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- Women in science
- ncb 13(5):489 (2011)
Nature Cell Biology | Editorial Women in science Journal name:Nature Cell BiologyVolume: 13,Page:489Year published:(2011)DOI:doi:10.1038/ncb0511-489aPublished online03 May 2011 Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. In the hundred years since the creation of International Women's Day, great strides have been made in gender equality, but recent analysis suggests the need for further changes to enhance the progression of women in science. View full text Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Focus on stem cells
- ncb 13(5):489 (2011)
Nature Cell Biology | Editorial Focus on stem cells Journal name:Nature Cell BiologyVolume: 13,Page:489Year published:(2011)DOI:doi:10.1038/ncb0511-489bPublished online03 May 2011 Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. This issue presents a series of specially commissioned articles that highlight exciting facets of stem cell research, including recent insights into the nature of pluripotency and how studying stem cells can increase our understanding of normal ageing and disease. View full text Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - The transcriptional and signalling networks of pluripotency
- ncb 13(5):490-496 (2011)
Nature Cell Biology | Review The transcriptional and signalling networks of pluripotency * Huck-Hui Ng1 * M. Azim Surani2 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:490–496Year published:(2011)DOI:doi:10.1038/ncb0511-490Published online03 May 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 Pluripotency and self-renewal are the hallmarks of embryonic stem cells. This state is maintained by a network of transcription factors and is influenced by specific signalling pathways. Current evidence indicates that multiple pluripotent states can exist in vitro. Here we review the recent advances in studying the transcriptional regulatory networks that define pluripotency, and elaborate on how manipulation of signalling pathways can modulate pluripotent states to varying degrees. View full text Figures at a glance * Figure 1: Crosstalk between transcriptional regulatory networks, epigenetic and non-coding RNA networks. Oct4 is one of the most well characterized transcription factors in ESCs. The Oct4 gene (top) is expressed in early embryos, pluripotent cell-lines and germ cells. Transcription factor binding site mapping studies showed that the regulatory regions of the Oct4 gene (light grey) are themselves bound by multiple transcription factors, including Oct4 itself. Oct4 is found to target genes involved in diverse cellular functions (transcriptional regulation, chromatin modifications, and post-transcriptional regulation through non-coding RNAs and microRNAs; bottom). * Figure 2: Interconversion of pluripotent states for mouse and human cells. () Plasticity of cell states for pluripotent mouse cell lines. Two major pluripotent cell lines (ESCs and EpiSCs) have been derived from early mouse embryos (in vivo). EGCs are also pluripotent cell lines converted from PGCs (indicated as yellow cells) harvested from the post-implantation embryos. In vitro, the ESC and EpiSC states are interconvertible through different methods. TFs; transcription factors. () The induction of alternative hESC states using transcription factors and/or small molecule inhibitors, as illustrated in each case (transcription factors on the left, medium conditions on the right). Reprogramming could be used to generate human iPSCs that do not require conventional hESC culture conditions but resemble mESCs in morphology. Direct conversion of hESCs into cells with alternative state can also be achieved using LIF and chemical inhibitors. Author information * Abstract * Author information Affiliations * Huck-Hui Ng is at the Gene Regulation Laboratory, Genome Institute of Singapore, Singapore 138672, Singapore; the Department of Biochemistry, National University of Singapore, Singapore 117597, Singapore; the Department of Biological Sciences, National University of Singapore, Singapore 117597, Singapore and the School of Biological Sciences, Nanyang Technological University, Singapore 639798, Singapore * Azim Surani is at the Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK * M. Azim Surani Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Huck-Hui Ng or * M. Azim Surani Author Details * Huck-Hui Ng Contact Huck-Hui Ng Search for this author in: * NPG journals * PubMed * Google Scholar * M. Azim Surani Contact M. Azim Surani Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Harnessing the potential of induced pluripotent stem cells for regenerative medicine
- ncb 13(5):497-505 (2011)
Nature Cell Biology | Review Harnessing the potential of induced pluripotent stem cells for regenerative medicine * Sean M. Wu1 * Konrad Hochedlinger2 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:497–505Year published:(2011)DOI:doi:10.1038/ncb0511-497Published online03 May 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 The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in regenerative medicine. View full text Figures at a glance * Figure 1: Schematic representation of the potential utility of iPSC technology in regenerative medicine. Introduction of reprogramming factors, such as Oct4, Sox2, Klf4 and c-Myc, into somatic cells of patients (for example, skin cells, keratinocytes or blood cells) gives rise to iPSCs. These patient-specific iPSCs can then be differentiated into a variety of specialized cell types for a potential use in disease modelling (top) or cell therapy (bottom). The concept behind disease modelling is to reproduce a cellular phenotype in cultured iPSC-derived cells as it occurs in the patient. Such a phenotype could be employed to model this disease for mechanistic studies as well as for large-scale drug screening efforts to identify compounds that could be used to treat any patient suffering from the same disease. The idea behind cell therapy is to generate autologous specialized cells from iPSCs for transplantation into individual patients. Shown in purple are the current limitations in using iPSC technology in regenerative medicine. * Figure 2: Xenogeneic rat-mouse chimaera to produce entirely iPSC-derived rat pancreas. The introduction of wild type rat iPSCs into Pdx1-deficient blastocyst-stage mouse embryos resulted in the generation of a chimaeric rat-mouse that harbours a rat iPSC-derived pancreas. This pancreas is expected to be composed entirely of rat iPSC-derived cells as the loss of Pdx1 in mouse embryos results in the complete absence of a developing pancreas. Author information * Abstract * Author information Affiliations * Sean M. Wu is at the Cardiovascular Research Center, Division of Cardiology, Massachusetts General Hospital, Boston 02114, Massachusetts, USA and the Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA * Konrad Hochedlinger is at the Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA; the Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, Massachusetts 02114, USA; the Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA and the Howard Hughes Medical Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA Competing financial interests K.H. is an advisor to iPierian, Inc. Corresponding authors Correspondence to: * Sean M. Wu or * Konrad Hochedlinger Author Details * Sean M. Wu Contact Sean M. Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Konrad Hochedlinger Contact Konrad Hochedlinger Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Emerging models and paradigms for stem cell ageing
- ncb 13(5):506-512 (2011)
Nature Cell Biology | Review Emerging models and paradigms for stem cell ageing * D. Leanne Jones1 * Thomas A. Rando2 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:506–512Year published:(2011)DOI:doi:10.1038/ncb0511-506Published online03 May 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 Ageing is accompanied by a progressive decline in stem cell function, resulting in less effective tissue homeostasis and repair. Here we discuss emerging invertebrate models that provide insights into molecular pathways of age-related stem cell dysfunction in mammals, and we present various paradigms of how stem cell functionality changes with age, including impaired self-renewal and aberrant differentiation potential. View full text Author information * Abstract * Author information Affiliations * D. Leanne Jones is at the Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA * Thomas A. Rando is at The Glenn Laboratories for the Biology of Aging and the Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford California 94305, USA, and the RR&D Center of Excellence and the Neurology Service, VAPAHCS, Palo Alto, California 94305, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thomas A. Rando Author Details * D. Leanne Jones Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas A. Rando Contact Thomas A. Rando Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Ferreting out stem cells from their niches
- ncb 13(5):513-518 (2011)
Nature Cell Biology | Perspective Ferreting out stem cells from their niches * Elaine Fuchs1 * Valerie Horsley2 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:513–518Year published:(2011)DOI:doi:10.1038/ncb0511-513Published online03 May 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 Over the past decade, it has become increasingly clear that many tissues have regenerative capabilities. The challenge has been to find the stem cells or progenitors that are responsible for tissue renewal and repair. The revolution in technological advances that permit sophisticated spatial, temporal and kinetic analyses of stem cells has allowed stem cell hunters to ferret out where stem cells live, and to monitor when they come and go from these hiding places. View full text Figures at a glance * Figure 1: Regulated expression of histone H2B–GFP to follow slow-cycling cells within a tissue. () Schematic representation of the genetic strategy to mark slow-cycling cells with GFP-labelled histone H2B. Transgenic mice harbouring a tissue-specific promoter driving the tetracycline-repressor (tetR)–VP16 transgene are crossed to transgenic mice expressing a tightly regulated tetracycline-responsive regulatory element (TRE)–mCMV–H2B–GFP element. Without doxycycline, H2B–GFP is uniformly present in the nuclei of all cells within the tissue of interest. With doxycycline, H2B–GFP expression is inhibited, resulting in a dilution of existing fluorescence by 2-fold with each division. By chasing for different time periods in presence of doxycycline, only slow-cycling stem cells or long-lasting, non-dividing, terminally differentiated cells of the tissue will remain labelled. Histology (left) and fluorescence activated cell sorting (FACS; right) analysis of H2B–GFP label retention in the hair follicle when Keratin 5 (K5) is the tissue-specific promoter. Left: a! t the start of new hair growth, green nuclei, reflective of LRCS, can be detected within the stem cells of the outer layer of the hair follicle bulge (the inner layer is marked by keratin 6; K6), as well as in the hair germ (HG) at the base of the bulge. Right: by analysing GFP expression during a 4-week chase, populations of cells exhibiting a 2-fold reduction in GFP fluorescence, reflective of cell division, can be detected by FACS. FACS analysis of H2B–GFP label retention in the immune system. Haematopoietic progenitors lose H2B–GFP retention after 56 weeks of chase, whereas 10% of stem cells retain label at this time point (green numbers and the lines represent percentage of cells expressing GFP). Images courtesy of Ya-Chieh Hsu, Tudorita Tumbar and Hanno Hock. * Figure 2: Genetic lineage tracing mediated by Cre recombinase in mammalian tissues. Schematic representation of the genetic strategy to lineage-trace stem cells. The genetic background of one animal is altered such that a specific promoter is used to induce Cre recombination in stem cells. These mice are crossed to a reporter animal harbouring a stop codon flanked by Cre-recombinogenic loxP sites upstream of a reporter gene, such as lacZ or GFP, under the control of the Rosa26 promoter. In mice expressing both genetic elements, Cre recombinase excises the stop codon, such that Rosa26 drives expression of the reporter in stem cells. Once marked in this way, all descendants propagate the expression of the reporter under Rosa26 control. () Genetic lineage tracing in the hair follicle using the Sox9 promoter driving expression of Cre recombinase and a Rosa26–lacZ reporter. Sox9 is expressed within the β4-integrin-positive developing hair follicles (left; image of mice at embryonic day (E) 18.5). In mice expressing Cre recombinase under the control of the Sox! 9 promoter, follicular bulge cells are labelled within 2 days and contribute to multiple follicle lineages after 21 days (middle; Epi, epidermis). These types of studies have demonstrated that the bulge is the bona fide niche for stem cells in the hair follicle. () Genetic lineage tracing in the intestine using the Lgr5 promoter driving an inducible Cre recombinase linked to the oestrogen receptor and a Rosa26–lacZ reporter. By inducing nuclear translocation of Cre with tamoxifen, Lgr5-expressing cells give rise to progeny that encompass all cell types in the intestinal crypt, as indicated by blue label (left). Images courtesy of Jonathan Nowak, Ya-Chieh Hsu and Nick Barker. * Figure 3: Multicolour Cre-recombinase-mediated reporter for marking stem cells and their progeny. () Schematic representation of the genetic strategy to mark stem cells with multiple fluorescent proteins. One animal harbours a transgene encoding a stem-cell-specific promoter driving Cre recombinase expression. These mice are crossed to a reporter animal that, under the control of the ubiquitous Rosa26 promoter, harbour a neomycin resistance gene flanked by Cre-recombinogenic loxP sites, and multiple genes, in sense and antisense orientations, encoding the fluorescent proteins, GFP, RFP, YFP and CFP (green, red, yellow and cyan fluorescent protein, respectively) that are flanked by Cre-recombinogenic loxP and inversion sites. () In mice expressing both genetic elements from , Cre recombinase stochastically excises and inverts at the loxP sites to generate the possible transgenes shown, and allow Rosa26 to drive expression of multiple combinations of fluorescent proteins in stem cells and their progeny. () Multicolour lineage tracing in the intestine using the Ah promoter ! to drive an inducible Cre recombinase. Following induction, Ah-expressing cells are marked with multiple colours in the intestinal crypt based on the stochastic recombinogenic activity of Cre recombinase. Arrows indicate the result of genetic events in (green, 1; yellow, 2; red, 3; blue, 4). The monoclonal nature of the crypts over time suggests that stem cells stochastically adopt stem cell fates. Image courtesy of Hugo Snippert and Hans Clevers. Author information * Abstract * Author information Affiliations * Elaine Fuchs is at the Howard Hughes Medical Institute, Mammalian Cell Biology and Development, the Rockefeller University, New York, New York 10065, USA * Valerie Horsley is at the Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Elaine Fuchs Author Details * Elaine Fuchs Contact Elaine Fuchs Search for this author in: * NPG journals * PubMed * Google Scholar * Valerie Horsley Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Squishy matter and active chemistry: understanding membrane organization
- ncb 13(5):519 (2011)
Nature Cell Biology | Turning Points Squishy matter and active chemistry: understanding membrane organization * Satyajit Mayor1Journal name:Nature Cell BiologyVolume: 13,Page:519Year published:(2011)DOI:doi:10.1038/ncb0511-519Published online03 May 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. I had just established my laboratory at the National Centre for Biological Sciences (NCBS) in Bangalore, and was recovering from the inevitable stress, when a very cheerful character dropped into my office. He asked if I would be able to explain the properties of cell membranes to him, as he was a physicist from the neighbouring Raman Research Institute, and had just moved from Madras to explore the scientific environs of Bangalore and to see if there were any biologists who knew anything about cell membranes. Just as I began to get rid of him, saying that the chemistry of the cell membrane was complex enough and I did not understand very much about the physical properties of cell membranes, he threw a squishy jelly-like object that looked like a lizard at the ceiling. To our amazement, it stuck there and refused to come down. Until then I had been quite unaware of the whole field of squishy (soft condensed) matter and if I had known it existed, I would have imagined that it! would help in the development of face creams and toothpaste. I certainly knew nothing about the huge strides made in this field in exploring 'active' systems, situated away from equilibrium. While we waited for the lizard to detach, a conversation ensued between Madan Rao and myself, which has led to an exciting dialogue that has lasted many years. And one that has been a turning point in my attempts to understand the connection between the chemistry of biomolecules and their behaviour in living cells. When I was an undergraduate, I went to an engineering school, the Indian Institute of Technology (IIT, Bombay), and found myself in the positivist world of engineering solutions where heat transfer efficiencies and an understanding of fluid mechanics were the order of the day. However, I found myself at odds with the dominant preoccupations in my academic environment and yearned to study biology. I began taking almost every course and elective offered that could provide a whiff of anything biological; I even equated chemistry with biology and immersed myself in the lore of physical and organic chemistry, equilibrium thermodynamics and chemical kinetics. For my undergraduate thesis, I joined the laboratory of (the late) Dr Anil Lala, an enthusiastic bioorganic chemist who was developing photoactivatable probes to study how proteins could insert into membranes. However, when I finally arrived in a pure biology laboratory for my doctorate at the Rockefeller University, I found ! it deeply intellectually disorienting: there seemed to be more to the story than a chemist's view of biological systems as assemblages of molecules subject to laws so neatly laid out in physical and organic chemistry textbooks. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Satyajit Mayor is at the National Centre for Biological Sciences, Bellary Road, Bangalore 560005, India Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Satyajit Mayor Author Details * Satyajit Mayor Contact Satyajit Mayor Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Murdoch Mitchison 1922–2011
- ncb 13(5):520 (2011)
Nature Cell Biology | Obituary Murdoch Mitchison 1922–2011 * Paul Nurse1Journal name:Nature Cell BiologyVolume: 13,Page:520Year published:(2011)DOI:doi:10.1038/ncb0511-520Published online03 May 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. J. M. Mitchison FRS, known to most as Murdoch, Professor of Zoology at the University of Edinburgh from 1963 to 1988, died in Edinburgh on 17 March 2011 aged 88 years. Murdoch was a scientist who marched to the beat of a different drum. Driven by a powerful and individual curiosity into how cells worked, he took little notice of what was current and fashionable, working only on what he judged to be important and interesting. As a consequence he was ahead of his time and was a founder of two fields of research: the development of fission yeast as a model for cell biology and of modern research into the cell cycle. His recognition that yeasts were good models for eukaryotic cell biology predated the subsequent explosion in yeast cell biology and his focus on the cell cycle helped rescue this area from a forgotten backwater, laying the foundations for the advances in understanding cell cycle control that occurred in the decades that followed. Murdoch came from a formidable academic and political family background. Born in 1922 in Oxford, his grandfather was the physiologist J. S. Haldane, his father Dick Mitchison was a Labour member of Parliament, his mother Naomi Mitchison was a prolific novelist, and his uncle J. B. Haldane was both a Marxist and a world-famous geneticist. His two brothers, Denis and Avrion, are also distinguished biologists, and the production of this trio of large scientific personalities led his mother to quip, so Murdoch told me, that "she was the only mother to have given birth to a third of a ton of Biology Professors". After school in Oxford and Winchester, and University at Cambridge, he joined Army Operational Research in 1941 and was engaged in the Italian campaign during the Second World War. He then returned to Cambridge, finishing his PhD in 1951, before moving to Edinburgh in 1953 where he worked for the rest of his life. He married in 1947 and his wife Rowy was an eminent Sc! ottish historian. At Cambridge he was present at the birth of molecular biology, co-publishing with Max Perutz and meeting Jim Watson who later dedicated his book The Double Helix to Murdoch's mother Naomi. But cell biology was Murdoch's research passion. His early work with Michael Swann was on the cleavage of sea urchin eggs and erythrocyte membranes. Murdoch liked improving microscopes and techniques, and developed an apparatus to measure membrane stiffness, showing there were cyclic changes in surface stiffness during the cell cycle of the sea urchin embryo, a phenomenon later confirmed in amphibian eggs. In the mid-1950s he shifted fields to investigate how cells grow during the cell cycle. He was attracted to fission yeast because it grows only in length during the cell cycle. This was an insightful choice of organism because a cell could be positioned in its cell cycle simply by its length. This is how fission yeast cell biology started. He went on to develop methods to prepare synchro! nous populations of cells by selecting small cells at the beginning of the cell cycle using gradient sedimentation centrifugation and elutriation, experimental advances which made fission yeast an organism of choice for studies of the cell cycle. Working mostly with his long-term experimental collaborator, Jim Creanor, and later with Bela Novak, he painstakingly investigated the patterns of increase for many components and processes during the cell cycle. In most cases they showed that the patterns were periodic rather than exponential, often with linear patterns of increase and rate changes. These experiments addressed the important issue of what limits growth. Murdoch's work defined this problem and we have yet to resolve what it means. Photo courtesy of Neil Mitchison His second major contribution was to reawaken interest in the cell cycle. The 1971 book The Biology of the Cell Cycle had major impact on researchers of the time. He critically reviewed the cell cycle field putting great emphasis on experimental data. Two concepts were particularly important. The first was the distinction he made between the DNA-division cycle, made up of S-phase mitosis and cell division, and the growth cycle, consisting of increases in other cellular components. He argued that progression through the DNA-division cycle in growing cells was dependent on cell growth. This concept had a significant role in developing rate-limiting cell cycle control models. The second concept was that the events of the DNA-division cycle were causally dependent, an early forerunner of checkpoint controls, and that the timing of these events could be determined either by causal dependencies or by master timing mechanisms. Again this had considerable influence on later developm! ents in the field of cell cycle control. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Affiliations * Royal Society and UK Centre for Medical Research and Innovation * Paul Nurse Corresponding author Correspondence to: * Paul Nurse Author Details * Paul Nurse Contact Paul Nurse Search for this author in: * NPG journals * PubMed * Google Scholar - Mitochondria unite to survive
