehicle as a control. The level of Golgi-associated vesicles was then quantified. The mean from three experim! ents with standard error is shown. () Anterograde transport through the Golgi stacks is inhibited by the anti-coatomer antibody. VSVG-ts045 was expressed in HeLa cells, and then allowed to accumulate at the pre-Golgi compartment by shifting cell incubation from 40 °C to 15 °C. Cells were then microinjected with the anti-coatomer antibody or with vehicle, and shifted to 20 °C for the times indicated. Co-localization of VSVG-ts045 with TGN46 was subsequently quantified. The mean from three experiments with standard error is shown. () Anterograde transport through the Golgi stacks is not affected by an anti-PLD2 antibody. A similar experiment was carried out as described above, except HeLa cells were microinjected with an anti-PLD2 antibody. The mean from three experiments with standard error is shown. * Figure 5: Characterizing cargo transport in COPI tubules. () COPI vesicles contain retrograde but not anterograde cargo. COPI vesicles were reconstituted from Golgi membrane that expressed either VSVG–Myc or VSVG–KDELR–Myc, followed by immunogold labelling for the Myc tag. Representative electron micrographs are shown (left); scale bar, 25 nm. Quantification was also carried out (right), with the mean and standard error from three experiments shown. () COPI tubules contain both anterograde and retrograde cargoes. The same experiment as described above was carried out, except COPI tubules were reconstituted by adding cPLA2α at the second stage. Representative electron micrographs are shown (left); scale bar, 25 nm. Quantification was also carried out (right), with the mean and standard error from three experiments shown. () VSVG is diffusely distributed along COPI tubules. The distribution of VSVG at the tip, base and stem of 30 tubules was quantified, and then expressed as a fraction of the total. The mean with standard ! error from three experiments is shown. () VSVG–KDELR is diffusely distributed along COPI tubules. A similar analysis as described above was carried out to track VSVG–KDELR. The mean from three experiments with standard error is shown. () Coatomer is concentrated at the tip and base of COPI tubules. Immunogold electron microscopy using the anti- α-COP antibody was carried out on reconstituted COPI tubules, with a representative electron micrograph shown (left); scale bar, 25 nm. The distribution of coatomer along three segments of tubular membrane (within 100 nm of the tip, within 100 nm of the base and in-between) was quantified for 30 tubules, and then expressed as a fraction of the total (right). The mean with standard error from three experiments is shown. () Comparison of electron-dense coating on COPI buds, vesicles and tubules. Representative high-resolution electron micrographs of all three membrane structures are shown; scale bar, 50 nm. The arrows high! light the thickness of the coating on the membrane structures.! () Comparison of membrane density. COPI vesicles, tubules or Golgi membrane was subjected to equilibrium centrifugation followed by immunoblotting for β-COP. A full scan of the gel is shown in Supplementary Fig. S6. Author information * Author information * Supplementary information Affiliations * Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA * Jia-Shu Yang, * Emilie Layre, * D. Branch Moody & * Victor W. Hsu * Telethon Institute of Genetics and Medicine, Via Pietro Castellino 111, 80131 Napoli, Italy * Carmen Valente, * Roman S. Polishchuk & * Alberto Luini * Institute of Protein Biochemistry, National Research Council, Via Pietro Castellino 111, 80131 Napoli, Italy * Carmen Valente, * Gabriele Turacchio, * Daniela Corda & * Alberto Luini * Department of Pediatrics, National Jewish Health, and Departments of Pathology and Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262, USA * Christina C. Leslie * Department of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195, USA * Michael H. Gelb * Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA * William J. Brown Contributions J-S.Y., C.V., R.S.P., G.T., E.L., C.C.L., M.H.G. and W.J.B participated in experimental work and data analysis. V.W.H., A.L., D.B.M. and D.C. participated in project planning and data analysis. V.W.H., A.L. and J-S.Y. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Alberto Luini or * Victor W. Hsu Author Details * Jia-Shu Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Carmen Valente Search for this author in: * NPG journals * PubMed * Google Scholar * Roman S. Polishchuk Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriele Turacchio Search for this author in: * NPG journals * PubMed * Google Scholar * Emilie Layre Search for this author in: * NPG journals * PubMed * Google Scholar * D. Branch Moody Search for this author in: * NPG journals * PubMed * Google Scholar * Christina C. Leslie Search for this author in: * NPG journals * PubMed * Google Scholar * Michael H. Gelb Search for this author in: * NPG journals * PubMed * Google Scholar * William J. Brown Search for this author in: * NPG journals * PubMed * Google Scholar * Daniela Corda Search for this author in: * NPG journals * PubMed * Google Scholar * Alberto Luini Contact Alberto Luini Search for this author in: * NPG journals * PubMed * Google Scholar * Victor W. Hsu Contact Victor W. Hsu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (570K) Supplementary Information Additional data - The SCF–FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication
- Nat Cell Biol 13(8):1004-1009 (2011)
Nature Cell Biology | Letter The SCF–FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication * Anja Puklowski1 * Yahya Homsi1 * Debora Keller2 * Martin May3 * Sangeeta Chauhan1 * Uta Kossatz1 * Viktor Grünwald4 * Stefan Kubicka5 * Andreas Pich3 * Michael P. Manns5 * Ingrid Hoffmann6 * Pierre Gönczy2 * Nisar P. Malek1, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1004–1009Year published:(2011)DOI:doi:10.1038/ncb2282Received30 July 2010Accepted19 May 2011Published online03 July 2011Corrected online06 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis1, 2. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4, 5) as a critical substrate of the SCF–FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF–FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degrada! tion during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6. View full text Figures at a glance * Figure 1: FBXW5 regulates centrosome duplication. () Quantification of mitotic spindles after transfecting HeLa or U2OS cells with the indicated siRNAs. Error bars represent ±s.d.from at least three experiments (n=3). The significance was calculated by a two-tailed t-test and scored always as follows: *P<0.05, **P<0.005. () Left, quantification of centrosomal number after transfecting HeLa or U2OS cells with the indicated siRNAs. Error bars represent ±s.d. from at least three experiments (n=3). Right, verification of FBXW5 knockdown by immunoblotting using the indicated antibodies. () Quantification of centrioles after transfecting HeLa with the indicated siRNAs (n=3). () Quantification of mitotic spindles together with centriole number after transfecting HeLa cells with the indicated siRNAs (n=3). () U2OS cells transfected with the indicated siRNAs were blocked in S phase with hydroxyurea (+HU) or aphidicolin (+Aphi). Centrosome numbers were quantified as in (n=3). () Reduction in centrosome numbers in HeLa cells after t! ransfecting a plasmid expressing FBXW5, compared with empty vector (n=3). () Centrosome overduplication after overexpression of the FBXW5ΔF-box deletion mutant. HeLa cells were transfected with the indicated plasmids and centrosome numbers were quantified as in (n=3). () U2OS cells were arrested in S phase and transfected with the indicated plasmids. Cells were treated and quantified as in (n=3). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 2: The APC/C regulates the stability of FBXW5 in mitosis. () Cell cycle regulation of FBXW5 in synchronized HeLa cells. Cells were blocked with thymidine for 24 h and released. Samples were taken every 2 h and analysed by immunoblotting. () FBXW5 binding to CDC20 depends on the D-box motif. HEK293 cells were transfected with plasmids encoding wild-type FBXW5 or a D-box-deleted mutant. After MG132 treatment, cells were collected and immunoprecipitated (IP) with antibodies against the indicated proteins, and immunoblotted with antibodies against FBXW5 or CDC20 (left panel). The input is shown in the right panel. () FBXW5 ubiquitylation depends on the D-box motif. Ubiquitylation was measured in cells expressing the indicated FBXW5 constructs. () Stabilization of the FBXW5ΔD-box mutant during mitosis. Left, quantification of the FBXW5 half-life during mitosis. Owing to the higher FBXW5ΔD-box expression levels, values above 100% are reached when compared with the wild-type protein. Right, representative western blots, which were u! sed for quantification. The significance was calculated by a two-tailed t-test and scored always as follows: *P<0.05, **P<0.005 (n=3). () Centrosome duplication is blocked by overexpression of a D-box mutant. HeLa cells were blocked in S-phase by a double thymidine block and released. At the indicated times after release, centrosomes were stained with γ-tubulin. Left, quantification of centrosome staining. Right, representative western blots. Error bars represent ± s.d. (n=3). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 3: HsSAS-6 is a substrate of FBXW5. () Interaction between FBXW5 and HsSAS-6 after overexpression of both proteins. Precipitated proteins were analysed by immunoblotting with antibodies against the indicated proteins. () FBXW5 and HsSAS-6 interaction can be detected after immunoprecipitation of endogenous proteins. Samples were treated with (+) or without (−) MG132 and subsequently analysed as in . () HsSAS-6 fails to bind to FBXW5ΔWD2/3. After MG132 treatment, cells were collected and immunoprecipitated with antibodies against the indicated proteins (upper panel). The input of the proteins is shown in the lower panel. () HsSAS-6 half-lives after overexpression of FBXW5ΔF-box or wild-type FBXW5; CHX, cycloheximide. Left, quantification of the FBXW5 half-life during mitosis. Right, representative western blots, which were used for quantification. The significance was calculated by a two-tailed t-test and scored always as follows: *P<0.05, **P<0.005 (n=3). () Determination of HsSAS-6 protein levels after dep! letion of endogenous FBXW5 by siRNA. Samples were analysed by immunoblotting with antibodies against the indicated proteins. () Ubiquitylation of HsSAS-6 after overexpression of the indicated proteins. His–ubiquitin-coupled HsSAS-6 was enriched by metal-affinity purification and analysed by western blotting with antibodies against the indicated proteins. () HsSAS-6 ubiquitylation depends on FBXW5 deletion mutants. Ubiquitylation was measured in cells expressing the indicated FBXW5 deletion mutants. Samples were treated as in . () HeLa cells were transfected with the indicated siRNA combinations. Centrioles were stained with anti-centrin antibody and quantified in mitotic cells. Error bars represent ± s.d. (n=3). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 4: PLK4 regulates FBXW5-dependent degradation of HsSAS-6. () Interaction between PLK4 and FBXW5 in HEK293 cells detected by immunoprecipitation against the indicated proteins and western blot analysis using antibodies against the indicated proteins. () HsSAS-6 half-life in the presence of either active or kinase-dead PLK4 after co-expression of FBXW5. Left, representative western blots, which were used for quantification. Right, quantification of the FBXW5 half-life. The significance was calculated by a two-tailed t-test and scored always as follows: *P<0.05, **P<0.005 (n=3). () HsSAS-6 ubiquitylation by FBXW5 is inhibited after co-expression of active PLK4. HEK293 cells were transfected with the indicated plasmids. His–ubiquitin-coupled HsSAS-6 was enriched by metal-affinity purification and analysed by western blotting. () Phosphorylation by PLK4 of the FBXW5S151A mutant is markedly reduced when compared with wild-type FBXW5. The FBXW5S151A mutant and FBXW5 wild-type protein were in vitro translated and subjected to immunopreci! pitation using FBXW5 antibody. Thereafter, they were phosphorylated in the presence of [γ- 32P]ATP using wild-type or kinase-dead PLK4. Top, reactions were subjected to SDS–PAGE and autoradiography. Bottom, quantification of FBXW5 wild type and S151A phosphorylation by PLK4 (n=3). () The FBXW5S151A mutant is able to ubiquitylate HsSAS-6 in the presence of PLK4. The significance was calculated by a two-tailed t-test and scored always as follows: **P<0.005 (n=3). () Expression of FBXW5S151A cannot induce centriole duplication. HeLa cells were synchronized with a single thymidine block. After the release, samples were taken at the next G1/S transition and analysed by immunofluorescence microscopy. Samples were stained for centrioles with anti-centrin and quantified as shown. The data show the mean of two experiments. Control indicates unmanipulated cells. () HeLa cells were transfected with the indicated siRNA combinations. Samples were analysed 48 h after siRNA transfe! ction. Centrioles were stained with anti-centrin antibody and ! quantified in mitotic cells. The mean of two