Latest Articles Include:
- Focus on Cell cycle and DNA damage
- Nat Cell Biol 13(10):1153 (2011)
Nature Cell Biology | Editorial Focus on Cell cycle and DNA damage Journal name:Nature Cell BiologyVolume: 13,Page:1153Year published:(2011)DOI:doi:10.1038/ncb2357aPublished online03 October 2011 How cells accurately duplicate and segregate their genetic information remains a topic of intense research. A series of specially commissioned articles in this issue presents recent insights into different aspects of the cell division cycle and genomic surveillance. Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg DNA replication, mitotic spindle formation, chromosome segregation and cytokinesis must be carefully controlled to ensure that all genetic information is passed over to the next cell generation. Although the core cell cycle machinery has been worked out, and the cyclin-dependent kinases and cyclins entered the textbooks a long time ago, we still lack a complete understanding of how events of the cell cycle are executed and coordinated. In mitosis, the microtubule-based spindle organizes duplicated chromosomes to allow the segregation of two identical sets to daughter cells. In most cells, centrosomes, each consisting of two centrioles, are the main organizers of microtubules, influencing spindle assembly and chromosome segregation. Centrosomes, like chromosomes, need to be replicated only once per cell cycle and segregated when the cell divides. Erich Nigg and Tim Stearns discuss recent insights into the centrosome life cycle, the potential role of centrosomes in genomic sta! bility and aspects of asymmetry in centriole architecture and segregation. Before separation occurs in anaphase, however, duplicated sister chromatids are held tightly together. The 'DNA glue' is provided by the cohesin complex, vital also for cohesion during DNA repair and replication. Mechanistically, cohesin has been suggested to form a ring that entraps the DNA, but the precise structure of the ring, and how it would come on and off DNA remains a mystery. Kim Nasmyth describes findings on cohesin function and proposes a model to explain cohesin loading onto — and dissociation from — DNA. Another aspect of cell cycle progression is the existence of safeguarding mechanisms called 'checkpoints' that ensure everything is in order before allowing the next event to proceed. The spindle checkpoint (or spindle assembly checkpoint, SAC) monitors the alignment of the full set of chromosomes on the mitotic spindle before anaphase can take place. In the twenty years since the isolation of the first spindle checkpoint genes in yeast, much has been learned about the molecular players in the SAC. In a Historical Perspective, Andrew Murray discusses these advances and outlines the most important questions that remain to be resolved. Repairing damaged DNA is crucial for genomic stability. Recent research has revealed that chromatin undergoes dramatic changes in response to DNA damage. This causes a massive accumulation of proteins in 'nuclear foci'. Jiri Lukas, Claudia Lukas and Jiri Bartek review the numerous post-translational modifications of chromatin proteins following DNA damage, and their potential biological function. In addition to these articles, an accompanying online library on this topic presents selected research papers and reviews from Nature journals. We thank our authors and reviewers for their contributions and hope that our readers will share our enthusiasm for this Focus issue. Additional data - UK Parliament comments on peer review
- Nat Cell Biol 13(10):1153 (2011)
Nature Cell Biology | Editorial UK Parliament comments on peer review Journal name:Nature Cell BiologyVolume: 13,Page:1153Year published:(2011)DOI:doi:10.1038/ncb2357bPublished online03 October 2011 Recognizing the importance of sound scientific advice to the government, the UK Parliament has examined the peer review system. Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In July, the House of Commons Science and Technology Committee published a comprehensive report "Peer review in scientific publication" based on input from researchers, funding bodies and publishers. Parliament's undertaking to understand and assess the value of the peer review process, which remains fundamental to ensuring quality in life science publications, should be applauded. The committee concludes that despite its flaws, pre-publication peer review is vital and cannot be dismantled. However, the report also highlights much-needed improvements to the process including training of early career scientists in peer review. The committee recognizes the crucial role of reviewers and their tremendous efforts and calls for better recognition of this work, but does not make any concrete proposals to this end. As we have previously discussed in these pages ("Reviewing refereeing"), we agree that this fundamental contribution should be appropriately acknowledged, not jus! t by journal editors and publishers, but also by tenure-granting committees, funding agencies and other bodies that evaluate researcher performance and their contributions to a field. The report also discusses avenues for reducing the burden on reviewers, such as editorial pre-screening, efforts to increase a journal's 'reviewer pool', and the possibility to transfer manuscripts between journals together with the referee reports. These approaches are indeed employed by Nature journals to facilitate a constructive and efficient peer review process for authors and referees alike. The committee rightly notes the important role of post-publication review through online commentary and of social media tools in communicating published work and discussing its merits and weaknesses. To this end, we have recently begun highlighting Faculty of 1000 coverage of our papers on our homepage. Of course, no analysis of peer review is complete without a discussion of impact factors or the pressures on researchers to publish in high-impact journals. The committee warns against using impact factors as a proxy for measuring the quality of a publication, and exhorts funders an! d research institutions to assess individual works. We agree; impact factors of journals are an imperfect proxy for measuring the significance of a study, and there is no substitute for evaluating an individual publication on its own merit. Finally, the report discusses research integrity. Journals, including this one, have some means to detect data manipulation (see "Guide to Authors: calling all authors!" and "Combating scientific misconduct"). However, the committee finds the oversight of research integrity at other levels unsatisfactory. Their call for the establishment of an oversight body in the UK, and their recommendation that research institutes have a formal process to deal with ethical issues should be implemented. In conclusion, the pre-publication peer review system, in spite of its deficiencies, is likely to remain an integral part of the dissemination of research findings. Not only does the scientific community rely on it, but scientific advice to governments and information to the public should be based on robust data. Additional data - The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries
- Nat Cell Biol 13(10):1154-1160 (2011)
Nature Cell Biology | Review The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries * Erich A. Nigg1 * Tim Stearns2 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1154–1160Year published:(2011)DOI:doi:10.1038/ncb2345Published online03 October 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 Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes. View full text Figures at a glance * Figure 1: Centriole biogenesis. This schematic representation of the centriole duplication cycle shows centrioles (green) and PCM (grey), with emphasis on two distinct linker structures. The G1–G2 tether (GGT; blue) connects the proximal ends of the two parental centrioles from G1 to late G2; it is important to ensure microtubule nucleation from a single microtubule organizing centre. The S–M Linker (SML; red) forms during S phase and connects the proximal end of the nascent procentriole to the lateral surface of the mother centriole. The removal of this tight connection in late M phase (disengagement) is an important element of cell cycle control of centriole duplication. Both the molecular components of the GGT and SML as well as the regulation of the formation and dissolution of these structures are expected to be distinct, although some PCM components are likely to be important for both GGT and SML. Also depicted are subdistal and distal appendages (triangles); although readily visible in electron ! micrographs during interphase, these appendages are difficult to visualize during M phase. In quiescent cells, the appendage-bearing centriole associates with the plasma membrane (PM) and acts as a basal body to form a primary cilium. Finally, in multi-ciliated epithelial cells, multiple centrioles form simultaneously from an amorphous structure termed the deuterosome (D). * Figure 2: Identification of SAS-6 as a key element of the centriolar cartwheel. () Immunolocalization of the SAS-6 protein (also known as Bld12p) to the central hub of the cartwheel in Chlamydomonas reinhardtii imaged by electron microscopy. Top images show longitudinal sections through wild-type centrioles; note the immunogold-labelling of the carthwheel by anti-SAS-6 antibodies (right). Bottom; immunogold-labelling of centriole in cross-section, showing that SAS-6 localizes to the central part of the cartwheel (right). Schematic representation (left) shows cartwheel in red. Scale bars,100 nm. Reproduced with permission from ref. 35. (). Structural model of a SAS-6 oligomer (upper panel) and rotary-metal-shadowing electron micrographs of the same structure (lower right panel; schematic representation of structure is shown on the left). Both images emphasize the importance of SAS-6 for conferring ninefold rotational symmetry to the centriole. (Reproduced with permission from ref. 38). * Figure 3: Centriole and centrosome asymmetries. Schematic representation of centrioles (green), with distal and subdistal appendages (triangles), G1–G2 tether (blue), S–M linker (red) and pericentriolar material associated with the base of each centriole. The centrioles are numbered to indicate their origin and age. The centriole marked '1' (centriole 1) is the older of the two centrioles in the G1 cell at the upper left. Centriole 2 formed in the previous cell cycle, as a procentriole adjacent to centriole 1. The centrioles in this cell are disengaged (no S–M linker), but tethered (G1–G2 tether). In S phase new procentrioles grow from each of centrioles 1 and 2 and elongate in G2 phase. These new centrioles (3 and 4) are engaged to their mother centrioles (1 and 2, respectively), but are otherwise equivalent. Centriole 2 acquires appendage proteins at the G2/M transition and appendages proper in the subsequent G1. The two centrosomes segregate at mitosis, with one cell receiving the 1, 3 pair and the other receiv! ing the 2, 4 pair. Although the centriole pairs are morphologically equivalent, there is a functional difference, in that the cell receiving the older mother centriole (centriole 1), is able to form a primary cilium earlier in the cell cycle than the other cell. The pericentriolar material at the base of each centriole is represented in different colours to indicate the possibility that proteins associated with the centrioles might be asymmetrically segregated at mitosis with them. Author information * Abstract * Author information Affiliations * E. A. Nigg is at the Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland * Erich A. Nigg * T. Stearns is at the Department of Biology, Stanford University, Stanford, California 94305 USA and the Department of Genetics, Stanford School of Medicine, Stanford, California 94305 USA * Tim Stearns Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Erich A. Nigg or * Tim Stearns Author Details * Erich A. Nigg Contact Erich A. Nigg Search for this author in: * NPG journals * PubMed * Google Scholar * Tim Stearns Contact Tim Stearns Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance
- Nat Cell Biol 13(10):1161-1169 (2011)
Nature Cell Biology | Review More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance * Jiri Lukas1 * Claudia Lukas1 * Jiri Bartek1, 2 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1161–1169Year published:(2011)DOI:doi:10.1038/ncb2344Published online03 October 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 Following the discovery in 1998 of γ-H2AX, the first histone modification induced by DNA damage1, interest in the changes to chromatin induced by DNA damage has exploded, and a vast amount of information has been generated. However, there has been a discrepancy between our rapidly advancing knowledge of how chromatin responds to DNA damage and the understanding of why cells mobilize large segments of chromatin to protect the genome against destabilizing effects posed by tiny DNA lesions. Recent research has provided insights into these issues and suggests that chromatin responses induced by DNA damage are not simply the accumulation of 'nuclear foci' but are mechanisms required to guard genome integrity. View full text Figures at a glance * Figure 1: Chromatin responses to DNA damage are orchestrated by a series of post-translational modifications. These include () poly(ADP-ribosyl)ation, () phosphorylation and () acetylation. Left panels depict the key protein complexes involved in a given modification together with the mechanisms regulating their recruitment to the sites of DNA damage. Right panels indicate the impact of a given modification on chromatin restructuring and/or recruitment of proteins to this compartment. Grey; poly(ADP-ribosyl)ation. Red; phosphorylation (MDC1 is depicted in dark red to highlight its central coordinating role in most steps of DNA-damage-induced chromatin development). Green; activities that alter chromatin compaction or topology. The activities that reverse a given modification are highlighted in black at the transition between left and right panels. * Figure 2: Post-translational modifications that affect chromatin organization and recruit additional genome caretakers to promote repair and suppress transcription in the vicinity of DNA lesions. () Ubiquitylation with SUMOylation. () Methylation. The layout and symbols in this Figure are as in Fig. 1. Yellow; enzymes involved in ubiquitylation and SUMOylation. Pink; enzymes involved in methylation. * Figure 3: Chromatin domains formed in G1 nuclei and enriched in 53BP1 protect DNA lesions generated by mitotic passage of under-replicated chromosomes. Owing to the paucity of replication origins at some genomic loci, such as the common fragile sites (CFS), fractions of genome may fail to complete DNA replication. Such chromosomes may enter mitosis, where the DNA within the under-replicated loci is converted to gaps or breaks through chromosome condensation or dissolution of ultrafine DNA bridges (UFB) by the BLM complex. A fraction of such lesions can be transmitted to daughter cells, where they are sequestered in large chromatin domains enriched in 53BP1 and other markers associated with the DNA-damage-modified chromatin. These chromatin domains have been proposed to shield the DNA lesions against adverse erosion by cellular nucleases and thus protect such loci until repair mechanisms become available. Image shows 53BP1 nuclear bodies in a pair of daughter cells shortly after cell division. Scale bar, 10 μm. * Figure 4: Chromatin modifications restrain unscheduled resection of DNA ends. () During G1, the CtIP nuclease initiates short-range DNA-end resection, which can expose microhomology regions required for alternative NHEJ (A-NHEJ, a backup repair mechanism to a 'classical' NHEJ, which does not require DNA-end processing). In some biological settings that involve DSB generation, such as the developing immune system, A-NHEJ can be detrimental because it competes with Artemis, the physiological nuclease required for assembly of antigen-receptor genes. The ATM-dependent phosphorylation of H2AX and the recruitment of its sensor MDC1 in the DSB-flanking chromatin inhibit CtIP from inducing unscheduled A-NHEJ events at the immunoglobulin loci. () During S and G2 phases, phosphorylation of CtIP by CDKs triggers its interaction with BRCA1. The resulting CtIP–BRCA1 complex becomes more 'processive' and, together with additional cellular nucleases and helicases, can generate longer regions of RPA-coated single-stranded DNA. This is essential for the homology-dir! ected DNA repair (homologous recombination) but excessive DSB resection might be detrimental. Assembly of 53BP1 and the RAP80–BRCA1 complex on the DSB-modified chromatin can counteract the CtIP–BRCA1-mediated stimulatory impact on DSB resection, suggesting that one function of histone-associated pools of 53BP1 and BRCA1 in genome surveillance includes protecting the DNA ends against excessive erosion that could otherwise undermine the genome integrity in the DSB-flanking regions. Author information * Abstract * Author information Affiliations * Jiri Lukas, Claudia Lukas and Jiri Bartek are at the Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark * Jiri Bartek is also at the Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Jiri Lukas or * Claudia Lukas or * Jiri Bartek Author Details * Jiri Lukas Contact Jiri Lukas Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia Lukas Contact Claudia Lukas Search for this author in: * NPG journals * PubMed * Google Scholar * Jiri Bartek Contact Jiri Bartek Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Cohesin: a catenase with separate entry and exit gates?