- ncb 13(5):521-522 (2011)
Nature Cell Biology | News and Views Mitochondria unite to survive * Craig Blackstone1 * Chuang-Rung Chang2 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:521–522Year published:(2011)DOI:doi:10.1038/ncb0511-521Published online03 May 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Starvation of animals or cells triggers autophagic degradation of cell contents to retrieve nutrients, but, paradoxically, mitochondria enlarge. This is now shown to result from inhibition of mitochondrial fission through PKA-mediated phosphorylation of the GTPase DRP1. Elongation of mitochondria optimizes ATP production and spares them from autophagy-mediated destruction. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Craig Blackstone is at the Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, 20892, USA * Chuang Rung Chang is at the Institute of Biotechnology and Department of Life Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan * Chuang-Rung Chang Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Craig Blackstone Author Details * Craig Blackstone Contact Craig Blackstone Search for this author in: * NPG journals * PubMed * Google Scholar * Chuang-Rung Chang Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - iPS cells forgive but do not forget
- ncb 13(5):523-525 (2011)
Nature Cell Biology | News and Views iPS cells forgive but do not forget * Maria J. Barrero1 * Juan Carlos Izpisua Belmonte1, 2 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:523–525Year published:(2011)DOI:doi:10.1038/ncb0511-523Published online03 May 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Induced pluripotent stem (iPS) cells offer the possibility to generate patient-specific cell types for use in regenerative medicine. However, a long-lasting question remains: are iPS and embryonic stem cells equivalent? iPS cells retain a transcriptional memory of their origin, which is now shown to endure with passages and to correlate with defects in the re-establishment of DNA methylation. Both selective pressure and genomic environment may account for these defects. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Maria J. Barrero and Juan Carlos Izpisua Belmonte are at The Center for Regenerative Medicine in Barcelona, Aiguader 88, E-08003 Barcelona, Spain * Juan Carlos Izpisua Belmonte is also at The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla, California 92037-1099, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Juan Carlos Izpisua Belmonte Author Details * Maria J. Barrero Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Carlos Izpisua Belmonte Contact Juan Carlos Izpisua Belmonte Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Transforming ER exit: protein secretion meets oncogenesis
- ncb 13(5):525-526 (2011)
Nature Cell Biology | News and Views Transforming ER exit: protein secretion meets oncogenesis * Silvere Pagant1 * Elizabeth Miller1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:525–526Year published:(2011)DOI:doi:10.1038/ncb0511-525Published online03 May 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. COPII-coated vesicles drive protein export from the endoplasmic reticulum (ER), although the regulation of this event, both spatially and kinetically, remains unclear. TFG is now defined as a factor that modulates recruitment of the coat and links ER sequestration of kinases to oncogenesis. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Silvere Pagant and Elizabeth Miller are in the Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue MC2456, New York, New York 10027, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Elizabeth Miller Author Details * Silvere Pagant Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth Miller Contact Elizabeth Miller Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Research highlights
- ncb 13(5):527 (2011)
Nature Cell Biology | Research Highlights Research highlights Journal name:Nature Cell BiologyVolume: 13,Page:527Year published:(2011)DOI:doi:10.1038/ncb0511-527Published online03 May 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. During cell migration, formation of cell protrusions involves Arp2/3 binding to the WAVE2 regulatory complex (WRC) to mediate actin nucleation. ERK is required for motility but its precise function remains unclear. Mendoza et al. now show that one of the roles of ERK (extracellular signal-regulated kinase) in cell migration is to regulate protrusion initiation by promoting WRC activation (Mol. Cell, 661–671; 2011). Visualization of cell protrusion formation revealed that ERK activity was necessary, and depended on the WRC, which co-localised with active ERK at cell protrusion edges. In contrast to previous reports, the authors found that ERK directly phosphorylates multiple sites in the WAVE2 and Abi1 components of the WRC. Inhibition of WAVE2 phosphorylation led to unregulated binding to Arp2/3 and actin, whereas inhibition of Abi1 phosphorylation suppressed these interactions, uncovering distinct modes of WRC regulation by ERK. In live cells, WRC mutants defective for ERK phosphorylation blocked cell protrusions, whereas their phosphomimetic counterparts had the converse effect. Notably, mutation of the Abi1 sites also affected cell migration, showing that Abi1 phosphorylation by ERK is essential for WRC activity in this context. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis
- ncb 13(5):529-540 (2011)
Nature Cell Biology | Article Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis * Romain Levayer1 * Anne Pelissier-Monier1 * Thomas Lecuit1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:529–540Year published:(2011)DOI:doi:10.1038/ncb2224Received31 August 2010Accepted14 February 2011Published online24 April 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 E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. Here we show that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis resulted in cell intercalation defects. We delineate a pathway that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercal! ating regions. Finally, we provide evidence that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin. View full text Figures at a glance * Figure 1: Polarization of the clathrin endocytic machinery at adherens junctions. Every image was taken in the ventrolateral region. Error bars, s.e.m. (,) αAP2 or clathrin light chain::GFP (Clc) at the level of adherens junctions (Junctions) and more basally (Basolateral). Scale bars, 5 μm. () Transmission electron microscopy sections at the level of adherens junctions. Coated pits (arrowhead) and coated vesicles (arrow) are located in the vicinity of SAJs (spot adherence junctions). Scale bar, 250 nm. () Number of coated pits or coated vesicles per 10 μm of membrane at adherens junctions or in lateral sections visualized by transmission electron microscopy. Distances in nanometres are the distances between coated vesicles and the closest membrane. () Single-plane acquisition of E-cad::GFP and Clc::mCherry in live embryos at the level of junctions showing clathrin-coated pits (arrowhead) and clathrin-coated vesicles (arrow). Scale bar, 250 nm. () Left: 2 μm projections of α-cat and αAP2 at adherens junctions. Scale bar, 5 μm. Right: re! lative mean junctional α-cat (left) or αAP2 (right) intensity according to the angle of the junctions (φ; 0°, parallel to the anteroposterior axis) in the ventrolateral (VL) and the anterior (Ant) region (see Methods). The reference categories for statistical tests are the 0°–15° junctions for α-cat and 75°–90° for αAP2 (−,P>0.05;*,P<0.05;**,P<0.01;***,P<0.001;****,P<10−4). () Left: single plane of E-cad::GFP and Clc::mCherry. Scale bar, 5 μm. Right: relative mean junctional clathrin intensity according to the angle of the junctions. The reference for the statistical tests is the 75°–90° category. () Single plane of perivitelline-injected Dextran Rhodamine. The white rectangle outlines the vertical junction shown in middle. Scale bar, 5 μm. Middle: still images from a time-lapse film of perivitelline Dextran Rhodamine. Black arrows indicate the passing of time in seconds. White arrowheads mark vesicle scission events. Scale bar, 1 μm. Right: a! verage number of scission events counted per vertical or trans! verse junction over 200 s (n, number of junctions; P<10−5). () Scheme of the localization of endocytosis proteins (left) and adherens complexes (right) in the ventrolateral region. * Figure 2: E-cad endocytosis controls its planar polarized distribution. Every image was taken in the ventrolateral region. Error bars, s.e.m. () Top: perivitelline-injected Dextran Rhodamine and E-cad::GFP after a filtering treatment. Arrowheads indicate examples of dextran-labelled vesicles containing E-cad::GFP. Scale bar, 5 μm. Bottom: still images from a time-lapse film showing a dextran budding vesicle containing E-cad::GFP. Scale bar, 1 μm. () Left: confocal micrographs of E-cad::GFP and Clc::mCherry at adherens junctions after water injection (H2O) or e-cad dsRNA injection. Note that the bright cytosolic spots of clathrin are probably not involved in endocytosis because they persist after downregulation of CME (Supplementary Fig. S2). Scale bars, 5 μm. Right: quantification of Clc junctional enrichment (P=3.3×10−5). () Left: confocal micrographs of perivitelline Dextran Rhodamine and E-cad::GFP in embryos injected with water or e-cad dsRNA. Right: quantification of the mean number of vesicles per cell, per 200 s (n, number o! f cells; P<10−5). () Left: 2 μm projections of confocal acquisitions of α-cat in yw or shi-ts embryos after a 30 min heat shock at 32 °C. Scale bars, 5 μm. Right: quantification of α-cat polarity for each genotype (φ, angle; 0°, parallel to anteroposterior axis). Statistical test were carried out between the 0°–15°junctions and the other categories (−,P>0.05;*,P<0.05;**,P<0.01;****,P<10−4). () Left: 2 μm projections of confocal acquisitions of E-cad after water or chlorpromazine injection. Scale bars, 5 μm. Right: quantification of E-cad polarity after water or chlorpromazine injection. () Left: 2 μm projections of confocal acquisitions of E-cad::GFP in wild-type and shi-ts embryos after a 20 min heat shock at 32 °C. Scale bars, 5 μm. Right: quantification of the increase in E-cad::GFP junctional signal intensity in shi-ts embryos (a.u., arbitrary units; P=1.5×10−5). * Figure 3: Blocking endocytosis affects junction remodelling. (,) A wild-type () and a shibire-ts () embryo at 32 °C at the onset (t=0) of GBE and 40 min later. The orange arrowhead marks the posterior of the germ band as it moves anteriorly along the dorsal (top) side of the embryos. Scale bar, 100 μm. () The length of the dorsally elongated germ band (l) was measured at t=40 min and normalized to the maximum extent of elongation (lmax) in multiple embryos from wild-type (blue, n=47) and shibire-ts (red, n=20) embryos. The distribution of the GBE phenotypes is shown on the graph. () Confocal micrographs from a time-lapse sequence showing E-cad::GFP in wild-type and shi-ts mutant embryos. Cells labelled with coloured dots are tracked in time to show cell intercalation. The double-headed arrow shows the anteroposterior elongation of cell clusters. Scale bars, 5 μm. () Type-1 (red), type-2 (yellow) and type-3 (red) configurations of cell contacts are tracked in a few cells undergoing intercalation in the control (top) and sh! i-ts mutants. Scale bars, 5 μm. () Type 1–3 configurations were quantified in wild-type and shi-ts embryos after 40 min at 32 °C and the distribution is shown. () Left: 4 μm projections of confocal acquisitions in the ventrolateral region of α-cat and Zipper (Myosin-II heavy chain) in yw and shi-ts embryos after a 30 min heat shock at 32 °C. Scale bars, 5 μm. Right: 4 μm projections of confocal acquisitions in the ventrolateral region of live wild-type and shi-ts embryos expressing Sqh::GFP after a 30 min heat shock at 32 °C. Scale bars, 5 μm. () 4 μm projections of confocal acquisitions in the ventrolateral region of live embryos expressing E-cad::GFP and Sqh::mCherry after water or chlorpromazine injection. Scale bars, 5 μm. () 4 μm projections of confocal acquisitions in the ventrolateral region of shi-ts embryos expressing Sqh::GFP after water or e-cad dsRNA injection and a 20 min heat shock at 32 °C. Scale bars, 5 μm. * Figure 4: Dia and Scar control different steps of E-cad endocytosis. Every image was taken in the ventrolateral region. Error bars, s.e.m. () Summary of the different characteristics of vesicles, tubules and CIVs. Clathrin is shown in green. CP: coated pit. () Left: confocal acquisitions of E-cad::GFP and Dextran Rhodamine at adherens junctions in wild-type, UAS-myr wasp, scar+/− and shit-ts (after a 30 min heat shock at 32 °C) embryos. Scale bars, 5 μm. Middle and right: corresponding quantification of the average number of vesicles per cell, per 200 s (middle) and the average number of tubules and CIVs per cell, per 200 s (right); n, number of cells. Statistical tests were carried out between wild-type and the other conditions (−,P>0.05;**,P<0.01;***,P<0.001;****,P<10−4, this code applies for the other dextran quantifications in this figure). () Top: confocal acquisitions of E-cad::GFP and Clc::mCherry in wild-type or scar+/− embryos. Scale bars, 5 μm. Bottom: quantification of junctional clathrin (P=0.825). () Top: ! confocal acquisitions of E-cad::GFP and Clc::mCherry in wild-type or shi-ts embryos after a 20 min heat shock at 32 °C. Scale bars, 5 μm. Bottom: quantification of junctional clathrin (P=0.958). () Top: confocal acquisitions at adherens junctions of E-cad::GFP and Dextran Rhodamine in UAS-diaCA embryos. Bottom: corresponding quantification of vesicles, tubules and CIVs (compared with wild type, shown in ). () Left: confocal acquisitions of junctional Dextran Rhodamine in yw and dia−/− embryos. Right: the corresponding quantification of vesicles, tubules and CIVs counted in 100 s. () Left: confocal acquisitions of Clc::mCherry in wild-type or dia−/− embryos. Scale bars, 5 μm. Note that a different insertion of Clc::mCherry was used in this experiment, compared with that for all other data in the paper. Right: quantification of junctional clathrin (P<10−5). * Figure 5: Dia and Myosin-II control clathrin and AP2 enrichment at adherens junctions. Unless specified, every image was taken in the ventrolateral region. Error bars, s.e.m. () Left: junctional E-cad::GFP and Clc::mCherry in wild-type, UAS-diaCA and UAS-myr wasp embryos. Scale bars, 5 μm. Right: the corresponding quantification of Clc junctional intensity (***, P=3.8×10−4UAS-diaCA versus wild type; *, P=0.011 UAS-myr wasp versus wild type). () Left: Clc::mCherry and Sqh::GFP (Myosin-II regulatory light chain::GFP) at adherens junctions in the dorsal cells of wild-type and UAS-diaCA embryos. Scale bars, 5 μm. Right: histogram showing the average ratio anterior/dorsal normalized Clc junctional intensity calculated for each embryo (one ratio per embryo, P<10−5). () Left: detailed view of Clc::mCherry andE-cad::GFP in wild-type and UAS-diaCA embryos showing the reduction of clathrin polarity. Scale bars, 5 μm. Right: the distribution of the clathrin relative intensity according to the angle of junctions (φ, junction angle; 0°, parallel to anteropo! sterior axis; **, P=0.0037). () Left: junctional E-cad::GFP and Clc::mCherry in embryos injected with water or Y-27632, a ROCK inhibitor. Scale bars, 5 μm. Right: quantification of junctional clathrin intensity (P=1.1×10−5). () Left: junctional E-cad::GFP and Dextran Rhodamine in embryos injected with water or Y-27632. Middle and right: quantification of the average number of vesicles per cell, per 200 s middle and average number of tubules and CIVs (right; n, number of cells; −,P>0.05;*,P<0.05;**,P<0.01;***,P<0.001;****,P<10−4). Scale bars, 5 μm. * Figure 6: Spatial control of E-cad endocytosis by RhoGEF2. Every image was taken in the ventrolateral region. Error bars, s.e.m. () 2 μm projections of junctional RhoGEF2, αAP2 and α-cat showing the planar polarity of RhoGEF2 and the subjunctional co-localization of αAP2 and RhoGEF2. Scale bar, 5 μm. () Relative mean junctional intensity of RhoGEF2, according to the angle of the junctions (φ, junction angle; 0°, parallel to anteroposterior axis). 75–90° junctions are the reference category for statistical tests (*,P<0.05;**,P<0.01;***,P<0.001;****,P<10−4). () Left: four example dot plots of junctional pixel intensity for αAP2 versus RhoGEF2, αAP2 normalized versus mean (RhoGEF2 normalized, α -cat normalized), αAP2 versus Neurotactin (negative control) and αAP2 Alexa 488 versus αAP2 Cy5 (positive control). Each dot corresponds to one junction pixel. The scattering of the dots indicates the pixel–pixel correlation between the two selected channels. Dotted lines are the linear fits of the clouds. Each cloud is e! xtracted from the analysis of one embryo. Right: the mean correlation coefficients (Mean corr. coef.) calculated for several dot plots (*,P<0.05;**,P<10−2;***,P<10−3;****,P<10−4). () Left: junctional E-cad::GFP and Clc::mCherry in wild-type embryos and embryos laid by rhoGEF2 6.5/+ mothers (rhoGEF2 6.5 is an antimorph dominant-negative allele of rhoGEF2). Scale bars, 5 μm. Right: clathrin junctional intensity (P=0.0016). () Top: junctional Dextran Rhodamine in wild-type and rhoGEF2−/− embryos. Scale bars, 5 μm. Bottom: average number of vesicles counted per cell, per 100 s (left); the same quantification for tubules and CIVs (right; n, number of cells; ****,P<10−4). () Left: 2 μm projections of junctional α -cat in wild-type, UAS-diaCA,dia−/− and rhoGEF2−/−. Note that the global intensity should not be compared between the different images because embryos were not necessarily stained with the same antibody mix. Scale bars, 5 μm. Right: qua! ntification of α-cat polarity. Statistical tests were carried! out between the mean wild-type value for 0°–15°junctions and each mutant condition (****,P<10−4). * Figure 7: IgG-induced E-cad clustering forces CME. () Schematic representation of the anti-E-cad injection assay. Perivitelline injection of an equimolar mix of rat anti-E-cad IgG and anti-rat IgG drives E-cad clustering. The control is the injection of an anti-rat IgG alone. In –, anti-E-cad represents perivitelline injection of an anti-E-cad and anti-rat IgG, and control represents anti-rat IgG alone. () Junctional co-localization of E-cad::GFP and anti-E-cad IgG. Arrowheads indicate examples of vesicles containing both E-cad::GFP and anti-E-cad. Scale bar, 5 μm. () Left: junctional Dextran Rhodamine visualized in two different regions (anterior and dorsal) on the same embryo after injection of anti-E-cad or anti-rat IgG alone (control). Scale bars, 5 μm. Right: histogram showing the average ratio of anterior/dorsal mean number of vesicles counted per cell, per 200 s (one ratio value per embryo); P=2.0×10−4; error bars, s.e.m. () Left: junctional Clc::mCherry visualized in two different regions (anterior and d! orsal) on the same embryo after injection of anti-E-cad or anti-rat IgG alone (control). Scale bars, 5 μm. Images on the right indicate a detailed view of a dorsal junction for each treatment. Clear labelling of the adherens junction can be seen only in embryos injected with anti-E-cad (bottom). Right: histogram showing the average ratio of anterior/dorsal mean Clc::mCherry junctional intensity (one ratio value per embryo); P=0.009; error bars, s.e.m. () Left: quantification of the mean number of vesicles counted per cell per 200 s in dia−/− embryos after injection of anti-E-cad or anti-rat IgG alone. Wild type corresponds to the experiment shown in Fig. 4b. Right: the same quantification for tubules and CIVs. () The same quantification after injection of Y-27632 + anti-E-cad or anti-rat IgG alone. The reference is water-injected embryos (corresponding to Supplementary Fig. S2b). n, number of cells; −,non-significant;*,P<0.05;**,P<0.01;****,P<10−4; error bars, ! s.e.m. * Figure 8: Model. () Summary of the sequence of events leading to E-cad endocytosis and the respective contribution of unbranched versus branched actin. () Summary of endocytosis and adhesive component patterning in the early gastrulating embryo. RhoGEF2 is present at the junctions everywhere except in the dorsal region. Moreover, RhoGEF2 is enriched in vertical junctions in the ventrolateral region, and is homogeneously distributed in the anterior region. The pattern is exactly the same for AP2 and clathrin. E-cad/β-cat/α-cat planar polarity is complementary to the RhoGEF2/AP2/clathrin polarized distribution. A, anterior; P, posterior. Author information * Abstract * Author information * Supplementary information Affiliations * IBDML, UMR6216 CNRS-Université de la Méditerranée, Campus de Luminy, case 907. 13288 Marseille Cedex 09, France * Romain Levayer, * Anne Pelissier-Monier & * Thomas Lecuit Contributions The experiments were conceived and planned by R.L., A.P. and T.L. A.P. made the initial observations that AP2 and Dyn were enriched at adherens junctions in early embryos, carried out the electron microscopy experiments (Fig. 1a–c) and showed that shi-ts is required for cell intercalation and GBE (Fig. 3a–g,g′). R. Levayer carried out all the other experiments. The data were analysed by R.L., A.P. and T.L. The manuscript was written by R.L. and T.L. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thomas Lecuit Author Details * Romain Levayer Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Pelissier-Monier Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Lecuit Contact Thomas Lecuit Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (3M) Supplementary Information * Supplementary Movie 2 (3M) Supplementary Information * Supplementary Movie 3 (2M) Supplementary Information * Supplementary Movie 4 (3M) Supplementary Information * Supplementary Movie 5 (3M) Supplementary Information * Supplementary Movie 6 (3M) Supplementary Information * Supplementary Movie 7 (2M) Supplementary Information * Supplementary Movie 8 (4M) Supplementary Information * Supplementary Movie 9 (5M) Supplementary Information * Supplementary Movie 10 (4M) Supplementary Information * Supplementary Movie 11 (2M) Supplementary Information * Supplementary Movie 12 (3M) Supplementary Information * Supplementary Movie 13 (2M) Supplementary Information * Supplementary Movie 14 (4M) Supplementary Information * Supplementary Movie 15 (3M) Supplementary Information * Supplementary Movie 16 (3M) Supplementary Information * Supplementary Movie 17 (3M) Supplementary Information * Supplementary Movie 18 (3M) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells
