Latest Articles Include:
- Scientific communication: Writing up
- Nat Cell Biol 13(11):1281 (2011)
Nature Cell Biology | Editorial Scientific communication: Writing up Journal name:Nature Cell BiologyVolume: 13,Page:1281Year published:(2011)DOI:doi:10.1038/ncb2375aPublished online02 November 2011 In the competitive world of scientific publishing, it is essential to communicate research findings in a clear and accessible manner. Scientists should develop the ability to write well-structured and compelling cover letters, manuscripts and rebuttal letters. Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Writing a manuscript involves compressing months or years of work, often performed by dozens of hands, into a single document. It can be a trying exercise, but concise and clear communication is important to effectively and efficiently share your data with the scientific community. Manuscripts submitted to Nature Cell Biology are first read by the primary handling editor and then discussed within the editorial team. We also pay close attention to the cover letter. The decision to send a manuscript for peer review is never a factor of the style or clarity of writing, but is instead determined by the advance provided for a broad cell biology audience in the context of the published literature (see also our previous editorial on peer review). However, a lucid and well-organized manuscript, accompanied by a coherent cover letter, can help convey the key elements of the research to the editors and external reviewers. A cover letter is the ideal place to communicate the novelty and potential interest of the dataset. Although the editor will assess the study's advance in light of the published literature, the author's perspective on how the findings fit within the context of the broader literature is also helpful. Therefore, a strong and cogent statement about how the study advances the field can enhance the overall presentation of a manuscript: over-interpreting or overhyping the data will not. The cover letter should also note whether related research, generated by the authors' own lab or by competitors, is under consideration at another journal. Finally, suggestions for potential referees or reviewers to exclude can also be listed. The manuscript text and figures should conform to the format guidelines in our 'Guide to Authors'. Before submission, authors might wish to solicit feedback from colleagues and collaborators, or enlist the services of a company that performs professional scientific editing (for example, MSC Scientific Editing), to ensure that the manuscript is clear and accessible, and that the data are interpreted in a balanced manner. Making figures can drive the non-computer-savvy — and even Adobe Illustrator savants — to the brink, but creating figures does not require advanced coursework in graphic design. Bang Wong, in his fascinating columns in Nature Methods, provides excellent suggestions for figure layout, colour combinations, and even more esoteric issues such as typography and the judicious use of arrows. In particular, in fairness to readers with colour blindness, we ask that red–green colour combinations be avoided when possible. Figures must be of sufficient resolution s! o that the editors, and potentially peer reviewers, can make sense of the data. If figure quality has suffered during online submission, higherresolution figures can be provided electronically or by post (as CDs/DVDs or glossy prints). We will ensure that the material is distributed to the referees. Data should not be manipulated or 'beautified' in a way that obscures or misrepresents the findings (see our previous editorial on data beautification and fraud and refer to our 'Guide to Authors' for current guidelines on image integrity). You've submitted your cover letter and manuscript, received encouraging reports from the referees, and made the appropriate revisions: now what? A rebuttal letter that provides a concise point-by-point response to referees' comments is essential. The rebuttal should outline precisely how the manuscript was revised to address the referees' concerns and guide them to the specific figure, text section or supplementary information item that was revised. Reviewing manuscripts requires considerable effort and time, and a coherent and detailed rebuttal letter can greatly assist referees in their task. Long-winded, opaque or vitriolic rebuttals carry the risk of antagonizing referees. For example, a letter that consolidates responses to similar issues raised by different referees reduces the clarity of the rebuttal, and makes efficient review of a revised manuscript that much more difficult. In some cases, we will ask an author to rewrite his or her rebuttal letter if it is deemed t! o be completely unsuitable. The accompanying cover letter, which is not shared with the referees, should provide an overview of the revisions and highlight any major changes in the overall conclusions. Once again, details of any related work under consideration elsewhere should be provided. After a manuscript is accepted for publication, the production editors ensure that the text and figures comply with journal style, but ultimately the responsibility for scientific communication rests with the authors. Writing clearly and concisely can help to convey the central aspects of a study and ensure that the results are accessible to specialists and non-specialists alike. Additional data - NCB tweets
- Nat Cell Biol 13(11):1281 (2011)