- Nat Cell Biol 13(10):1170-1177 (2011)
Nature Cell Biology | Review Cohesin: a catenase with separate entry and exit gates? * Kim Nasmyth1Journal name:Nature Cell BiologyVolume: 13,Pages:1170–1177Year published:(2011)DOI:doi:10.1038/ncb2349Published online03 October 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 Cohesin confers both intrachromatid and interchromatid cohesion through formation of a tripartite ring within which DNA is thought to be entrapped. Here, I discuss what is known about the four stages of the cohesin ring cycle using the ring model as an intellectual framework. I postulate that cohesin loading onto chromosomes, catalysed by a separate complex called kollerin, is mediated by the entry of DNA into cohesin rings, whereas dissociation, catalysed by Wapl and several other cohesin subunits (an activity that will be called releasin here), is mediated by the subsequent exit of DNA. I suggest that the ring's entry and exit gates may be separate, with the former and latter taking place at Smc1–Smc3 and Smc3–kleisin interfaces, respectively. Establishment of cohesion during S phase involves neutralization of releasin through acetylation of Smc3 at a site close to the putative exit gate of DNA, which locks rings shut until opened irreversibly by kleisin cleavage throu! gh the action of separase, an event that triggers the metaphase to anaphase transition. View full text Figures at a glance * Figure 1: Structure of the cohesin ring. Cohesin is built around a pair of rod-shaped Smc proteins, Smc1 (red) and Smc3 (blue), that possess dimerization domains at one end and ABC-like ATPases at the other. Heterotypic interactions between the dimerization domains creates V-shaped Smc1–Smc3 heterodimers. Tripartite rings are formed through association of the Smc1–Smc3 heterodimer with an α-kleisin subunit (yellow) whose N- and C-terminal domains bind to the nucleotide-binding domains (NBDs) of Smc3 and Smc1, respectively, and whose central domain binds Scc3/SA and Pds5 subunits. Pds5 in turn binds Wapl. A, B and S refer to Walker A, Walker B and signature motifs, respectively (for explanation see Box 1). * Figure 2: Three types of ring model. () A single monomeric cohesin ring entraps sister DNAs. (, ) Alternatively, cohesin forms dimeric rings, either by virtue of N- and C-terminal kleisin domains binding to NBDs from different Smc1–Smc3 heterodimers () or through cohesin ring concatenation (). Another view (not shown) is that a series of Smc1–Smc3 heterodimers are linked together by kleisin interactions to form an oligomeric bracelet that winds around sisters and does not in fact form a closed ring106. () Another proposal is that sister DNAs are entrapped inside separate cohesin rings that are connected because both bind to the same Scc3/SA subunit — a handcuff model107. The finding that chemical cross-linking of the tripartite ring's three interfaces is sufficient to trap sister DNAs from circular minichromosomes from yeast within a structure resistant to protein denaturation30 is consistent with the monomeric ring model, difficult to reconcile with dimeric rings, but inconsistent with the handcuff and b! racelet models. * Figure 3: (a) Cohesin's association and dissociation from chromatin. Soluble cohesin rings are loaded onto chromatin by the Scc2–Scc4 loading complex, here named kollerin. Cohesin dissociation is mediated by Wapl–Pds5, here called releasin. Acetylation of the Smc3 NBD (marked by AC2) mediated by the Eco1 family of acetyl transferases (CoAT) is coupled to replication and in mammalian cells promotes recruitment of soronin to cohesin-bound chromatin. It is thought that Smc3 acetylation and/or sororin binding abrogates releasin activity, blocking cohesin's dissociation from chromatin. Hence, the slow, if non-existent, dissociation of such complexes from chromatin (dotted arrow). () The dual gate hypothesis. According to this, kollerin promotes cohesin loading onto chromatin by facilitating entry of DNA inside cohesin rings via a transiently opened Smc1–Smc3 hinge, while releasin triggers dissociation by facilitating the exit of DNA via a transiently opened Smc3–kleisin interface, a process regulated by Smc3 acetylation. Author information * Abstract * Author information Affiliations * Kim Nasmyth is at University of Oxford, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, UK Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Kim Nasmyth Author Details * Kim Nasmyth Contact Kim Nasmyth Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - A brief history of error
- Nat Cell Biol 13(10):1178-1182 (2011)
Nature Cell Biology | Historical Perspective A brief history of error * Andrew W. Murray1Journal name:Nature Cell BiologyVolume: 13,Pages:1178–1182Year published:(2011)DOI:doi:10.1038/ncb2348Published online03 October 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 spindle checkpoint monitors chromosome alignment on the mitotic and meiotic spindle. When the checkpoint detects errors, it arrests progress of the cell cycle while it attempts to correct the mistakes. This perspective will present a brief history summarizing what we know about the checkpoint, and a list of questions we must answer before we understand it. View full text Figures at a glance * Figure 1: Tension at kinetochores regulates their attachment to microtubules. Nicklas and Koch12 micromanipulated meiotic chromosomes so that the paternal and maternal chromosomes were attached to the same pole of the meiotic spindle. In the absence of opposing forces on the two kinetochores (black circles), one of the two kinetochores detaches from the pole. If it reattaches to the same pole, the cycle is repeated, but if it attaches to the opposite pole, the kinetochores experience tension again and the attachment becomes stable. Experimentally applying tension, using the micromanipulation needle, produces the same stabilization. * Figure 2: A model that unifies aspects of different models for the spindle checkpoint. At the attached kinetochore, Mad1(green) binds Mad2 (red) in the closed conformation, but the binding of p31-comet (black) to Mad2 prevents this complex from catalytically activating other molecules of Mad2. Interaction of the Mad1–Mad2 complex with the kinetochore that has lost its microtubule leads to the displacement of p31-comet and allows the conversion of other molecules of Mad2 into the closed conformation. These and a complex of Bub3 and Mad3/BUBR1 bind to Cdc20 to form the mitotic checkpoint complex (MCC), which binds to and inhibits the anaphase promoting complex (APC). The schematic representation for the structure of the APC is loosely based on ref. 60. Author information * Abstract * Author information Affiliations * Andrew W. Murray is at Harvard University, Molecular and Cellular Biology, 52 Oxford Street, Northwest Science Building, Cambridge, Massachusetts 02138, USA Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Andrew W. Murray Author Details * Andrew W. Murray Contact Andrew W. Murray Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Spindle positioning: going against the actin flow
- Nat Cell Biol 13(10):1183-1185 (2011)
Article preview View full access options Nature Cell Biology | News and Views Spindle positioning: going against the actin flow * Marie-Hélène Verlhac1Journal name:Nature Cell BiologyVolume: 13,Pages:1183–1185Year published:(2011)DOI:doi:10.1038/ncb2352Published online03 October 2011 Successful completion of meiosis in vertebrate oocytes requires the localization and maintenance of the meiotic spindle at the cell cortex. Arp2/3-nucleated actin filaments are now shown to flow away from the cortex overlying the spindle, resulting in cytoplasmic streaming, which maintains the spindle in its asymmetric position. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Marie-Hélène Verlhac is at the Collège de France, Center for Interdisciplinary Research in Biology (CIRB), UMR-CNRS7241/INSERM-U1050, 11 place Marcelin Berthelot, 75005 Paris, France and the Memolife Laboratory of Excellence and Paris Science Lettre Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Marie-Hélène Verlhac Author Details * Marie-Hélène Verlhac Contact Marie-Hélène Verlhac Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Making sense of glycosphingolipids in epithelial polarity
- Nat Cell Biol 13(10):1185-1187 (2011)
Article preview View full access options Nature Cell Biology | News and Views Making sense of glycosphingolipids in epithelial polarity * Vincent Hyenne1 * Michel Labouesse1 * Affiliations * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1185–1187Year published:(2011)DOI:doi:10.1038/ncb2350Published online18 September 2011 A potential role for glycosphingolipids and lipid rafts in apical sorting was initially met with enthusiasm, but genetic analysis has since provided little support for it. A report now establishes that glycosphingolipids mediate apical sorting, and specifically help maintain apicobasal polarity in Caenorhabditis elegans. Figures at a glance * Figure 1: Glycosphingolipids maintain C. elegans epithelial polarity. () Simplified view of an epithelial cell with its two membrane domains separated by cell–cell junctions, with the key members of the pathway that establish polarity. PtdIns(4,5)P2, as well Crumbs and the aPKC–PAR-3–PAR-6 complex, are important determinants of apical polarity, whereas PtdIns(3,4,5)P3 and Scrib (known as LET-413 in C. elegans) associate with basolateral membrane regions. () In wild-type C. elegans larvae, the tubular intestine generally comprises two juxtaposed epithelial cells. Cortical ERM-1 is apical; LET-413 is basolateral. () On inhibition of GSL synthesis, multiple ectopic lumens form still flanked by junctions. * Figure 2: Simplified representation of glycosphingolipid biosynthetic pathways. Arrows are indicative of a relationship rather than a precise description of individual enzymatic steps. Some of the genes identified by Zhang et al. are: pod-2, encoding acetyl-CoA-carboxylase; let-767, encoding steroid-dehydrogenase/3-ketoacyl-CoA-reductase; acs-1, encoding long-chain-fatty-acid-acyl-CoA ligase; sptl-1, encoding serine-palmitoyl-transferase (SPT); and cgt-1/3, encoding ceramideglucosyltransferases. Myriocin is an inhibitor of SPT enzymes. Lipid biosynthesis in C. elegans and vertebrates may involve slightly different steps, and most enzymatic activities are inferred from homology. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Cell Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * IGBMC, Development and Stem Cells Program, CNRS (UMR7104), INSERM (U964), Université de Strasbourg, 1 rue Laurent Fries, BP10142, 67400 Illkirch, France * Vincent Hyenne & * Michel Labouesse Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Vincent Hyenne or * Michel Labouesse Author Details * Vincent Hyenne Contact Vincent Hyenne Search for this author in: * NPG journals * PubMed * Google Scholar * Michel Labouesse Contact Michel Labouesse Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis
- Nat Cell Biol 13(10):1189-1201 (2011)
Nature Cell Biology | Article Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis * Hongjie Zhang1 * Nessy Abraham1 * Liakot A. Khan1 * David H. Hall2 * John T. Fleming1 * Verena Göbel1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1189–1201Year published:(2011)DOI:doi:10.1038/ncb2328Received29 December 2010Accepted28 July 2011Published online18 September 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 Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings ide! ntify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane. View full text Figures at a glance * Figure 1: Apicobasal polarity conversion and ectopic lumen formation in the intestines of lipid-biosynthetic-enzyme-depleted animals. (See Supplementary Fig. S1 for anatomy and phenotypic details.) () Top, schematic representation of the mature single-layered C. elegans intestine where all apical membranes form the lumenal surface. The outlined area is magnified below. Bottom, epifluorescence dissecting micrographs of live animals (as identified in screen) showing displacement of the membrane–cytoskeleton linker ERM-1::GFP (ref. 57) from the apical (lumenal) membrane in wild type (left; for brevity, transgenic marker strains will be denoted as wild type) to the basolateral membrane in lipid-biosynthetic-enzyme-depleted animal (right; left arrow: lateral side, right arrow: basal side; note wild-type intercalation pattern). Representative sptl-1(RNAi) L1 are shown; pod-2-, let-767- and acs-1 RNAi copy the phenotype (Supplementary Fig. S1b). Here and below, standard RNAi conditions (Methods) are shown unless indicated otherwise; anterior is left and dorsal up. () Typical phenotype development in RNAi L1 lar! vae. Confocal microscopy sections showing initial wild-type ERM-1::GFP placement (top left); ERM-1::GFP decrease from apical, and displacement to basolateral, membranes and enrichment in apicolateral angles (arrows; top right); multiple small lateral ectopic lumens (bottom left); ERM-1::GFP fully displaced from the central lumen to large lateral ectopic lumens (bottom right, arrows bracket central lumen area where ERM-1::GFP is missing). () TEM micrographs of intestinal cross-sections of wild-type (top left) and sptl-1(RNAi) L1 larvae (all others). Oval lumen (L) with dense microvilli (long white arrows) and tightly adjacent terminal web (arrowheads) in wild-type; deformed main lumen in RNAi animals with either short (long white arrows) or absent microvilli (right-middle inset, yellow arrows), dehiscence of the terminal web (arrowheads) and ectopic lateral lumens (EL) with stunted microvilli (long white arrows). Intact apical junctions (short arrows) in both wild-type and R! NAi animals; note excess junctions between ectopic lateral lum! ens in RNAi animals; N, nucleus. Upper right image shows INT I. () Model of the multiple-lumen intestinal phenotype. Early ectopic lumen development (, bottom-left image) in otherwise wild-type intestine is shown. Twenty intestinal cells are arranged in bilateral symmetry to form nine INT rings (I—IX; INT I contains four cells); rings twist along the anterior–posterior axis. View is from the anterior left lateral aspect58. * Figure 2: Apicobasolateral membrane and apical junction components in lipid-biosynthetic-enzyme-depleted intestines. (Supplementary Fig. S4 shows additional components.) (–) Apical membrane. () Left to right: confocal microscopy image of actin (phalloidin) overlay (purple) with intermediate filaments (IFB-2) at apical, and LET-413 at basolateral, membranes; Nomarski images: early phenotype lacks visible changes, later stage shows lateral lumens (arrows). () Left to right: apical actin/ERM-1 overlay (yellow); displaced basolaterally (yellow, lower arrow) and cytoplasmically (red, upper arrow); cortical actin (ACT-5)/IFB-2 overlay displaced to lateralized lumenal membranes (turquoise, left arrow) and cytoplasm (green, right arrow). () Left to right: apical submembraneous IFB-2; unravelling from lumen; displaced to lateralized lumenal membranes (arrow indicates central-lumenal IFB-2 contiguity). () Left to right: OPT-2, a transmembraneous oligopeptide transporter, positioned apical to IFB-2 (green without overlay); displaced basolaterally (arrow); displaced lateral to lateralized IFB-2 (gre! en without overlay; note displacement to, but not expansion into, the lateral domain (arrow)). () Left to right: IFB2/PAR-6 overlay (turquoise); cytoplasmic PAR-6 displacement at early and late stage (green). (–) Basolateral membrane. () Left to right: ICB4 stains an unknown panmembraneous marker in wild type (wt); apicobasolateral ERM-1/ICB4 overlay (turquoise, arrow) in early-stage mutant (RNAi) phenotype (mt); ICB4 subapical accumulation (blue, arrow) in late-stage mutant; same image with separated ICB4 for clarity. () NHX-7, a basolateral integral membrane Na+/H+ pump, retained at basolateral membranes, but partially cytoplasmically displaced in late-stage mutant. () Basolateral LET-413, a junction-mediated polarity determinant59, excluded from apical membrane in wild type (no IFB2 overlap) and also from lateralized ectopic lumenal membrane in mutant, but not displaced from the basolateral membrane (arrow). (,) Apical junctions. The junction integrity molecule AJM-1 a! nd the adherens junction component HMP-1 remain contiguous at ! apicolateral boundaries in both wild type and mutant (arrowheads), but additionally surround nascent lateral lumens in mutant (arrows; note absence of fragmented junction pattern). () Feeding of dsRed-labelled bacteria fails to label ectopic lumens (arrows) or the cytoplasm of mt (same sections in all). Confocal images of representative L1 intestinal sections are shown: pod-2-, let-767-, acs-1- and sptl-1 RNAi cause the same mislocalization/localization of all markers (N>40 for each marker). * Figure 3: Lipid biosynthesis perturbations reversibly shift apicobasal domain identities and lumen position on expanding intestinal membranes in situ. () Tracking ERM-1::GFP displacement/placement after lipid-biosynthesis inhibition (0 h, hatching of let-767(RNAi) progeny, not shown): 6 h, apical (excretory canal visible in this image, arrow); 12 h, as puncta in cytoplasm; 15 h, as puncta at basal membranes; 18 h, entire basolateral membranes outlined; 21 h, enriched at apicolateral angle and disappearing from apical membrane; 24 h, at lateral lumens in apicolateral angle; 30 h, at multiple ectopic lumens along lateral membranes; 33 h, at enlarging ectopic lumens; 36h, at separate lateral lumens, almost lost from original apical membrane (arrowheads bracket central lumen area, dotted line indicates missing apical ERM-1::GFP). () Tracking ERM-1::GFP replacement/placement subsequent to lipid biosynthesis restoration (0 h, removal from let-767RNAi): 0 h, at multiple lateral lumens; 3 h, at smaller lateral lumens; 6 h, at lateral lumens and cytoplasmic; 12 h, cytoplasmic (blurred outlines of small l! ateral lumens still visible); 18 h, fully cytoplasmic, reappearing at widened contiguous apical membrane (hidden behind excretory canal, arrows); 24 h, at the centring apical/lumenal membrane, lateral lumens no longer visible; 30 h, at the wide central apical membrane and lateral membranes, now without lumens; 36 h, at the fully restored although undulating apical membrane; 42 h, at the apical membrane in the extended epithelium. The 42 h image encompasses 2 INT rings whereas the 0 h image includes all 9 INT rings: difference reflects epithelial expansion (note stage progression to L2 (30 h) and L3 (42 h); compare with L1 arrest 36 h post-RNAi induction ()). Representative animals are shown, N>200; confocal sections or projections on the level of the intestine: full projections in to encompass all ERM-1::GFP; arrows indicate superimposed excretory canals. * Figure 4: Germline mutations in fatty-acid-biosynthetic enzymes cause intestinal tubulogenesis defects that are rescued with exogenous fatty acids. An allelic series of let-767 point mutations26 includes: s2176, suggested null (early larval lethality); s2819, moderately severe (mid-larval lethality); and s2464, less severe allele (reaches adulthood with lethal progeny (maternal-effect)). () Top, confocal image of s2819 with early basolateral ERM-1::GFP displacement; bottom, s2176 with early ectopic lumen formation (let-767(s2167/s2819) dpy-17(e164) unc-32(e189)III; sDp3(III;f); fgEx13(perm-1::erm-1::gfp rol-6(su1006))). Lateral ERM-1::GFP displacement and ectopic lumen formation are obscured in the Dpy background with widened intestinal lumen and body shape. () Penetrance and expressivity of tubulogenesis defects (speed of development and number and size of lateral lumens) increase with allelic severity. Higher penetrance by RNAi than in s2176 indicates maternal product requirement. All animals were evaluated 30 h post-hatching. Mean±s.d. shown, n=5 (N>200 animals per experiment) for mutants and n=5 (N>1,000 animals ! per experiment) for RNAi animals. () Rescue with exogenous lipids supplied by food. mmBCFAs partially rescue let-767-, fully rescue acs-1-, but not pod-2(RNAi) tubulogenesis defects; straight-chain LCFAs partially rescue let-767-, fully rescue pod-2-, but not acs-1(RNAi). sptl-1(RNAi) defects are not rescued with either mmBCFAs or straight-chain LCFAs. Experimental and control animals were evaluated 60 h post-feeding with fatty acids/solvents (Methods). Mean±s.d. is shown, n=5 (N>200animals per experiment), *P<0.05 and ***P<0.001, two-tailed t-test. () Top, moderately severe let-767(s2819) allele (later stage of phenotype development than in ); bottom, almost fully rescued with exogenous mmBCFAs. * Figure 5: Tubular polarity requires saturated LCFA biosynthesis. Conserved fatty-acid-biosynthesis pathways are shown56. POD-2 and FASN-1 catalyse the first steps in saturated small- and medium-chain-fatty-acid biosynthesis (top: acetyl–CoA shown as primer example for even-numbered SFAs, isovaleryl-CoA for odd-numbered mmBCFAs), subsequently elongated to LCFAs by elongases (middle: this area was probed in greater detail and has been expanded; see text), variably desaturated by desaturases (right). All products are precursors for complex lipids (bottom). The presence or absence of polarity defects (wild-type (wt) or mutant (mt) indicated throughout by image insets) was evaluated in RNAi animals and/or germline mutants (see Supplementary Table S1 for specific genes targeted (N=162), most of which lacked polarity defects; see Supplementary Fig. S6 for phenotypes). Identified enzymes are shown in blue, and corresponding genes are shown in red. fasn-1(RNAi) was not identified here because of its early arrest (pod-2 and fasn-1 are the only li! pid-biosynthetic enzymes previously shown to affect polarity, at the first-cell stage; the mediating lipid compound was not identified25). The precise pathway location of acs-1 is not known. Decreasing cholesterol levels to trace amounts did not produce the phenotype (not shown; C. elegans is a cholesterol auxotroph, but requires such low amounts of cholesterol