- ncb 13(5):541-549 (2011)
Nature Cell Biology | Article Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells * Yuki Ohi1, 2, 3, 12 * Han Qin1, 2, 3 * Chibo Hong4 * Laure Blouin1, 2, 3 * Jose M. Polo5, 6 * Tingxia Guo3, 7 * Zhongxia Qi8 * Sara L. Downey4 * Philip D. Manos6, 9 * Derrick J. Rossi6, 9, 10 * Jingwei Yu8 * Matthias Hebrok3, 7 * Konrad Hochedlinger5, 6 * Joseph F. Costello4 * Jun S. Song11, 12 * Miguel Ramalho-Santos1, 2, 3 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:541–549Year published:(2011)DOI:doi:10.1038/ncb2239Received14 February 2011Accepted16 March 2011Published online17 April 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 Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports indicate that there may be important differences between them. We carried out a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low-passage iPS cells analysed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 (chrom! osome 9 open reading frame 64) reduces the efficiency of human iPS cell generation, indicating that somatic memory genes may be functionally relevant during reprogramming. View full text Figures at a glance * Figure 1: Pluripotency validation for the derived Hep-iPS cells used for the microarray studies. () The three Hep-iPS clones used in this analysis showed strong, positive immunostaining for all analysed specific markers for human ES (hES) cells. SSEA, stage-specific embryonic antigen. Tra1-81, tumour rejection antigen 1-81. Scale bar, 300 μm. () All Hep-iPS clones showed high expression levels of endogenous pluripotency markers and negligible levels of transgene expression by quantitative rtPCR. Values were standardized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ubiquitin B (Ubb), then normalized to H9 ES cells (endogenous) or 5-factor-infected hepatocytes+doxycycline (Hep-inf+dox) at 4 days (viral). Data are from triplicate reactions. Error bars represent standard deviations. () All Hep-iPS clones formed embryoid bodies in vitro when grown under non-attachment conditions. Shown here are d8 embryoid bodies and control ES-cell-derived embryoid bodies. Scale bar, 200 μm. () Pluripotency of the Hep-iPS cell clones was further confirmed by their ability t! o form teratomas in vivo, comprised of tissues derived from all three germ layers. (i) Neural tissue (ectoderm). (ii) Striated muscle and adipocytes (mesoderm). (iii) Gut-like epithelium (endoderm). Also see Supplementary Fig. S2 for pluripotency validation of Fib-iPS cells used for the microarray analysis. Mel-iPS cells have previously been described17. * Figure 2: Multiple cell types undergo extensive transcriptional reprogramming to the human iPS cell state. () Average-linkage hierarchical clustering of the RMA-normalized expression profiles shows that the replicate data cluster together tightly, confirming the reproducibility of the experiments, and that the somatic cells have been successfully reprogrammed. EB, embryoid bodies. () The box plot of log expression fold changes for all RefSeq genes further shows that the iPS cells have been reprogrammed to closely resemble the transcriptional profiles of ES cells. The black centre line represents the median. The upper and lower edges of the box represent the first and third quartiles, and they define the inter-quartile range. Outliers farther than 1.5 times the inter-quartile range from the box are shown as circles. * Figure 3: iPS cells retain a transcriptional memory of the original somatic cell. () LOESS curves fitted to the scatter plots of t -test log P values for hepatocyte and hepatocyte-derived iPS cells: −log(P) and log(P) are plotted for fold changes greater than 1 and less than 1, respectively. The black line is a curve fitted to our data, and other curves are fitted to the 1,000 bootstrap simulation data sets obtained by assuming identically distributed iPS and ES cell expression levels. The black line shows clear deviation from the null hypothesis iPS=ES and thus reflects the trend that the transcriptional memory of the originating cell type is retained in low-passage iPS cells: genes that were higher (or lower) in the somatic cell than in ES cells tend to be significantly repressed (or induced) during reprogramming, but nevertheless remain higher (or lower) in iPS cells than in ES cells. () Box plots of expression levels for 191 genes that are higher in both iPS cells and somatic cells relative to ES cells (upper right corner genes in ) and 391 genes th! at are lower in both iPS cells and somatic cells relative to ES cells (lower left corner genes) at a t -test P -value cutoff of 0.01. The plots illustrate progressive convergence of somatic gene expression towards the ES cell state. () Top, Venn diagrams for progressively reprogrammed genes (somatic>iPS>ES or somatic - TFG-1 function in protein secretion and oncogenesis
- ncb 13(5):550-558 (2011)
Nature Cell Biology | Article TFG-1 function in protein secretion and oncogenesis * Kristen Witte1, 4 * Amber L. Schuh1, 4 * Jan Hegermann2 * Ali Sarkeshik3 * Jonathan R. Mayers1 * Katrin Schwarze2 * John R. Yates III3 * Stefan Eimer2 * Anjon Audhya1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:550–558Year published:(2011)DOI:doi:10.1038/ncb2225Received23 August 2010Accepted07 February 2011Published online10 April 2011Corrected online11 April 2011 Abstract * Abstract * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Export of proteins from the endoplasmic reticulum in COPII-coated vesicles occurs at defined sites that contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the endoplasmic reticulum in Caenorhabditis elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers that facilitate the co-assembly of SEC-16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a marked decline in both SEC-16 and COPII levels at endoplasmic reticulum exit sites. The sequence encoding the amino terminus of human TFG has been previously identified in chromosome translocation events involving two protein kinases, which created a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to endoplasmic reticulum exit sites, where they prematurely phosphorylate substrates during endoplasmic reticulum export. Our f! indings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis. View full text Figures at a glance * Figure 1: C. elegans TFG-1 interacts with the endoplasmic reticulum exit-site component SEC-16. () Schematic representation of human (Hs) and C. elegans (Ce) Sec16 isoforms. The central conserved domain (CCD) is highlighted in each protein. () SEC-16 was immunoprecipitated from C. elegans embryo extract and blotted with anti-TFG-1 antibodies (n=3). A mock immunoprecipitation (IP) assay was conducted in parallel using rabbit immunoglobulin G. () TFG-1 was immunoprecipitated from C. elegans embryo extract and blotted with anti-SEC-16 antibodies (n=3). A mock immunoprecipitation assay was conducted in parallel using rabbit immunoglobulin G. () GST alone and GST-tagged full-length SEC-16 were immobilized on glutathione agarose beads, which were incubated with an extract generated from Escherichia coli expressing recombinant TFG-1. Following a series of washes, proteins were eluted using reduced glutathione, separated by SDS–PAGE and either Coomassie blue stained (top) or immunoblotted using TFG-1 antibodies (bottom). () Polyhistidine-tagged full-length and truncated form! s of TFG-1, either encoding amino acids 1–195 (TFG-1(N)) or 196–486 (TFG-1(C)), were purified from E. coli onto nickel affinity resin and incubated with freshly prepared whole-worm extract (n=3). Imidazole-eluted proteins were separated by SDS–PAGE, Coomassie blue stained (top) and blotted with anti-SEC-16 antibodies (bottom). Uncropped scans of gels and immunoblots are provided in Supplementary Fig. S6. * Figure 2: TFG-1 localizes to endoplasmic reticulum exit sites that are juxtaposed to the Golgi. () Dissected C. elegans gonads were fixed and stained using Cy2-labelled anti-TFG-1 and Cy3-labelled anti-SEC-16 antibodies (n=8). Both individual and merged images of proximal oocytes with TFG-1 in green and SEC-16 in red are shown (scale bar, 10 μm). The upper right image is the area outlined in the panel below magnified sixfold (scale bar, 2 μm). Also shown is a schematic representation of the C. elegans reproductive system, which includes a syncytial stem-cell niche in the distal gonad (light-green highlighted area) and proximal oocytes that have undergone cellularization (dark-green highlighted area). (,) Lowicryl sections of C. elegans oocytes were stained with antibodies against TFG-1 or a combination of TFG-1 and SEC-13 antibodies. Arrows highlight Golgi cisternae. Large arrowheads point out 15 nm gold particles associated with immunoreactive TFG-1, and small arrowheads highlight 5 nm gold particles associated with SEC-13. Scale bars, 100 nm. An inset is ! provided in to clearly show the distribution of 5 nm particles at higher magnification (inset scale bar, 15 nm). In addition, a three-dimensional reconstruction of TFG-1 immunolocalization is shown. The image was generated using the software Reconstruct from serial 50 nm thin sections. Vesicles were reconstructed using the sphere setting, and all other components (endoplasmic reticulum, ERGIC, coats, Golgi stacks) were generated using the Boissonnat surface setting. Light grey, endoplasmic reticulum; dark grey, COPII coat; orange, endoplasmic-reticulum-derived transport vesicles and ERGIC; red, green and blue, Golgi cisternae, from cis to trans, respectively. () An electron micrograph illustrating two endoplasmic reticulum exit sites and adjacent Golgi complexes in the most proximal oocyte of an animal following high-pressure freezing and freeze substitution (scale bar, 500 nm). On the right is a three-dimensional reconstruction of the same pair of Golgi complexes a! nd associated endoplasmic reticulum exit sites. The endoplasmi! c reticulum exit sites are surrounded by vesicles that fuse to form the ERGIC. Light grey, endoplasmic reticulum; dark grey, COPII coat; orange, endoplasmic-reticulum-derived transport vesicles and ERGIC; yellow, red and blue, Golgi cisternae, from cis to trans, respectively. * Figure 3: TFG-1 regulates SEC-16 levels on endoplasmic reticulum exit sites. () In the proximal gonad, a 300 nm section of the early secretory pathway (endoplasmic reticulum exit sites, ERGIC and Golgi) was analysed by electron tomography. Endoplasmic reticulum exit sites are highlighted by arrowheads. On the left are individual sections from the tomographic stack. On the right are two orthogonal views of the tomogram following three-dimensional reconstruction. Light grey, endoplasmic reticulum; black, COPII coat; orange and yellow, endoplasmic-reticulum-derived transport vesicles and ERGIC; green, red, blue, Golgi cisternae; diffuse grey, not further resolvable matrix. Scale bar, 100 nm. () An electron micrograph illustrating endoplasmic reticulum exit sites and adjacent Golgi complexes in the distal gonad following high-pressure freezing and freeze substitution. An arrowhead highlights the presence of a budding vesicle from smooth endoplasmic reticulum (scale bar, 100 nm). Below is a three-dimensional reconstruction of the same Golgi complexe! s and associated endoplasmic reticulum exit sites. () Dissected gonads from control, TFG-1-depleted and SEC-16-depleted animals were fixed and stained using Cy2-labelled anti-TFG-1 and Cy3-labelled anti-SEC-16 antibodies. Individual and merged images of the distal gonad with TFG-1 in green and SEC-16 in red are shown (scale bar, 10 μm). () The fluorescence intensity of SEC-16 in the distal gonad was measured in control and TFG-1-depleted animals, and intensities were segregated into low, medium and high thresholds. To establish individual thresholds, a histogram of fluorescence intensities was equally divided into three regions, and the number of endoplasmic reticulum exit sites within each area was calculated. The histogram indicates the percentages of all endoplasmic reticulum exit sites that fall within specific thresholds. For each condition, at least 1,000 unique endoplasmic reticulum exit sites were examined. Error bars represent mean±s.e.m.; 10 different animals.! **P<0.01 when compared with control, calculated using a paire! d Student t -test. () Western blots of extracts prepared from animals depleted of TFG-1 by RNAi (n=3). Serial dilutions of extracts prepared from control animals were loaded to quantify depletion levels. Blotting with anti-CAR-1 antibodies was carried out to control loading. Uncropped scans of the immunoblots are provided in Supplementary Fig. S6. * Figure 4: The N terminus of TFG-1 mediates its oligomerization. The results presented in each panel are representative of at least three individual experiments. In all cases, the intensities of each band were measured to identify the peak elution fraction, which was used to calculate either a Stokes radius or sedimentation value, depending on the experiment. () Western blots using SEC-16 or TFG-1 antibodies of C. elegans embryo extract fractionated on a Superose 6 gel filtration chromatography column. The peaks corresponding to SEC-16 and TFG-1 partially overlap. A Stokes radius was calculated for each protein on the basis of comparison with the elution profiles of known standards. (–) Recombinant polyhistidine-tagged TFG-1 or fragments of TFG-1 described in Fig. 1e were expressed and purified from E. coli extracts using nickel resin. A Coomassie-blue-stained gel of the peak elution fractions after fractionation of the recombinant proteins on a Superose 6 gel filtration chromatography column is shown. Proteins were fractionated on a 10! –30% glycerol gradient, and S values were calculated on the basis of the location of characterized standards run on a parallel gradient. () Western blots of control and TFG-1-depleted C. elegans whole-worm extracts fractionated on a Superose 6 gel filtration column and probed with SEC-16 or SEC-13 antibodies. Fractionation of HGRS-1, a component of ESCRT-0 (endosomal sorting complex required for transport-0), was examined in both control and TFG-1-depleted conditions to ensure that gel filtration profiles were directly comparable. Stokes radii were calculated for each protein on the basis of comparison with the elution profiles of known standards. Uncropped scans of gels and immunoblots are provided in Supplementary Fig. S6. * Figure 5: TFG-1 is required for COPII recruitment and protein secretion. () Dissected C. elegans gonads were fixed and stained using Cy2-labelled anti-SEC-13 and Cy3-labelled anti-SEC-16 antibodies (n=15). Merged images of the distal gonad with SEC-13 in green and SEC-16 in red are shown on the left (scale bar, 10 μm). Panels to the right are magnified ×5 views of the outlined area in the adjacent panel (scale bar, 2 μm). () Histogram showing the average ratio of SEC-13 to SEC-16 fluorescence intensities in control and TFG-1-depleted animals. For each condition, at least 250 unique endoplasmic reticulum exit sites in the distal gonad were examined. Error bars represent mean±s.e.m.; six different animals. No statistically significant difference was observed, on the basis of a calculation using a paired Student t -test. () Swept-field confocal optics were used to image anaesthetized control (n=15) and TFG-1-depleted (n=15) adult animals expressing GFP–SNB-1 and mCherry–PH. Scale bar, 10 μm. () Electron micrographs illustrating the ea! rly secretory pathway in the most proximal oocyte of control and TFG-1-depleted animals following high-pressure freezing and freeze substitution (scale bar, 100 nm). Arrowheads highlight endoplasmic reticulum exit sites. Below each micrograph is a three-dimensional reconstruction of the same regions. Light grey, endoplasmic reticulum; dark grey, COPII coat; orange, endoplasmic-reticulum-derived transport vesicles and ERGIC; green, red and blue, Golgi cisternae, from cis to trans, respectively. * Figure 6: Human TFG functions at endoplasmic reticulum exit sites and binds to Sec16. () Swept-field confocal optics were used to image HeLa cells that had been transiently transfected with GFP–TFG and mCherry–Sec16B (n=42). Representative colour overlays of mCherry–Sec16B (red) and GFP–TFG (green) are shown. Scale bar, 10 μm. () Swept-field confocal optics were used to monitor the recovery of GFP–TFG after photobleaching (n=15). A threefold-magnified view of the outlined region where GFP–TFG was bleached is shown below. Times are in seconds relative to the bleach. Scale bars, 10 μm (top) and 1 μm (bottom). () Graph showing the average percentage of GFP–TFG and mCherry–Sec16B fluorescence recovered as a function of time in seconds relative to the bleach (error bars represent means±s.e.m. for each time; n=15 different cells for each fluorescent fusion protein). () Western blots of HeLa cell extract fractionated on a Superose 6 gel filtration chromatography column (n=3). A Stokes radius was calculated for human TFG on the basis of comp! arison with the elution profiles of known standards. (,) A GST-tagged, truncated form of human TFG, amino acids 1–193, was expressed and purified from E. coli extracts using glutathione agarose (n=3), and the GST tag was subsequently cleaved using PreScission Protease before loading onto a gel filtration column or glycerol gradient. A Coomassie-blue-stained gel of the peak elution fractions after fractionation of the recombinant protein, referred to as TFG(N), on a Superose 6 gel filtration chromatography column is shown (). The protein was also fractionated on a 10–30% glycerol gradient (), and an S value was calculated on the basis of the location of characterized standards run on a parallel gradient (n=3). () Antibodies directed against mCherry were used to immunoprecipitate mCherry–Sec16B from HeLa cells transiently transfected with GFP–TFG or a GFP fusion to the N terminus of TFG referred to as GFP–TFG(N) (n=3). Isolated proteins were separated by SDS–PAGE ! and immunoblotted (IB) with anti-TFG and anti-GFP antibodies. ! Uncropped scans of gels and immunoblots are provided in Supplementary Fig. S6. * Figure 7: Targeting of the NTRK1 kinase domain to endoplasmic reticulum exit sites is sufficient to activate NTRK1-mediated downstream signalling. (–) Swept-field confocal optics were used to image HeLa cells that had been transiently transfected with mCherry–Sec16B and GFP fusions to either the N terminus of TFG, referred to as GFP–TFG(N) (n=18 ), the transmembrane and kinase domains of NTRK1, referred to as GFP–NTRK(C) (n=15), or a TFG(N)–NTRK1(C) fusion (n=28), which is equivalent to the oncogene characterized previously21. Representative colour overlays of mCherry–Sec16B (red) and GFP fusions (green) are shown. Scale bar, 10 μm. () Histogram showing the percentage of co-localization between the GFP fusions described above and mCherry–Sec16B (error bars represent means±s.e.m. for each condition; n=15 different cells for each condition and at least 800 unique endoplasmic reticulum exit sites were examined). () Extracts from hTERT-RPE1 cells stably transfected with GFP alone (control) or various GFP fusions to the NTRK1 transmembrane and kinase domains (as indicated) were separated by SDS–PAGE and ! blotted using a phospho-specific ERK1–ERK2 antibody and a pan-ERK1–ERK2 antibody. Uncropped scans of immunoblots are provided in Supplementary Fig. S6. Change history * Abstract * Change history * Author information * Supplementary informationCorrigendum 11 April 2011In the version of this Article initially published online, the title was incorrect. The error has been corrected in the HTML and PDF versions of the article. Author information * Abstract * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Kristen Witte & * Amber L. Schuh Affiliations * Department of Biomolecular Chemistry, University of Wisconsin–Madison Medical School, 1300 University Avenue, Madison, Wisconsin 53706, USA * Kristen Witte, * Amber L. Schuh, * Jonathan R. Mayers & * Anjon Audhya * European Neuroscience Institute and Center for Molecular Physiology of the Brain (CMPB), 37077 Goettingen, Germany * Jan Hegermann, * Katrin Schwarze & * Stefan Eimer * Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA * Ali Sarkeshik & * John R. Yates III Contributions K.W., A.L.S., S.E. and A.A. conceived and designed experiments. K.W., A.L.S., A.S., J.H., J.R.M., K.S., S.E. and A.A. carried out experiments and analysed data. S.E., J.R.Y. and A.A. contributed reagents, materials and analysis tools. A.A. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Anjon Audhya Author Details * Kristen Witte Search for this author in: * NPG journals * PubMed * Google Scholar * Amber L. Schuh Search for this author in: * NPG journals * PubMed * Google Scholar * Jan Hegermann Search for this author in: * NPG journals * PubMed * Google Scholar * Ali Sarkeshik Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan R. Mayers Search for this author in: * NPG journals * PubMed * Google Scholar * Katrin Schwarze Search for this author in: * NPG journals * PubMed * Google Scholar * John R. Yates III Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Eimer Search for this author in: * NPG journals * PubMed * Google Scholar * Anjon Audhya Contact Anjon Audhya Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Change history * Author information * Supplementary information Movies * Supplementary Movie 1 (1M) Supplementary Information * Supplementary Movie 2 (3M) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - ARFGAP1 promotes AP-2-dependent endocytosis