experiments is shown. Uncropped images of blots are shown in Supplementary Fig. S8. Change history * Change history * Author information * Supplementary informationCorrigendum 06 July 2011In the version of this article initially published online, all proteins should have been upper case. This has been corrected in both the HTML and PDF versions of the article. Author information * Change history * Author information * Supplementary information Affiliations * Institute for Molecular Biology, Hannover Medical School, Carl Neuberg Strasse 1, 30625 Hannover, Germany * Anja Puklowski, * Yahya Homsi, * Sangeeta Chauhan, * Uta Kossatz & * Nisar P. Malek * School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Swiss Institute for Experimental Cancer Research (ISREC), CH-1015 Lausanne, Switzerland * Debora Keller & * Pierre Gönczy * Institute of Pharmacology and Toxicology, Hannover Medical School, Carl Neuberg Strasse 1, 30625 Hannover, Germany * Martin May & * Andreas Pich * Department of Hematology, Hannover Medical School, Carl Neuberg Strasse 1, 30625 Hannover, Germany * Viktor Grünwald * Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl Neuberg Strasse 1, 30625 Hannover, Germany * Stefan Kubicka, * Michael P. Manns & * Nisar P. Malek * German Cancer Research Center, DKFZ, Heidelberg, Germany * Ingrid Hoffmann Contributions A.P., Y.H., D.K., M.M., S.C., U.K. and A.P. carried out experiments, analysed data and designed figures; V.G., S.K., A.P., M.P.M., I.H., P.G. and N.P.M. designed experiments, analysed data, designed figures and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Nisar P. Malek Author Details * Anja Puklowski Search for this author in: * NPG journals * PubMed * Google Scholar * Yahya Homsi Search for this author in: * NPG journals * PubMed * Google Scholar * Debora Keller Search for this author in: * NPG journals * PubMed * Google Scholar * Martin May Search for this author in: * NPG journals * PubMed * Google Scholar * Sangeeta Chauhan Search for this author in: * NPG journals * PubMed * Google Scholar * Uta Kossatz Search for this author in: * NPG journals * PubMed * Google Scholar * Viktor Grünwald Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Kubicka Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Pich Search for this author in: * NPG journals * PubMed * Google Scholar * Michael P. Manns Search for this author in: * NPG journals * PubMed * Google Scholar * Ingrid Hoffmann Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre Gönczy Search for this author in: * NPG journals * PubMed * Google Scholar * Nisar P. Malek Contact Nisar P. Malek Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Change history * Author information * Supplementary information PDF files * Supplementary Information (1700K) Supplementary Information Additional data - RasGRF suppresses Cdc42-mediated tumour cell movement, cytoskeletal dynamics and transformation
- Nat Cell Biol 13(8):1010 (2011)
Nature Cell Biology | Erratum RasGRF suppresses Cdc42-mediated tumour cell movement, cytoskeletal dynamics and transformation * Fernando Calvo * Victoria Sanz-Moreno * Lorena Agudo-Ibáñez * Fredrik Wallberg * Erik Sahai * Christopher J. Marshall * Piero CrespoJournal name:Nature Cell BiologyVolume: 13,Page:1010Year published:(2011)DOI:doi:10.1038/ncb2319Published online01 August 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Cell Biol.13, 819–826 (2011); published online 19 June 2011; corrected after print 15 July 2011 In the version of this article initially published online and in print, the panel on the lower right-hand side of Fig. 3a corresponding to the anti-SOS blot was incorrectly duplicated and shown in place of the anti-Cdc42 blot. The correct panels are shown below. These errors have also been corrected in the HTML and PDF versions of the article. Additional data Author Details * Fernando Calvo Search for this author in: * NPG journals * PubMed * Google Scholar * Victoria Sanz-Moreno Search for this author in: * NPG journals * PubMed * Google Scholar * Lorena Agudo-Ibáñez Search for this author in: * NPG journals * PubMed * Google Scholar * Fredrik Wallberg Search for this author in: * NPG journals * PubMed * Google Scholar * Erik Sahai Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J. Marshall Search for this author in: * NPG journals * PubMed * Google Scholar * Piero Crespo Search for this author in: * NPG journals * PubMed * Google Scholar
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