that its role as a structural membrane lipid has been questioned60). * Figure 6: Fatty-acid biosynthesis determines tubular polarity through sphingolipid synthesis. () Ceramide: the sphingoid base (LCB) is linked to an LCFA through an amide bond (22:0 hydroxy-15 methyl-2-aminohexadec-4-en-1, 3-diol is shown). () Reversal of polarity conversion, induced by fatty-acid-biosynthetic-enzyme depletion, requires sphingolipid synthesis. Note, fumonisin (with and without sptl-1 RNAi), but not sptl-1(RNAi) alone (or myriocin, not shown), suppresses pod-2(RNAi) polarity reversal. Thus, POD-2 uses LCBs generated during its suppression, indicating that it predominantly generates ceramide LCFAs. Conversely, acs-1- and let-767(RNAi) polarity reversal is suppressed by sptl-1 alone, indicating that they lack such LCBs and thus contribute to their synthesis. This is consistent with the role of let-767 and acs-1 in mmBCFA synthesis, and the role of pod-2 and let-767 in straight LCFA synthesis23, 24, 25 (the LCB requires mmBCFAs; ref. 31) and with the rescue experiments shown in Fig. 4b. Conditions of sphingolipid biosynthesis reduction were titrated to pr! event induction of the phenotype on their own (Methods; under these conditions, sptl-1(RNAi) is enhanced by fumonisin). For comparison, identical RNAi conditions were used throughout: different reversal efficiencies thus reflect phenotype severity (varies with each enzyme, see Supplementary Figs S1 and S6; for example, standard acs-1 RNAi induces a strong phenotype, less easily reversed). Mean±s.d. is shown, n=4 (N>60 animals per experiment) *P<0.05, **P<0.01 and ***P<0.001, two-tailed t-test. () Dominant genetic interactions between fatty-acid and sphingolipid-biosynthetic enzymes. let-767(s2819);sDp3 are wild type; thus, any increase of polarity defects it induces in RNAi animals demonstrates enhancement. pod-2 and sptl-1 RNAi were titrated to generate no phenotypes, cgt-1;cgt-3(RNAi) was induced at standard conditions. Mean±s.d. is shown, n=5 (N>300animals per experiment), *P<0.05, **P<0.01, two-tailed t-test. () Ion chromatogram of wild-type worm extracts shown for th! e [M+H]+ m/z 250.3u fragment of their common d17:1 sphingoid b! ase. Interpretation of mass ions from mass spectra of each peak: 2.24 min:d17:1ceramides (Cer), 2.48 min:d17:1hydroxyceramides (Cer–OH), 6.27 min:d17:1glucosylhydroxyceramides (GlcCer–OH), 7.65 min:d17:1sphingomyelins (SM), 8.19 min:d17:1 hydroxysphingomyelins (SM–OH). Here and below: synchronized wild-type or enriched RNAi L1 larvae were collected from ~800 plates each (Methods). () Decreased sphingolipid levels in both sphingolipid- and fatty-acid-biosynthesis-depleted animals. Relative amounts are plotted (C21-26–Cer, –Cer–OH, -GlcCer–OH, C21–23,25-SM and–SM–OH; Supplementary Table S3). Bars indicate median; wild type arbitrarily set at 1; dots represent individual compounds. Concentrations calculated from the mass-spectrometry-derived response ratio of compound area/internal standard area of the corresponding period per sample dry weight. * Figure 7: Tubular polarity requires CGTs and GSLs. () Conserved sphingolipid metabolic pathways are shown56. Left, de novo biosynthesis; right, salvage pathway; bottom, complex membrane sphingolipid biosynthesis. Presence/absence of polarity defects were assessed in RNAi animals and/or germline mutants (see Supplementary Table S2 for specific genes targeted (N=85), most of which lacked polarity defects; see Supplementary Fig. S6 for phenotypes). Identified enzymes are shown in blue, and corresponding genes are shown in red. The precise location of sphingolipid fatty-acid hydroxylase is unknown. () sptl-1-, cgt-1(tm999);cgt-3- and let-767(RNAi) tubulogenesis defects are partially rescued by exogenous sphingolipids. A sphingolipid standard mixture, containing ten different sphingolipid compounds, was fed with RNAi bacteria (Methods). The percentage of animals with tubulogenesis defects was evaluated 64–66 h after feeding with lipids or solvents (control). Mean±s.d. is shown, n=5 (N>200animals per experiment), *P<0.05, two! -tailed t-test. () NBD–C6-GlcCer (NBD–GSL) supplementation attenuates ectopic lumen formation in sptl-1-, let-767- and cgt-1(tm999);cgt-3-(RNAi) L1 intestines. Animals were evaluated for 'severe' (discontinuous central lumen/large ectopic lumens) versus 'mild' (partially contiguous central lumen/small ectopic lumens) phenotypes 5 and 6 days after feeding with GSLs or solvents (control; supplied with RNAi bacteria). For sptl-1 RNAi, which induces the strongest phenotype, 'severe' was defined as arrest/intestinal disintegration and 'mild' as mobile/intestine not disintegrated. Confocal microscopy sections of representative animals are shown. Mean±s.d., n=5 (N>200animals per experiment), *P<0.05, two-tailed t-test. () The arthro-series of GlcCer derivatives is dispensable for the function of GlcCer in tubular polarity. The first steps of insect-type GlcCer-derivative biosynthesis are shown, and corresponding C. elegans orthologues are on the left39. The in! set demonstrates intact intestinal lumen (purple) in the presu! med null allele bre-3(ye28) combined with bre-3(RNAi) (animals grow to fertile adults)39. N>2,000. () Accumulated mass spectra of wild-type GlcCer–OH with an elution peak of 6.27 min (Fig. 6d). GlcCer with m/z 787.2, with a C17 sphingoid base and a hydroxylated saturated C22 fatty-acid chain, was present in greatest abundance (inset). Hydroxyacylamines with saturated C21 (m/z773.4) and C23-26 carbon chains (m/z 801.3, 815.4, 829.1, and 843.3, respectively) were present in low abundance. Peak m/z 769.2 may represent [M-4H] C21:0-d17:1 and peak m/z 797.3 may represent [M-4H] C23:0-d17:1 GlcCer–OH compounds. * Figure 8: Subcellular localization of exogenous sphingolipids and the effects of sphingolipid-biosynthesis suppression on vesicular trafficking. () BODIPY–C5-Cer (top) and NBD–C6-GlcCer (NBD–GSL, bottom), supplied with food, localize to intestinal vesicles (arrowheads) and apical/lumenal plasma membranes (arrows) of wild-type animals. Over time, both exogenous lipids accumulate in larger vesicles (NBD–GSL-fed animal is shown immediately after lipid supplementation; Methods). Lumenal membrane lipids are distinguishable from intra-lumenal lipids in NBD–GSL-fed animal. Left, confocal microscopy images; right, corresponding Nomarski images. () Low-temperature interference with trafficking61, 62 inhibits polarity conversion (compare Supplementary Fig. S8). At 12 °C, the let-767(s2176) typical moderate polarity defects are suppressed and animals progress beyond L1 arrest to L2/L3. Developmental stages (larval: L1—4, adult: Ad) at 12 and 22 °C on days 3, 6, 9 and 12 post-bleaching are shown beneath the bars (Methods). Mean±s.d. shown, n=4 (N>120 animals per experiment) **P<0.01, ***P<0.001, two-tailed t-t! est. () TEM L1 intestinal cross-sections (Supplementary Fig. S8 shows whole intestines): vesicle abundance and variety in wild type (left; including lipid-storage vesicles (arrows) and yolk vesicles (arrowheads)); vesicle paucity and profusion of distended endoplasmic reticular and Golgi membranes (arrow shows example) in sptl-1(RNAi) (right; note lumen deformation and missing apical microvilli). L, lumen; Nc, nucleus. () Apical endosome populations become depleted during polarity conversion. Left to right, progression to late-stage polarity defect (representative L1 intestines shown): lumenal-membrane-associated RAB-11 (arrow) is lost; pancytoplasmic RAB-11-positive vesicles are first mildly enlarged (arrowheads), then mostly lost. Pancytoplasmic RAB-5-positive vesicles are more slowly and less severely depleted (arrowhead indicates occasionally enlarged subapical vesicles). L1-specific subapical RAB-7-positive vesicle aggregates (arrow) are lost; the pancytoplasmic fracti! on is unchanged or moderately decreased. Pancytoplasmic RAB-10! -positive vesicles are moderately decreased in number. The evenly dispersed vesicular MANS-positive Golgi stacks (GFP::MANS) aggregate basolaterally in linear rather than vesicular structures (arrow; compare text). () BODIPY–C5-Cer displacement to ectopic lateral lumenal membranes in sptl-1(RNAi)- (top) and cgt-1(tm999)/cgt-3(RNAi) L1 intestines (bottom; confocal images left, corresponding Nomarski images right). Cer-positive ectopic lumenal membranes show lateral membrane connections (outlined by dots (top)) and are distinct from cytoplasmic vesicles; arrows indicate main lumen. All confocal images in this figure are collected at settings limiting autofluorescent vesicle interference. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA * Hongjie Zhang, * Nessy Abraham, * Liakot A. Khan, * John T. Fleming & * Verena Göbel * Center for C. elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA * David H. Hall Contributions H.Z. generated and assembled most of the data and contributed to project design, data analysis and writing of the manuscript. N.A. participated in most experiments, carried out the glycosylation screen and contributed to experimental design and data analysis. L.A.K. contributed to the genetic interaction experiments. D.H.H. and J.T.F. contributed to electron microscopy experiments and J.T.F. to writing of the manuscript. V.G. conceived and directed the project, participated in experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Verena Göbel Author Details * Hongjie Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Nessy Abraham Search for this author in: * NPG journals * PubMed * Google Scholar * Liakot A. Khan Search for this author in: * NPG journals * PubMed * Google Scholar * David H. Hall Search for this author in: * NPG journals * PubMed * Google Scholar * John T. Fleming Search for this author in: * NPG journals * PubMed * Google Scholar * Verena Göbel Contact Verena Göbel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (1400K) Supplementary Information Excel files * Supplementary Table 1 (116K) Supplementary Information * Supplementary Table 2 (48K) Supplementary Information * Supplementary Table 3 (45K) Supplementary Information * Supplementary Table 4 (40K) Supplementary Information * Supplementary Table 5 (22K) Supplementary Information Additional data - VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling
- Nat Cell Biol 13(10):1202-1213 (2011)
Nature Cell Biology | Article VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling * Tuomas Tammela1, 9 * Georgia Zarkada1, 9 * Harri Nurmi1 * Lars Jakobsson2, 10 * Krista Heinolainen1 * Denis Tvorogov1 * Wei Zheng1 * Claudio A. Franco2 * Aino Murtomäki1 * Evelyn Aranda3 * Naoyuki Miura4 * Seppo Ylä-Herttuala5 * Marcus Fruttiger6 * Taija Mäkinen1, 10 * Anne Eichmann7 * Jeffrey W. Pollard3 * Holger Gerhardt2, 8 * Kari Alitalo1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1202–1213Year published:(2011)DOI:doi:10.1038/ncb2331Received24 May 2010Accepted03 August 2011Published online11 September 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 Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2+/−;Vegfr3+/− compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-der! ived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts. View full text Figures at a glance * Figure 1: Blood vascular hyperplasia and excessive filopodia projection in mice with a targeted deletion of Vegfr3 in the endothelium. (,) Visualization of blood vessels by isolectin B4 (iB4) staining of Vegfr3iΔEC and wild-type littermate retinas at P5. Yellow dots indicate filopodia at the vascular front in . Scale bars, 100 μm () and 50 μm (). (–) Quantitative analysis of the retinas shown in and . () iB4-positive surface area normalized to total area. () Number of vessel branching points. () Number of filopodia per length of vascular front. () BrdU-positive cells per iB4 area (see Supplementary Fig. S2). In all cases, Cre activity was induced for 48 h before the mice were killed. – show data from one litter containing 5 Vegfr3iΔEC and 3 wild-type mice. () Data from one litter containing 3 Vegfr3iΔEC and 4 wild-type mice. (,) Endomucin staining of E11.5 mouse hindbrains after Cre induction for 24 h before the mice were killed. Yellow asterisks indicate the hindbrain midline in , and yellow dots indicate filopodia in . Scale bars, 100 μm () and 20 μm (). (–) Quantitative analysis ! of the Vegfr3iΔECand wild-type hindbrains; n=3 Vegfr3iΔEC and 5 wild-type embryos. () Endomucin-positive surface area normalized to total area. () Number of vessel branching points in the subventricular side. () Number of vessel sprouts in the pial side (see Supplementary Fig. S3). () PECAM-1 staining of LLC tumour xenografts 11 days after implantation into Vegfr3iΔEC or wild-type littermate mice. Scale bar, 50 μm. () Quantification of PECAM-1-positive area in the tumours shown in ; n=5 Vegfr3iΔEC and 5 wild-type mice. () Fold increase in vascular area 4 days after transduction with adenoviral vectors encoding VEGF (AdVEGF), normalized to AdVEGF-B in Vegfr3iΔEC versus wild-type mice (see Supplementary Fig. S4); n=3 ears per group. **P<0.005, *P<0.05. Error bars, s.e.m. * Figure 2: Role of VEGFR-3 tyrosine kinase activity in angiogenesis. () Intra-embryonic injection of FITC–dextran (green) into the cardiac outflow tract at E11.5 showing homogeneous perfusion of the embryo. Scale bar, 200 μm. (,) Immunoprecipitation (IP) of VEGFR-3 () or VEGFR-2 () of embryos stimulated with VEGF, VEGF-C or BSA followed by western blotting (WB) for phosphotyrosine (pY), VEGFR-3 (R3) or VEGFR-2 (R2). N=9 () and 8 () embryos per lane. () Immunoprecipitation of VEGFR-2 from hBECs transduced with pMX–VEGFR3–StreptagII retrovirus. Adherent cells were stimulated with VEGF-C, whereas detached cells were replated on collagen I or poly-l-lysine, and subjected to the indicated inhibitors. Uncropped images of blots are shown in Supplementary Fig. S9a. () Schematic illustration showing the expected VEGFR-3 activity following the indicated genetic perturbations of Vegfr3. () iB4 staining of mouse retinas at P5 48 h after 4-OHT administration. A, artery; V, vein. Scale bar, 100 μm. (–) Quantitative analysis of the retinas s! hown in . () Isolectin B4 (iB4)-positive surface area normalized to total area. () Number of vessel branching points. () Number of filopodia per length of vascular front. Data pooled from 4 litters containing altogether 8 iΔEC/iΔEC, 4 iΔEC/KD, 6 +/iΔEC, 5 KD/+ and 7 wild-type pups. *P<0.05. Error bars, s.e.m. * Figure 3: An increased level of VEGFR-2 signalling contributes to vascular hyperplasia in Vegfr3iΔEC retinas. () Isolectin B4 staining (in green) of Vegfr3iΔEC retinas after treatment with VEGFR-3- or VEGFR-2-blocking antibodies during P3–P5. Non-specific rat IgG was used as a control. Arrowheads indicate abnormally thick vessels. Scale bar, 100 μm. () Statistical analysis showing the percentage vessel area increase in Vegfr3iΔEC versus wild-type littermate mice in every treatment group (individual experiments; n=4, 5 and 4 Vegfr3iΔEC pups treated with anti-VEGFR-3, anti-VEGFR-2 and IgG, respectively; and 6, 3 and 5 wild-type pups treated with anti-VEGFR-3, anti-VEGFR-2 and IgG, respectively). () qRT-PCR analysis of Vegfr1 gene (also known as Flt1) expression; n=4 Vegfr3iΔEC and 3 wild-type pups. In all analyses of the retina, Cre activity was induced for 48 h before the mice were killed. *P<0.05, ***P<0.001. Error bars, s.e.m. () Cultured HUVECs subjected to siRNA-mediated silencing of VEGFR3 expression (VEGFR3 siRNA) and stimulation with VEGF for the indicated times. VE! GFR-2 was immunoprecipitated (IP) followed by immunoblotting (IB) for phosphotyrosine (pY) and VEGFR-2. Numbers below the blots indicate relative intensities of pY to VEGFR-2, normalized to control siRNA at the same time point. Note the increased pVEGFR-2 signal at 30 min and 60 min (red). Immunoprecipitation and western blot analysis for VEGFR-3 from the same lysates is shown below. Uncropped images of blots are shown in Supplementary Fig. S9b. * Figure 4: A decreased level of Notch signalling underlies excessive angiogenesis in Vegfr3iΔEC retinas. () Fold changes in Hey1, Hey2, Nrarp and Dll4 mRNA levels in the retinas of Vegfr3iΔEC and wild-type littermate pups at P5. mRNA levels were normalized to Cadh5 to compensate for the increased endothelial cell numbers in Vegfr3iΔEC retinas. *P<0.05; n=4 Vegfr3iΔEC and 3 wild-type pups. Error bars, s.e.m. (,) Vessel area quantification () and isolectin B4 (iB4) staining () of Vegfr3iΔEC and wild-type littermate retinas at P5 following administration of Jagged1 peptide mimetics (Jag1) or scrambled peptides (SC-Jag1) and 4-OHT for 48 h. Scale bar, 100 μm. ***P<0.001; n=3 Vegfr3iΔEC and 4 wild-type pups treated with SC-Jag1 and 4 Vegfr3iΔEC and 4 wild-type pups treated with Jag1. Data pooled from 2 individual experiments. Error bars, s.e.m. () A 10 day chimaeric embryoid body derived from wild-type DsRed-expressing embryonic stem cells (red), mixed in a 1:1 ratio with embryonic stem cells having one functional Vegfr3 allele (Vegfr3+/LacZ) and stained for iB4 (green)! . Red arrowheads indicate tip cells of wild-type origin; green arrowheads point to Vegfr3 heterozygous cells. Scale bar, 200 μm. () High-magnification image of a sprout showing a mosaic distribution of the cells. DNA in blue. Scale bar, 20 μm. (,) Quantification of the tip cell genotype in all sprouts (; 65.89%±2.5% s.e.m.; n=621 sprouts), in sprouts that exhibited a 1:1 contribution of wild-type and Vegfr3+/LacZ cells (; 61.8%±1.8% s.e.m.; n=360 sprouts) and in sprouts with a 1:1 contribution of wild-type and Vegfr3+/LacZ cells following treatment with DAPT (; 53.7%±2.7% s.e.m.; n=325 sprouts). **P<0.01, **P<0.05. Error bars, s.e.m. () Mosaic retina of a P5.5 pup derived from a wild-type blastocyst injected with Vegfr3+/LacZ embryonic stem cells and stained for iB4. β-galactosidase activity (in black, arrow) indicates a Vegfr3+/LacZ cell. Scale bar, 50 μm. * Figure 5: Vegfc haploinsufficiency leads to instability of sprout fusion points and inefficient angiogenesis. () Isolectin B4 (iB4) staining (green) of retinas from Vegfc+/− mice and their wild-type littermates at P5. (–) Quantitative analysis of the retinas shown in ; data pooled from two litters containing altogether 6 Vegfc+/− and 9 wild-type pups. () iB4-positive surface area normalized to total area. () Extent of vascular plexus migration from the optic stalk (OS). () Number of vessel branching points. () Number of sprouts. () Filopodia per length of vascular front. () Fold changes in Hey1, Hey2 and Nrarp mRNA levels analysed by qRT-PCR in the retinas of Vegfc+/− and wild-type pups at P5 (data pooled from two litters containing altogether 7 Vegfc+/− and 6 wild-type pups). () Number of failed fusions per vascular loop in the retinas of Vegfc+/− and Vegfc+/+ pups at P5 (n=6 Vegfc+/− and 9 wild-type pups, data pooled from 2 litters). () iB4 (green) and collagen IV (red) staining of Vegfc+/− or wild-type littermate retinas at P5. Arrowheads indicate empty basement m! embrane sleeves. () iB4 (white), VEGF-C (red) and Tie2 (green) immunostaining in wild-type mouse retinas at P5. Arrows indicate VEGF-C- and Tie2-positive macrophages at the angiogenic front. () iB4 staining (green) of P5 retinas of op/op pups and op/+ littermate controls. (–) Quantitative analysis of the retinas shown in ; n=5 op/op and 4 op/+ pups. Dashed line in and indicates a similar distance from the optic stalk (OS). () iB4-positive surface area normalized to total area. () Extent of vascular plexus migration from the optic stalk. () Number of vessel branching points. () Number of sprouts. () Fold changes in Hey1, Hey2 and Nrarp mRNA levels analysed by qRT-PCR in the retinas of op/op pups and op/+ pups at P5 (n=5 op/op and 3 op/+ pups). Scale bars, 100 μm (,) and 50 μm (,). *P<0.05, **P<0.01, ***P<0.001. Error bars, s.e.m. * Figure 6: VEGF-C promotes Notch signalling in endothelial cells through VEGFR-3 and PI(3)K. (–) Fold changes in Notch target gene and DLL4 levels in hBECs stimulated with 200 ng ml−1 VEGF-C, and treated with Dll4-Fc conditioned medium (), transfected with VEGFR3 siRNA or control siRNA (), in conditions where 50% of hBECs express membrane-bound Dll4 (Dll4-TM; ) or treated with the PI(3)K inhibitor LY294002 (). Cells were stimulated for 1 h before lysis. Expression of GAPDH was used as the normalization control. Note the successful transduction of hBECs with retroviruses encoding Dll4-TM in , as evaluated by qRT-PCR. () Fold increase in PI(3)K activity in VEGFR3 versus control silenced hBECs after stimulation with VEGF-C (100 ng ml−1) for 15 min. Data pooled from 2 individual experiments, each containing 3 replicates. * denotes Pvalues versus control group (*P<0.05, **P<0.01, ***P<0.001) and # denotes P values between groups (#P<0.05, ##P<0.01). Error bars, s.e.m. * Figure 7: VEGFR-3 interacts with the transcription factor FoxC2 to control angiogenesis. () Fold change in the level of FOXC2 mRNA expression following stimulation of hBECs with 200 ng ml−1 VEGF-C (n=3 plates per group). () Immunostaining for FoxC2 (red) and isolectin B4 (iB4; green) in Vegfr3iΔEC and wild-type littermate pups at P5. Arrowheads indicate FoxC2-negative tip cells. () Quantification of FoxC2-positive nuclei from the retinas shown in . Nuclei in the area of iB4-positive endothelial cells were quantified at the angiogenic front (n=3 pups per group). (,) Fold change in the level of Foxc2 mRNA expression in Vegfr3iΔEC and wild-type littermate retinas (), and in Vegfc+/− or wild-type littermate retinas () at P5 (n=3 pups per group). () iB4 staining (white) in Foxc2+/−;Vegfr3+/−, Foxc2+/−, Vegfr3+/− or wild-type littermate retinas at P5. Yellow dots in the lower panels indicate filopodia. (–) Quantitative analysis of the retinas shown in . () iB4-positive surface area normalized to total area. () Number of vessel branching points. () ! Filopodia per length of vascular front. Data pooled from 2 litters; n=3 Foxc2+/−;Vegfr3+/−, 4 Foxc2+/−, 4 Vegfr3+/− and 4 wild-type pups. Scale bars, 50 μm. *P<0.05, **P<0.01, ***P<0.001. Error bars, s.e.m. () Schematic of VEGF-C-expressing macrophages in vessel anastomosis and branch maintenance during developmental angiogenesis. Initially, 2 tip cells that lead vascular sprouts are chaperoned to fuse by a macrophage (green). VEGF-C expression (purple) ensues in the macrophage, activating VEGFR-3 in the tip cells, which leads to the expression of Notch target genes and decreased sensitivity to the VEGF gradient in the cells. Vegfr3 loss-of-function (LOF) leads to decreased Notch signalling. A simplified summary of the 'active' (green) and 'passive' (red) signalling pathways originating from VEGFR-3 is shown in the upper left corner. Only the 'active' pathway is targetable by inhibitors. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Tuomas Tammela & * Georgia Zarkada Affiliations * Molecular/Cancer Biology Laboratory, Institute for Molecular Medicine Finland, Research Programs Unit and Department of Pathology, Haartman Institute, Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland * Tuomas Tammela, * Georgia Zarkada, * Harri Nurmi, * Krista Heinolainen, * Denis Tvorogov, * Wei Zheng, * Aino Murtomäki, * Taija Mäkinen & * Kari Alitalo * Vascular Biology Laboratory, London Research Institute—Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK * Lars Jakobsson, * Claudio A. Franco & * Holger Gerhardt * Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, New York 10461, USA * Evelyn Aranda & * Jeffrey W. Pollard * Department of Biochemistry, Hamamatsu University School of Medicine, 431-3192 Hamamatsu, Japan * Naoyuki Miura * A. I. Virtanen Institute and Department of Medicine, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland * Seppo Ylä-Herttuala * Institute of Ophthalmology, University College London, London EC1V 9EL, UK * Marcus Fruttiger * Institut National de la Santé et de la Recherche Médicale U833, Collège de France, 11 Place Marcelin Berthelot, 75005 Paris, France * Anne Eichmann * Vascular Patterning Laboratory, Vesalius Research Center, VIB, Campus Gasthuisberg, B-3000 Leuven, Belgium * Holger Gerhardt * Present addresses: Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles Väg 2, SE171 77 Stockholm, Sweden (L.K.); Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK (T.M.) * Lars Jakobsson & * Taija Mäkinen Contributions T.T. and G.Z. designed, directed and carried out experiments and data analysis, as well as interpreted results, and wrote the paper; H.N. designed and carried out cell culture and biochemistry experiments, and analysed data; L.J. carried out three-dimensional embryoid body sprouting experiments and analysed data; K.H. carried out cell culture, morphometry of retinal vessels and qRT-PCR, and analysed data; D.T. carried out biochemistry experiments and analysed data; W.Z. produced and validated Notch ligand and inhibitor proteins; C.A.F. carried out three-dimensional embryoid body sprouting experiments and analysed data; A.M. carried out retina experiments and analysed data; E.A. provided op/op retinas and carried out genotyping; N.M. generated FoxC2 antibodies; S.Y-H. generated adenoviral vectors; M.F. generated PdgfbCreERT2 mice; T.M. generated Vegfr3flox/floxmice; A.E. analysed retinas of Vegfr3+/LacZ mice; J.W.P. provided op/op retinas; H.G. directed experiments, interpret! ed results and helped write the paper; K.A. designed and directed experiments, interpreted results and wrote the paper. Competing financial interests K.A. is the chairman of the Scientific Advisory Board of Circadian. Corresponding author Correspondence to: * Kari Alitalo Author Details * Tuomas Tammela Search for this author in: * NPG journals * PubMed * Google Scholar * Georgia Zarkada Search for this author in: * NPG journals * PubMed * Google Scholar * Harri Nurmi Search for this author in: * NPG journals * PubMed * Google Scholar * Lars Jakobsson Search for this author in: * NPG journals * PubMed * Google Scholar * Krista Heinolainen Search for this author in: * NPG journals * PubMed * Google Scholar * Denis Tvorogov Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Zheng Search for this author in: * NPG journals * PubMed * Google Scholar * Claudio A. Franco Search for this author in: * NPG journals * PubMed * Google Scholar * Aino Murtomäki Search for this author in: * NPG journals * PubMed * Google Scholar * Evelyn Aranda Search for this author in: * NPG journals * PubMed * Google Scholar * Naoyuki Miura Search for this author in: * NPG journals * PubMed * Google Scholar * Seppo Ylä-Herttuala Search for this author in: * NPG journals * PubMed * Google Scholar * Marcus Fruttiger Search for this author in: * NPG journals * PubMed * Google Scholar * Taija Mäkinen Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Eichmann Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey W. Pollard Search for this author in: * NPG journals * PubMed * Google Scholar * Holger Gerhardt Search for this author in: * NPG journals * PubMed * Google Scholar * Kari Alitalo Contact Kari Alitalo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2600K) Supplementary Information Additional data - Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity
- Nat Cell Biol 13(10):1214-1223 (2011)
Nature Cell Biology | Article Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity * Tse-Chun Kuo1, 9 * Chun-Ting Chen1, 9 * Desiree Baron1 * Tamer T. Onder2, 3, 4, 5 * Sabine Loewer2, 3, 4, 5 * Sandra Almeida6 * Cara M. Weismann1, 6 * Ping Xu1 * Jean-Marie Houghton7 * Fen-Biao Gao6 * George Q. Daley2, 3, 4, 5, 8 * Stephen Doxsey1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1214–1223Year published:(2011)DOI:doi:10.1038/ncb2332Received23 August 2010Accepted03 August 2011Published online11 September 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 midbody is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. Midbodies persist in cells long after division as midbody derivatives (MBds), but their fate is unclear. Here we show that MBds are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells and potential cancer 'stem cells' in vivo and in vitro. MBd loss accompanies stem-cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor NBR1 to the midbody protein CEP55. Differentiating cells and normal dividing cells do not accumulate MBds and possess high autophagic activity. Stem cells and cancer cells accumulate MBds by evading autophagosome encapsulation and exhibit low autophagic activity. MBd enrichment enhances reprogramming to induced pluripotent stem cells and increases the in vitro tumorigenicity of cancer cells! . These results indicate unexpected roles for MBds in stem cells and cancer 'stem cells'. View full text Figures at a glance * Figure 1: MBds accumulate within cells. (,) Multiple MBds associate with a PC3 cell () and a B-lymphoblast (). Insets: MBd labelling () and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries (). MKLP1, MBd marker (,; red); CD44, membrane (; green); DAPI, DNA (; blue). Scale bar, 5 μm () and 2 μm (). (,) Three-dimensional reconstructions of polarized cells in a monolayer () and a HeLa cell () show intracellular MBds. () ZO-1, tight junction; MKLP1, MBds. Scale bar, 2 μm. Enlargement (bottom panel) of the box in the top panel shows five MBds (arrows). () Wheat-germ agglutinin, plasma membrane (red); MKLP1–GFP, MBds (green); DAPI, DNA (blue). Scale bar, 5 μm. () Electron micrograph of an MBd in a permeabilized MCF-7 cell showing immungold labelling with MKLP1 antibodies. Inset, lower magnification of the MBd (boxed) in the cell; nucleus, right. Scale bar, 200 nm. () Time-lapse images during extracellular trypsin treatment of HeLa cells show retention ! of most MBds (MKLP1–GFP, red). Two MBds (yellow arrows) are lost on treatment, indicating digestion and/or dissociation. Time (h:min) post trypsin. Scale bar, 5 μm. () Two-day co-cultures of HeLa cells expressing either MKLP1–GFP (MBd marker) or cytosolic RFP. Green MBds (arrows) associated with red cells (asterisk) indicate post-mitotic transfer of MBds between cells. Scale bar, 10 μm. * Figure 2: MBds are preferentially inherited by the cell with the older centrosome. () The CETN1–GFP signal is brighter in the upper centrosome/spindle pole of a mitotic spindle. The merged differential interference contrast microscopy image with CETN1–GFP labelling at two centrosomes shows a metaphase chromosome. Insets: enlargements and semi-quantitative integrated intensity profiles of centrioles. Scale bar, 5 μm. () The brighter CETN1–GFP signal represents the older centrosome, as it co-stains more intensely for hCenexin1 and remains more intense throughout cell division (Supplementary Fig. S1a). Scale bar, 5 μm. Lower left, merge. (,) Time-lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells () and hESCs (). Cells were imaged at the indicated times (h:min) from telophase by phase-contrast microscopy () and from metaphase by differential interference contrast microscopy (). Middle panel of () and upper left panel of (): CETN1–GFP at centrosomes; enlargements and! integrated intensity profiles show that the daughter cell with the older centrosome (, upper; , lower) inherits the MBd (lower right images in ,). Mitotic midbody and MBds (,; arrows). MKLP1, MBd marker (red); α-tubulin, mitotic midbody and cell-boundary marker (green); DAPI, DNA (blue). Scale bars, 10 μm (,). * Figure 3: MBds accumulate in stem cells in vivo and in vitro. () A histological section through mouse seminiferous tubules labelled for MKLP1 shows several MKLP1+ puncta in cells of the basal layer where stem cells reside. Scale bar, 20 μm. Inset: Enlargement of the cell marked by the arrow. (,) Electron micrographs of mitotic midbody (, arrow) and multiple midbody-like structures in interphase cells with similar shapes and sizes in a juxtanuclear position (, arrows) in basal cells of mouse seminiferous tubules. N, nucleus. Scale bars, 1 μm. () Representative planes of a neural progenitor cell in the ventricular zone (Sox2+, bottom left panel) of an E13.5 mouse brain show that an intracellular MBd (asterisk) is associated with the ventricle-facing daughter in the asymmetrically dividing cell (top row). The bottom row emphasizes the position of paired chromosomes in a dividing anaphase cell. CD133, midbody/MBd marker (green); Na/K-ATPase, cell-border marker (red); DRAQ5, DNA (blue); DAPI, DNA. v, ventricle. Scale bar, 5 μm. Not! e that abscission occurs apically in these cells. () A histological section through a hair follicle (left, phase-contrast microscopy) stained for the stem-cell marker keratin 15 (K15) to identify the bulge region (dotted box), the stem-cell niche. DNA stain (DAPI) and the phase-contrast microscopy image show full follicle architecture. () Upper panels show MBd-accumulating cells in the bulge region (boxed) co-labelled with K15 and MKLP1. Enlargements (lower panels) of the boxed region highlight a cell with four MBds (asterisks). N, nucleus. Scale bar, 5 μm. (–) Quantitative analysis and representative images show a decrease in MBd-accumulating cells on the differentiation of pluripotent stem cells () to fibroblast-like cells (), and an increase in MBd-accumulating cells after reprogramming differentiated cells () to induced pluripotent stem cells (). Numbers refer to mean±s.d., n=3. MKLP1, MBds; ZO-1, tight junctions; α-tubulin, microtubules; Aurora B, midbodies. Sca! le bar, 10 μm. * Figure 4: MBd accumulation is high in stem cells and subpopulations of cancer cells and does not correlate with cell doubling time. () Above: percentages of cells that accumulate MBds (>1) in a range of different cell types, as indicated. Below: doubling times of representative cell lines aligned with MBd-accumulation data. Data are presented as mean±s.d.; cell lines are examined in triplicate (MCF-10A, DLD-1, MCF-10AT, MCF-7, H1 and H9) or quadruplicate (e.v. B6 MEFs, HeLa, SAOS-2 and MCF-10CA1a), except hRPE-1 (n=6), U2OS (n=7) and NCC-IT (n=8). Horizontal line, cell lines with different MBd accumulation potentials (14-fold) but similar doubling times. () Cells pulse-chased with EdU show a decrease in EdU intensity (x axis) over time (y axis), reflecting dilution of dye after cell divisions. (,) After a 96 h chase period, EdU levels were compared between cells with MBd numbers of >1, 1 and 0 (y axis) in HeLa () and SAOS-2 cells (). In both cases, no significant differences were noted (, P=0.2101; , P=0.5609, one-way analysis of variance, with at least 800 cells analysed for each experiment, n=3), in! dicating similar cycling rates among different subpopulations of cells. (–) Each graph is a representative experiment. Cells analysed are shown by green points, the medians are depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range. * Figure 5: MBds in stem and cancer cells evade membrane encapsulation and lysosomal degradation. () Depiction of FPP assay. Digitonin selectively permeabilizes the plasma membrane but not internal membranes. Proteinase K degrades cytoplasmic components but membranous compartments remain intact. Under these conditions, MKLP1–GFP-labelled MBds (blue ellipse) in the cytoplasm will be degraded whereas those inside membrane-bound compartments will not. () MBds in MBd-poor hRPE-1 cells are largely protected (~90% in membranous compartments, 10 cells analysed), whereas most MBds in HeLa cells are not (~27%, 11 cells analysed), and are thus degraded in cytoplasm. Scale bar, 5 μm. () Presence of MBds in lysosomes on chloroquine or E64d–pepstatin A (E64d–PepA) inhibition in hRPE-1 and HeLa cells, but not in MCF-7 and H9 hESCs. Chloroquine treatment of H9 hESCs is not included as it caused differentiation and cell death. A representative image of hRPE-1 cells inhibited by chloroquine is shown, depicting two MBds inside lysosomes. MKLP1 and LAMP2 are used as MBd (red) and ! lysosome (green) markers, respectively. DAPI, DNA (blue). n=100 MBds per treatment in each of the biological triplicates. Scale bar, 5 μm. () Percentage of MBd+ cells (MBd levels), percentage of MBds within lysosomes and percentage of cells exiting cytokinesis following synchronization. MKLP1 and LAMP2 are used as markers as in . Note that MBds are transferred into only one of the two nascent daughter cells after abscission (Fig. 2d), so a 50% maximum will be expected for MBd+ cells. The peak of MBds transferred to cells is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h. () Both chloroquine and E64d–PepA treatments increase the percentage of MBd+ cells in hRPE-1 cells and HeLa cells (chloroquine, P=0.0021 and P=0.0187, respectively; E64d–PepA, P=0.0022 and P=0.0043, respectively; n=3 for all experiments). In contrast, lysosomal inhibition has no detectable effect on hESCs (H1, H9) or MCF-7 cancer cells. DMSO, dimethylsulphoxide. Data are p! resented as mean±s.d. (–), except mean±s.e.m. in hESCs (). * Figure 6: Autophagy controls intracellular MBd levels. () Single-plane confocal microscopy images of MBds within LC3-positive autophagosomes in MEFs expressing GFP–LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MBd markers: Cep55, MKLP1 or MgcRacGAP. Autophagosomes: GFP–LC3 or LC3. Note that MKLP1 (blue) and MgcRacGAP (red) are co-localized (magenta) in the autophagosome (green), indicating that MBds are sorted into autophagosomes. Scale bars, 2 μm. () Decreasing autophagy levels by deletion of the Atg5 gene (left) or depletion of ATG7 by siRNA (right) significantly increases the percentage of MBd+ cells (P=0.0019 and P=0.021, respectively, n=3). Immunoblots confirm loss of the Atg5–Atg12 conjugation in mutant cells and depletion of ATG7 (asterisk). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. () Rapamycin (Rapa) and LiCl co-treatment induces autophagy and decreases the percentage of MBd+ cells (left, HeLa; P=0.0056, n=3). Immunoblots showing increased LC3-II levels confirm autophagy induction. I! nduction of autophagy by overexpression of Flag-tagged BECN1 reduces the percentage of MBd+ cells (right, MCF-7; P=0.0008, n=4). Ctrl, control. () Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d–PepA. U, uninhibited. I, inhibited. Below, the average of the percentage change in LC3-II levels after lysosomal inhibition from three experiments. α-tubulin, loading control. () Quantification of autophagic flux from three experiments in different cell lines. Normal dividing cells (MBd poor) typically have high autophagic flux, whereas stem and cancer cells (MBd rich) have low autophagic flux. The data are presented as mean±s.d. (–). Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 7: NBR1 is a receptor for targeting MBds to the autophagy pathway. () Single-plane confocal microscopy images showing co-localization of the MBds and the autophagic receptor NBR1 in U2OS cells and p62-deleted MEFs. MBd markers: MKLP1 or Cep55. Scale bar, 2 μm. () The percentage of MBd+ cells is significantly increased following the depletion of NBR1 (P=0.022, n=3), but not another autophagic receptor, p62. Co-depletion of NBR1 and p62 does not further increase MBd levels over NBR1 depletion alone. () Deletion of the p62 gene does not affect the percentage of MBd+ cells. For and , immunoblots verify protein loss. () Co-immunoprecipitation (IP) reveals that CEP55 and NBR1 form a complex. Precipitated proteins and 5% of the input material (Input) were analysed by immunoblotting (IB) with antibodies against NBR1 or CEP55. (–) Overexpression of CEP55–eGFP (enhanced green fluorescent protein) increases the percentage of MBd+ cells (; P=0.0007, n=3) and the percentage of NBR1-negative MBds (; P=0.0568, n=3), presumably by sequestering NBR1 ! (red) away from MBds in cells expressing CEP55–eGFP (green) as shown in , and consequently preventing MBd degradation. The dotted box in is enlarged (top right panel), and the labelling of NBR1 and CEP55–eGFP (middle and bottom right panels) are also presented. DAPI, DNA (blue). Scale bar, 5 μm. The data are presented as mean±s.d. (,,,). Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 8: MBd enrichment increases reprogramming efficiency and enhances in vitro tumorigenicity. (–) Reprogramming is more efficient after MBd enrichment. Differentiated cells (dH1f) and embryonic fibroblasts (IMR90) are reprogrammed after stable expression of either NBR1 shRNA or shNT. Emerging iPSC colonies are scored on the basis of TRA-1-60 expression37. Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation (,, dH1f, 3.1±0.5-fold, n=15, P=0.00035; IMR90, 3.4±0.8-fold, n=3, P=0.02; data are mean±s.e.m.) but insignificant changes in autophagic activity () over shNT control. () Representative plates with TRA-1-60-immunostained iPSC colonies. The immunoblot (, top) and densitometry (, bottom; percentage of autophagic flux) show representative results (n=3); α-tubulin, loading control. () MCF-7 side-population (SP) cells have a significantly higher percentage of MBd+ cells over the non-side-population (MP; P=0.0015, n=3; data are mean±s.d.). (,) MBd enrichment in cancer cells leads to increased anchorage-independent growth. MKLP1�! ��GFP-expressing HeLa cells are separated into 'MBd-high' and 'MBd-low' subpopulations. An increase in the 'MBd high' over 'MBd low' ratio is associated with an increase in soft-agar colony formation (). No significant difference was observed when the enrichment of the MBd-high subpopulation was less than threefold. More soft-agar colonies are formed when MBds are enriched by NBR1 depletion (NBR1 shRNA) in HeLa (, left; P=0.0012, n=3) and mouse 134-4 cells (, right; P=0.0086, n=3); control, shNT. Data are mean±s.d., and the colony number (,) is the sum of INT-violet-stained colonies from ten random fields. () Model for MBd fate in cells. The newly formed MBd is preferentially inherited by the daughter cell with the older centrosome (top panel). The inherited MBd (black ring) is recognized by binding of the NBR1 autophagic receptor (grey circle) with the midbody protein CEP55 (magenta). The MBd is then encapsulated by the autophagosome (yellow circle), and d! egraded after fusion of autophagosome and lysosome (red circle! ) in differentiated cells. This pathway prevents MBd accumulation. In contrast, stem cells efficiently accumulate MBds through successive divisions and evasion of NBR1-mediated autophagy. Furthermore, differentiated and stem cells possess overall high and low autophagic activity, respectively. Uncropped images of blots are shown in Supplementary Fig. S6. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Tse-Chun Kuo & * Chun-Ting Chen Affiliations * Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA * Tse-Chun Kuo, * Chun-Ting Chen, * Desiree Baron, * Cara M. Weismann, * Ping Xu & * Stephen Doxsey * Stem Cell Program, Children's Hospital Boston, Boston, Massachusetts 02115, USA * Tamer T. Onder, * Sabine Loewer & * George Q. Daley * Stem Cell Transplantation Program, Division of Pediatric Hematology and Oncology, Manton Center for Orphan Disease Research, Children's Hospital Boston and Dana Farber Cancer Institute, Boston, Massachusetts 02115, USA * Tamer T. Onder, * Sabine Loewer & * George Q. Daley * Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA * Tamer T. Onder, * Sabine Loewer & * George Q. Daley * Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA * Tamer T. Onder, * Sabine Loewer & * George Q. Daley * Departments of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA * Sandra Almeida, * Cara M. Weismann & * Fen-Biao Gao * Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA * Jean-Marie Houghton * Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA * George Q. Daley Contributions C-T.C. and S.D. conceived the project and wrote the manuscript with the help of T-C.K. and D.B. The experiments on the inheritance and localization of MBds as well as some for MBd degradation were conducted by C-T.C. The experiments on MBd accumulation were conducted by C-T.C. with the help of T-C.K. and C.M.W. Investigation of the mechanisms for MBd degradation was conceived by T-C.K. and S.D., and much of the work executed by T-C.K. Autophagic flux assay, soft-agar assay of FACS-isolated cells and MBd localization in neural progenitors were conducted by D.B., who contributed substantially to the work and intellectual input on multiple aspects of this project. The reprogramming assay was conducted and analysed by T-C.K., T.T.O and S.L. The preparation of hESCs for live imaging was conducted by S.A. Tissue preparation was assisted by P.X. and J.M.H. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stephen Doxsey Author Details * Tse-Chun Kuo Search for this author in: * NPG journals * PubMed * Google Scholar * Chun-Ting Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Desiree Baron Search for this author in: * NPG journals * PubMed * Google Scholar * Tamer T. Onder Search for this author in: * NPG journals * PubMed * Google Scholar * Sabine Loewer Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra Almeida Search for this author in: * NPG journals * PubMed * Google Scholar * Cara M. Weismann Search for this author in: * NPG journals * PubMed * Google Scholar * Ping Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Marie Houghton Search for this author in: * NPG journals * PubMed * Google Scholar * Fen-Biao Gao Search for this author in: * NPG journals * PubMed * Google Scholar * George Q. Daley Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen Doxsey Contact Stephen Doxsey Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (3800K) Supplementary Information Additional data - Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase
- Nat Cell Biol 13(10):1224-1233 (2011)
Nature Cell Biology | Article Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase * Kambiz N. Alavian1, 11 * Hongmei Li1, 11 * Leon Collis2, 11 * Laura Bonanni3 * Lu Zeng1 * Silvio Sacchetti1, 4 * Emma Lazrove1 * Panah Nabili1 * Benjamin Flaherty1 * Morven Graham5 * Yingbei Chen6 * Shanta M. Messerli2 * Maria A. Mariggio4 * Christoph Rahner5 * Ewan McNay7 * Gordon C. Shore8 * Peter J. S. Smith2, 9 * J. Marie Hardwick6, 10 * Elizabeth A. Jonas1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1224–1233Year published:(2011)DOI:doi:10.1038/ncb2330Received07 June 2011Accepted02 August 2011Published online18 September 2011Corrected online27 September 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 Anti-apoptotic Bcl2 family proteins such as Bcl-xL protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-xL enhances the efficiency of energy metabolism. Our evidence indicates that Bcl-xLinteracts directly with the β-subunit of the F1FO ATP synthase, decreasing an ion leak within the F1FO ATPase complex and thereby increasing net transport of H+ by F1FO during F1FO ATPase activity. By patch clamping submitochondrial vesicles enriched in F1FO ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-xL activity increases the membrane leak conductance. In addition, recombinant Bcl-xL protein directly increases the level of ATPase activity of purified synthase complexes, and inhibition of endogenous Bcl-xL decreases the level of F1FO enzymatic activity. Our findings indicate that increased mitochondrial efficiency contributes to the! enhanced synaptic efficacy found in Bcl-xL-expressing neurons. View full text Figures at a glance * Figure 1: Cellular ATP levels are altered by Bcl-xL overexpression or depletion in hippocampal neurons. () ATP levels as measured by firefly luciferin–luciferase luminescence intensity at 7 days after transduction with lentivirus constructs. The luminescence level was normalized to the protein level in each individual well (N=8 wells, ***P<0.0001). At least three independent experiments of different cultures showed similar results. Ctl, control. () Western blot for endogenous Bcl-xL protein. Cell lysates prepared from non-transduced control hippocampal neuron cultures, scrambled (Scr.)-shRNA-expressing neuron cultures and Bcl-xL-shRNA-expressing neuron cultures at 4 days after viral transduction. GAPDH serves as a loading control. () ATP levels as measured by firefly luciferin–luciferase luminescence intensity in control cultures or cultures expressing Bcl-xL shRNA or scrambled shRNA at 4 days after viral transduction. The luminescence intensity was normalized to the protein level in each individual well (N=11 for each condition, representing two independent cultures; *P<0! .03). () ATP levels as measured by firefly luciferin–luciferase luminescence intensity in control cultures or cultures exposed for 12–18 h to ABT-737 (ABT) at the indicated concentrations. The luminescence intensity was normalized to the protein level in each individual well (N=15 for each condition, **P<0.004 ***P<0.0001, three independent cultures). () Example image of a neuron expressing a CSCW2–luciferase lentiviral vector. Light is produced in response to the application of 1 mM luciferin. Shown are phase, luminescent and overlay images. In the pseudocolour images, blue is low luminescence and yellow is high luminescence. Scale bar, 20 μm. () Group data for the amount of ATP represented by the luminescence intensity per coverslip of living hippocampal neurons at 7 days after transfection with the indicated constructs. The light levels were normalized to the average of light levels in mito-GFP control cells. Living neurons were transfected with a CSCW2–lu! ciferase–IG lentivirus vector and mito-GFP or GFP–Bcl-xL (! N=8 coverslips from at least three independent cultures for mito-GFP-expressing neurons, N=10 coverslips from at least three independent cultures of GFP–Bcl-xL-expressing neurons, *P<0.02). () Lactate levels in medium surrounding GFP–Bcl-xL-expressing neurons, compared with GFP-expressing controls, after 12 h in physiological (5 mM) glucose medium (one culture, N=3replicates for each condition *P<0.02). For all panels error bars indicate s.e.m. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 2: Bcl-xL alters oxygen uptake by neurons. Resting Bcl-xL-overexpressing and Bcl-xL-depleted neurons have altered oxygen uptake. () Photomicrograph of a self-referencing amperometric O2 microsensor positioned next to a single hippocampal neuron. Scale bar, 20 μm. () Group data for basal respiration in GFP–Bcl-xL-expressing neurons, compared with mito-GFP-expressing neurons, at 7 days after transfection (N=28mito-GFP-expressing neurons, N=26 GFP–Bcl-xL-expressing neurons; ***P<0.0005). The experiment was repeated in five different cultures from five different animals. () Representative traces of oxygen flux levels of single neurons. For neuronal flux measurements, a self-referencing amperometric O2 microsensor was placed within 1 μm of the cell surface. For the background measurement, the electrode was moved 200 μm from the cell surface. Scr., scramble; Ctl, control. () Group data for basal respiration levels of single non-expressing neurons, or neurons expressing Bcl-xL shRNA or scrambled shRNA at 7 days! after viral transduction (N=10replicates for the control and Bcl-xL shRNA, N=9 replicates for scrambled shRNA; at least three independent cultures were used for studies; *P<0.05). () Group data for basal respiration levels of single control neurons or neurons exposed for 18 h to 10 μM ABT-737 (ABT; N=26 for control and N=19 for ABT-737; **P<0.002; at least three different cultures for each group). () Representative traces of oxygen flux levels of single neurons expressing Bcl-xL–GFP or mito-GFP, while resting (left), stimulated with 90 mM KCl (middle) and after the addition of 5 mg ml−1 oligomycin (right). All values were normalized against the average oxygen flux of the same neuron at the resting flux level. () Group data for the oxygen flux of single cultured neurons expressing Bcl-xL–GFP or mito-GFP. The leak-subtracted oxygen flux was divided by the peak oxygen flux measured during neuronal activity. Neurons were studied from four independent cultures ! (N=9mito-GFP control neurons, N=12 GFP–Bcl-xL-expressing neu! rons; *P<0.04). () Luminescence of firefly luciferase in cultured hippocampal neurons exposed or not to 1 μM ABT-737 for 5 min and subsequently stimulated with 90 mM KCl for 90 s (N=12 wells per group; ***P<0.0001; two different cultures for each condition). Measurement of stimulated wells was taken 5 min after washout of high K. For all panels error bars indicate s.e.m. * Figure 3: Bcl-xL is expressed in the mitochondrial inner membrane and interacts with ATP synthase. () Immuno-electron micrographs from cultured neurons overexpressing Bcl-xL at 7 days after viral transduction. Bcl-xL immunoreactivity in the outer membrane (left, arrow) and the inner membrane cristae (right, arrow) are shown. Scale bars, 200 nm. () Immuno-electron micrographs prepared from untreated rat brain (large balls, Bcl-xL; small balls, MnSOD). () Average number of immunogold particles per electron micrograph representing Bcl-xL protein in the outer versus inner membrane (N=30 micrographs). Error bars indicate s.e.m. () Reciprocal immunoblots (IB) of co-immunoprecipitation (IP) of Bcl-xL and the ATP synthase β-subunit from purified rat brain ATP synthase complex. Antibodies are as indicated (immunoblot; N=3). Top, the precipitating antibodies were IgG and Bcl-xL. The right lane represents the whole-cell lysate. Bottom, the precipitating antibodies were IgG and the ATP synthase β-subunit. The right lane represents the whole-cell lysate. () Top, immunoprecipitatio! n of the Myc–Flag-tagged ATP synthase subunits (α, β, b, c, δ, d, ε, γ, and OSCP), precipitated using the anti-Flag affinity gel and immunoblotted using anti-Myc tag antibody. Ctl, control. Bottom, western blot analysis, using anti-Bcl-xL antibody, on the immunoprecipitated samples. () Top, immunoprecipitation of the Myc–Flag-tagged ATP synthase subunit β, precipitated using the anti-Flag affinity gel and immunoblotted using anti-Myc antibody. Cells were pre-exposed for 12 h to 1 μM ABT-737 (ABT) or vehicle. Bottom, western blot analysis, using anti-Bcl-xL antibody on the immunoprecipitated samples. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 4: Bcl-xL protein regulates ATPase activity. () Luminescence intensity of firefly luciferin–luciferase activity in the presence of ATP. N=3 wells without F1FO ATP synthase (blank); N=3 wells F1FO ATP synthase plus the FO inhibitor oligomycin (Oligo.; 5 mg ml−1); N=6 wells synthase plus recombinant Bcl-xL protein (0.045–0.79 mg protein per millilitre); N=9 wells synthase plus control (Ctl) protein (0.05 mg ml−1 BSA, F1FO ATP synthase concentration for all experiments was 4 mg protein per millilitre). *P<0.05, **P<0.005, ***P<0.0005. The experiments on Bcl-xL versus control were repeated and confirmed on five different experimental days using at least two different F1FO ATPase vesicle preparations (from two different animals). () F1FO ATPase activity of purified F1FO ATP synthase in the absence and presence of ABT-737 (ABT; 20 μM). Data are shown as the percentage change in fluorescence intensity over time (N=3 for each condition; ***P<0.0008, **P<0.003, *P<0.04). Experiments were repeated on thr! ee independent isolations with similar results. () F1FO ATPase activity of the purified F1FO ATP synthase vesicles in the presence of the indicated recombinant proteins or reagents as a function of the rate of decrease in NADH fluorescence intensity (see Methods). Left, N=3samples in each condition; **P<0.002, *P<0.04; assay carried out with similar results on two independent F1FO ATP synthase isolations. Inact., inactive. Right, N=7 samples for each condition; assay carried out with similar results on two independent isolations ***P<0.0001. Obato., obatoclax. For all panels error bars indicate s.e.m. * Figure 5: ATP-sensitive H+ ion sequestration into F1FO ATPase vesicles (SMVs) is attenuated by Bcl-xL inhibitors, and by oligomycin and FCCP. () Arrangement of an F1FO ATPase vesicle exposed to the fluorescent pH indicator, ACMA. ATP binds to F1 to activate ATP hydrolysis and drives H+ ions through FO, decreasing the ACMA fluorescence intensity. Bcl-xL inhibitors produce a H+ leak out of the F1FO ATPase membrane, perhaps at the site of the F1FO ATPase itself, resulting in an increase in ACMA fluorescence intensity. Oligomycin blocks the movement of H+ ions through FO, and thus prevents a drop in the ACMA fluorescence intensity. FCCP is a H+ ionophore that causes the leakage of H+ out of the SMV. () Example traces of fluorescence intensity changes of the ACMA indicator over time in the presence of F1FO ATPase vesicles (N=3 samples for each condition, repeated three times; comparing effects of reagents in the presence of ATP with the effect of ATP alone, *P<0.05; **P<0.01; ***P<0.0001, one-way analysis of variance, ANOVA). Obato., obatoclax; ABT, ABT-737; Ctl, control. () Group data showing the peak effect on relati! ve fluorescence intensity (percentage of control). The control represents the fluorescence of the ACMA indicator in the presence of SMVs before the addition of ATP (N=3 samples for each group; *P<0.05; ***P<0.0001, one-way ANOVA). Oligo., oligomycin. This study was repeated at least three times on different batches of SMVs with similar results. Error bars indicate s.e.m. * Figure 6: Pharmacological inhibition or depletion of Bcl-xL reverses leak closure in patch-clamp recordings of isolated ATP F1FO ATPase vesicles. () Example SMV patch-clamp recording at the indicated voltage before and after ATP and ATP+ABT-737 exposure. The dashed line represents 0 pA. Ctl, control; ABT, ABT-737. () Group data of membrane conductances of all recordings such as shown in (SMV recordings from left to right, N=30, 23, 19, 7; **P<0.002, ***P<0.0009). The last bar shows experiments in which the Bcl-xL inhibitor was added to patches in the absence of ATP. () Example SMV patch-clamp recording at the indicated voltage before and after ATP and ATP plus obatoclax (Obato.) exposure. The dashed line represents 0 pA. () Group data of membrane conductances of all recordings such as shown in (SMV recordings from left to right, N=23, 9, 15, 14; **P<0.004, *P<0.04). The last bar shows experiments in which the Bcl-xL inhibitor was added to patches in the absence of ATP. () Western blot for endogenous Bcl-xL protein. Cell lysates prepared from non-transduced control hippocampal neuron cultures, scrambled-shRNA-expre! ssing neuron cultures and Bcl-xL-shRNA-expressing neuron cultures at 4 days after transduction. The protein concentration was controlled by immunoblotting for GAPDH. () SMV patch-clamp recordings before and after the addition of 0.5 mM ATP. SMVs were prepared from hippocampal neurons expressing control (scrambled) shRNA at 4 days after transduction. () Group data from all recordings of control (scrambled, Scr.) shRNA or Bcl-xL shRNA. Shown is the membrane leak conductance remaining after the addition of ATP as a percentage of the initial conductance before the addition of ATP (N=5 control recordings, N=7 Bcl-xL-shRNA recordings; *P<0.03). () SMV patch-clamp recordings before and after the addition of ATP. SMVs were prepared from hippocampal neurons expressing Bcl-xL shRNA at 4 days after transduction. For all panels error bars indicate s.e.m. Uncropped images of blots are shown in Supplementary Fig. S8. Change history * Abstract * Change history * Author information * Supplementary informationCorrected online 27 September 2011In the version of this article initially published online and in print, the affiliation denoted by number 4 was incorrect. Author information * Abstract * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to the work * Kambiz N. Alavian, * Hongmei Li & * Leon Collis Affiliations * Department of Internal Medicine, Yale University, New Haven, Connecticut 06520, USA * Kambiz N. Alavian, * Hongmei Li, * Lu Zeng, * Silvio Sacchetti, * Emma Lazrove, * Panah Nabili, * Benjamin Flaherty & * Elizabeth A. Jonas * Biocurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA * Leon Collis, * Shanta M. Messerli & * Peter J. S. Smith * Department of Oncology and Neuroscience and Aging Research Center, University G.D'Annunzio of Chieti-Pescara, I-66013 Chieti, Italy * Laura Bonanni * Department of Neuroscience and Imaging, and Ce.S.I. Aging Research Center, Universitá G.D'Annunzio of Chieti-Pescara, I-66013 Chieti, Italy * Silvio Sacchetti & * Maria A. Mariggio * Department of Cell Biology, Yale University, New Haven, Connecticut 06520, USA * Morven Graham & * Christoph Rahner * Department of Pharmacology and Molecular Sciences, Johns Hopkins, Baltimore, Maryland 21205, USA * Yingbei Chen & * J. Marie Hardwick * Behavioural Neuroscience and Center for Neuroscience Research, University at Albany, New York 12222, USA * Ewan McNay * Gemin X Pharmaceuticals, Montréal, Quebec H2X 2H7, Canada * Gordon C. Shore * Institute for Life Sciences, University of Southampton, SO17 1BJ, UK * Peter J. S. Smith * Department of Molecular Microbiology and Immunology, Johns Hopkins, Baltimore, Maryland 21205, USA * J. Marie Hardwick Contributions K.N.A. and E.A.J. conceived the project, carried out most of the experiments, analysed the data and prepared the manuscript. H.L. and L.C. contributed experiments to Figs 1 and 2. L.B. contributed experiments to Fig. 6. L.Z., S.S. and M.A.M. contributed to Fig. 4. E.L. and P.N. contributed to Fig. 3. B.F. helped with Fig. 6. M.G. and C.R. contributed experiments to Fig. 3 and Supplementary Fig. S2. S.M.M. and E.M. contributed to Fig. 1. Y.C. and G.C.S. contributed to discussion. P.J.S.S. provided experimental design and discussion for Figs 1 and 2. J.M.H. designed Bcl-xL immunolocalization experiments, and contributed intellectually as well as in manuscript preparation. Competing financial interests G.C. Shore is a shareholder in Gemin X Pharmaceuticals Inc. Corresponding author Correspondence to: * Elizabeth A. Jonas Author Details * Kambiz N. Alavian Search for this author in: * NPG journals * PubMed * Google Scholar * Hongmei Li Search for this author in: * NPG journals * PubMed * Google Scholar * Leon Collis Search for this author in: * NPG journals * PubMed * Google Scholar * Laura Bonanni Search for this author in: * NPG journals * PubMed * Google Scholar * Lu Zeng Search for this author in: * NPG journals * PubMed * Google Scholar * Silvio Sacchetti Search for this author in: * NPG journals * PubMed * Google Scholar * Emma Lazrove Search for this author in: * NPG journals * PubMed * Google Scholar * Panah Nabili Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin Flaherty Search for this author in: * NPG journals * PubMed * Google Scholar * Morven Graham Search for this author in: * NPG journals * PubMed * Google Scholar * Yingbei Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Shanta M. Messerli Search for this author in: * NPG journals * PubMed * Google Scholar * Maria A. Mariggio Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph Rahner Search for this author in: * NPG journals * PubMed * Google Scholar * Ewan McNay Search for this author in: * NPG journals * PubMed * Google Scholar * Gordon C. Shore Search for this author in: * NPG journals * PubMed * Google Scholar * Peter J. S. Smith Search for this author in: * NPG journals * PubMed * Google Scholar * J. Marie Hardwick Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth A. Jonas Contact Elizabeth A. Jonas Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data - APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment
- Nat Cell Biol 13(10):1234-1243 (2011)