- ncb 13(5):559-567 (2011)
Nature Cell Biology | Article ARFGAP1 promotes AP-2-dependent endocytosis * Ming Bai1, 10 * Helge Gad2, 3, 10 * Gabriele Turacchio2, 4, 10 * Emanuele Cocucci5 * Jia-Shu Yang1 * Jian Li1 * Galina V. Beznoussenko2 * Zhongzhen Nie6 * Ruibai Luo7 * Lianwu Fu8 * James F. Collawn8 * Tomas Kirchhausen5 * Alberto Luini2, 9 * Victor W. Hsu1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:559–567Year published:(2011)DOI:doi:10.1038/ncb2221Received28 September 2010Accepted03 February 2011Published online17 April 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 COPI (coat protein I) and the clathrin–AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport. View full text Figures at a glance * Figure 1: Interactions with ARFGAP1 and effects of its knockdown. () Detection of proteins interacting with ARFGAP1 using a GST-pulldown assay. GST–ARFGAP1 was incubated with cytosol and associated proteins were analysed by Coomassie blue staining. () Detection of ARFGAP1 interactions with coat components using a GST-pulldown assay. GST–ARFGAP1 was incubated with cytosol and immunoblotted for the indicated proteins. ARFGAP1 interacts with components of AP-2, and also with previously known interacting proteins that are components of the COPI complex. CHC, clathrin heavy chain. () Tf uptake is reduced by siRNA against ARFGAP1. BSC-1 cells were bound with fluorescently labelled Tf and assessed for the level of internalized Tf at 10 min. The mean from three experiments with standard error is shown. The difference between the two conditions is significant (P<0.05). () EGF uptake is not markedly affected by siRNA against ARFGAP1. BSC-1 cells were bound with fluorescently labelled EGF and assessed for the level of internalized EGF at 10 m! in. The mean from three experiments with standard error is shown. The difference between the two conditions is insignificant (P>0.05). () LDL uptake is not markedly affected by siRNA against ARFGAP1. BSC-1 cells were bound with fluorescently labelled LDL and assessed for the level of internalized LDL at 10 min. The mean from three experiments with standard error is shown. The difference between the two conditions is insignificant (P>0.05). Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 2: Distinct requirements for ARFGAP1 binding to coatomer versus AP-2 and clathrin. () Interaction of different ARFGAP1 truncation mutants with coatomer. The different forms of GST–ARFGAP1 were bound to beads, incubated with cytosol and immunoblotted for the indicated proteins. GST fusion proteins were detected by Coomassie blue staining. () Interaction of different truncation mutants of ARFGAP1 with AP-2 or clathrin. Pulldown experiments were carried out as described in . () Interaction of different point mutants of ARFGAP1 with clathrin, AP-2 or coatomer. Pulldown experiments were carried out as described in . () Interaction of additional mutants of ARFGAP1 with clathrin, AP-2 or coatomer. Pulldown experiments were carried out as described in . () Interactions of ARFGAP1 with coatomer, AP-2 and clathrin are summarized. The catalytic domain of ARFGAP1 is shown in white. Asterisks indicate W-based motifs. Degree of association between ARFGAP1 form and indicated coat component: none/minor (−), moderate (+), strong (++). Uncropped images of blots are show! n in Supplementary Fig. S6. * Figure 3: Disrupting the interaction between ARFGAP1 and either coatomer or AP-2 leads to selective disruption in transport pathways. () Pulldown assays to assess direct binding of ARFGAP1 to AP-2. Different forms of GST–ARFGAP1 were bound to beads, incubated with purified AP-2 adaptors and immunoblotted for the indicated proteins. GST fusion proteins were detected by Coomassie blue staining. () Pulldown assays to assess direct binding of ARFGAP1 to clathrin. The different forms of GST–ARFGAP1 were bound to beads, incubated with purified clathrin triskelia and immunoblotted for the indicated proteins. GST fusion proteins were detected by Coomassie blue staining. () Sequential incubations reveal that the AP-2 adaptor is needed to link ARFGAP1 to the clathrin triskelion. Different forms of GST–ARFGAP1 were bound to beads, and incubated with purified AP-2 followed by incubation with purified clathrin. Beads were analysed by immunoblotting for the indicated proteins. GST fusion proteins were detected by Coomassie blue staining. () Rescue of defective Tf uptake induced by the depletion of ARFGAP1. BSC-1 c! ells stably expressing shRNA against ARFGAP1 were transfected with the different forms of rat ARFGAP1 as indicated, and uptake of biotin–Tf at 10 min was quantified. The mean from three experiments with standard error is shown. The differences among conditions of ARFGAP1 shRNA, FWW and EDE are not significant (P>0.05). The difference between this group and all other conditions is significant (P<0.05). () Rescue of defective COPI transport induced by the depletion of ARFGAP1. BSC-1 cells stably expressing shRNA against ARFGAP1 were transfected with the different forms of rat ARFGAP1 as indicated. The redistribution of VSVG–KDELR from the Golgi to the endoplasmic reticulum at 30 min was then quantified. The mean from three experiments with standard error is shown. The differences among conditions of wild type, FWW and EDE are not significant (P>0.05). The difference between this group and conditions of shARFGAP1 and 1–400 is significant (P<0.05). Uncropped images of! blots are shown in Supplementary Fig. S6. * Figure 4: Surveying transport pathways affected by the depletion of ARFGAP1. () The surface level of TfR is not affected by ARFGAP1 depletion. An antibody that recognizes the extracellular domain of TfR was bound to BSC-1 cells, followed by quantification. The mean from three experiments with standard error is shown. The difference between the two conditions is insignificant (P>0.05). () TfR recycling is reduced by siRNA against ARFGAP1. BSC-1 cells were treated with siRNA against ARFGAP1 and the level of internal biotin–Tf was quantified at the indicated time points. The mean from three experiments with standard error is shown. The difference between the two conditions (except time=0) is significant (P<0.05). () Rescue of defective TfR recycling induced by the depletion of ARFGAP1. BSC-1 cells stably expressing shRNA against ARFGAP1 were transfected with the different forms of rat ARFGAP1 indicated. The level of internal biotin–Tf that remained after 10 min of recycling was then quantified. The mean with standard error from three experiments i! s shown. The difference between cells expressing ARFGAP1 shRNA and all other conditions is significant (P<0.05). () Depletion of ARFGAP1 inhibits transport from the endoplasmic reticulum to the TGN. VSVG-ts045–GFP was transfected into BSC-1 cells, followed by quantification of its co-localization with a TGN marker (TGN46). The mean from three experiments with standard error is shown. The difference at all time points is significant (P<0.05). () Depletion of ARFGAP1 does not affect transport from the endoplasmic reticulum to the cis side of the Golgi. VSVG-ts045–GFP was transfected into BSC-1 cells, followed by quantification of its co-localization with a cis-Golgi marker (giantin). The mean from three experiments with standard error is shown. The difference at all time points is insignificant (P>0.05). () Depletion of ARFGAP1 does not affect transport from the TGN to the plasma membrane (PM). VSVG-ts045–GFP was transfected into BSC-1 cells, and then accumulated at the! TGN. Cells were then shifted from 20 to 32 °C to allow tra! nsport to the plasma membrane. Arrival of VSVG at the plasma membrane was detected through co-localization with fluorescently labelled CTB (which was bound to the cell surface). The mean from three experiments with standard error is shown. The difference at all time points is insignificant (P>0.05). * Figure 5: The role of ARFGAP1 in coated-pit formation. () Co-localization of ARFGAP1 with clathrin in coated pits. BSC-1 cells transfected with GFP-tagged ARFGAP1 and mCherry-tagged clathrin light chain were examined by TIR-FM. The merged view shows co-localization of the two proteins; scale bar, 2 μm. Insets highlight examples of coated pits found to have both ARFGAP1 (green) and clathrin (red). () Dynamic association of ARFGAP1 with clathrin in coated pits. BSC-1 cells were transfected with GFP–ARFGAP1 and mCherry-tagged clathrin light chain and examined by TIR-FM with live-cell imaging. Two examples are shown, with images captured every 3 s; scale bars, 1 μm. () Depletion of ARFGAP1 reduces the level of coated pits. HeLa cells were treated with the indicated siRNA conditions. All forms of clathrin-coated (CC) intermediates were counted and then divided by the length of the plasma membrane. Seven cells were randomly selected from each condition to obtain the mean with standard deviation. The difference between the tw! o conditions is significant (P<0.05). () Depletion of ARFGAP1 inhibits the endocytosis of CFTR. CFBE41o- cells expressing wild-type CFTR were polarized and treated with the indicated siRNA conditions. The level of internalized CFTR was then quantified. The mean with standard error from three experiments is shown. The difference between the two conditions (except time=0) is significant (P<0.05). () Depletion of ARFGAP1 does not induce accumulation of a particular stage of coated pit. The different stages of coated-pit formation, with representative images for each stage shown (left; bar, 100 nm), were detected by electron microscopy, and then quantified. The mean from three experiments with standard deviation is shown (right). The difference between corresponding stages is insignificant (P>0.05). * Figure 6: ARFGAP1 and AP-2 interact specifically with surface TfR. () Endogenous surface TfR interacts with endogenous forms of ARFGAP1 and AP-2 in vivo. Biotin-labelled Tf was bound to the surface of HeLa cells, followed by isolation using streptavidin beads and then immunoblotting for the indicated proteins. () GFP-tagged ARFGAP1 associates with endogenous forms of surface TfR and AP-2. BSC-1 cells were transfected with GFP-tagged ARFGAP1. Cells were then bound with biotin-labelled Tf, followed by incubation of cell lysate with streptavidin beads and immunoblotting for the indicated proteins. () Surface TfR does not associate with ARFGAP-2 or ARFGAP3. HeLa cells were transfected with Myc-tagged ARFGAP1, or (HA)-tagged forms of ARFGAP-2 or ARFGAP3. Cells were bound with biotin-labelled Tf. Cell lysis was followed by incubation of cell lysate with streptavidin beads and immunoblotting for the indicated proteins. ARFGAPs were detected using antibodies against the epitope tag. () A surface form of LDLR does not associate with ARFGAP1. HeLa ce! lls stably expressing CD8-LDLR were incubated with anti-CD8 antibody. After this surface binding, cells were lysed and incubated with protein A beads, followed by immunoblotting for the indicated proteins. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 7: Characterizing the binding of TfR by ARFGAP1 and AP-2. () Pulldown assays were carried out in which the indicated TfR constructs were incubated with recombinant ARFGAP1, followed by immunoblotting for ARFGAP1. GST fusion proteins were detected by Coomassie blue staining. () Pulldown assays were carried out in which the indicated TfR constructs were incubated with recombinant ARFGAP1, followed by immunoblotting for ARFGAP1. GST fusion proteins were detected by Coomassie blue staining. Point mutations within particular truncation constructs are indicated within parentheses. () Pulldown assays were carried out in which the indicated TfR constructs were incubated with purified AP-2, followed by immunoblotting for β2 -adaptin. GST fusion proteins were detected by Coomassie blue staining. Point mutations within particular truncation constructs are indicated within parentheses. () Effects of TfR mutations on its association with ARFGAP1 and AP-2. Biotin-labelled Tf was bound to the surface of mutant CHO (TRVb) cells expressing differe! nt full-length TfR forms (wild type or with the indicated point mutations) followed by isolation using streptavidin beads and immunoblotting for the proteins indicated. The '4A' construct contains alanine substitutions at residues 12, 13, 22 and 23 of TfR. () Association of surface TfR with AP-2 requires ARFGAP1. BSC-1 cells were treated with siRNA against ARFGAP1. Biotin-labelled Tf was bound to the surface of BSC-1 cells, followed by isolation using streptavidin beads and immunoblotting for the endogenous proteins indicated. Cell lysates were also directly immunoblotted for proteins in the indicated conditions. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 8: The GAP activity of ARFGAP1 promotes TfR endocytosis. () The GAP activity of ARFGAP1 is important for TfR endocytosis. BSC-1 cells stably expressing shRNA against ARFGAP1 were transfected with the indicated constructs and uptake of biotin–Tf at 10 min was quantified. The mean with standard error from three experiments is shown. The difference between the condition of shRNA and rescue by the wild type is significant (P<0.05). The difference between the condition of shRNA and rescue by R50K is insignificant (P>0.05). () Depletion of ARF6 inhibits Tf uptake. BSC-1 cells were treated with siRNA against ARF6 and uptake of biotin–Tf at 10 min was quantified. The mean with standard error from three experiments is shown. The difference between the two conditions is significant (P<0.05). () AP-2 enhances GAP activity of ARFGAP1 towards ARF6. The GAP assay was carried out using ARF6 as the substrate, and either with (triangles) or without (squares) AP-2. The mean from three experiments with standard error is shown. () Binding of ! ARF6 to TfR is activation dependent. The functional activation of ARF6 was confirmed using the effector domain of GGA3 (Golgi-localized, γ-adaptin ear-containing, ARF-binding protein 3) in a pulldown experiment (left panel). ARF6 forms were also incubated with GST–TfR in another pulldown experiment (right panel). () GAP activity of ARFGAP1 optimizes the binding of AP-2 to TfR. GST–TfR was incubated sequentially with ARF6 (containing different bound nucleotides, as indicated), followed by ARFGAP1 and then AP-2. () Deactivation of ARF6 by ARFGAP1 releases ARF6 from binding to TfR. ARF6 loaded with GTP forms, as indicated, were incubated with GST–TfR along with ARFGAP1 in a pulldown experiment. () Activation-dependent binding of ARF6 to surface TfR. BSC-1 cells were transfected with point-mutant forms of ARF6 as indicated. Surface TfR was isolated through biotin–Tf binding to cell surface, followed by incubation with streptavidin beads and immunoblotting was carried o! ut for the indicated proteins. () The constitutively activated! form of ARF6 (Q67L) reduces the binding of AP-2 to surface TfR. BSC-1 cells were transfected with ARF6Q67L or mock transfected. Surface TfR was isolated as described in , followed by immunoblotting for the indicated associated proteins. Uncropped images of blots are shown in Supplementary Fig. S8. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Ming Bai, * Helge Gad & * Gabriele Turacchio Affiliations * Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA * Ming Bai, * Jia-Shu Yang, * Jian Li & * Victor W. Hsu * Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy * Helge Gad, * Gabriele Turacchio, * Galina V. Beznoussenko & * Alberto Luini * Department of Genetics, Microbiology and Toxicology, Stockholm University, 10691 Stockholm, Sweden * Helge Gad * Institute of Protein Biochemistry, National Research Council, Via Pietro, Castellino 111, 80131 Naples, Italy * Gabriele Turacchio * Department of Cell Biology, Harvard Medical School, and Immune Disease Institute, Boston, Massachusetts 02115, USA * Emanuele Cocucci & * Tomas Kirchhausen * Department of Urology, University of Florida College of Medicine, Gainesville, Florida 32610, USA * Zhongzhen Nie * Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA * Ruibai Luo * Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA * Lianwu Fu & * James F. Collawn * Telethon Institute of Genetics and Medicine, Via Pietro Castellino 111, 80131 Napoli, Italy * Alberto Luini Contributions M.B., H.G., G.T., E.C., J-S.Y., J.L., G.V.B., Z.N., L.F. and R.L. carried out experiments and data analyses. V.W.H., A.L., T.K. and J.F.C. supervised the work. V.W.H., A.L., H.G. and M.B. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Victor W. Hsu Author Details * Ming Bai Search for this author in: * NPG journals * PubMed * Google Scholar * Helge Gad Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriele Turacchio Search for this author in: * NPG journals * PubMed * Google Scholar * Emanuele Cocucci Search for this author in: * NPG journals * PubMed * Google Scholar * Jia-Shu Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Jian Li Search for this author in: * NPG journals * PubMed * Google Scholar * Galina V. Beznoussenko Search for this author in: * NPG journals * PubMed * Google Scholar * Zhongzhen Nie Search for this author in: * NPG journals * PubMed * Google Scholar * Ruibai Luo Search for this author in: * NPG journals * PubMed * Google Scholar * Lianwu Fu Search for this author in: * NPG journals * PubMed * Google Scholar * James F. Collawn Search for this author in: * NPG journals * PubMed * Google Scholar * Tomas Kirchhausen Search for this author in: * NPG journals * PubMed * Google Scholar * 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 * Abstract * Author information * Supplementary information PDF files * Supplementary Information (600K) Supplementary Information Additional data - Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease
- ncb 13(5):568-579 (2011)