Nature Cell Biology | Editorial NCB tweets Journal name:Nature Cell BiologyVolume: 13,Page:1281Year published:(2011)DOI:doi:10.1038/ncb2375bPublished online02 November 2011 Nature Cell Biology editors highlight research and news relevant to cell and developmental biologists. Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg As this issue of the journal went to press, we started tweeting. We hope to draw attention to recent research published in this journal (such as new content published online, special projects and conference attendance by editors) as well as research from across Nature Publishing Group and beyond. In addition, we will also tweet news items of interest to our readers, including new developments in science policy and funding, issues affecting graduate students and postdocs, and other topics that are featured in our editorial pages. Follow us @naturecellbio and do join in the conversation. Additional data - Nuclear actin and myosins: Life without filaments
- Nat Cell Biol 13(11):1282-1288 (2011)
Nature Cell Biology | Review Nuclear actin and myosins: Life without filaments * Primal de Lanerolle1 * Leonid Serebryannyy1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1282–1288Year published:(2011)DOI:doi:10.1038/ncb2364Published online02 November 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 Actin and myosin are major components of the cell cytoskeleton, with structural and regulatory functions that affect many essential cellular processes. Although they were traditionally thought to function only in the cytoplasm, it is now well accepted that actin and multiple myosins are found in the nucleus. Increasing evidence on their functional roles has highlighted the importance of these proteins in the nuclear compartment. View full text Figures at a glance * Figure 1: The roles of actin in the nucleus. Monomeric G-actin can polymerize to form canonical actin filaments (F-actin) in the cytoplasm. Actin in complex with cofilin can enter the nucleus via nuclear pore complexes (NPC), while profilin–actin complexes are exported via exportin 6. Nuclear actin seems to be highly dynamic. It can interact with all three polymerases and engage in transcription, it can undergo post-translational modifications, such as SUMOylation, or it can form actin polymers (shown as groups of three or fewer monomers to differentiate from F-actin in the cytoplasm). Helical rod bundles consisting of actin and cofilin have been detected in the nucleus under various stressful conditions and in the disease intranuclear rod myopathy. The formation of actin rods or polymers and the recruitment of nuclear myosin I or myosin Va seem to be important in viral replication. LINC complexes, which bind to lamins inside the nucleus and to actin filaments in the cytoplasm, seem to be important in outside-in nucl! ear signalling. The cytoplasmic actin cytoskeleton can also influence nuclear activity. The transcription factors PREP2, YY1 and MAL bind to cytoplasmic actin and changes in actin polymerization can sequester or release these factors so that they can enter the nucleus to regulate transcription. Steroid receptors that translocate to the nucleus following binding of their activating ligands also interact with actin, actin binding proteins and myosins, and act as transcriptional co-activators of genes regulated by steroid hormones. * Figure 2: Myosin in the nucleus. Myosins are involved in many nuclear functions. NMI and actin are important for transcription by RNA polymerase II and I, through structural roles, motor functions or a combination of the two. Myosin VI is also implicated in transcription by RNA polymerase II (see also Fig. 3a). The motor activity of NMI and actin is also critical for positioning and organizing chromatin and the expression of oestrogen-receptor-activated genes. In the nucleolus, NMI and Myosin Vb are localized in a transcription-dependent manner to the dense fibrillar component, while actin is in fibrillar centres. Myosins are also part of nuclear scaffolding complexes containing lamins and emerin. The latter associates with actin, NMI, myosin II heavy chain, spectrin and others, thus providing a link to the nucleoskeleton and chromatin. Myosin XVIb may regulate the cell cycle by interacting with cyclin A and proliferating cell nuclear antigen (PCNA), whereas binding to protein phosphatase 1 (PP1) may contro! l its activity and its nuclear transport through the nuclear pore complex (NPC). Adult and embryonic myosin II have also been found in the nucleus, and have functions in regulation of gene expression and differentiation, respectively. Myosin XVIIIb has also been implicated in the transcription of genes required for differentiation. Myosin Va is found in speckles and is thought to be involved in RNA processing. * Figure 3: Actin and myosins in transcription. () Transcription by RNA polymerase II. Actin interacts with RNA polymerase II and is necessary to form the pre-initiation complex. The actin-binding protein WASP has been discovered in the nucleus and modulates transcription independently or with the ARP2/3 complex. Actin is needed to enhance the activity of several chromatin remodelling proteins and the elongation machinery, including BAF, PCAF, Cdk9, P300 and P-TEFB, following phosphorylation (P) of the C-terminal domain of the polymerase. Actin also induces the activity of hnRNP U, a post-transcriptional modifier of mRNA. Nuclear myosin I (NMI) is needed to create the first phosphodiester bond in transcription initiation. Myosin VI (MVI), a backwards motor, has also been implicated in transcription, posing the intriguing possibility that NMI and MVI have counteracting motor functions (represented by white arrows). Myosin Va has been discovered in nuclear speckles, suggesting a role in splicing. () Transcription by RNA pol! ymerase I. Actin also binds to polymerase I and a polymerized form of actin is suggested to be necessary for rRNA transcription. NMI is recruited by phosphorylated (P) TIF-1A and the binding of the phospho-TIF-1A–NMI complex results in an initiation-competent form of polymerase I. NMI also has a role in chromatin remodelling by the WSTF–SNF2h complex and rRNA maturation. Actin is proposed to act in concert with NMI, either as a structural complex or a supplemental motor in transcription. Myosin Vb has also been implicated as a regulator of polymerase I transcription. Author information * Abstract * Author information Affiliations * Primal de Lanerolle and Leonid Serebryannyy are in the Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Primal de Lanerolle Author Details * Primal de Lanerolle Contact Primal de Lanerolle Search for this author in: * NPG journals * PubMed * Google Scholar * Leonid Serebryannyy Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Misfolded proteins driven to destruction by Hul5