Nature Cell Biology | Article APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment * Jörg Mansfeld1 * Philippe Collin1, 3 * Mark O. Collins2, 3 * Jyoti S. Choudhary2 * Jonathon Pines1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1234–1243Year published:(2011)DOI:doi:10.1038/ncb2347Received06 June 2011Accepted17 August 2011Published online18 September 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 Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the anaphase-promoting complex, or cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important for the understanding of how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator CDC20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated CDC20 remain 'locked' onto the APC/C, which prevents the ubiquitylation and degradation of cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of CDC20 and MCCs on the APC/C to allow the SAC to respond ! to the attachment state of kinetochores. View full text Figures at a glance * Figure 1: APC15 is a subunit of the human APC/C. () APC4 was immunoprecipitated (IP) from asynchronously growing HeLa cells and the co-precipitating APC/C subunits were analysed by immunoblotting with the indicated antibodies. () HeLa cells were synchronized at different stages of the cell cycle and analysed as in . Asyn., asynchronous. () Typical size-exclusion chromatography of an extract from prometaphase-arrested HeLa cells. The fractions were analysed by immunoblotting (V0, void volume) with the indicated antibodies. () Top, anti-APC4 immunoprecipitations using extracts from prometaphase-arrested HeLa cells that were treated with 50 nM of the indicated siRNAs for 72 h. Note that depleting APC6 or APC8 causes the APC/C to dissociate into two subcomplexes13, one of which was precipitated with anti-APC4 and the other with anti-APC3 (see schematic, bottom, and Supplementary Fig. S2b). AB, antibody. Molecular mass markers are shown on the right of each panel. The results in each panel are representative of at least thr! ee experiments. Uncropped images of blots are shown in Supplementary Fig. S10. * Figure 2: APC15 is an APC/C subunit required for timely entry into anaphase. () HeLa (top) or RPE1 (bottom) cells were treated for 85 h with 50 nM of the indicated siRNAs before analysis by immunoblotting. Molecular mass markers are shown on the right. The results are representative of three experiments. () The time from NEBD to anaphase of asynchronously growing RPE1 cyclin-B1–Venus cells, treated as in , was determined by fluorescence and phase-contrast microscopy. Scatter dot blots show the mean (red line) of the indicated number of cells from three experiments (P<0.0001 versus GAPDH siRNA for each oligonucleotide, Supplementary Table S1). () Montage of representative images showing RPE1 cyclin-B1–Venus/H2B–mRuby-expressing cells treated as in using APC15 oligonucleotide 4. NEBD set to 0 min. Scale bar, 10 μm. (,) Single-cell destruction assays of asynchronously growing RPE1 cyclin-B1–Venus () and RPE1 cyclin-A2–Venus () cells after 85 h siRNA treatment. Images were captured every 3 min and the total cell fluorescence intens! ity was measured. Fluorescence intensities were normalized to NEBD. Error bars indicate s.d. from 25 cells for GAPDH siRNA and APC15 siRNA and 15 cells for APC3 siRNA from three experiments. Only cells that arrested for more than 100 min were quantified in APC3-siRNA experiments. The asterisk indicates the time point before the beginning of anaphase in GAPDH-siRNA-treated cells. Uncropped images of blots are shown in Supplementary Fig. S10. * Figure 3: APC15 depletion causes a SAC-dependent delay in mitosis but does not affect APC/C activity. (,) The timing from NEBD to anaphase of asynchronously growing RPE1 cyclin-B1–Venus cells treated for 85 h with the indicated siRNAs was determined by microscopy. GAPDH siRNA and APC15 siRNA are identical to Fig. 2b as the data are derived from the same experiments. Dimethylsulphoxide (DMSO) or 0.5 μM reversine was added as indicated. Scatter dot blots show the mean (red line) of the indicated number of cells from three experiments (P<0.0001 for APC15 siRNA versus GAPDH siRNA in and , Supplementary Table S1). Open circles indicate the minimal time that cells were arrested when NEBD or anaphase was not observed during the experiment. (,) Cells were treated as in Fig. 2d,e but with 0.5 μM reversine added to the medium before imaging. Error bars indicate s.d. from 50 cells for GAPDH siRNA and APC15 siRNA, 10 cells for APC3 siRNA from three experiments () or 25 cells for GAPDH siRNA and APC15 siRNA, and 50 cells for GAPDH siRNA+DMA from two experiments, respectively ()! . () Left, top, autoradiography of an in vitro ubiquitylation assay using interphase APC/C purified from GAPDH- and APC15-depleted cells. The APC/C was immunoprecipitated using anti-APC3 antibodies, activated with Cdh1, and probed for its ability to ubiquitylate cyclin B1 (amino acids 1–86). Cyclin-B1–Ub(n), ubiquitylated cyclin B1. Bottom, western blot analysis (WB) of the ubiquitylation assay done in parallel. Molecular mass markers are shown on the right. Right, the quantification shows cyclin-B1 ubiquitylation normalized to GAPDH-siRNA APC/C (mean±s.e.m. from three experiments). Uncropped images of blots are shown in Supplementary Fig. S10. * Figure 4: APC15 is required for the turnover of MAD2, BUBR1, BUB3 and CDC20 on the APC/C during prometaphase. (,) Top, anti-APC3 () and anti-CDC20 () immunoprecipitates (IP) from HeLa cells arrested in prometaphase and treated with the indicated siRNAs for 85 h were analysed by immunoblotting. The amounts of MCC proteins that precipitated with APC3 or CDC20 were analysed by quantitative immunoblotting and normalized to GAPDH-siRNA treatment. Molecular mass markers are shown on the right. Bottom, histograms indicate the mean±s.e.m. from six experiments. () Anti-MAD2 immunoblots of fractions from size-exclusion chromatography analyses of HeLa cell extracts treated as in . Molecular mass markers are shown on the right. The complete immunoblot analyses are shown in Supplementary Fig. S5a–c. The results are representative of two experiments. Uncropped images of blots are shown in Supplementary Fig. S10. * Figure 5: APC15 is required for the release of MAD2, BUBR1 and BUB3 from the APC/C after the SAC had been satisfied. () Timing from NEBD to anaphase in the presence of 4 μM ZM447439 in RPE1 cyclin-B1–Venus cells treated as in Fig. 3b. Scatter dot blots show the mean (red line) of the indicated number of cells from three experiments (P<0.0001 for APC15 siRNA versus GAPDH siRNA, Supplementary Table S1). Open circles indicate the minimal time that cells were arrested when NEBD or anaphase was not observed during the experiment. Adding 4 μM ZM447439 slowed progress from NEBD to anaphase by a factor of ~1.5 independently of siRNA treatment (compare Figs 5a and 3a). A similar delay has previously been reported in RPE1 cells43 and might reflect the extended time ZM447439-treated cells spend in prometaphase31. () HeLa cells were released from a DMA-block into fresh medium containing 0.5 μM reversine and 10 μM MG132. Samples were collected at the indicated times and the APC/C was immunoprecipitated (IP) using anti-APC3 antibodies before analysis by immunoblotting with the indicated an! tibodies. Molecular mass markers are shown on the right. The quantification of three experiments is shown in Supplementary Fig. S6b. () Top, autoradiography of in vitro ubiquitylation assays using APC/C purified from GAPDH-siRNA- and APC15-siRNA-treated HeLa cells arrested in prometaphase. Cyclin-B1–Ub(n), ubiquitylated cyclin B1. Bottom, the amounts of SAC proteins bound to the APC/C were determined by immunoblotting of an in vitro ubiquitylation assay done in parallel. The asterisk denotes His–CDC20 that binds to the APC/C; the lower band is endogenous CDC20. Molecular mass markers are shown on the right. WB, western blot. The quantification of two experiments is shown in Supplementary Fig. S6e. () Quantification of ubiquitylated cyclin B1 (amino acids 1–86) normalized to control reactions from three experiments (mean±s.e.m.). Uncropped images of blots are shown in Supplementary Fig. S10. * Figure 6: Ubiquitylation of CDC20 is not required to release MCCs from the APC/C. () Immunoblot analysis of CDC20 ubiquitylation from the experiments shown in Fig. 4a. LC denotes the light chain of CDC20 antibodies. Molecular mass markers are shown on the right. () Ubiquitylated CDC20 was analysed by quantitative western blotting from four experiments shown in Fig. 4a. To determine the relative amount of ubiquitylated CDC20 on the APC/C the ratio of CDC20 modified with one to four ubiquitin molecules (Ub1–4, indicated by asterisks) following GAPDH-siRNA- versus APC15-siRNA- or p31comet treatment was determined and normalized to the ratio of unmodified CDC20. The value for GAPDH-siRNA-treated cells was set to 1. The bars indicate the mean±s.e.m. of four experiments. () Stable cell lines expressing siRNA-resistant Flag–CDC20 wild type or Flag–CDC20K485/490R from an inducible promoter were treated with CDC20-siRNA oligonucleotides for 72 h. The cells were arrested with DMA, and then released into fresh medium containing 0.5 μM reversine and 10 ! μM MG132. Samples were collected at the indicated times and the APC/C was immunoprecipitated (IP) using anti-APC3 antibodies before immunoblot analysis with the indicated antibodies. Molecular mass markers are shown on the right. () The amount of MCC components bound to the APC/C during the DMA arrest (t=0) was determined for CDC20K485/490R and normalized to wild-type CDC20. () The amount of MCC components bound to the APC/C 30 minutes after release from the DMA-block was normalized to time zero (t=0). The bars indicate the mean±s.e.m. of three experiments for and . Uncropped images of blots are shown in Supplementary Fig. S10. * Figure 7: Ubiquitylation contributes to the release of MCCs from the APC/C. () Immunoblot analysis with the indicated antibodies of total cell extracts from DMA-arrested HeLa cells that were treated with the indicated siRNAs using a double transfection protocol (see Methods). Molecular mass markers are shown on the right. () Immunoblot analysis of anti-APC4 immunoprecipitates (IP) from cell extracts shown in with the indicated antibodies. () Quantification of the amount of APC/C-bound SAC proteins during a DMA-arrest shown in and (t=0) relative to GAPDH-siRNA-treated APC/C. The bars indicate the mean±s.e.m. of six experiments for and of four experiments for . () HeLa cells from were released for 30 min into fresh medium containing 0.5 μM reversine and 10 μM MG132 and the APC/C was precipitated by APC4 antibodies before immunoblot analysis with the indicated antibodies. () HeLa cells treated with the indicated siRNAs were arrested as in (t=0) and released as in (t=30). Anti-APC3 immunoprecipitates from the indicated times were analysed with ! the indicated antibodies. The quantification of MCC binding before the release is shown in . The asterisk denotes the UbcH10 signal derived from the first immunodetection. Molecular mass markers are shown on the right. Uncropped images of blots are shown in Supplementary Fig. S10. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally * Philippe Collin & * Mark O. Collins Affiliations * The Gurdon Institute and Department of Zoology, Tennis Court Road, Cambridge CB2 1QN, UK * Jörg Mansfeld, * Philippe Collin & * Jonathon Pines * Proteomic Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK * Mark O. Collins & * Jyoti S. Choudhary Contributions J.M. carried out all of the experiments, P.C. generated the RPE1 cyclin-A2– and B1–Venus knock-in cell lines, M.O.C. and J.S.C. carried out the mass spectrometry that identified APC15 and the CDC20 ubiquitylation site. J.M. and J.P. designed the experiments and wrote the paper. All authors contributed to the interpretation of the results. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jonathon Pines Author Details * Jörg Mansfeld Search for this author in: * NPG journals * PubMed * Google Scholar * Philippe Collin Search for this author in: * NPG journals * PubMed * Google Scholar * Mark O. Collins Search for this author in: * NPG journals * PubMed * Google Scholar * Jyoti S. Choudhary Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathon Pines Contact Jonathon Pines Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2500K) Supplementary Information Excel files * Supplementary Table 1 (47K) Supplementary Information * Supplementary Table 2 (30K) Supplementary Information Additional data - Notch post-translationally regulates β-catenin protein in stem and progenitor cells
- Nat Cell Biol 13(10):1244-1251 (2011)
Nature Cell Biology | Letter Notch post-translationally regulates β-catenin protein in stem and progenitor cells * Chulan Kwon1, 2, 3 * Paul Cheng1, 3 * Isabelle N. King1 * Peter Andersen2 * Lincoln Shenje2 * Vishal Nigam1, 4 * Deepak Srivastava1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1244–1251Year published:(2011)DOI:doi:10.1038/ncb2313Received09 September 2010Accepted05 July 2011Published online14 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-catenin signalling often intersect in stem and progenitor cells and regulate each other transcriptionally. The biological outcome of signalling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here, we show that membrane-bound Notch physically associates with unphosphorylated (active) β-catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-catenin protein. Notch-dependent regulation of β-catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase- 3β-dependent activity of the β-catenin destruction complex. It did, however, require the endocytic adaptor protein Numb and lysosomal activity. This study reveals a previously unrecognized function of Notch in neg! atively titrating active β-catenin protein levels in stem and progenitor cells. View full text Figures at a glance * Figure 1: Notch negatively regulates active β-catenin in stem cells independently of RBP-J. () Western analysis of ESCs transfected with control or Notch1 (N1) siRNA with active (Act), phospho- (Ser 37) or total β-catenin antibodies that detect N-terminal-dephosphorylated β-catenin. (,) Relative β-catenin/TCF-directed luciferase activity in ESCs () or NSCs () transfected with control siRNA or siRNA against Notch1 or Notch1–4 (N1–4). β-catenin/TCF activity was measured by co-transfecting cells with a luciferase reporter downstream of multiple TCF binding sites (Topflash). A mutant reporter (Fopflash) exhibited negligible activity in all luciferase assays done in this study. () Relative RBP-J expression levels by qPCR in ESCs after transfection with control or RBP-J siRNA, determined by qPCR. () Western analysis of ESCs transfected with control or RBP-J siRNA (50 or 100 nM) with Act β-cat antibodies. () Transverse sections of control, Notch1 knockout (KO) (IslCre;Notch1tm2Rko) or RBP-J KO (IslCre;RBP-Jflox/flox) embryos stained with haematoxylin and eosin ! (H&E) (top) or Isl1 antibody (green, bottom) at embryonic day 9.5, at the level of the outflow tract (ot). The asterisks indicate precardiac mesoderm containing CPCs. 4,6-diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. The cutting plane is indicated by a dotted line (left). Scale bars, 100 μm. All luciferase values were normalized to Renilla activity (mean±s.d.; n=4;*P<0.01). P values were determined using a two-tailed Student t-test, type II (see Methods). Gapdh antibody was used as a loading control. Numbers on western blots correspond to relative quantification. h, head; ht, heart tube. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 2: Notch1 negatively regulates active β-catenin in ESCs and physically interacts with β-catenin. () Relative expression of β-catenin and Cyclin D1 mRNA in ESCs transfected with control or Notch1 siRNA (100 nM), determined by qPCR. () Western analysis of ESCs transfected with control plasmids or plasmids encoding N1ICD (100 or 300 ng) and cultured with BIO. Gapdh antibody was used as a loading control. () Relative β-catenin/TCF luciferase activity of BIO-treated ESCs transfected with control or N1ICD±MAML or RBP-J siRNA. () Relative β-catenin/TCF luciferase activity of ESCs transfected with control or Notch1 siRNA and cultured with or without BIO. (,) Transverse sections of control, IslCre;β-catenin(ex3)loxP (Act β-cat) or IslCre;β-catenin(ex3)loxP;Gt(ROSA)26Sortm1(Notch1)Dam/J (Act β-cat; N1ICD overexpression) embryos at embryonic day 9.5, stained with H&E () or β-catenin antibody (red, ). The asterisks indicate precardiac mesoderm containing CPCs (). Scale bars, 100 μm () or 25 μm (). DAPI (blue) was used to counterstain the nuclei (). nt, neural tub! e; ot, outflow tract; pe, pharyngeal endoderm; ec, pharyngeal ectoderm; pm, precardiac mesoderm. (,) ESCs treated with or without BIO () or SW480 (human colon cancer) cells (,) were transfected with expression constructs for Myc (−) or N1ICD–Myc (+), immunoprecipitated (IP) with anti-Myc antibody and immunoblotted (IB) with β-catenin antibody recognizing its carboxy terminus (), dephosphorylated (active) form or the phosphorylated N-terminus (). Notch expression was detected with anti-Myc antibody (). () Schematic representation of Notch1 deletion constructs and their interactions with β-catenin. TM, transmembrane domain; R, RAM domain; ANK, Ankyrin repeats; TA, transactivation domain; P, PEST domain. () Co-IP of BIO-treated ESCs with the Notch1 deletion constructs shown in using the antibodies indicated. Arrowheads indicate Notch1 expression. () Relative β-catenin/TCF-dependent luciferase activity when compared with control (dashed line) of BIO-treated ESCs transfec! ted with the Notch constructs shown in . BIO was used at 2 �! �M. All qPCR or luciferase values were normalized to Gapdh or Renilla activity, respectively. (mean±s.d.; n=4;*P<0.01.) P values were determined using a two-tailed Student t-test, type II (see Methods). Numbers on western blots correspond to relative quantification. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 3: Membrane-bound Notch1 negatively regulates active β-catenin levels through Numb and Numb-like in stem cells. () Schematic representation of a cleavage-site-mutated tethered form of Notch1 (V1774L, top) and western analysis of ESCs transfected with plasmids encoding Myc-tagged wild-type Notch1 or tethered Notch1 (V1774L) and blotted with anti-Myc antibody (bottom), showing the lack of a cleaved protein band. () Relative RBP-J-responsive luciferase activity of mESCs transfected with control plasmids or plasmids encoding tethered Notch1 or N1ICD. () Relative β-catenin/TCF luciferase activity of ESCs transfected with the control or tethered Notch1 (V1774L) construct shown in or treated with Dkk1 (50 ng ml−1). () BIO-treated ESCs transfected with control or tethered Notch1–Myc constructs immunoprecipitated (IP) with anti-Myc antibody and immunoblotted (IB) with β-catenin antibody. Notch1 expression was detected with anti-Myc antibody. () Western analysis of active or total β-catenin in BIO-treated ESCs transfected with control plasmids or plasmids encoding tethered Notch1. ()! Percentage of Brachyury–GFP+ cells after 3 days of differentiation of mouse ESCs with tethered Notch (V1774L) or control in the presence or absence of BIO (0.5 μM; mean±s.d.; n=4;*P<0.01). () Relative β-catenin/TCF luciferase activity of ESCs or NSCs treated with increasing doses of DAPT for 72–96 h. () Western analysis of active β-catenin in mouse or human ESCs, NSCs or bone marrow MSCs treated with increasing doses of DAPT (0, 25, 50 or 100 μM) for 72–96 h. () Percentage of Brachyury–GFP+ cells after 3.5 days of differentiation of mouse ESCs with control or 75 μm DAPT (mean±s.d.; n=4;*P<0.01). () Relative β-catenin/TCF luciferase activity of ESCs treated with control or batimastat, an α-secretase inhibitor. () Relative β-catenin/TCF luciferase activity of wild-type ESCs (control) or ESCs with ligand-binding-site-deleted Notch1 (Notch1lbd/lbd) treated with Wnt3a. All luciferase values were normalized to Renilla activity (mean±s.d.; n=4;*P<0.01! ). P values were determined using a two-tailed Student t-test,! type II (see Methods). Gapdh antibody was used as a loading control. Numbers on western blots correspond to relative quantification. BIO was used at 2 μM. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 4: Notch-mediated degradation of β-catenin requires Numb and lysosomal activity. () Human colon cancer cells (SW480) transfected with pcDNA–Myc or tethered Notch (V1774L)–Myc constructs, immunoprecipitated (IP) with anti-Myc antibody and immunoblotted (IB) with anti-Numb antibody. Expression of tethered Notch was detected with anti-Myc antibody; expression of pcDNA–Myc was confirmed by PCR. () Relative β-catenin/TCF luciferase activity of ESCs transfected with control plasmids or plasmids encoding N1ICD or tethered Notch (V1774L) in the presence or absence of Numb/Numbl siRNA and cultured in BIO for 72 h. () Western analysis of active β-catenin in ESCs transfected with control plasmids or plasmids encoding tethered Notch (V1774L) in the presence or absence of Numb/Numbl siRNA. () Western analysis of active β-catenin in ESCs with control or DAPT treatment in the presence or absence of bafilomycin A1, which inhibits lysosomal activity. All luciferase values were normalized to Renilla activity (mean±s.d.; n=4;*P<0.01; NS, not significant). P val! ues were determined using a two-tailed Student t-test, type II (see Methods). Gapdh antibody was used as a loading control. Numbers on western blots correspond to relative quantification. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 5: GSIs negatively regulate Wnt signalling and cell expansion in colon cancer cells by blocking Notch cleavage. () Western analysis of active β-catenin in SW480 human colon cancer cells transfected with control siRNA or siRNA against Notch1–4 (100 nM each). () Relative β-catenin/TCF luciferase activity of SW480 cells treated with increasing doses of DAPT for 96 h. () Western analysis of β-catenin levels in SW480 and a second colon cancer cell line, HT-29, treated with increasing doses (0, 25, 50 or 100 μM) of DAPT for 96 h. () Relative number of SW480 cells treated with DAPT (50 or 100 μM) for 72 h (mean±s.d.; n=4;*P<0.01). () Western analysis of active β-catenin levels in SW480 cells with increasing DAPT in the presence or absence of proteasome inhibitor (PI) MG-132 (5 nM) for 72 h. Fewer PI-treated cells were loaded in the right-hand lane because they exhibit higher levels of β-catenin. () Notch/RBP-J luciferase reporter activity (multimerized RBP-J binding sites) of SW480 cells treated with increasing doses of ibuprofen. () Relative β-catenin/TCF lucifera! se activity of SW480 cells treated with ibuprofen for 72 h. () Western analysis of active β-catenin in SW480 cells treated with ibuprofen (200 μM) for 72 h. () Western analysis of active β-catenin in SW480 cells with control or ibuprofen (200 μM) treatment transfected with Notch1–4 (100 nM each) siRNA. Gapdh antibody was used as a loading control. All luciferase values were normalized to Renilla activity (mean±s.d.; n=4;*P<0.01). P values were determined using a two-tailed Student t-test, type II (see Methods). Numbers on western blots correspond to relative quantification. () Model for post-translational regulation of β-catenin protein by Notch. In the absence of Wnt, the destruction complex of Axin, APC and GSK3β phosphorylates β-catenin, leading to its proteasomal degradation (left). When the destruction complex is inactivated by Wnts, dephosphorylated (active) β-catenin functions as a transcriptional activator with LEF/TCF. We show that active β-ca! tenin protein levels can be negatively regulated by interactio! n with Notch in a Numb-dependent manner, involving the lysosome. Notch-mediated degradation of β-catenin is independent of the APC-dependent destruction complex. FZD, frizzled; LRP, low-density lipoprotein receptor-related protein; Dsh, dishevelled; NECD, Notch extracellular domain; NICD, Notch intracellular domain. Uncropped images of blots are shown in Supplementary Fig. S8. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Chulan Kwon & * Paul Cheng Affiliations * Gladstone Institute of Cardiovascular Disease and Departments of Pediatrics and Biochemistry & Biophysics, University of California, San Francisco, 1650 Owens Street, San Francisco, California 94158, USA * Chulan Kwon, * Paul Cheng, * Isabelle N. King, * Vishal Nigam & * Deepak Srivastava * Division of Cardiology, Department of Medicine, Johns Hopkins University, 720 Rutland Avenue, Ross 954B, Baltimore, Maryland 21205, USA * Chulan Kwon, * Peter Andersen & * Lincoln Shenje * Present address: Division of Cardiology, Department of Pediatrics, University of California, San Diego 92903, USA * Vishal Nigam Contributions C.K. designed, carried out and supervised in vivo and in vitro work and wrote the manuscript. P.C. designed and carried out in vivo and in vitro work and wrote the manuscript. I.N.K. carried out Notch Co-IP and western analyses. P.A. cultured embryonic stem cells and carried out luciferase assays. L.S. carried out immunocytochemistry and confocal microscopy. V.N. isolated mesenchymal stem cells and carried out western analyses. D.S. designed and supervised this work and wrote the manuscript. Competing financial interests D.S. is a scientific co-founder of iPierian Inc. and is a member of the Scientific Advisory Board of iPierian Inc. and RegeneRx Pharmaceuticals. Corresponding authors Correspondence to: * Chulan Kwon or * Deepak Srivastava Author Details * Chulan Kwon Contact Chulan Kwon Search for this author in: * NPG journals * PubMed * Google Scholar * Paul Cheng Search for this author in: * NPG journals * PubMed * Google Scholar * Isabelle N. King Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Andersen Search for this author in: * NPG journals * PubMed * Google Scholar * Lincoln Shenje Search for this author in: * NPG journals * PubMed * Google Scholar * Vishal Nigam Search for this author in: * NPG journals * PubMed * Google Scholar * Deepak Srivastava Contact Deepak Srivastava 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 - Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes
- Nat Cell Biol 13(10):1252-1258 (2011)
Nature Cell Biology | Letter Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes * Kexi Yi1 * Jay R. Unruh1 * Manqi Deng2 * Brian D. Slaughter1 * Boris Rubinstein1 * Rong Li1, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1252–1258Year published:(2011)DOI:doi:10.1038/ncb2320Received18 March 2011Accepted13 July 2011Published online28 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap1, 2, 3. Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of bal! anced forces governed by the Arp2/3 complex. View full text Figures at a glance * Figure 1: Inhibition of Arp2/3-complex activity disrupts asymmetric MII spindle position. () Representative images of MII spindle position after different drug treatments. The Arp2/3 inhibitor CK-666 (50 μM) induced spindle detachment from the cortex towards the cell centre. The four leftmost panels show the effects of blebbistatin and nocodazole on CK-666-induced spindle detachment. Scale bar, 10 μm. () Time-lapse imaging of chromosome movement in MII oocytes treated with 50 μM CK-666 with and without 100 μM blebbistatin. Scale bar, 10 μm. () Quantification of spindle detachment percentage after various treatments as indicated. Data are mean±s.e.m. from three experiments, 22–52 oocytes per experiment. * Figure 2: Ran signalling regulates cortical localization of the Arp2/3 complex. () Cortical cap localization of the Arp2/3 complex, as determined by anti-Arp2 immunostaining and Arp3–eGFP expression. In the anti-Arp2 panel, blue shows the DAPI (4,6-diamidino-2-phenylindole) staining of chromatin. Scale bar, 10 μm. () Quantification of spindle detachment percentage after RanT24N mutant protein microinjection. () Confocal micrographs showing Arp2 and N-WASP dislocalized in RanT24N-injected oocytes, but not in oocytes injected with wild-type Ran or buffer. In this experiment, nocodazole was used to prevent chromosome detachment from the cortex. Blue shows the DAPI staining of chromatin (DNA). Scale bar, 10 μm. () Quantification of cortical actin cap intensities stained with fluorescently labelled phalloidin in Ran-injected oocytes in the presence of nocodazole. n=28 (Ran wild type), 41 (RanT24N) and 26 (buffer). The box range represents s.e.m.; whiskers show s.d.; the small square is the mean; and the line inside the box is the median. * Figure 3: The Arp2/3 complex is required for most F-actin assembly in the cortical cap and for myosin-II ring maintenance. () Representative images showing actin and myosin-II localization by phalloidin staining and myosin-II immunostaining, respectively. All actin or myosin-II images were acquired in the same way so that their intensities can be compared across different conditions. The images were pseudo-coloured after data acquisition, and the images shown in the same row were from the same oocyte. Scale bar, 10 μm. Two classes of staining pattern, representing 60.3% and 39.7% of the total, were observed for CK-666-treated oocytes on the basis of spindle position. The myosin-II cortical ring appears as two intensity peaks flanking the actin cap (arrows, also see Supplementary Fig. S3a), whereas only a myosin-II cap was observed after CK-666 treatment. DNA, DAPI staining; MT, microtubule staining. () Quantification of cortical actin intensities in CK-666-treated and control oocytes (see Methods). The intensity trace of each group is the mean from 10 oocytes. () Peptide 2CA, but not 2(CAW55A! ), disrupted the cortical localization of Arp2 and the actin cap. Two classes of staining pattern, representing 46.9% and 53.1% of the total, are shown for 2CA-injected oocytes on the basis of spindle position. Arp2 localization and the actin cap were disrupted even in oocytes with spindles remaining attached. Scale bar, 10 μm. () Quantification of cortical actin cap intensities in 2CA-, 2(CAW55A)- and H2O-injected oocytes. n=34 (H2O), 22 (2CA) and 18 (2(CAW55A)). Box plots are as described in the caption of Fig. 2d. * Figure 4: Cytoplasmic streaming powered by Arp2/3-complex-dependent actin flow generates a net pushing force on the spindle. () Kymograph (left), generated along the red line shown in the right panel, showing continuous F-actin flow (GFP fluorescence streaks from the cortex towards the oocyte interior, arrow pointing to one example) away from the cortical cap. Movie duration, 1,600 s. Scale bar, 10 μm. () Vector map of actin flow (from a UtrCH–GFP movie, top) in an MII oocyte obtained using STICS analysis. Heat bar unit, μm min−1. The lower panel is a time projection of a DIC movie (Supplementary Movie S6) showing a swirling pattern of cytoplasmic particles. Scale bar, 10 μm. () Top, montage showing chromatin (blue) movement towards the cortex after spindle disassembly by nocodazole. Scale bar, 10 μm. Bottom, kymograph (left) along the red line in the right panel, of the same movie. For this kymograph and also for the one in , to enhance the contrast of the transmitted light particles for the kymograph, an edge-detection (Sobel) filter was applied, resulting in white-coloured edg! es and cytoplasmic particles. Note the similar angle of the chromatin streak before arriving at the cortex to that of the streak by a cytoplasmic particle (white arrow), indicating that these entities were moving at similar rates. Movie duration, 2,670 s. Scale bars, 10 μm. () Kymograph (left), along the red line in the right panel, showing the spindle migrating back towards the cortex after drug wash-out in a CK-666-treated oocyte. Movie duration, 3,450 s. Note again, a cytoplasmic particle (white arrow) moving at the same speed as the chromatin. Scale bar, 10 μm. () Vector maps of cytoplasmic streaming generated by STICS in MII oocytes with different spindle orientations (as depicted in the illustration above each panel) relative to the confocal plane. Note the similar flow patterns in these different oocytes. Pink shows the Hoechst staining of chromatin. Scale bars, 10 μm. * Figure 5: Myosin-II-dependent cortical cap contraction drives the MII spindle away from the cortex in the absence of Arp2/3 activity. () Vector maps of reverse cytoplasmic streaming in a CK-666-treated oocyte (top) and blocking of this reverse streaming by blebbistatin (bottom). Heat bar unit, μm min−1. The middle panel is a time projection of the DIC movie of the same oocytes as in the top panel, showing a swirl pattern of cytoplasmic particles. Scale bar, 10 μm. () Kymograph (left) showing the spindle/chromatin (blue) movement away from the cortex at a rate similar to that of cytoplasmic particles (white streak, red arrow) after CK-666 addition. Movie duration, 2,070 s. The position of the line for kymograph generation is shown in the right panel. Scale bars, 10 μm. () Kymograph (left) generated along a line through the cortex of a UtrCH–GFP-expressing oocyte (dotted line, right panel), showing actin cap contraction after CK-666 addition. The yellow arrow in the right panel corresponds to the left edge of the kymograph. Movie duration, 2,245 s. Scale bars, 10 μm. Note the movement of ! actin structures (streaks, white arrows) from both sides towards the centre. () Kymograph (left) generated along a line through the cortex of a Texas-red–Con-A-labelled oocyte (dotted line, right panels, blue shows the Hoechst staining of chromatin) showing cortical cap contraction after CK-666 addition without (top) or with blebbistatin (middle), or with nocodazole (bottom). The yellow arrows in the right panels correspond to the left edges of the kymographs. The cap regions showed low-intensity ConA staining as marked. Movie durations, 4,395 s (top), 4,375 s (middle) and 2,035 s (bottom). Scale bars, 10 μm. Author information * Author information * Supplementary information Affiliations * Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA * Kexi Yi, * Jay R. Unruh, * Brian D. Slaughter, * Boris Rubinstein & * Rong Li * Department of Obstetrics and Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA * Manqi Deng * Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA * Rong Li Contributions K.Y. and R.L. designed the experiments, interpreted results and prepared the manuscript; K.Y. carried out all of the experiments; J.R.U. carried out STICS analysis with assistance from B.D.S. and also contributed to other image analysis; M.D. assisted in the initial experimental set-up. B.R. carried out the numerical simulations; R.L. conceived and supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Rong Li Author Details * Kexi Yi Search for this author in: * NPG journals * PubMed * Google Scholar * Jay R. Unruh Search for this author in: * NPG journals * PubMed * Google Scholar * Manqi Deng Search for this author in: * NPG journals * PubMed * Google Scholar * Brian D. Slaughter Search for this author in: * NPG journals * PubMed * Google Scholar * Boris Rubinstein Search for this author in: * NPG journals * PubMed * Google Scholar * Rong Li Contact Rong Li Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1,600K) Supplementary Information Movies * Supplementary Movie 1 (200K) Supplementary Information * Supplementary Movie 2 (280K) Supplementary Information * Supplementary Movie 3 (4M) Supplementary Information * Supplementary Movie 4 (2M) Supplementary Information * Supplementary Movie 5 (800K) Supplementary Information * Supplementary Movie 6 (900K) Supplementary Information * Supplementary Movie 7 (2M) Supplementary Information * Supplementary Movie 8 (1M) Supplementary Information * Supplementary Movie 9 (2,600K) Supplementary Information * Supplementary Movie 10 (1,200K) Supplementary Information * Supplementary Movie 11 (3M) Supplementary Information * Supplementary Movie 12 (1,300K) Supplementary Information * Supplementary Movie 13 (1,700K) Supplementary Information * Supplementary Movie 14 (800K) Supplementary Information Additional data - Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart
- Nat Cell Biol 13(10):1259-1264 (2011)
Nature Cell Biology | Letter Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart * Marcus Braun1, 2, 4 * Zdenek Lansky3, 4 * Gero Fink1, 2, 4 * Felix Ruhnow1, 2 * Stefan Diez1, 2 * Marcel E. Janson3 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1259–1264Year published:(2011)DOI:doi:10.1038/ncb2323Received08 March 2011Accepted20 July 2011Published online04 September 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Short regions of overlap between ends of antiparallel microtubules are central elements within bipolar microtubule arrays. Although their formation requires motors1, recent in vitro studies demonstrated that stable overlaps cannot be generated by molecular motors alone. Motors either slide microtubules along each other until complete separation2, 3, 4 or, in the presence of opposing motors, generate oscillatory movements5, 6, 7. Here, we show that Ase1, a member of the conserved MAP65/PRC1 family of microtubule-bundling proteins, enables the formation of stable antiparallel overlaps through adaptive braking of Kinesin-14-driven microtubule–microtubule sliding. As overlapping microtubules start to slide apart, Ase1 molecules become compacted in the shrinking overlap and the sliding velocity gradually decreases in a dose-dependent manner. Compaction is driven by moving microtubule ends that act as barriers to Ase1 diffusion. Quantitative modelling showed that the molecular o! ff-rate of Ase1 is sufficiently low to enable persistent overlap stabilization over tens of minutes. The finding of adaptive braking demonstrates that sliding can be slowed down locally to stabilize overlaps at the centre of bipolar arrays, whereas sliding proceeds elsewhere to enable network self-organization. View full text Figures at a glance * Figure 1: Ase1 slows Ncd-driven microtubule sliding. () Schematic representation of Ncd-driven sliding of a transport microtubule (MT) along a surface-immobilized template microtubule in the presence of Ase1. () Time-lapse fluorescence micrographs of transport microtubule motion before and after the addition of 0.39 nM Ase1–GFP at t=1.2 min (the Ncd concentration is kept constant at 0.29 nM). The short transport microtubule on the right presumably has a parallel orientation and therefore is not moved by Ncd (ref. 3). The schematic diagram illustrates microtubule orientations and positions at the start of the experiment (template microtubule is dim red and transport microtubules are bright red; black marks indicate plus-ends). () Multi-channel kymographs of the experiment shown in ; the asterisk denotes the addition of Ase1–GFP. () Multi-channel kymographs of microtubule sliding with GFP–Ncd and non-labelled Ase1. () Quantification of transport microtubule sliding velocity and Ase1–GFP intensity in the overlap of ! the experiment in and . * Figure 2: Ase1 prevents antiparallel microtubules from sliding completely apart. () Typical multi-channel kymographs showing the slowdown of microtubule (MT) sliding, as well as the distribution of Ase1–GFP, when a transport microtubule starts to slide apart from the template microtubule. The dashed lines indicate the position of the template microtubule minus-end. () Ase1–GFP density (right axis) and velocity of the transport microtubule (left axis) before and during overlap shrinkage. The dashed line indicates the start of separation. The brief slowdown around −3 min coincides with the crossing of an additional template microtubule (Supplementary Movie S2). () Ase1–GFP profiles along the dashed (before the start of microtubule separation, left panel) and the solid green line (during microtubule separation, right panel) in . () Typical multi-channel kymographs showing the GFP–Ncd distribution during slowdown of microtubule sliding. * Figure 3: Moving microtubule ends constitute barriers for Ase1 diffusion. () One-dimensional diffusion of a transport microtubule (MT) relative to an immobilized template microtubule at low Ase1–GFP concentration. () Diffusion of single Ase1–GFP molecules within a moving microtubule overlap. The solid arrow shows the sliding velocity of the transport microtubule, the dashed arrow shows Ase1–GFP drift and the arrowheads show Ase1–GFP tracking to the trailing end of the microtubule overlap. The plus-end of the template microtubule is indicated by the dashed lines. * Figure 4: Quantitative description of microtubule overlap dynamics. () Instantaneous microtubule sliding velocity as function of Ase1–GFP density for 127 transport microtubules (including 27 measurements in the absence of Ase1–GFP). Within each bin, values that belong to the same microtubule were first averaged (grey circles; bin width is 50 AU μm−1). Black dots denote averaged values of the grey data in each bin (±s.e.m.). The red line shows a linear fit to the average velocities within the bins (see Methods). () Normalized Ase1–GFP density ρ/ρ0 versus L0/L, the inverse of the normalized overlap length. Grey circles represent time points from 51 events of transport microtubule stalling at template ends. Black dots are binned averages (±s.e.m.) of the grey data. The red line shows a linear fit of equation (2) in the Methods to the black data. Densities are derived from measured velocities using the linear relationship established in (see Methods). () Multi-colour kymographs showing the formation of stable microtubule overlap! s between template microtubule (dim red) and transport microtubule (bright red) in the presence of Ase1–GFP (green). Experimental data (left) and model constructed from artificially generated microscope images (right). () Normalized overlap lengths L/L0 for the event in (experimental data in black and model calculations in red). The measured sliding velocity v0 and the length of the transport microtubule, L0, are indicated. The dashed pale red curves indicate model calculations for different values of v0. () Normalized overlap length L/L0 at t0+3 minversus Ase1–GFP density ρ0 for all events where overlap shrinkage lasted at least 4 min (n=32). The grey area indicates model outcomes for a corresponding range of L0 and kon (Methods). Red points indicate the steady-state events shown in Supplementary Fig. S5b. () Schematic representation of the motorized formation of stable antiparallel microtubule overlaps in the presence of Ase1. i, Ncd in the overlap slides microtu! bules in the presence of Ase1 at low density. ii, During micro! tubule separation, Ase1 becomes compacted through molecular sweeping, whereas the density of Ncd does not change. As a result microtubule sliding progressively slows down. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Marcus Braun, * Zdenek Lansky & * Gero Fink Affiliations * Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, 01307 Dresden, Germany * Marcus Braun, * Gero Fink, * Felix Ruhnow & * Stefan Diez * B CUBE, Technische Universität Dresden, Arnoldstr. 18, 01307 Dresden, Germany * Marcus Braun, * Gero Fink, * Felix Ruhnow & * Stefan Diez * Laboratory of Plant Cell Biology, Wageningen University, Droevendaalsesteeg, 6708 PB Wageningen, The Netherlands * Zdenek Lansky & * Marcel E. Janson Contributions M.B., Z.L., G.F., S.D. and M.E.J. designed the experiments; M.B., Z.L. and G.F. carried out the experiments; M.B., Z.L., G.F. and F.R. analysed the data; Z.L. and M.E.J. developed the model; M.B., Z.L., M.E.J. and S.D. wrote the manuscript; M.E.J. and S.D. initiated the research and supervised the work. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Stefan Diez or * Marcel E. Janson Author Details * Marcus Braun Search for this author in: * NPG journals * PubMed * Google Scholar * Zdenek Lansky Search for this author in: * NPG journals * PubMed * Google Scholar * Gero Fink Search for this author in: * NPG journals * PubMed * Google Scholar * Felix Ruhnow Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Diez Contact Stefan Diez Search for this author in: * NPG journals * PubMed * Google Scholar * Marcel E. Janson Contact Marcel E. Janson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Movies * Supplementary Movie 1 (67K) Supplementary Information * Supplementary Movie 2 (43K) Supplementary Information * Supplementary Movie 3 (400K) Supplementary Information * Supplementary Movie 4 (1200K) Supplementary Information * Supplementary Movie 5 (74K) Supplementary Information Additional data - Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase
- Nat Cell Biol 13(10):1265-1271 (2011)