Nature Cell Biology | Article Cdk5-mediated phosphorylation of endophilin B1 is required for induced autophagy in models of Parkinson's disease * Alan S. L. Wong1, 2, 3 * Rebecca H. K. Lee1, 2, 3 * Anthony Y. Cheung1, 2, 3 * Patrick K. Yeung4 * Sookja K. Chung4, 5 * Zelda H. Cheung1, 2, 3 * Nancy Y. Ip1, 2, 3 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:568–579Year published:(2011)DOI:doi:10.1038/ncb2217Received02 September 2010Accepted27 January 2011Published online17 April 2011Corrected online21 April 2011 Abstract * Abstract * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that is increasingly implicated in various neurodegenerative diseases. Deregulated Cdk5 activity has been associated with neuronal death, but the underlying mechanisms are not well understood. Here we report an unexpected role for Cdk5 in the regulation of induced autophagy in neurons. We have identified endophilin B1 (EndoB1) as a Cdk5 substrate, and show that Cdk5-mediated phosphorylation of EndoB1 is required for autophagy induction in starved neurons. Furthermore, phosphorylation of EndoB1 facilitates EndoB1 dimerization and recruitment of UVRAG (UV radiation resistance-associated gene). More importantly, Cdk5-mediated phosphorylation of EndoB1 is essential for autophagy induction and neuronal loss in models of Parkinson's disease. Our findings not only establish Cdk5 as a critical regulator of autophagy induction, but also reveal a role for Cdk5 and EndoB1 in the pathophysiology of Parkinson's disease thr! ough modulating autophagy. View full text Figures at a glance * Figure 1: EndoB1 is a substrate of Cdk5/p35. () p35 interacts with EndoB1 in a GST pulldown assay. (,) EndoB1 co-immunoprecipitates (IP) with p35 in COS-7 cell lysates expressing p35 and full-length EndoB1 () and adult rat brain lysate (). () Putative Cdk5 phosphorylation site(s) in the EndoB1 sequence. Two proline-directed threonine residues (arrows) are present with a Cdk5 consensus site found at Thr 145 (underlined). () Dose-dependent phosphorylation of EndoB1 by Cdk5/p35 in an in vitro kinase assay. () Cdk5/p35 phosphorylates EndoB1 at Thr 145 in an in vitro kinase assay. () Cdk5/p35 phosphorylates EndoB1 at Thr 145 in COS-7 cells. () Cdk5 phosphorylates EndoB1 at Thr 145 in Cdk5+/+, but not Cdk5−/− mouse brains. Quantification of p-Thr145-EndoB1 level is shown in the right panel. Data are means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 2: Cdk5-mediated phosphorylation of EndoB1 is required for starvation-induced autophagy in neurons. () Left: starvation increases the level of RFP–LC3 puncta (arrows) in rat cortical neurons. Middle: quantification of the percentage of cells with RFP–LC3 puncta. Right: pretreatment with 10 mM 3-MA attenuates the starvation-induced increase in LC3-II level in neurons. Data are means ± s.e.m.; n=3. Samples were run on the same gel and spliced together to create the image shown. () Knockdown of EndoB1 abolishes the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP–LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=3. () Starvation enhances Cdk5 activity (top) and increases the level of Thr 145-phosphorylated EndoB1 in neurons (top and bottom). Histone H1 was used as a Cdk5 substrate. Data are means ± s.e.m.; n=3. (,) Pretreatment with Ros () or knockdown of Cdk5 () attenuates starvation-induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons. Data are means ± s.e.m.; n=3 for and n=8 for . DMSO, dim! ethylsulphoxide. () Overexpression of EndoB1T145A significantly reduces the starvation-induced increase in LC3-II levels (left) and the percentage of cells with RFP–LC3 puncta (right) in neurons. Data are means ± s.e.m.; n=6. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 3: Thr 145 phosphorylation of EndoB1 does not affect its lipid binding and co-localization with Atg5. () Left: lipid binding of EndoB1 is inhibited by mutating the five lysine/arginine residues between amino acids 176 and 183 of EndoB1 to glutamic acid (5E mutant). Right: purified GST protein was included as a negative control. S, supernatant fraction; P, pellet fraction that contains the lipid-bound proteins. (,) The 5E mutant of EndoB1 fails to dimerize () and interact with UVRAG () in 293T cells. () Overexpression of the 5E mutant of EndoB1 blocks starvation-induced autophagy in cortical neurons. Data are means ± s.e.m.; n=3. () Mutation of Thr 145 to alanine (T145A) or glutamic acid (T145E) has negligible effect on the lipid-binding property of EndoB1. Conversely, the 5E mutant is less able to bind lipid. The right panel shows quantified data as means ± s.e.m.; n=3. () Starvation increases co-localization of GFP–EndoB1 (green) with Atg5 (red) in neurons. The T145A mutation of GFP–EndoB1 does not affect its co-localization with Atg5-positive entities. Arrows denote ! GFP–EndoB1- and Atg5- positive vesicles. The right panel shows quantified data as means ± s.e.m.; n=9. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 4: Cdk5-mediated Thr 145 phosphorylation of EndoB1 regulates its dimerization and interaction with UVRAG/Beclin 1. () Overexpression of Cdk5/p35 enhances EndoB1 dimerization in 293T cells. Samples were run on the same gel and spliced together to create the image shown. () Thr 145 phosphorylation of EndoB1 facilitates its dimerization in 293T cells. () Diminished interaction between EndoB1 and UVRAG in Cdk5−/− mouse brain lysates. (,) Thr 145 phosphorylation of EndoB1 enhances its interaction with UVRAG () and Beclin 1 () in 293T cells. (,) Gel-filtration analysis on the membrane fraction of mouse brain lysates. Fractions obtained were subjected to immunoblot analysis (), or immunoprecipitation followed by immunoblotting (). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 5: Cdk5-mediated Thr 145 phosphorylation of EndoB1 is involved in autophagy triggered by MPTP. () MPTP administration decreases the numbers of tyrosine hydroxylase (TH)- and cresyl violet (CV)-positive neurons in the substantia nigra. Representative images of tyrosine hydroxylase-stained brain sections are shown. Right panels show quantified data as means ± s.e.m.; n=9 for tyrosine hydroxylase and n=3 for cresyl violet. () MPTP administration upregulates the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the midbrain. Lower panels show quantified data as means ± s.e.m.; n=3. () Knockdown of EndoB1 or Cdk5 inhibits MPP+-induced autophagy in cultured neurons. Data are means ± s.e.m.; n=5. () Cdk5 mediates the MPP+-induced EndoB1 phosphorylation at Thr 145 and the MPP+-induced increase in the LC3-II level in neurons. () Overexpression of EndoB1T145A attenuates MPP+-induced autophagy in neurons. Data are means ± s.e.m.; n=7. () MPTP fails to increase LC3-II and Thr 145-phosphorylated EndoB1 levels in the midbrain of p35−/− mice. The concomitant reduction in! tyrosine hydroxylase level triggered by MPTP injection is also markedly attenuated. Right panels show quantified data as means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 6: Thr 145 phosphorylation of EndoB1 by Cdk5 contributes to α-synucleinA53T mutant-induced autophagy. () Upregulation of the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the cerebellum and striatum of α-synucleinA53T transgenic mice. Right panels show quantified data as means ± s.e.m.; n=3. () Overexpression of α-synucleinA53T mutant upregulates LC3-II level and Thr 145-phosphorylated EndoB1 in cultured neurons. Lower panel shows quantified data as means ± s.e.m.; n=3. () Knockdown of EndoB1 or Cdk5 inhibits α-synucleinA53T mutant-induced autophagy induction in neurons. Data are means ± s.e.m.; n=7. () Cdk5 mediates α-synucleinA53T mutant-induced Thr 145 phosphorylation of EndoB1 and the increase of LC3-II in neurons. () Overexpression of EndoB1T145A attenuates α-synucleinA53T mutant-induced autophagy in neurons. Data are means ± s.e.m.; n=7. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 7: Cdk5-mediated Thr 145 phosphorylation of EndoB1 is required for MPP+- and α-synucleinA53T mutant-induced neuronal death. () Pretreatment with 3-MA, but not z-VAD-fmk, significantly reduces MPP+-induced neuronal death. Data are means ± s.e.m.; n=6. () Knockdown of Atg5 expression abrogates MPP+-induced neuronal loss, whereas it reduces neuronal survival in untreated cells. Data are means ± s.e.m.; n=3. () Knockdown of EndoB1 or Cdk5 expression abolishes MPP+-induced neuronal death. Data are means ± s.e.m.; n=8. () Overexpression of EndoB1T145A inhibits MPP+-induced neuronal death. Data are means ± s.e.m.; n=5. () Pretreatment with 3-MA, but not z-VAD-fmk, reduces α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6. () Knockdown of Atg5 expression abolishes α-synucleinA53T mutant-induced neuronal death, whereas it reduces neuronal survival in vector-transfected cells. Data are means ± s.e.m.; n=3. () Knockdown of EndoB1 or Cdk5 expression abolishes α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=4. () Expression of EndoB1T145A inhibits ! α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6. * Figure 8: Proposed model for the role of Cdk5 and EndoB1 in induced autophagy and neuronal death. Cdk5-mediated Thr 145 phosphorylation of EndoB1 is required for induced autophagy and cell death in neurons. Starvation, treatment with MPTP/MPP+ and overexpression of the α-synucleinA53T mutant increase the level of EndoB1 Thr 145 phosphorylation by Cdk5/p35 in neurons. Phosphorylation of EndoB1 promotes its dimerization and recruitment of the UVRAG/Beclin 1 complex to induce autophagy. Change history * Abstract * Change history * Author information * Supplementary informationErratum 21 April 2011In the version of this article initially published online and in print, there were some errors in Fig. 3b. These errors have been corrected in the HTML and PDF versions of the article. Author information * Abstract * Change history * Author information * Supplementary information Affiliations * Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China * Alan S. L. Wong, * Rebecca H. K. Lee, * Anthony Y. Cheung, * Zelda H. Cheung & * Nancy Y. Ip * Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China * Alan S. L. Wong, * Rebecca H. K. Lee, * Anthony Y. Cheung, * Zelda H. Cheung & * Nancy Y. Ip * State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China * Alan S. L. Wong, * Rebecca H. K. Lee, * Anthony Y. Cheung, * Zelda H. Cheung & * Nancy Y. Ip * Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China * Patrick K. Yeung & * Sookja K. Chung * Research Centre of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China * Sookja K. Chung Contributions A.S.L.W., R.H.K.L., A.Y.C. and P.K.Y. carried out the experiments. A.S.L.W., R.H.K.L., A.Y.C., P.K.Y., Z.H.C. and N.Y.I. planned the studies, designed the experiments and interpreted the results. S.K.C., Z.H.C. and N.Y.I. provided reagents and resources. A.S.L.W., Z.H.C. and N.Y.I. wrote the manuscript. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Zelda H. Cheung or * Nancy Y. Ip Author Details * Alan S. L. Wong Search for this author in: * NPG journals * PubMed * Google Scholar * Rebecca H. K. Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony Y. Cheung Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick K. Yeung Search for this author in: * NPG journals * PubMed * Google Scholar * Sookja K. Chung Search for this author in: * NPG journals * PubMed * Google Scholar * Zelda H. Cheung Contact Zelda H. Cheung Search for this author in: * NPG journals * PubMed * Google Scholar * Nancy Y. Ip Contact Nancy Y. Ip Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Exocyst function regulated by effector phosphorylation
- ncb 13(5):580-588 (2011)
Nature Cell Biology | Article Exocyst function regulated by effector phosphorylation * Xiao-Wei Chen1, 2 * Dara Leto1, 3 * Junyu Xiao1, 4 * John Goss5 * Qian Wang4 * Jordan A. Shavit1, 6, 7 * Tingting Xiong1, 2 * Genggeng Yu1 * David Ginsburg1, 6, 7, 8 * Derek Toomre5 * Zhaohui Xu1, 4 * Alan R. Saltiel1, 2, 3, 6 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:580–588Year published:(2011)DOI:doi:10.1038/ncb2226Received12 April 2010Accepted17 February 2011Published online24 April 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 The exocyst complex tethers vesicles at sites of fusion through interactions with small GTPases. The G protein RalA resides on Glut4 vesicles, and binds to the exocyst after activation by insulin, but must then disengage to ensure continuous exocytosis. Here we report that, after recognition of the exocyst by activated RalA, disengagement occurs through phosphorylation of its effector Sec5, rather than RalA inactivation. Sec5 undergoes phosphorylation in the G-protein binding domain, allosterically reducing RalA interaction. The phosphorylation event is catalysed by protein kinase C and is reversed by an exocyst-associated phosphatase. Introduction of Sec5 bearing mutations of the phosphorylation site to either alanine or aspartate disrupts insulin-stimulated Glut4 exocytosis, as well as other trafficking processes in polarized epithelial cells and during development of zebrafish embryos. The exocyst thus serves as a 'gatekeeper' for exocytic vesicles through a circuit o! f engagement, disengagement and re-engagement with G proteins. View full text Figures at a glance * Figure 1: RalA promotes exocyst function independently of GTP hydrolysis. () Constitutively active RalA enhances glucose uptake. 3T3-L1 adipocytes were infected with lentivirus expressing GFP or RalAG23V and subjected to glucose-uptake assay. The error bars represent the s.e.m. of triplicate samples. Asterisk, P<0.01. The experiment shown was representative of six independent repeats. () Constitutively active RalA upregulated glucose transport after depletion of endogenous RalA. 3T3-L1 adipocytes were pretreated for 72 h with RalA untranslated region siRNA by electroporation; GFP or constitutively active RalA (RalA G23V) was introduced into these cells by lentiviral expression. Cells were maintained in the basal state and subjected to glucose-uptake assay as in . The error bars represent the s.e.m.*, P<0.05 () Constitutively active RalA does not increase proximal insulin signalling or expression levels of glucose transporters. Cell lysates from were subjected to western blotting. The upper bands in RalA blotting represent exogenous forms. IB: im! munoblotting. () Generation of active RalA mutants uncoupled from RalBP1 or the exocyst. Indicated Flag-tagged RalA mutants were expressed in adipocytes by lentivirus, and subjected to immunoprecipitation (IP) and western blotting with indicated antibodies. () Active RalA promotes glucose uptake through the exocyst. Adipocytes were infected with lentivirus expressing indicated proteins and subjected to glucose-uptake assay. The error bars represent the s.e.m. of triplicate samples. Asterisk, P<0.05; double asterisk, P<0.001. The experiment shown was representative of three independent repeats. () Uncoupling RalA from the exocyst does not affect GTP hydrolysis of the protein. Cells expressing the indicated RalA proteins were lysed and subjected to pulldown assay; RalA bound to GST protein (active) and in cell lysates (total) was determined by western blotting. When indicated, cell lysates were loaded with 1 mM GDP or 200 μM GTPγS. In the case of active RalA or RalA loa! ded with GTPγS, only 10% of the pulldown was subjected to wes! tern blotting analysis to ensure linear range signals. () Quantification of RalA GTP loading from four independent experiments as carried out in . The error bars represent the s.d. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 2: The RalA-interacting exocyst subunit Sec5 undergoes hormone-stimulated phosphorylation in its effector domain. () Inhibition of cellular phosphatase causes a mobility shift of Sec5. 3T3-L1 adipocytes were treated with 10 nM Calyculin A for 30 min; cell lysates were subjected to SDS–PAGE and western blotting with indicated antibodies. () 32P metabolic labelling of Sec5. Cos-1 cells expressing the indicated constructs were labelled with 1 mCi of ortho-phosphate (32P) for 2 h. Cell lysates were subjected to immunoprecipitation (IP) with the indicated antibodies. After SDS–PAGE, phosphate incorporation was detected by autoradiography, whereas total protein was determined by western blotting with specific antibodies. When indicated, cells were treated with 10 ng μl−1 EGF for 5 min. () EGF recruits Sec5 to plasma membrane ruffles. Cos-1 cells expressing HA-tagged Sec5 were treated with dimethylsulphoxide (DMSO) or 10 ng μl−1 EGF for 5 min before being subjected to immunostaining with the indicated antibodies. Images were at the same magnification; bar=10 μm! . () Schematic representation of Sec5 truncation mutants. () The RBD of Sec5 undergoes hormone-stimulated phosphorylation. Cos-1 cells expressing the indicated constructs were treated with EGF as in , and subjected to immunoprecipitation and western blotting with the indicated antibodies. () Mapping the phosphorylation site on Sec5 to Ser 89. Wild-type and alanine-substituted RBDs were expressed in Cos-1 cells, and immunoprecipitated with HA antibody before being subjected to western blotting with the p-motif antibody. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 3: Sec5 effector-domain phosphorylation disengages the G protein RalA during vesicle targeting. () The phosphorylation-mimetic substitution S89D inhibits Sec5 RBD binding to active RalA. Cos-1 cells expressing the indicated RBD constructs were lysed and subjected to pulldown with immobilized GST–RalAG23V. RBD bound to RalA or present in total cell lysates was determined by western blotting. () Direct inhibition of RalA interaction by phospho-mimetic RBDS89D. Recombinant RBDs of the indicated forms were purified from Escherichia coli and immobilized on GSH beads before being used to pull down endogenous RalA from cell lysates. RalA bound to immobilized RBD or present in total cell lysates was determined by western blotting (WB). () Replacement of endogenous Sec5 by exogenous copies of the protein. Adipocytes were infected with lentivirus expressing the indicated Myc-tagged Sec5 for 3 weeks before being subjected to cell lysis and immunoprecipitation (IP) with Myc antibody. Immunoprecipitates and total cell lysates were fractioned by SDS–PAGE and were subjected to we! stern blotting analysis with indicated antibodies. The arrows indicate exogenous Sec5 (upper band) and endogenous Sec5 (lower band). () Perturbation of Sec5 Ser 89 phosphorylation disrupts plasma membrane fusion of Glut4. Adipocytes were co-infected with lentivirus expressing the indicated Sec5 constructs and the Myc–Glut4–eGFP reporter at a 6:1 ratio for 3 weeks. Cells were starved for 4 h before stimulation with vehicle or 100 ng μl−1 insulin for 20 min and fixed with 10% formalin for 10 min without permeabilization before being subjected to immunostaining with Myc (red) antibody and confocal microscopy. Images were at the same magnification; bar =10 μm. () Quantification of five different experiments (n=5 ) carried out as in . A minimum of 400 cells in total were counted for each situation. The error bars represent the s.d. Asterisk: P<0.01. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 4: PKC catalyses Sec5 effector-domain phosphorylation, which negatively regulates RalA interaction. () Recognition of the phospho-mimetic S89D substitution by p-Ser 89 antibody. Indicated GST fusion proteins purified from E.coli were blotted with p-Ser 89 antibody after SDS–PAGE. () PKC inhibitors abolish Sec5 RBD Ser 89 phosphorylation. Cos-1 cells expressing HA-tagged Sec5 RBD were treated with the indicated inhibitors. Phosphorylation was detected with the p-Ser 89 antibody after anti-HA immunoprecipitation. () PKCα stimulates Sec5 Ser 89 phosphorylation in vivo. Cos-1 cells expressing the indicated constructs were stimulated as described. Phosphorylation was detected with p-Ser 89 antibody after anti-HA immunoprecipitation. () TPA induces phosphorylation of endogenous Sec5. Cos-1 cells were stimulated as in before being subjected to immunoprecipitation and western blotting with the indicated antibody. () Loss of PKC activity inhibits Glut4 exocytosis. 3T3-L1 adipocytes expressing Myc–Glut4–eGFP reporter were transfected with control siRNA or Rictor siRNA, or tre! ated with Gö6850 or BAPTA-AM to inhibit PKC activity. Cells were processed as in Fig. 3d for confocal microscopy. Images were of the same magnification; bar=10 μm. () Quantification of four independent experiments (n=4) carried out as in . A total of 400 cells were counted for each situation. Errors represent the s.d. Asterisk: P<0.001. KD: knockdown. () Direct phosphorylation of Sec5 Ser 89 by PKC isozymes. GST fragments containing Sec5 Ser 89 (or S89A) were incubated with the indicated PKC isozymes. Phosphorylation was detected by 32P incorporation and p-Ser 89 western blotting. () PKC phosphorylation directly inhibits RalA interaction with Sec5 RBD. Wild-type or S89A GST–Sec5 RBD was incubated without or with recombinant PKCα before being immobilized on GSH beads, then used to pull down endogenous RalA from cell lysates. () PKC phosphorylates Sec5 to inhibit RalA interaction in vivo. Cos-1 cells transfected with HA-tagged Sec5 full length (FL) or RBD, Flag–RalAG! 23VK47E and wild-type GST-PKCα were subjected to anti-HA immu! noprecipitation antibody after the indicated stimulation. () Ser 89 is required for phosphorylation-dependent disengagement of RalA–Sec5 interaction in vivo. Cos-1 cells transfected with HA-tagged wild-type Sec5 or RBDS89A, Flag–RalAG23V and GST–PKCα were stimulated as in , and subjected to immunoprecipitation and western blotting with the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 5: Sec5 phosphorylation contributes to a cyclic regulatory loop on recognition by RalA. () Sec5 Ser 89 phosphorylation is concentrated at the abscission site during cytokinesis. Cos-1 cells were stained with antibodies against Sec8 (green) and p-Ser 89 Sec5 (red), and subjected to confocal microscopy. When indicated, cells were stained with an equal amount of antibodies plus p-Ser 89 antigenic peptide. Images were at the same magnification; bar =10 μm. () Structural illustration of spatial positioning of Ser 89 in Sec5 RBD. Figures were generated from the structure of Sec5 RBD (Protein Data Bank ID: 1HK6) using Pymol software (DeLano Scientific LLC). () D73S substitution alleviates the inhibitory effects on RalA interaction observed with RBDS89D. Lysates of Cos-1 cells expressing indicated constructs were incubated with immobilized GST–RalAG23V. RBD bound to RalA or present in total cell lysates was determined by western blotting. () Reconstitution of phosphorylation-dependent disengagement between RalA and Sec5. Upper: Schematic representation of the hypo! thetical sequential events during phosphorylation-induced disengagement. Lower: A complex between recombinant His–RalA loaded with GTPγS and GST–Sec5 RBD was incubated with PKC, and was immobilized by a GSH column. RalA bound to Sec5, or released into supernatant, was determined by western blotting. () Loss of Sec5 phosphorylation leads to accumulation of binding with endogenous RalA. Cos-1 cells expressing the indicated constructs were stimulated as indicated before being subjected to anti-HA immunoprecipitation. Bound RalA was detected by western blotting using protein-A-conjugated secondary antibody. () Total internal reflection fluorescent microscopy to evaluate the role of Sec5 phosphorylation. Representative images from Cos-1 cells overexpressing pHluorin–TfR and wild-type Sec5 (upper left) or Sec5S89A (lower left). Scale bar=10 μm. Representative fusion events are marked by red boxes 1 and 2, or red boxes 3 and 4. Upper right, time-series images of sample f! usion events (1 and 2) for Sec5 wild type and (3 and 4) for Se! c5S89A. Each panel=5 μm. Lower right, pHluorin–TfR fusion events per area (μm2) per time (min) observed in Sec5 wild type and Sec5S89A Cos cells. Over 1,500 fusion events from three different experiments (n=3 ) were quantified. The error bars represent the s.d. Asterisk: P<0.05. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 6: Sec5 phosphorylation contributes to cellular organization and organism development. () Perturbation of the Sec5 phosphorylation cycle leads to defects in epithelial cells. MDCK cells stably expressing the indicated HA–Sec5 proteins were fixed in formalin and stained with ZO-1(green), HA (red) and 4,6-diamidino-2-phenylindole (DAPI; blue). Images were at the same magnification; bar =10 μm. () Quantification of multinucleated cells expressing the indicated form of Sec5 from three different experiments (n=3) carried out as in . A minimum of 600 cells in total were counted. Error bars represent the s.e.m. Right: Western blotting to confirm replacement of endogenous Sec5 with cell lysates prepared from the same experiments. () Perturbation of the Sec5 phosphorylation cycle in zebrafish embryos causes severe deformation and lethality. Zebrafish embryos were injected with 0.5 mM MO oligonucleotides alone or in combination with indicated mRNAs encoding different versions of mouse Sec5 around cell stages 4–8. Embryos were monitored up to 96 hpf. () Quanti! fication of embryos with normal morphology or only minimal degeneration as in . A minimum of ~150 embryos in total were phenotyped in a double-blind manner from three or four independent experiments (n=4 for MO alone and wild type, n=3 for mutants). The error bars represent the s.d. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 7: Proposed model describing the role of the cyclic phosphorylation of Sec5 in an engagement–disengagement cycle between the exocyst and its upstream G protein. After its activation, the vesicular GTPase RalA interacts with the exocyst protein Sec5, in the process catalysing the engagement between cargo vesicles and the exocyst. Sec5 subsequently undergoes PKC-dependent phosphorylation within the RBD. This phosphorylation allosterically reduces the affinity of its interaction with RalA, resulting in the release of the vesicle from the exocyst so that it can fuse with the plasma membrane. Sec5 then undergoes dephosphorylation by a resident phosphatase, recycling the exocyst for the next round of vesicle recognition. Author information * Abstract * Author information * Supplementary information Affiliations * Life Sciences Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA * Xiao-Wei Chen, * Dara Leto, * Junyu Xiao, * Jordan A. Shavit, * Tingting Xiong, * Genggeng Yu, * David Ginsburg, * Zhaohui Xu & * Alan R. Saltiel * Department of Molecular and Integrative Physiology, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA * Xiao-Wei Chen, * Tingting Xiong & * Alan R. Saltiel * Program of Cellular and Molecular Biology, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA * Dara Leto & * Alan R. Saltiel * Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA * Junyu Xiao, * Qian Wang & * Zhaohui Xu * Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA * John Goss & * Derek Toomre * Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA * Jordan A. Shavit, * David Ginsburg & * Alan R. Saltiel * Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA * Jordan A. Shavit & * David Ginsburg * Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA * David Ginsburg Contributions X.W.C., D.L., J.X., J.G., Q.W., T.X. and G.Y. carried out experiments; X.W.C., J.A.S., D.G., D.T., Z.X. and A.R.S. analysed the data; X.W.C., Q.W., D.G., D.T., Z.X. and A.R.S. designed experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alan R. Saltiel Author Details * Xiao-Wei Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Dara Leto Search for this author in: * NPG journals * PubMed * Google Scholar * Junyu Xiao Search for this author in: * NPG journals * PubMed * Google Scholar * John Goss Search for this author in: * NPG journals * PubMed * Google Scholar * Qian Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Jordan A. Shavit Search for this author in: * NPG journals * PubMed * Google Scholar * Tingting Xiong Search for this author in: * NPG journals * PubMed * Google Scholar * Genggeng Yu Search for this author in: * NPG journals * PubMed * Google Scholar * David Ginsburg Search for this author in: * NPG journals * PubMed * Google Scholar * Derek Toomre Search for this author in: * NPG journals * PubMed * Google Scholar * Zhaohui Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Alan R. Saltiel Contact Alan R. Saltiel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (1M) Supplementary Information * Supplementary Movie 2 (2M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - During autophagy mitochondria elongate, are spared from degradation and sustain cell viability
- ncb 13(5):589-598 (2011)
Nature Cell Biology | Article During autophagy mitochondria elongate, are spared from degradation and sustain cell viability * Ligia C. Gomes1, 2, 3 * Giulietta Di Benedetto2, 4 * Luca Scorrano1, 2, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:589–598Year published:(2011)DOI:doi:10.1038/ncb2220Received24 May 2010Accepted31 January 2011Published online10 April 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 A plethora of cellular processes, including apoptosis, depend on regulated changes in mitochondrial shape and ultrastructure. The role of mitochondria and of their morphology during autophagy, a bulk degradation and recycling process of eukaryotic cells' constituents, is not well understood. Here we show that mitochondrial morphology determines the cellular response to macroautophagy. When autophagy is triggered, mitochondria elongate in vitro and in vivo. During starvation, cellular cyclic AMP levels increase and protein kinase A (PKA) is activated. PKA in turn phosphorylates the pro-fission dynamin-related protein 1 (DRP1), which is therefore retained in the cytoplasm, leading to unopposed mitochondrial fusion. Elongated mitochondria are spared from autophagic degradation, possess more cristae, increased levels of dimerization and activity of ATP synthase, and maintain ATP production. Conversely, when elongation is genetically or pharmacologically blocked, mitochondria c! onsume ATP, precipitating starvation-induced death. Thus, regulated changes in mitochondrial morphology determine the fate of the cell during autophagy. View full text Figures at a glance * Figure 1: Mitochondrial elongation in response to autophagy. () Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP. Where indicated, cells were starved for 2.5 h. Scale bar, 20 μm. () Morphometric analysis of mitochondrial shape. Experiments were carried out as in . Data represent mean ± s.e.m. of three independent experiments (n=100 cells per condition in each experiment). () Top left, 48 h after transfection with the indicated siRNA, MEFs were lysed and 25 μg samples of proteins were separated by DS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. Top right and bottom, representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP. After a further 24 h, confocal micrographs were acquired. Scale bar, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S8. () Morphometric analysis of mitochondrial shape. Experiments wer! e carried out as in . Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition in each experiment). () Representative images of mitochondrial fusion over time (indicated in min). MEFs were co-transfected with mtPAGFP and mtRFP and after 24 h, mtPAGFP was photoactivated in a region of interest and cells were imaged by real-time confocal microscopy. Where indicated, MEFs were starved for 2.5 h. Scale bar, 20 μm. See also Supplementary Movies S1 and S2. (,) Quantification of mitochondrial fusion in MEFs of the indicated genotype. Experiments were carried out as in . Data represent mean ± s.e.m. of four independent experiments. () Representative electron micrographs of muscle (longitudinal sections; four top micrographs) and liver (four bottom micrographs) from CD1 mice. Where indicated, mice were fasted for 12 h. The outlined regions of micrographs from muscle samples are magnified ×7 in the images below. Scale bars, 2 μm. * Figure 2: Increased phosphorylation of Ser 637 of DRP1 during autophagy. (–) Levels of mitochondria-shaping proteins during starvation. Protein samples (20 μg) from MEFs of the indicated genotype were separated by SDS–PAGE and immunoblotted with the indicated antibodies. Cells were starved for the indicated times. () Association of DRP1 with mitochondria during starvation. Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS–PAGE and immunoblotted with the indicated antibodies. () Levels of Ser 637 phosphorylation of DRP1 during starvation. Equal amounts of cell lysates from wild-type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS–PAGE and immunoblotted with the indicated antibodies. () Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation. Experiments were carried out as in . Data are normalized to total levels of DRP1 and represent the mean ± s! .e.m. of three independent experiments. () MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS–PAGE and immunoblotted using the indicated antibodies. () HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS–PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8. * Figure 3: Mitochondrial elongation during starvation is mediated by the cAMP–PKA axis. () Pseudocolour-coded images of EPACI–camps FRET from real-time imaging of wild-type MEFs transfected with EPACI–camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates F1/F0 ratio. See also Supplementary Movie S3. () Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in . Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments. () Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8. () Representative images of the effect of H8! 9 on mitochondrial morphology on starvation. Wild-type and Mfn2−/−MEFs were transfected with mtYFP, and after 24 h confocal micrographs were acquired. Where indicated, cells were starved for 2.5 h and 20 μM H89 was added. Scale bar, 20 μm. () Morphometric analysis. Experiments were carried out as in . Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition). () Starvation-induced mitochondrial elongation depends on Ser 637 of DRP1. Representative confocal micrographs of mitochondrial morphology of wild-type MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. () Morphometric analysis of mitochondrial shape. Experiments were carried out as in . Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition). () Representative conf! ocal micrographs of mitochondrial morphology of Drp1−/− ME! Fs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. () Morphometric analysis of mitochondrial shape. Experiments were carried out as in . Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition). * Figure 4: Elongated mitochondria are spared from degradation during starvation. (–) MEFs of the indicated genotype were treated as indicated, counted and 2.7×105 cells were lysed. Lysates were separated by SDS–PAGE and immunoblotted using the indicated antibodies. () Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype. One representative experiment of five independent repetitions performed as in – is shown. () MEFs of the indicated genotype starved for 5 h were treated where indicated with 0.5 μM wortmannin (Wortm.). Lysates from 2.7×105 cells were separated by SDS–PAGE and immunoblotted with the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8. * Figure 5: Mitochondrial elongation sustains cellular ATP production and viability during autophagy. (,) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype. Where indicated, cells were starved for 5 h before TMRM loading. Where indicated (arrows), 2.5 μg ml−1 oligomycin and 2 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were added. Data represent mean ± s.e.m. of seven independent experiments. () Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments. () Mitochondrial ATP measured in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments and are normalized to the initial value. (,) Cells of the indicated genotype were starved for the indicated times. Viability was determined by flow cytometry as the percentage of annexin-V- and propidium iodide (PI)-negati! ve events. Data represent mean ± s.e.m. of five independent experiments. () MEFs of the indicated genotype were starved for 2.5 h. Where indicated, cells were treated with 20 μM H89. Viability was determined as in ,. Data represent mean ± s.e.m. of five independent experiments. () Cells of the indicated genotype were starved for the indicated times. Viability was determined as in ,. Data represent mean ± s.e.m. of five independent experiments. () Drp1−/− MEFs were transfected with the indicated plasmids and after 24 h starved for 5 h where indicated. Cell death was determined by flow cytometry as the percentage of YFP- and propidium-iodide-positive events. Data represent mean ± s.e.m. of four independent experiments. (,) MEFs of the indicated genotype were starved for 5 h in the presence of 2.5 μg ml−1 oligomycin where indicated. Viability was determined as in ,. Data represent mean ± s.e.m. of five independent experiments. * Figure 6: Mitochondrial elongation during starvation is associated with dimerization and activation of ATPase. () Blue-native electrophoresis analysis of ATPase dimerization and activity. Cells of the indicated genotype were treated as indicated and 500 μg of total cell extract was solubilized with 4% digitonin and proteins were separated by blue-native PAGE. ATPase activity was measured in-gel (top) and ATPase levels were measured by immunoblotting for the indicated antibody (bottom). (,) Quantitative analysis of the ratio of levels () and activity () between dimeric and monomeric forms of ATPase, assessed by densitometric analysis of gels from experiments performed as in . Data represent mean ± s.e.m. of five independent experiments. () Experiments were carried out as in , but with cells of the indicated genotype. * Figure 7: Density of cristae increases in mitochondria elongated during starvation. () Representative electron micrographs of cells of the indicated genotype starved where indicated for 5 h, fixed and processed for electron microscopy. Scale bar, 2 μm. () Representative electron micrographs of randomly selected mitochondria from cells of the indicated genotype. Where indicated, cells were starved for 5 h. Scale bars, 0.5 μm. () Morphometric analysis of cristae density in cells of the indicated genotype. Experiments were carried out as in . The number of cristae in 50 randomly selected mitochondria of the indicated genotype was normalized for the calculated surface of the organelle. Data represent mean ± s.e.m. of five independent experiments. * Figure 8: Mitochondrial elongation induced by PKA determines cell fate during starvation. The diagrams depict the cascade of mitochondrial elongation triggered during starvation and its role in determining cell fate. Top row: mitochondrial elongation protects it from organelle degradation and allows maintenance of ATP levels. Bottom row: when mitochondrial elongation is impaired, mitochondria are degraded and the remaining organelles consume cellular ATP, precipitating cell death. Author information * Abstract * Author information * Supplementary information Affiliations * Dulbecco-Telethon Institute, Via Orus 2, 35129 Padova, Italy * Ligia C. Gomes & * Luca Scorrano * Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padova, Italy * Ligia C. Gomes, * Giulietta Di Benedetto & * Luca Scorrano * PhD Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal * Ligia C. Gomes * CNR Institute for Neurosciences, Section of Padova, Via G. Colombo 3, 35129 Padova, Italy * Giulietta Di Benedetto * Department of Cell Physiology and Medicine, University of Geneva, 1 Rue M. Servet, 1211 Geneve, Switzerland * Luca Scorrano Contributions L.C.G. and L.S. conceived research, analysed data and wrote the manuscript. L.C.G., G.D.B. and L.S. carried out experiments and analysed data. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Luca Scorrano Author Details * Ligia C. Gomes Search for this author in: * NPG journals * PubMed * Google Scholar * Giulietta Di Benedetto Search for this author in: * NPG journals * PubMed * Google Scholar * Luca Scorrano Contact Luca Scorrano 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 (6M) Supplementary Information * Supplementary Movie 3 (18M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - Meiotic homologue alignment and its quality surveillance are controlled by mouse HORMAD1
- ncb 13(5):599-610 (2011)
Nature Cell Biology | Article Meiotic homologue alignment and its quality surveillance are controlled by mouse HORMAD1 * Katrin Daniel1 * Julian Lange2 * Khaled Hached3, 4 * Jun Fu5 * Konstantinos Anastassiadis6 * Ignasi Roig2, 10 * Howard J. Cooke7 * A. Francis Stewart5 * Katja Wassmann3, 4 * Maria Jasin8 * Scott Keeney2, 9 * Attila Tóth1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:599–610Year published:(2011)DOI:doi:10.1038/ncb2213Received15 October 2010Accepted20 January 2011Published online10 April 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 Meiotic crossover formation between homologous chromosomes (homologues) entails DNA double-strand break (DSB) formation, homology search using DSB ends, and synaptonemal-complex formation coupled with DSB repair. Meiotic progression must be prevented until DSB repair and homologue alignment are completed, to avoid the formation of aneuploid gametes. Here we show that mouse HORMAD1 ensures that sufficient numbers of processed DSBs are available for successful homology search. HORMAD1 is needed for normal synaptonemal-complex formation and for the efficient recruitment of ATR checkpoint kinase activity to unsynapsed chromatin. The latter phenomenon was proposed to be important in meiotic prophase checkpoints in both sexes. Consistent with this hypothesis, HORMAD1 is essential for the elimination of synaptonemal-complex-defective oocytes. Synaptonemal-complex formation results in HORMAD1 depletion from chromosome axes. Thus, we propose that the synaptonemal complex and HORMAD1 ! are key components of a negative feedback loop that coordinates meiotic progression with homologue alignment: HORMAD1 promotes homologue alignment and synaptonemal-complex formation, and synaptonemal complexes downregulate HORMAD1 function, thereby permitting progression past meiotic prophase checkpoints. View full text Figures at a glance * Figure 1: HORMAD1 promotes synaptonemal-complex formation independently of DSB-dependent processes. (–) Images of SYCP3 (chromosome axis) and SYCP1 (synaptonemal-complex transverse filament), detected by immunofluorescence microscopy on nuclear spreads of wild-type zygotene (), wild-type pachytene () and mutant zygotene–pachytene (– left) spermatocytes collected from 14-week-old mice. (– right) The frequency distribution of synaptonemal-complex stretches in mutant zygotene–pachytene spermatocytes. () Synaptonemal-complex formation is never completed on all autosomes in Hormad1−/− cells, and unlike in meiocytes with a mutated synaptonemal-complex transverse filament, in which unsynapsed chromosomes align along their length8, 9, 10, 11, unsynapsed chromosomes do not align in Hormad1−/− spermatocytes. Nevertheless, robust stretches of synaptonemal complex frequently form between chromosomes that seem homologous on the basis of their similar axis lengths. Insets, enlarged view of a partially synapsed autosome: unsynapsed axes (arrowheads) are of similar lengt! hs. () In contrast, synaptonemal complexes connect multiple non-homologous axes, thereby creating a meshwork of interconnected axes in the Spo11−/− mutant, in which strand invasion and homology search are not possible owing to the lack of DSBs. Insets, enlarged view of chromosome axes: arrowheads mark unsynapsed, arrows mark synapsed axes. () Both the number and the length of SYCP1 stretches are lower in Hormad1−/− Spo11−/− spermatocytes, relative to the single mutants. Most (61% in n=144 cells) of the remaining SYCP1 stretches are unambiguously linked to a single chromosome axis (arrowheads), indicating that SYCP1 stretches do not necessarily mark inter-chromosomal synaptonemal complexes. Scale bars, 10 μm. * Figure 2: Numbers of early-, intermediate- and late-recombination-protein foci are lower in the absence of HORMAD1 in prophase meiocytes. (–) SYCP3 and either RAD51 (), DMC1 (), RPA () or MSH4 () are detected on nuclear spreads of typical early–mid-zygotene wild-type and Hormad1−/− spermatocytes from 16-day-old mice. Scale bars, 10 μm. (–) Numbers of early (RAD51, ; DMC1, ) and intermediate (RPA, ; MSH4, ) recombination protein foci during leptotene (le) and early–mid-zygotene (e-zy) in wild-type and Hormad1−/−, late zygotene (l-zy) and pachytene (pa) in wild-type and zygotene–pachytene (zy-pa) in Hormad1−/− spermatocytes. Median numbers of foci are marked. During the comparable early–mid-zygotene stage, a threefold to sixfold reduction (highly significant by Mann–Whitney test) is observed in the numbers of recombination protein foci in the mutant relative to wild-type spermatocytes. (–) The numbers of crossover marker MLH1 foci are lower in the absence of HORMAD1 in oocytes. () SYCP3 (chromosome axis), SYCE2 (synaptonemal-complex central element) and MLH1 were detected by immuno! fluorescence microscopy in nuclear spreads of Hormad1+/− and Hormad1−/− oocytes from 19.5 dpc fetuses (a stage when most oocytes are in the late pachytene or diplotene stage in wild-type mice). Fewer chromosome-axis-associated MLH1 foci are detected in Hormad1−/− oocytes than in Hormad1+/− oocytes. Note that most MLH1 foci (78%, n=51 cells) are observed on synapsed axes in the mutant. Scale bars, 10 μm. () Scatter plot shows positive correlation (Spearman's r=0.72,n=30) between the number of MLH1 foci and the number of synaptonemal-complex (SC) stretches (immunostaining for SYCE1 or SYCE2) in Hormad1−/− oocytes, indicating that DSBs may be repaired as crossovers preferentially in chromosome regions that align and synapse, or that synapsis occurs preferentially where crossovers are successfully designated. () The number of chromosome-axis-associated MLH1 foci is approximately threefold lower in Hormad1−/− oocytes from 18.5 to 19.5 dpc fetuses, relati! ve to Hormad1+/− oocytes. Median numbers of foci are marked ! by horizontal lines. * Figure 3: Amounts of SPO11–oligonucleotide complexes in testes are lower in the absence of HORMAD1. (–) Levels of SPO11–oligonucleotide complexes and SPO11 protein were examined in the testes of the indicated mouse strains. (,) Measurement of SPO11–oligonucleotide complexes in testes of adult 14-week-old () and juvenile 14-dpp () mice. SPO11–oligonucleotide complexes were immunoprecipitated with or without anti-SPO11 antibodies, and covalently linked oligonucleotides were radioactively labelled. Each sample represents one-testis-equivalent SPO11–oligonucleotide complexes. Bars mark SPO11-specific signals and asterisks indicate nonspecific labelling of a contaminant in the terminal deoxytransferase preparations. Ig-hc marks an artefactual radioactive signal attributable to the presence of immunoglobulin heavy chain (Ig-hc in ). Quantified radioactive signals in the mutants have been background-corrected and normalized: in signals are normalized to the adult wild-type control; in Dmc1−/− and Hormad1−/− signals are normalized to their littermate Dmc1+/+ and ! Hormad1+/+ controls, respectively (see Methods). Blots of immunoprecipitates from and were probed with anti-SPO11 antibodies in and , respectively. () In wild-type adults, two alternative forms of SPO11 (α and β ) are present5. Total SPO11 amounts are similar in Dmc1−/− and Hormad1−/− Dmc1−/− mutants, and are lower in the mutants than in wild-type controls. Only SPO11β, the form that appears early in meiosis, is detected in the mutants. Arrowhead marks the immunoglobulin heavy chain (bleached out signal). Mid-pachytene spermatogenic block in the mutants () is the likely cause of the lower SPO11 amounts in Dmc1−/− and Hormad1−/− Dmc1−/− testes, and of the lower SPO11–oligonucleotide amounts in Dmc1−/− testes12, 43, 44. () In juveniles, total SPO11 protein levels are low and only the long β form of SPO11 (arrow) is detectable5. For full-scan gel images of –, see Supplementary Fig. S10. () DNA was detected by DAPI, and apoptosis was detected! by an IF–TUNEL assay on cryosections of testes of 15-week-o! ld mice. Dmc1−/− and Hormad1−/− Dmc1−/− spermatocytes undergo apoptosis in stage-IV tubules, as identified by the concomitant presence of intermediate spermatogonia (In), late-prometaphase intermediate spermatogonia (La–In), mitotic intermediate spermatogonia (m) and spermatogonia B (SgB). Both non-apoptotic (sc) and apoptotic (asc) spermatocytes are present in the stage-IV tubules shown. Spermatocytes are fully eliminated on progression to stage V (data not shown). Scale bars, 20 μm. * Figure 4: HORMAD1 is required for sex-body and pseudo-sex-body formation in the Spo11+/+ and Spo11−/− backgrounds, respectively. () SYCP3 (chromosome axis), SYCE2 (synaptonemal-complex central element) and γH2AX were detected in nuclear spreads of wild-type late-zygotene and pachytene, and Hormad1−/− zygotene–pachytene spermatocytes collected from 16-day-old mice. Matched-exposure images of γH2AX are shown. In wild-type late-zygotene spermatocytes, γH2AX chromatin domains associate with unsynapsed chromosome axes. In wild-type pachytene cells, unsynapsed regions of X and Y sex-chromosome axes (marked by x and y) are surrounded by one large γH2AX-rich chromatin domain, the sex body. Anti-γH2AX staining is patchy in most (59%) Hormad1−/− spermatocytes, with no clear correlation between lack of synapsis and γH2AX localization (third row). A few large γH2AX-rich chromatin domains form in a small number of Hormad1−/− spermatocytes (41%, n=512 ), but only a subset of unsynapsed axes overlap with γH2AX-rich chromatin, and synapsed axes overlapping with γH2AX-rich chromatin domains are ! also observed regularly (bottom row). Arrowheads mark two unsynapsed axes in wild-type late-zygotene spermatocytes and in each mutant cell type. Scale bars, 10 μm. () Matched-exposure images of SYCP3 (chromosome axis), SYCP1 (synaptonemal-complex transverse filament) and γH2AX in nuclear spreads of Spo11−/−, Hormad1−/− Spo11−/− and Syce2−/− Spo11−/− spermatocytes of adult (9-week-old) mice. Large γH2AX-rich chromatin domains (pseudo-sex bodies marked by arrowheads) frequently form in Spo11−/− and Syce2−/− Spo11−/− spermatocytes. Scale bars, 10 μm. () Quantification of pseudo-sex-body formation in spermatocytes with full-length chromosome axes (collected from 24-day-old mice). The percentage of cells with no pseudo-sex body (cells without PSB) or with one to three clear pseudo-sex bodies (cells with PSB) is shown. () Quantification of the γH2AX signal in spermatocytes with full-length chromosome axes (collected from 24-day-old mice). ! There is a significant reduction in total nuclear γH2AXamount! s in spermatocytes from Hormad1−/− Spo11−/− mice, relative to spermatocytes from Spo11−/− and Syce2−/− Spo11−/− mice (Mann–Whitney test). * Figure 5: HORMAD1 is required for efficient accumulation of ATR, TOPBP1 and BRCA1 on chromatin in the absence of programmed DSBs. (–) Matched-exposure images of SYCP3 (chromosome axis), γH2AX and either ATR (), TOPBP1 () or BRCA1 () in nuclear spreads of spermatocytes collected from 24-day-old mice. Spo11−/− and Syce2−/− Spo11−/− spermatocytes are shown with cloud-like ATR () or TOPBP1 () accumulation, or with BRCA1 localized to axes () in γH2AX-marked pseudo-sex bodies. In and , Hormad1−/− Spo11−/− cells are shown without a pseudo-sex body and without ATR or TOPBP1 accumulation on chromatin, respectively. In , a Hormad1−/− Spo11−/− cell is shown with a pseudo-sex body, within which no BRCA1 association with the axis was detected. Note that only a small number of Hormad1−/− Spo11−/− spermatocytes show pseudo-sex-body-like accumulation of γH2AX (Fig. 4c). Scale bars, 10 μm. (–) Frequency of cloud-like ATR () and TOPBP1 () accumulation and frequency of BRCA1 association with axes () in spermatocytes with fully formed chromosome axes. ATR- and TOPBP1-rich chro! matin domains are frequently observed in Spo11−/− and Syce2−/− Spo11−/− spermatocytes, and BRCA1 association with axes is also observed in these mutants. ATR and TOPBP1 are virtually absent from chromatin in most of the Hormad1−/− Spo11−/− cells, and BRCA1 localization to axes was never observed in Hormad1−/− Spo11−/− spermatocytes. * Figure 6: Lack of HORMAD1 allows survival of oocytes in the synaptonemal-complex-defective Spo11−/− mutant. () NOBOX (postnatal oocyte marker) was detected by immunofluorescence microscopy on cryosections of ovaries from 6-week-old mice. DNA was detected by DAPI. Oocytes in primordial (pd), primary (pr) and secondary (s) follicular stages are shown in wild-type, Hormad1−/− and Hormad1−/− Spo11−/− ovaries. In the synaptonemal-complex-defective Spo11−/− mutant, oocyte numbers are much lower. A lower-magnification section of a Spo11−/− ovary is shown to better illustrate the absence of oocytes. Scale bars, 50 μm. () Sum of oocyte numbers on every tenth section of sectioned-through ovary pairs at the indicated ages. Each data point represents a mouse. () Fraction of apoptotic oocytes in the ovaries of 1-day-old (1 dpp) mice of indicated genotypes. Horizontal lines show medians in and . * Figure 7: Lower numbers of chiasmata form in Hormad1−/− oocytes. () Centromeres were detected by immunofluorescence microscopy and DNA was detected by propidium iodide staining on nuclear spreads of in vitro-matured metaphase stage oocytes. In wild-type cells, 20 pairs of chromosomes are connected by chiasmata. In the Hormad1−/− mutant, many chromosomes do not have chiasmata (one such chromosome is marked by an arrowhead). Arrow marks a pair of chromosomes connected by a chiasma in the mutant oocyte. Note that bivalents are symmetrical and chiasmata form between chromosomes of identical length in the mutant, indicating that crossover formation took place between homologous chromosomes. Chromosomes scatter over a larger area during nuclear spreading in the mutant; therefore, it was not possible to include all 40 chromosomes of a meiosis I oocyte in the image. Scale bars, 10 μm. () The average numbers of paired chromosomes connected by chiasmata (marked by line) are nearly threefold lower in in vitro-matured Hormad1−/− oocytes, r! elative to wild-type oocytes. (,) mRNAs encoding β -tubulin–GFP and histone-H2B–RFP were injected into wild-type () and Hormad1−/− () oocytes during the germinal vesicle stage (prophase), and the oocytes were matured in vitro. The fluorescent proteins in the oocytes were imaged 5.5 h after germinal vesicle break down (GVBD), at a time when wild-type oocytes are in the first meiotic metaphase, and 17.5 h after GVBD, at a time when wild-type oocytes are arrested in the second meiotic metaphase. A polar body (marked by arrowhead in ), which was extruded 7 h after GVBD (Supplementary Movie S1), is observed next to the metaphase II stage wild-type oocyte at the 17.5 h time point. In the Hormad1−/− oocyte (), the meiotic spindle is abnormally long and chromosomes fail to align at both time points. Meiotic anaphase did not take place in the Hormad1−/− oocyte shown (Supplementary Movie S2), and no polar body can be observed at either time point (). Scale ba! rs, 20 μm. * Figure 8: Model for meiotic progression: negative feedback loop of HORMAD1 and synaptonemal-complex coordinates homology search and meiotic progression. Processes, activation–promotion and inhibition are marked by solid black arrows, red dotted arrows and blue flat-ended dashed arrows, respectively. HORMAD1 associates with forming chromosome axes at the beginning of meiosis, where it promotes DSB formation and/or processing of DSBs. It thereby ensures that adequate numbers of single-stranded DSBs are available for homology search. As part of the homology search process, DSB ends strand-invade into homologues. Multiple strand-invasion events along the length of chromosomes lead to full alignment of pairs of homologues, which is a prerequisite for the completion of synaptonemal-complex formation. HORMAD1 also promotes synaptonemal-complex formation through a mechanism that is independent of homology search. Synaptonemal-complex formation leads to depletion of HORMAD1 from axes and downregulation of HORMAD1 function40. Hence, in spermatocytes, full autosomal synaptonemal-complex formation leads to a restriction of HORMAD1 and! ATR activity to sex chromosomes, thereby promoting efficient silencing of sex chromosomes, which is a prerequisite for progression beyond mid-pachytene13, 60, 61. In oocytes, completion of synaptonemal-complex formation on all chromosomes leads to complete inactivation of HORMAD1, which in turn leads to downregulation of ATR and MSUC. As sustained ATR activity and/or sustained MSUC is believed to block progression beyond meiotic prophase13, full synaptonemal-complex formation and HORMAD1 inactivation link successful homologue alignment with progression beyond meiotic prophase. Note that successful DSB repair is probably also required for full downregulation of ATR activity and for meiotic progression in oocytes (for the sake of simplicity, this branch of the prophase checkpoint is not shown in the model). Author information * Abstract * Author information * Supplementary information Affiliations * Institute of Physiological Chemistry, Technische Universität Dresden, Fiedlerstrasse 42, 01307 Dresden, Germany * Katrin Daniel & * Attila Tóth * Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA * Julian Lange, * Ignasi Roig & * Scott Keeney * CNRS UMR7622 Biologie du Développement, 9 quai St Bernard, Paris, 75005, France * Khaled Hached & * Katja Wassmann * UPMC Paris 6, 9 quai St Bernard, Paris, 75005, France * Khaled Hached & * Katja Wassmann * Genomics, BioInnovationsZentrum, Technische Universität Dresden, Am Tatzberg 47, 01307 Dresden, Germany * Jun Fu & * A. Francis Stewart * Center for Regenerative Therapies Dresden, BioInnovationsZentrum, Technische Universität Dresden, Am Tatzberg 47, 01307 Dresden, Germany * Konstantinos Anastassiadis * MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK * Howard J. Cooke * Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA * Maria Jasin * Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA * Scott Keeney * Present address: Unitat de Citologia i Histologia, Departamento Biologia Cellular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193, Spain * Ignasi Roig Contributions K.D. designed, carried out and analysed most of the experiments; J.L., I.R., M.J. and S.K. contributed with SPO11–oligonucleotide measurements; J.F., K.A. and A.F.S. designed and generated the targeting construct and targeted embryonic stem cells; K.H. and K.W. carried out oocyte-maturation experiments and oocyte video microscopy; H.J.C. provided the Syce2+/− mouse, A.T. was involved in oocyte-maturation experiments and oocyte counts, helped K.D. in experimental design and wrote the paper together with K.D. All authors were involved in discussions and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Attila Tóth Author Details * Katrin Daniel Search for this author in: * NPG journals * PubMed * Google Scholar * Julian Lange Search for this author in: * NPG journals * PubMed * Google Scholar * Khaled Hached Search for this author in: * NPG journals * PubMed * Google Scholar * Jun Fu Search for this author in: * NPG journals * PubMed * Google Scholar * Konstantinos Anastassiadis Search for this author in: * NPG journals * PubMed * Google Scholar * Ignasi Roig Search for this author in: * NPG journals * PubMed * Google Scholar * Howard J. Cooke Search for this author in: * NPG journals * PubMed * Google Scholar * A. Francis Stewart Search for this author in: * NPG journals * PubMed * Google Scholar * Katja Wassmann Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Jasin Search for this author in: * NPG journals * PubMed * Google Scholar * Scott Keeney Search for this author in: * NPG journals * PubMed * Google Scholar * Attila Tóth Contact Attila Tóth Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (2M) Supplementary Information * Supplementary Movie 2 (2M) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Auxin triggers a genetic switch
- ncb 13(5):611-615 (2011)
Nature Cell Biology | Letter Auxin triggers a genetic switch * Steffen Lau1 * Ive De Smet1, 2, 4 * Martina Kolb1 * Hans Meinhardt3 * Gerd Jürgens1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:611–615Year published:(2011)DOI:doi:10.1038/ncb2212Received28 June 2010Accepted20 January 2011Published online10 April 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cell specification in development requires robust gene-regulatory responses to transient signals. In plants, the small signalling molecule auxin has been implicated in diverse developmental processes1, 2. Auxin promotes the degradation of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) inhibitors that prevent AUXIN RESPONSE FACTOR (ARF) transcription factors from regulating their target genes1, 3. However, the precise role of auxin in patterning has remained unclear, the view of auxin acting as a morphogen is controversial4, 5 and the transcriptional control of the ARF genes themselves is barely explored6. Here, we demonstrate by experimental and computational analyses that the Arabidopsis ARF protein MONOPTEROS (MP) controls its own expression and the expression of its AUX/IAA inhibitor BODENLOS (BDL), with auxin acting as a threshold-specific trigger by promoting the degradation of the inhibitor. Our results suggest a general mechanism for how the transient accumulation of auxin acti! vates self-sustaining or hysteretic feedback systems of interacting auxin-response proteins that, similarly to other genetic switches, result in unequivocal developmental responses. View full text Figures at a glance * Figure 1: Regulation of BDL and MP expression by auxin (1-naphthylacetic acid, NAA), MP and BDL. (–) Analysis of BDL (–) and MP expression (–) in Arabidopsis protoplasts (–,–) and seedlings (,) using luminescence assays (,,,) and quantitative real-time PCR (,,,). Protoplasts and seedlings were treated with NAA, CHX or DEX and/or expressed MP, bdl or MPΔIII/IV as indicated; protoplasts (,) and seedlings (,) expressed GR:MPΔIII/IV (not indicated in the panels). () Auxin, MP and MPΔIII/IV induce the expression of pBDL::LUC, whereas stabilized BDL (bdl) blocks pBDL::LUC induction by MP ± auxin. () MPΔIII/IV induces the expression of pBDL::BDL:LUC. (,) Relative LUC transcript levels (pBDL::LUC) in protoplasts (), and relative BDL transcript levels in seedlings () as determined by quantitative real-time PCR, after cycloheximide (CHX) ± dexamethasone (DEX) treatment causing nuclear uptake of GR:MPΔIII/IV. () Auxin, MP and MPΔIII/IV induce the expression of pMP::LUC, whereas stabilized BDL (bdl) blocks pMP::LUC induction by MP ± auxin. () MPΔIII/IV induces t! he expression of pMP::MP:LUC. (,) Relative LUC transcript levels (pMP::LUC) in protoplasts () and relative MP transcript levels in seedlings (), as determined by quantitative real-time PCR after cycloheximide ± dexamethasone treatment causing nuclear uptake of GR:MPΔIII/IV. Values represent mean ± s.e.m. *, statistically significant differences for values when compared with the control (− indicates no NAA treatment and no effector DNA; CHX, cycloheximide treatment), or as indicated by black lines, as determined by Student's t -test (P<0.05); for statistical details, see Supplementary Table S2. * Figure 2: Auxin-regulated MP expression in planta. (–) pMP::n3xGFP expression is altered in gnom mutant embryos (,), compared with wild-type embryos (,). Depicted are transition-stage embryos (,) and torpedo-stage embryos (,). Of 47 gnom mutant embryos, 28% exhibited the expression pattern shown. Insets in and show pDR5::GFP expression in wild-type and gnom mutant embryos, respectively. () Treatment with 1-naphthylphthalamic acid affected pMP::n3xGFP expression in ovule-cultured embryos in a way comparable to gnom mutant embryos (compare with and ). (,) Auxin (2,4-dichlorophenoxyacetic acid) treatment caused ectopic expression of pMP::n3xGFP in Arabidopsis seedling roots. Optical section through the root of a control () and an auxin-treated seedling (). Ectopic gene expression (white arrowheads) and epidermis (Ep) are indicated. Insets in , and show respective differential-interference contrast images. Scale bars, 20 μm. * Figure 3: MP–BDL regulatory circuitry. () Schematic representation of the interactions between MP, BDL and auxin. MP and BDL in rectangles, MP and BDL genes; encircled MP and BDL, respective proteins; dashed arrows, expression (transcription + translation); solid green arrows, transcriptional activation; solid red line (coming from BDL), protein inhibition; solid blue line, stimulation of protein degradation. (,) Simulation of the switch-like activation of MP and BDL under the influence of auxin. The plots show MP (green), BDL (red) and auxin (blue) concentrations in a single cell as a function of time. () One-step increase and decrease of auxin levels, respectively. () Two-step increase and decrease of auxin levels, respectively, to illustrate the hysteresis. * Figure 4: Gene activation by MP. (,) Induction of pBDL::LUC () and pDR5::LUC () by dexamethasone-induced GR:MPΔIII/IV. Assays were carried out in the presence of bdl to reduce the background activity of pBDL::LUC and pDR5::LUC, respectively. Values represent mean ± s.e.m. For statistical details, see Supplementary Table S2. Author information * Author information * Supplementary information Affiliations * Department of Cell Biology, Max Planck Institute for Developmental Biology, Spemannstraße 35, D-72076 Tübingen, Germany * Steffen Lau, * Ive De Smet, * Martina Kolb & * Gerd Jürgens * Center for Plant Molecular Biology, University of Tübingen, Auf der Morgenstelle 3, D-72076 Tübingen, Germany * Ive De Smet & * Gerd Jürgens * Max Planck Institute for Developmental Biology, Spemannstraße 35, D-72076 Tübingen, Germany * Hans Meinhardt * Present address: Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Loughborough LE12 5RD, UK * Ive De Smet Contributions S.L. and G.J. designed the project, S.L., I.D.S., H.M. and M.K. carried out the research, and S.L., I.D.S., H.M. and G.J. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gerd Jürgens Author Details * Steffen Lau Search for this author in: * NPG journals * PubMed * Google Scholar * Ive De Smet Search for this author in: * NPG journals * PubMed * Google Scholar * Martina Kolb Search for this author in: * NPG journals * PubMed * Google Scholar * Hans Meinhardt Search for this author in: * NPG journals * PubMed * Google Scholar * Gerd Jürgens Contact Gerd Jürgens Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (700K) Supplementary Information Additional data - Coordinated transcriptional regulation underlying the circadian clock in Arabidopsis
- ncb 13(5):616-622 (2011)