Nature Cell Biology | Letter Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase * Emily A. Foley1 * Maria Maldonado1 * Tarun M. Kapoor1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1265–1271Year published:(2011)DOI:doi:10.1038/ncb2327Received31 May 2011Accepted25 July 2011Published online28 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore–microtubule attachments (K-fibres) on sister chromatids that have attached to opposite spindle poles (bi-oriented)1. In many instances, phosphorylation correlates with K-fibre destabilization2, 3, 4, 5, 6, 7. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes when compared with bi-oriented chromosomes, which have stable attachments2, 8. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore–microtubule interactions can be stabilized when kinase levels are high. Here we show that the generation of stable K-fibres depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibres are destabilized ! and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the level of phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, we find that chemical inhibition of Aurora or Plk1 restores K-fibres in B56-PP2A-depleted cells. Our findings reveal that PP2A, an essential tumour suppressor9, tunes the balance of phosphorylation to promote chromosome–spindle interactions during cell division. View full text Figures at a glance * Figure 1: Microtubule-sensitive targeting of PP2A to centromeres/kinetochores during cell division. () Schematic representation of showing the scaffold, catalytic and regulatory subunits of PP2A. () Maximum-intensity confocal projections show distributions of GFP–scaffold expressed in an RPE1 cell at mitosis (top). Centrosome (arrow) and centromere (arrowhead) localizations are indicated. DIC images (bottom) show chromosomes in the same cell. () Immunofluorescence micrographs of a maximum-intensity projection of an RPE1 cell expressing GFP–scaffold fixed and stained for kinetochores (CREST, red), GFP (green) and DNA (blue, only shown in overlay). () Maximum-intensity projection of the optical sections spanning the outlined regions in enlarged ×2 with DNA omitted. Plotted is the intensity profile of the CREST (red) and GFP (green) signal measured along a line (white) drawn across the centromere. () Maximum-intensity confocal projections of GFP–scaffold distribution and chromosomes (DIC) in a cell arrested at metaphase (10 μM MG132), and imaged live at the indicate! d times relative to the addition of nocodazole (3.2 μM, time zero). Scale bars, 5 μm. * Figure 2: Microtubule-sensitive targeting of B56 regulatory subunits to centromeres/kinetochores. () Maximum-intensity confocal projections show distributions of GFP in different cell lines stably expressing GFP–B56α–ε proteins. () RPE1 cells stably expressing a GFP fusion of the indicated B56 regulatory subunit were arrested at metaphase (10 μM MG132) and imaged live before and after the addition of nocodazole (3.2 μM, time zero). Maximum-intensity confocal projections show GFP distribution, and DIC images show chromosomes before nocodazole addition. Spindle pole targeting was observed in MG132-arrested cells (asterisk). () Cells in MG132 (10 μM) were treated with nocodazole (Noc.; 3.2 μM, bottom) or control solvent (DMSO, top) for 5 min and processed for immunofluorescence microscopy. Equivalently scaled maximum-intensity projections of tubulin, DNA, kinetochores (CREST) and B56α staining are shown. Scale bars, 5 μm. * Figure 3: B56-PP2A is required for stable kinetochore–microtubule attachments and chromosome alignment. () Analysis of GFP–scaffold levels at centromeres/kinetochores after B56-PP2A-siRNA treatment. An RPE1 cell line expressing GFP–scaffold was transfected with control or either of two B56-PP2A-siRNA pools (1, 2) and treated with nocodazole (3.2 μM, 60 min) before processing for immunofluorescence microscopy. The GFP signal at centromeres/kinetochores was measured, processed and normalized to the average value in cells treated with control siRNA. An intensity distribution histogram is plotted from one experiment. B56-PP2A siRNA decreased scaffold targeting to 0.52±0.05 (pool 1) or 0.55±0.05 (pool 2) relative to control cells (mean±s.e.m., 4 experiments, >50 centromeres/kinetochores from 5 cells per condition, per time). (–) K-fibre defects in B56-PP2A-siRNA cells. () The frequency of pre-anaphase mitotic cells with few or no K-fibres. (,) Rescue of siRNA phenotype by stable overexpression of siRNA-resistant B56α or B56β. Cells were arrested in mitosis with noco! dazole (0.32 μM, 2.5 h) and released into MG132 (10 μM, 40 min) before cold-treatment and fixation. () Cold-stable microtubules in a control- and B56-PP2A (pool 2)-siRNA-treated cell. Insets show ×2 enlargement of the outlined regions. () The frequency of K-fibre defects. (,) Chromosome alignment defects in B56-PP2A-siRNA cells. Control- or B56-PP2A-siRNA-treated cells were arrested with MG132 (10 μM, 60 min). () Example of chromosome alignment defects in B56-PP2A (pool 2)-treated cells versus control cells. () The frequency of mitotic cells with misaligned chromosomes. () Cohesion is preserved in B56-PP2A-siRNA cells. Chromosome spreads were prepared from nocodazole-arrested cells (3.2 μM, 4 h) treated with either of two B56-PP2A-siRNA pools. The fraction of paired chromatids from two experiments is shown. () Chromosome spreads were prepared from control- and B56-PP2A (pool 2)-siRNA-treated RPE1 cells arrested as in . Equivalently scaled Sgo1 images an! d an overlay with DNA and kinetochores are shown. Images show ! maximum-intensity projections with tubulin or Sgo1 (green), DNA (blue) and kinetochores (CREST, red). Scale bars, 5 μm. Bars show mean±s.e.m. (n=3 experiments, >80 cells per condition per time). * Figure 4: B56-PP2A depletion increases the level of phosphorylation of Aurora B substrates and Aurora inhibition suppresses the B56-PP2A-siRNA phenotype. (–) RPE1 cells were transfected with control or B56-PP2A siRNA (pool 2) and incubated with nocodazole (3.2 μM, 60 min) before fixation (,), or fixed (), and stained using the indicated antibodies. Images are maximum-intensity projections with equivalent scaling. The signal at kinetochores was measured, processed and normalized to the average value in control-siRNA cells. Histograms show intensity distributions from one experiment. () KMN network targeting is preserved in B56-PP2A-siRNA cells. In B56-PP2A-siRNA cells, the mean kinetochore staining intensities were calculated for Dsn1 (0.85±0.11), Knl1 (1.41±0.23) and Hec1 (1.05±0.08) relative to control cells (n=2–3 experiments, >75 kinetochores from 5 cells per condition, per time). (,) Analysis of Aurora B substrate phosphorylation. () In prometaphase cells, kinetochores with an inter-kinetochore stretch of 1.2–1.5 μM were analysed. In B56-PP2A-siRNA cells, the mean phosho-Dsn1 and phospho-Knl1 intensity wa! s 1.79±0.32 and 2.26±0.07, respectively (n=2 experiments, >50 kinetochores from 5 cells per condition, per time). () In nocodazole-treated B56-PP2A-siRNA cells, the mean intensity of phospho-Dsn1 and phospho-Knl1 was 1.03±0.13 and 1.21±0.15, respectively, relative to control cells (3 experiments, >60 kinetochores from 5 cells per condition per experiment). (,) RPE1 cells treated with control or either of two pools of B56-PP2A siRNA (1, 2) were incubated in MG132 (10 μm, 15 min), followed by the addition of hesperadin (50 nM) or ZM447439 (1 μM) or control solvent (DMSO) for 45 min. () The frequency of mitotic cells with few or no cold-stable K-fibres is plotted (n=3 experiments, >80 cells per condition per time). () Maximum-intensity projection of tubulin (green) and an overlay with kinetochores (CREST, red) in B56-PP2A-siRNA (pool 2) cells treated with the indicated inhibitor. Insets are ×2 enlargement of the optical sections spanning the outlined regions. ! Scale bars, 5 μm. Mean±s.e.m. provided. * Figure 5: B56-PP2A regulates Plk1 substrate phosphorylation and Plk1 targeting to the kinetochore, and Plk1 inhibition suppresses the B56-PP2A-siRNA phenotype. (,) RPE1 cells transfected with control or either of two B56-PP2A siRNA pools (1, 2) were incubated in MG132 (10 μM, 15 min), followed by the addition of BI2536 (40 nM) or DMSO for 45 min. () The frequency of mitotic cells with few or absent cold-stable K-fibres (n=3 experiments, >80 cells per condition per time). () Maximum-intensity projection of tubulin (green) and an overlay with kinetochores (CREST, red) in B56-PP2A-siRNA (pool 2) cells treated with DMSO or BI2536 (40 nM). Insets are ×3 enlargement of the optical sections spanning the outlined centromeres. () RPE1 cells transfected with control or B56-PP2A siRNA (pool2) were fixed and stained. Maximum-intensity projections with equivalent scaling are shown. () RPE1 cells or a cell line expressing siRNA-resistant B56β was transfected with control or B56-PP2A (pool2) siRNA and incubated with nocodazole (3.2 μM, 60 min) before processing for immunofluorescence microscopy. The signal at kinetochores was me! asured, processed and normalized to the average value in control-siRNA cells. Histograms show intensity distributions from one experiment. In B56-PP2A-siRNA cells, the mean kinetochore intensity for BubR1 (0.84±0.24), phospho-BubR1 (3.54±1.41) and Plk1 (2.57±0.32) was calculated relative to control cells (n=3–6 experiments, >60 kinetochores from 5 cells per condition, per time). In the cell line overexpressing siRNA-resistant B56β, Plk1 targeting was similar after B56-PP2A-siRNA (1.08±0.12) or control-siRNA treatment (0.97±0.11), relative to RPE1 cells treated with control siRNA (n=3 experiments, >60 kinetochores from 5 cells per condition, per time). Scale bars, 5 μm. Mean±s.e.m. provided. () Schematic model showing how B56-PP2A localization to centromeres (white oval) and kinetochores (grey) may promote microtubule binding. Author information * Author information * Supplementary information Affiliations * Laboratory of Chemistry and Cell Biology, Rockefeller University, 1230 York Avenue, New York, New York 10065, USA * Emily A. Foley, * Maria Maldonado & * Tarun M. Kapoor Contributions E.A.F. and T.M.K. designed the experiments and wrote the paper. E.A.F. carried out essentially all of the experiments. M.M. contributed to the live-cell imaging. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tarun M. Kapoor Author Details * Emily A. Foley Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Maldonado Search for this author in: * NPG journals * PubMed * Google Scholar * Tarun M. Kapoor Contact Tarun M. Kapoor Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (2400K) Supplementary Information Additional data - NF-κB controls energy homeostasis and metabolic adaptation by upregulating mitochondrial respiration
- Nat Cell Biol 13(10):1272-1279 (2011)
Nature Cell Biology | Letter NF-κB controls energy homeostasis and metabolic adaptation by upregulating mitochondrial respiration * Claudio Mauro1, 7 * Shi Chi Leow1, 2, 7 * Elena Anso3 * Sonia Rocha4 * Anil K. Thotakura1 * Laura Tornatore1 * Marta Moretti1, 5 * Enrico De Smaele5 * Amer A. Beg6 * Vinay Tergaonkar2 * Navdeep S. Chandel3 * Guido Franzoso1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1272–1279Year published:(2011)DOI:doi:10.1038/ncb2324Received10 June 2011Accepted22 July 2011Published online28 August 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cell proliferation is a metabolically demanding process1, 2. It requires active reprogramming of cellular bioenergetic pathways towards glucose metabolism to support anabolic growth1, 2. NF-κB/Rel transcription factors coordinate many of the signals that drive proliferation during immunity, inflammation and oncogenesis3, but whether NF-κB regulates the metabolic reprogramming required for cell division during these processes is unknown. Here, we report that NF-κB organizes energy metabolism networks by controlling the balance between the utilization of glycolysis and mitochondrial respiration. NF-κB inhibition causes cellular reprogramming to aerobic glycolysis under basal conditions and induces necrosis on glucose starvation. The metabolic reorganization that results from NF-κB inhibition overcomes the requirement for tumour suppressor mutation in oncogenic transformation and impairs metabolic adaptation in cancer in vivo. This NF-κB-dependent metabolic pathway involv! es stimulation of oxidative phosphorylation through upregulation of mitochondrial synthesis of cytochrome c oxidase 2 (SCO2; ref. 4). Our findings identify NF-κB as a physiological regulator of mitochondrial respiration and establish a role for NF-κB in metabolic adaptation in normal cells and cancer. View full text Figures at a glance * Figure 1: NF-κB counters reprogramming to aerobic glycolysis and promotes metabolic adaptation to nutrient starvation. (–) Glucose consumption (, left), lactate production (), ATP concentration () and oxygen consumption () in immortalized MEFs expressing non-specific (ns) or RelA-specific shRNAs, under normal culture conditions. (), Right, western blots with the cells in –, showing levels of RelA (knockdown efficiency), c-Rel and β-actin (knockdown specificity). () Left, viability of immortalized MEFs expressing non-specific or RelA shRNAs after glucose starvation (GS). Middle, images of representative cells. Right, western blots with non-specific and RelA shRNA cells. Similar results were obtained using two additional non-specific shRNAs (ns2, shc003v), two additional RelA-specific shRNAs and eGFP-specific, luciferase-specific, laminA/C-specific and cyclophilinB-specific shRNAs. (–) Lactate production (), oxygen consumption () and ATP concentration () in cells treated as in . Lactate values in after day 4 should be interpreted with caution, owing to massive necrosis in RelA-shRNA cel! ls. (,) Survival of the cells in after 4-day glucose starvation, either alone (GS) or together with serum deprivation (GS+SD; ) or rapamycin (GS+Rapa; , left). () Right, representative images. In – the values denote mean±s.e.m. n=3 (–,,,–); n=4 (); n=10 (). In , *P<0.05; **P<0.01. Scale bars: 50 μm. * Figure 2: p53 mediates NF-κB-dependent protection against glucose starvation. () Western blots with antibodies against total or Ser15-phosphorylated (P) p53 (equivalent to mouse p53-Ser18) in non-specific (ns)- and RelA-shRNA-expressing immortalized MEFs after glucose starvation (GS). exp, exposure. () qRT-PCR with RelA- or p53-specific primers and RNAs from immortalized MEFs expressing the shRNAs shown. (,) Chromatin immunoprecipitation with antibodies against RelA, qPCR primers specific for κB-containing regions of the p53 and IκBα promoters or control genomic regions (control 1–3) and extracts from untreated (UT), TNFα-treated or glucose-starved immortalized wild-type (,) and RelA−/− (RelA knockout; ) MEFs. (,) Survival of early passage RelA−/− () and p53−/− (p53 knockout; ) MEFs expressing exogenous eGFP, mouse (m)-p53 or human (h)-RelA before and after glucose starvation (left). qRT-PCR with RNAs from the same cells (0 h; right). (,) Lactate production and oxygen consumption in RelA−/− MEFs expressing exogenous eGFP or mou! se p53 in the presence of normal medium (NM) and during glucose starvation (GS). In – the values denote mean±s.e.m. n=3 (–); n=15 (). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 3: SCO2 mediates NF-κB-dependent protection against glucose-starvation-induced PCD. () qRT-PCR with RNAs from non-specific (ns)- or RelA-shRNA-expressing immortalized MEFs under basal conditions. p53 metabolic targets referred to in the text, but not specified: TP53-induced glycolysis and apoptosis regulator18 (TIGAR), guanidinoacetate methyl transferase13 (GAMT), glutaminase 2 (GLS2; ref. 19). () Top, survival of early passage RelA−/− MEFs expressing exogenous eGFP or mouse (m)-SCO2 after glucose starvation (GS). () Left, survival of immortalized MEFs expressing the indicated shRNAs after glucose starvation. () Bottom and () Right, qRT-PCR showing relative Sco2 and RelA expression in the same cells (0 h). In – the values denote mean±s.e.m. (n=3). * Figure 4: RelA suppresses oncogenic transformation by regulating energy metabolism. () Images of representative early passage RelA−/−, p53−/− and wild-type MEFs infected with pWPT–eGFP or pWPT–H-Ras(V12), showing transformation features (that is, higher density, spindled morphology) in mutant cells, but not in wild-type cells. () Fold change in cell numbers in H-Ras(V12)-expressing MEFs relative to the respective eGFP-expressing controls after a 48 h culture. () Top, percentage change in colony numbers with H-Ras(V12)-transformed cells relative to H-Ras(V12)-infected p53−/− MEFs. Bottom, images of representative colonies. (,) Percentage change in colony numbers with H-Ras(V12)-transformed early passage RelA−/− MEFs expressing eGFP, mouse (m)-p53 or m-SCO2 and H-Ras(V12)-transformed early passage wild-type MEFs expressing eGFP relative to eGFP-expressing RelA−/− MEFs (), and H-Ras(V12)-transformed early passage wild-type MEFs expressing non-specific (ns), RelA, p53 or Sco2 shRNAs relative to RelA-shRNA-expressing MEFs (). (,) Percen! tage change in lactate production () and glucose consumption () in H-Ras(V12)-transformed MEFs relative to the respective eGFP-expressing controls (see also Supplementary Fig. S1a,b, absolute levels in the absence of H-Ras(V12)). The data in ,, and are from the cells in . In – the values denote mean±s.e.m. n=3 (,,); n=4 (,). Scale bars: 50 μm. * Figure 5: NF-κB promotes metabolic adaptation in cancer in vivo. () Left, viability of CT-26 cells expressing non-specific (ns) or RelA shRNAs before and after glucose starvation (GS). Right, western blots showing RelA-knockdown efficiency. () Survival of the cells in after a 4-day treatment with glucose starvation, metformin (MET) or glucose starvation plus metformin. () Growth of CT-26 tumours expressing non-specific or RelA shRNAs in nude mice treated with metformin or PBS. () Images of representative tumours from . () qRT-PCR showing the relative RelA and Sco2 levels in tumours isolated from two representative metformin-treated mice at day 14. In – and the values denote mean±s.e.m. n=3 (,,); n=9 (); *P<0.05; **P<0.01. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Claudio Mauro & * Shi Chi Leow Affiliations * Section of Inflammation and Signal Transduction, Department of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK * Claudio Mauro, * Shi Chi Leow, * Anil K. Thotakura, * Laura Tornatore, * Marta Moretti & * Guido Franzoso * Laboratory of NF-κB Signaling, Proteos, Singapore 138673, Singapore * Shi Chi Leow & * Vinay Tergaonkar * Department of Medicine, Division of Pulmonary and Critical Care Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA * Elena Anso & * Navdeep S. Chandel * Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK * Sonia Rocha * Department of Experimental Medicine, Sapienza University, Rome 00161, Italy * Marta Moretti & * Enrico De Smaele * Department of Immunology, Moffitt Cancer Center, Tampa, Florida 33612, USA * Amer A. Beg Contributions C.M. first observed the glucose addiction exhibited by RelA-null cells. C.M. and S.C.L. carried out the further experimental characterization of this phenomenon and most of the analyses shown. E.A. and S.R. carried out the oxygen consumption assays. A.K.T. carried out the cell-cycle analysis and helped with in vivo studies. L.T. carried out the immunoblot analyses of apoptosis and autophagy and the in vitro metabolic analyses of CT-26 cells. E.D.S. and A.A.B. generated the early passage p53−/− and RelA−/− MEFs, respectively, as well as the early passage wild-type controls from littermates. G.F., C.M. and S.C.L. wrote the manuscript and conceived the experiments. N.S.C. and V.T. contributed to the design of some of the experiments and made substantial critical revision to the manuscript. C.M., E.A., A.K.T., L.T. and M.M. carried out the experiments during revision of the manuscript. All authors discussed and revised the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Guido Franzoso Author Details * Claudio Mauro Search for this author in: * NPG journals * PubMed * Google Scholar * Shi Chi Leow Search for this author in: * NPG journals * PubMed * Google Scholar * Elena Anso Search for this author in: * NPG journals * PubMed * Google Scholar * Sonia Rocha Search for this author in: * NPG journals * PubMed * Google Scholar * Anil K. Thotakura Search for this author in: * NPG journals * PubMed * Google Scholar * Laura Tornatore Search for this author in: * NPG journals * PubMed * Google Scholar * Marta Moretti Search for this author in: * NPG journals * PubMed * Google Scholar * Enrico De Smaele Search for this author in: * NPG journals * PubMed * Google Scholar * Amer A. Beg Search for this author in: * NPG journals * PubMed * Google Scholar * Vinay Tergaonkar Search for this author in: * NPG journals * PubMed * Google Scholar * Navdeep S. Chandel Search for this author in: * NPG journals * PubMed * Google Scholar * Guido Franzoso Contact Guido Franzoso Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1700K) Supplementary Information Excel files * Supplementary Table 1 (22K) Supplementary Information * Supplementary Table 2 (11K) Supplementary Information Additional data - Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway
- Nat Cell Biol 13(10):1280 (2011)
Nature Cell Biology | Corrigendum Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway * Brice Marcet * Benoît Chevalier * Guillaume Luxardi * Christelle Coraux * Laure-Emmanuelle Zaragosi * Marie Cibois * Karine Robbe-Sermesant * Thomas Jolly * Bruno Cardinaud * Chimène Moreilhon * Lisa Giovannini-Chami * Béatrice Nawrocki-Raby * Philippe Birembaut * Rainer Waldmann * Laurent Kodjabachian * Pascal BarbryJournal name:Nature Cell BiologyVolume: 13,Page:1280Year published:(2011)DOI:doi:10.1038/ncb2358Published online03 October 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, 693–699 (2011); published online 22 May 2011; corrected after print 14 September 2011 In the version of this Letter initially published online and in print, an article by Lizé et al. (Cell Cycle, 4579–4583; 2010), which reports that miR-449 microRNAs accumulate during mucociliary differentiation of human airway epithelia, was inadvertently omitted from the references list. On pages 1–2, the following text has replaced the previous text: "miR-449a, miR-449b and miR-449c (collectively named miR-449), constitute by far the most strongly induced microRNAs during epithelium differentiation in both species. Although representing less than 0.01% of all microRNA sequences in proliferating HAECs, miR-449 accounted for more than 8% of the microRNA reads in differentiated HAECs (Fig. 1a and Supplementary Fig. S1c,d; see also ref. 13)." The omitted reference has now been added to the reference list: 13. Lizé, M., Herr, C., Klimke, A., Bals, R. & Dobbelstein, M. MicroRNA 449a levels increase by several orders of magnitude during mucociliary differentiation of airway epithelia. Cell Cycle, 4579–4583 (2010). References 13–40 have been changed to 14–41, respectively. Additional data Author Details * Brice Marcet Search for this author in: * NPG journals * PubMed * Google Scholar * Benoît Chevalier Search for this author in: * NPG journals * PubMed * Google Scholar * Guillaume Luxardi Search for this author in: * NPG journals * PubMed * Google Scholar * Christelle Coraux Search for this author in: * NPG journals * PubMed * Google Scholar * Laure-Emmanuelle Zaragosi Search for this author in: * NPG journals * PubMed * Google Scholar * Marie Cibois Search for this author in: * NPG journals * PubMed * Google Scholar * Karine Robbe-Sermesant Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Jolly Search for this author in: * NPG journals * PubMed * Google Scholar * Bruno Cardinaud Search for this author in: * NPG journals * PubMed * Google Scholar * Chimène Moreilhon Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa Giovannini-Chami Search for this author in: * NPG journals * PubMed * Google Scholar * Béatrice Nawrocki-Raby Search for this author in: * NPG journals * PubMed * Google Scholar * Philippe Birembaut Search for this author in: * NPG journals * PubMed * Google Scholar * Rainer Waldmann Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent Kodjabachian Search for this author in: * NPG journals * PubMed * Google Scholar * Pascal Barbry Search for this author in: * NPG journals * PubMed * Google Scholar
No comments:
Post a Comment