Nature Cell Biology | Letter Coordinated transcriptional regulation underlying the circadian clock in Arabidopsis * Gang Li1 * Hamad Siddiqui2, 8 * Yibo Teng3, 8 * Rongcheng Lin4, 8 * Xiang-yuan Wan5 * Jigang Li1 * On-Sun Lau1 * Xinhao Ouyang1 * Mingqiu Dai1 * Jianmin Wan6 * Paul F. Devlin2 * Xing Wang Deng1, 5 * Haiyang Wang1, 5, 6, 7 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:616–622Year published:(2011)DOI:doi:10.1038/ncb2219Received07 September 2010Accepted28 January 2011Published online17 April 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The circadian clock controls many metabolic, developmental and physiological processes in a time-of-day-specific manner in both plants and animals1, 2. The photoreceptors involved in the perception of light and entrainment of the circadian clock have been well characterized in plants3. However, how light signals are transduced from the photoreceptors to the central circadian oscillator, and how the rhythmic expression pattern of a clock gene is generated and maintained by diurnal light signals remain unclear. Here, we show that in Arabidopsis thaliana, FHY3, FAR1 and HY5, three positive regulators of the phytochrome A signalling pathway, directly bind to the promoter of ELF4, a proposed component of the central oscillator, and activate its expression during the day, whereas the circadian-controlled CCA1 and LHY proteins directly suppress ELF4 expression periodically at dawn through physical interactions with these transcription-promoting factors. Our findings provide evidenc! e that a set of light- and circadian-regulated transcription factors act directly and coordinately at the ELF4 promoter to regulate its cyclic expression, and establish a potential molecular link connecting the environmental light–dark cycle to the central oscillator. View full text Figures at a glance * Figure 1: FHY3 and FAR1 are essential for circadian gene expression and flowering-time control. (–) qRT–PCR analyses showing that cyclic expression of CCA1, LHY and ELF4 was reduced in fhy3 and far1 mutants in continuous white-light (LL) conditions (mean±s.d., n=3). (,) Bioluminescence assays showing that expression of CAT3::LUC and TOC1::LUC reporters was arrhythmic in the fhy3 mutants in continuous white-light conditions. () Expression of CAB2::LUC showed a reduction in amplitude and lengthening of period. c.p.s., counts per second (mean±s.e.m., n=18). The white and grey bars in each panel indicate the subjective day and night, respectively. (,) fhy3 mutants flowered early under both long-day (16 h light/8 h dark) and short-day (8 h light/16 h dark) conditions. The numbers of rosette leaves at the time of the first flower opening are shown (mean±s.d. , n=19 for fhy3-9, n=30 for other lines; , n=24). No-0; Nossen ecotype, wild type. * Figure 2: FHY3, FAR1, CCA1, LHY and HY5 directly bind to distinct cis elements in the ELF4 promoter in vitro and in vivo. () Top panel, schematic representation of the position and nucleotide sequence of various cis-elements in the ELF4 promoter. wt, wild-type; m, mutant. Bottom panels, yeast one-hybrid assays showing that CCA1, LHY, FHY3, FAR1 and HY5 activate the LacZ reporter genes driven by the full-length wild-type ELF4 promoter, but not LacZ reporter genes driven by the ELF4 promoter with mutations in the specific cis elements. mEE, mutation of all three EE elements (EE-1, EE-2 and EE-3). AD; activation domain. () ChIP–qPCR assays showing that the ELF4 promoter fragments containing the putative FHY3-, FAR1-, HY5- and CCA1-binding sites are specifically enriched. A carboxy-terminal exon region (Exon) of ELF4 and Actin were used as the negative controls for the ChIP–qPCR experiment (mean±s.d., n=3). Col-0; Colombia ecotype, wild type. () Overexpression of ELF4 (ELF4-OX) rescued the early-flowering phenotype of the fhy3 mutant under long-day conditions. B2 and F2 are two independent tra! nsgenic lines (mean±s.d., n=19 for No-0 and B2; n=15 for fhy3-4 and F2). * Figure 3: FHY3, FAR1 and HY5 activate ELF4 expression, whereas CCA1 and LHY suppress ELF4 expression. (,) Bioluminescence assays showing that ELF4::LUC expression was reduced in fhy3 far1 () and hy5 hyh () mutants. Wild-type controls; No-0, Nossen ecotype; Ws, Wassilewskija ecotype. () Bioluminescence assays showing that the amplitude and rhythmic pattern of ELF4 expression were altered by mutations in specific cis elements (FBSm::LUC and ACEm::LUC). c.p.s., counts per second. Data are means±s.d., n=25. Data are representative of four independent lines. () qRT–PCR assays showing that ELF4 expression is reduced in the CCA1-OX transgenic plants. The white and grey bars in each panel indicate the subjective day and night, respectively (mean±s.d., n=3). LL, continuous white-light conditions. Col-0; Colombia ecotype, wild type. (,) Transient expression assays showing that FHY3, FAR1 and HY5 upregulate (), whereas CCA1 and LHY downregulate (), ELF4 expression in Nicotiana benthamiana leaves. CK, empty vector control. Relative expression of ELF4::LUC was normalized to 35S::REN ! (internal control) (LUC/REN, mean±s.d., n=3). * Figure 4: CCA1 and LHY suppress the transcriptional activation activity of FHY3, FAR1 and HY5 through direct physical interaction. () Yeast two-hybrid assays showing that FHY3, but not FAR1, interacts with CCA1, LHY and HY5 in vitro(mean±s.d., n=5). () Co-immunoprecipitation assays showing that CCA1 specifically associates with FHY3 in vivo. Anti-RPT5 antibodies were used as a negative control for the immunoprecipitation experiment. () LCI assays showing that the N termini of FHY3 and CCA1 mediate the interaction. nLUC and cLUC, N-terminal or C-terminal fragment of firefly luciferase, respectively; FHY3N and CCA1N, N-terminal fragment of FHY3 and CCA1, respectively. () CCA1 and LHY suppress the activation activity of FHY3, FAR1 and HY5 on ELF4 expression in Nicotiana benthamiana leaves. Relative expression of ELF4::LUC was normalized to 35S::REN (internal control; LUC/REN, mean±s.d., n=3). () ChIP–qPCR assays showing that the increasing CCA1 protein level at ZT12 in CCA1-OX plants suppresses the binding of FHY3 to the ELF4 promoter fragment containing the FBS cis element. An exon region of ELF4 was ! used as a negative control in the ChIP–qPCR assay (mean±s.d., n=3). * Figure 5: FHY3, FAR1, HY5, CCA1 and LHY coordinately regulate the cyclic expression of ELF4. () qRT–PCR analysis of FHY3 and FAR1 genes. Arabidopsis wild-type (No-0 ecotype) seedlings were entrained in cycles of 12 h light/12 h dark conditions for 7 days, and then released into continuous white-light conditions (LL) for 3 days. Expression of FHY3 and FAR1 is normalized to the expression of a ubiquitin gene. Data are means±s.d., n=3. (,) Bioluminescence assays showing that the expression pattern of FHY3::FHY3–LUC is regulated by diurnal (; long day, LD) conditions and the circadian clock (). c.p.s., counts per second. Data are means±s.e.m., n=16. () A model depicting the coordinated antagonistic regulation of ELF4 expression by positively acting and negatively acting transcription factors at dawn and dusk. CCA1 and LHY can suppress ELF4 expression by themselves or by suppressing the activation activities of FHY3 and FAR1. Arrow: positive regulation; bar: negative regulation; X: blocking effect. () qRT–PCR analyses showing that ELF4 expression peaks higher! and earlier in the cca1 lhy double-mutant background, compared with wild-type plants. Arabidopsis wild-type (Ws) and cca1 lhy double-mutant seedlings were entrained in cycles of 12 h light/12 h dark conditions for 4 days, and followed by 1 day of long-day (top panel) or continuous white-light (bottom panel) conditions. Expression of ELF4 is normalized to the expression of an actin gene. The white and grey (or dark) bars at the bottom indicate the subjective light and dark period, respectively. Data are means±s.e.m., n=3. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Hamad Siddiqui, * Yibo Teng & * Rongcheng Lin Affiliations * Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA * Gang Li, * Jigang Li, * On-Sun Lau, * Xinhao Ouyang, * Mingqiu Dai, * Xing Wang Deng & * Haiyang Wang * School of Biological Sciences, Royal Holloway University of London, Egham TW20 0EX, UK * Hamad Siddiqui & * Paul F. Devlin * College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China * Yibo Teng * Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China * Rongcheng Lin * National Engineering Research Center for Crop Molecular Design, Beijing 100085, China * Xiang-yuan Wan, * Xing Wang Deng & * Haiyang Wang * Institute of Crop Sciences, Chinese Academy of Agriculture Sciences, Beijing 100081, China * Jianmin Wan & * Haiyang Wang * Hunan Hybrid Rice Research Center, Hunan, 410125, China * Haiyang Wang Contributions P.F.D., X.W.D., J.W. and H.W. contributed to project design. R.L. recorded the phenotype of fhy3 mutants and generated FHY3–LUC reporter lines. X-Y.W. generated 3×Flag–3×HA transgenic lines. H.S. carried out the bioluminescence analyses of all the LUC reporter lines. Y.T. carried out the yeast assays, qRT–PCR and generated ELF4-OX transgenic lines. G.L. carried out all other experiments. G.L., P.F.D. and H.W. wrote the manuscript. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Haiyang Wang Author Details * Gang Li Search for this author in: * NPG journals * PubMed * Google Scholar * Hamad Siddiqui Search for this author in: * NPG journals * PubMed * Google Scholar * Yibo Teng Search for this author in: * NPG journals * PubMed * Google Scholar * Rongcheng Lin Search for this author in: * NPG journals * PubMed * Google Scholar * Xiang-yuan Wan Search for this author in: * NPG journals * PubMed * Google Scholar * Jigang Li Search for this author in: * NPG journals * PubMed * Google Scholar * On-Sun Lau Search for this author in: * NPG journals * PubMed * Google Scholar * Xinhao Ouyang Search for this author in: * NPG journals * PubMed * Google Scholar * Mingqiu Dai Search for this author in: * NPG journals * PubMed * Google Scholar * Jianmin Wan Search for this author in: * NPG journals * PubMed * Google Scholar * Paul F. Devlin Search for this author in: * NPG journals * PubMed * Google Scholar * Xing Wang Deng Search for this author in: * NPG journals * PubMed * Google Scholar * Haiyang Wang Contact Haiyang Wang Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (700K) Supplementary Information Additional data - RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling
- ncb 13(5):623-629 (2011)
Nature Cell Biology | Letter RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling * Yue Zhang1 * Shanming Liu1 * Craig Mickanin1 * Yan Feng1 * Olga Charlat1 * Gregory A. Michaud1 * Markus Schirle1 * Xiaoying Shi1 * Marc Hild1 * Andreas Bauer1 * Vic E. Myer1 * Peter M. Finan1 * Jeffery A. Porter1 * Shih-Min A. Huang1, 2 * Feng Cong1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:623–629Year published:(2011)DOI:doi:10.1038/ncb2222Received10 May 2010Accepted04 February 2011Published online10 April 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The Wnt/β-catenin signalling pathway plays essential roles in embryonic development and adult tissue homeostasis, and deregulation of this pathway has been linked to cancer. Axin is a concentration-limiting component of the β-catenin destruction complex, and its stability is regulated by tankyrase. However, the molecular mechanism by which tankyrase-dependent poly(ADP-ribosyl)ation (PARsylation) is coupled to ubiquitylation and degradation of axin remains undefined. Here, we identify RNF146, a RING-domain E3 ubiquitin ligase, as a positive regulator of Wnt signalling. RNF146 promotes Wnt signalling by mediating tankyrase-dependent degradation of axin. Mechanistically, RNF146 directly interacts with poly(ADP-ribose) through its WWE domain, and promotes degradation of PARsylated proteins. Using proteomics approaches, we have identified BLZF1 and CASC3 as further substrates targeted by tankyrase and RNF146 for degradation. Thus, identification of RNF146 as a PARsylation-direc! ted E3 ligase establishes a molecular paradigm that links tankyrase-dependent PARsylation to ubiquitylation. RNF146-dependent protein degradation may emerge as a major mechanism by which tankyrase exerts its function. View full text Figures at a glance * Figure 1: RNF146 positively regulates Wnt signalling by affecting the protein level of axin. () Depletion of RNF146 specifically inhibits the Wnt3a-induced STF reporter, but not the TNF-α -induced NF-κB reporter in HEK293 cells. Error bars denote the s.d. between four replicates. () Depletion of RNF146 blocks Wnt3a-induced accumulation of cytosolic β-catenin in HEK293 cells. Co-depletion of TNKS1 and TNKS2 was used as a control. () Depletion of RNF146 abolishes Wnt3a-induced axin2 upregulation. Error bars denote the s.d. between triplicates. () Depletion of RNF146 increases the protein level of axin and TNKS1/2 in HEK293 cells. The asterisk indicates a background band. () Knockdown of Drosophila RNF146 (CG8786) using independent dsRNAs increases the protein level of HA-tagged Drosophila axin in Drosophila S2 cells. dsRNA against white was used as a control. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 2: Interaction between the WWE domain and PAR is essential for RNF146-dependent regulation of axin in vivo. () Schematic representation of the domain structure of RNF146. () Expression of siRNA-resistant wild-type RNF146, but not RNF146 mutants with either the RING domain or the WWE domain deleted (ΔRING and ΔWWE), prevents RNF146-siRNA-induced stabilization of axin1 and TNKS1/2 in HEK293 cells. () Immunoprecipitation (IP) of RNF146 and RNF146R161A, but not RNF146ΔWWE or RNF146R163A, by anti-PAR antibodies in HEK293 cells. WB, western blot; TCL, total cell lysates. () Dot-blot analysis of PAR-binding activities of GST–WWE proteins with BSA used as a control. () Binding of PAR with wild-type WWE, but not WWER163A, as shown by surface plasmon resonance. () Axin1 and RNF146 interact with each other in a PARsylation-dependent and WWE-domain-dependent manner. The asterisk indicates a nonspecific band. () Expression of RNF146R161A, but not RNF146R163A, prevents RNF146-siRNA-induced stabilization of axin1 and TNKS1/2 in HEK293 cells. Uncropped images of blots are shown in Supplement! ary Fig. S7. * Figure 3: RNF146 is required for PARsylation-dependent degradation of axin and tankyrase in vivo. () Enrichment of PARsylated axin1 using GST–WWE in a sequential pulldown analysis. Straight immunoprecipitation with anti-Flag antibody was used as a control. (,) Inhibition of tankyrase by XAV939 or depletion of RNF146 using DOX-inducible RNF146 shRNA increases the protein levels of axin () and tankyrase () in HEK293 cells. XAV939 reduces, whereas depletion of RNF146 markedly increases, the amount of axin () or tankyrase () pulled down by GST–WWE. GST–WWER163A was used as a control. DMSO, dimethylsulphoxide. () RNF146 is required for tankyrase-dependent ubiquitylation and degradation of axin in a compound wash-off experiment. () RNF146 siRNA stabilizes axin2 in SW480 cells in a pulse-chase analysis. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 4: PARsylation-dependent ubiquitylation in vitro. () Auto-ubiquitylation of RNF146 in an in vitro ubiquitylation assay. () PAR increases auto-ubiquitylation of RNF146, but not RNF146R163A. () Time course of PAR-enhanced auto-ubiquitylation of RNF146. () Ubiquitylation of TNKS2 by wild-type RNF146, but not RNF146R163A, in a PARsylation-dependent manner. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 5: BLZF1 is identified as a substrate of tankyrase and RNF146 using quantitative mass spectrometry. () A quantitative proteomics approach to identify substrates of RNF146 through combining RNF146 knockdown and GST–WWE pulldown. () A scatter plot depicting proteins identified and quantified in a quantitative proteomics experiment. Proteins significantly enriched in the DOX-treated condition are highlighted as potential substrates of RNF146. () Inhibition of tankyrase by either XAV939 or IWR-1 increases the protein level of GFP–BLZF1 in SW480 cells. () Depletion of RNF146 or co-depletion of TNKS1 and TNKS2 increases the protein level of GFP–BLZF1. () XAV939 decreases, whereas RNF146 siRNA markedly increases, the amount of GFP–BLZF1 pulled down by GST–WWE, which is presumably PARsylated. () The TBD of BLZF1 is evolutionarily conserved. () Deletion of the TBD abolishes the interaction between TNKS1 and BLZF1 in a co-immunoprecipitation assay. () Deletion of the TBD stabilizes GFP–BLZF1 in SW480 cells. Uncropped images of blots are shown in Supplementary Fig. S7. Author information * Author information * Supplementary information Affiliations * Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA * Yue Zhang, * Shanming Liu, * Craig Mickanin, * Yan Feng, * Olga Charlat, * Gregory A. Michaud, * Markus Schirle, * Xiaoying Shi, * Marc Hild, * Andreas Bauer, * Vic E. Myer, * Peter M. Finan, * Jeffery A. Porter, * Shih-Min A. Huang & * Feng Cong * Present address: Sanofi-Aventis Oncology, Cambridge, Massachusetts 02139, USA * Shih-Min A. Huang Contributions Y.Z., C.M., Y.F., G.A.M., M.S., M.H., A.B., V.E.M, P.M.F., J.A.P., S-M.A.H and F.C. conceived and designed the study. Y.Z., S.L., C.M., Y.F., O.C., G.A.M., M.S., X.S. and F.C. designed and implemented experiments. Y.Z. and F.C. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Feng Cong Author Details * Yue Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Shanming Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Craig Mickanin Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Feng Search for this author in: * NPG journals * PubMed * Google Scholar * Olga Charlat Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory A. Michaud Search for this author in: * NPG journals * PubMed * Google Scholar * Markus Schirle Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaoying Shi Search for this author in: * NPG journals * PubMed * Google Scholar * Marc Hild Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Bauer Search for this author in: * NPG journals * PubMed * Google Scholar * Vic E. Myer Search for this author in: * NPG journals * PubMed * Google Scholar * Peter M. Finan Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffery A. Porter Search for this author in: * NPG journals * PubMed * Google Scholar * Shih-Min A. Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Feng Cong Contact Feng Cong Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Other * Supplementary Table 1 (15K) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - Mechanoreception in motile flagella of Chlamydomonas
- ncb 13(5):630-632 (2011)
Nature Cell Biology | Brief Communication Mechanoreception in motile flagella of Chlamydomonas * Kenta Fujiu1 * Yoshitaka Nakayama2, 3 * Hidetoshi Iida3 * Masahiro Sokabe1, 4 * Kenjiro Yoshimura1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:630–632Year published:(2011)DOI:doi:10.1038/ncb2214Received22 October 2010Accepted20 January 2011Published online10 April 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 Ciliates and flagellates temporarily swim backwards on collision by generating a mechanoreceptor potential. Although this potential has been shown to be associated with cilia in Paramecium, the molecular entity of the mechanoreceptor has remained unknown. Here we show that Chlamydomonas cells express TRP11, a member of the TRP (transient receptor potential) subfamily V, in the proximal region of the flagella, and that suppression of TRP11 expression results in loss of the avoiding reaction. The results indicate that Chlamydomonas flagella exhibit mechanosensitivity, despite constant motility, by localizing the mechanoreceptor in the proximal region, where active bending is restricted. View full text Author information * Abstract * Author information * Supplementary information Affiliations * ICORP-SORST-Cell Mechanosensing Project, Japan Science and Technology Agency, Nagoya, Aichi 466-8550, Japan * Kenta Fujiu, * Masahiro Sokabe & * Kenjiro Yoshimura * Division of Structural Biosciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan * Yoshitaka Nakayama & * Kenjiro Yoshimura * Department of Biology, Tokyo Gakugei University, Koganei, Tokyo 184-8501, Japan * Yoshitaka Nakayama & * Hidetoshi Iida * Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan * Masahiro Sokabe Contributions K.F., Y. N. and K.Y. carried out the experiments; K.F. and K.Y. designed the study; M.S. and H.I. gave conceptual advice; K.Y. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kenjiro Yoshimura Author Details * Kenta Fujiu Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshitaka Nakayama Search for this author in: * NPG journals * PubMed * Google Scholar * Hidetoshi Iida Search for this author in: * NPG journals * PubMed * Google Scholar * Masahiro Sokabe Search for this author in: * NPG journals * PubMed * Google Scholar * Kenjiro Yoshimura Contact Kenjiro Yoshimura Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (400K) Supplementary Information Additional data - How APC/C–Cdc20 changes its substrate specificity in mitosis
- ncb 13(5):633 (2011)
Nature Cell Biology | Corrigendum How APC/C–Cdc20 changes its substrate specificity in mitosis * Daisuke Izawa * Jonathon PinesJournal name:Nature Cell BiologyVolume: 13,Page:633Year published:(2011)DOI:doi:10.1038/ncb0511-633aPublished online03 May 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, 223–233 (2011); published online 20 February 2011; corrected after print 31 March 2011 In the version of this article initially published online and in print, Table 1 was incorrect, as figures for the time from NEBD to the start of Cyclin B1 destruction were incorrect by a factor of three. This error has been corrected in both the HTML and PDF versions of the article. Additional data Author Details * Daisuke Izawa Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathon Pines Search for this author in: * NPG journals * PubMed * Google Scholar - Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation
- ncb 13(5):633 (2011)
Nature Cell Biology | Erratum Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation * Maria Maldonado * Tarun M. KapoorJournal name:Nature Cell BiologyVolume: 13,Page:633Year published:(2011)DOI:doi:10.1038/ncb0511-633bPublished online03 May 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, 475–482 (2011); published online 13 March 2011; corrected after print 24 March 2011 In the version of this article initially published online and in print, Figure 5b was incorrectly labelled. This error has been corrected in both the HTML and PDF versions of the article. Additional data Author Details * Maria Maldonado Search for this author in: * NPG journals * PubMed * Google Scholar * Tarun M. Kapoor Search for this author in: * NPG journals * PubMed * Google Scholar
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