- Nat Cell Biol 13(11):1290-1292 (2011)
Article preview View full access options Nature Cell Biology | News and Views Misfolded proteins driven to destruction by Hul5 * Daniel Finley1Journal name:Nature Cell BiologyVolume: 13,Pages:1290–1292Year published:(2011)DOI:doi:10.1038/ncb2371Published online09 October 2011 Misfolded proteins are potentially toxic and are therefore subjected to highly selective degradation by the ubiquitin–proteasome system. The identification of the Hul5 ubiquitin ligase as a major mediator of such 'quality-control' ubiquitylation following heat shock raises new questions about the design of these pathways. 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 * Daniel Finley is at the Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Daniel Finley Author Details * Daniel Finley Contact Daniel Finley Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - SHARPINing integrin inhibition
- Nat Cell Biol 13(11):1292-1293 (2011)
Article preview View full access options Nature Cell Biology | News and Views SHARPINing integrin inhibition * Mark D. Bass1Journal name:Nature Cell BiologyVolume: 13,Pages:1292–1293Year published:(2011)DOI:doi:10.1038/ncb2368Published online02 November 2011 The activity state of integrins is crucial for cell adhesion, migration and differentiation, and is regulated predominantly by protein interactions of the integrin β cytoplasmic domain. SHARPIN is now shown to negatively regulate integrin activation by binding the α-integrin subunit and interfering with the association of the β cytodomain with activating proteins. 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 * Mark D. Bass is at the School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Mark D. Bass Author Details * Mark D. Bass Contact Mark D. Bass Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Gene bookmarking accelerates the kinetics of post-mitotic transcriptional re-activation
- Nat Cell Biol 13(11):1295-1304 (2011)
Nature Cell Biology | Article Gene bookmarking accelerates the kinetics of post-mitotic transcriptional re-activation * Rui Zhao1, 2 * Tetsuya Nakamura3, 5 * Yu Fu1, 4, 5 * Zsolt Lazar1 * David L. Spector1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1295–1304Year published:(2011)DOI:doi:10.1038/ncb2341Received07 March 2011Accepted10 August 2011Published online09 October 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 Although transmission of the gene expression program from mother to daughter cells has been suggested to be mediated by gene bookmarking, the precise mechanism by which bookmarking mediates post-mitotic transcriptional re-activation has been unclear. Here, we used a real-time gene expression system to quantitatively demonstrate that transcriptional activation of the same genetic locus occurs with a significantly more rapid kinetics in post-mitotic cells versus interphase cells. RNA polymerase II large subunit (Pol II) and bromodomain protein 4 (BRD4) were recruited to the locus in a different sequential order on interphase initiation versus post-mitotic re-activation resulting from the recognition by BRD4 of increased levels of histone H4 Lys 5 acetylation (H4K5ac) on the previously activated locus. BRD4 accelerated the dynamics of messenger RNA synthesis by de-compacting chromatin and hence facilitating transcriptional re-activation. Using a real-time quantitative approach,! we identified differences in the kinetics of transcriptional activation between interphase and post-mitotic cells that are mediated by a chromatin-based epigenetic mechanism. View full text Figures at a glance * Figure 1: Transcriptional activation of the same locus exhibits faster kinetics during post-mitotic activation than in the previous interphase. () Schematic diagram of the gene expression system. Binding of LacI-fluorescent protein to the lac operator repeats results in visualization of the gene locus. pTet-On expression in the presence of doxycycline (Dox) induces gene expression driven by the minimal CMV promoter. MS2-fluorescent protein binds to the MS2 stem loop repeats, rendering visualization of the transcribed mRNA. Two-hundred copies of the gene expression cassette are stably integrated as a transgene array at human 1p36 in U2OS-2-6-3 cells. The diagram is not drawn to scale. () Interphase induction (left). Following doxycycline induction, mCherry–Pol II and MS2–YFP were recruited to the locus (right, arrowheads). Image stacks were acquired every 3 min. 0' indicates the first time point where Pol II is detectable at the locus. Scale bars, 10 μm (main panel) and 2 μm (inset). () Post-mitotic re-activation (left). Cells were induced with doxycycline overnight before imaging. Cells with an active ! locus were followed (right). Both Pol II and MS2–YFP dissociate from the locus on entry into mitosis. On exit from mitosis both fusion proteins were recruited to the daughter loci (white arrowheads indicate the presence of signal; yellow arrowheads indicate an absence of signal). Images were taken every 2 min. Scale bars, 10 μm (main panel) and 2 μm (inset). () Quantitative analysis showed that Pol II recruitment was 13 times more rapid during post-mitotic re-activation, compared with interphase induction. The Pol II signal at the locus was quantified across the imaging session for each cell and the average curve was generated (left) as described in the Methods. The rising time (right) of the Pol II signal for interphase induction and post-mitotic re-activation was plotted as mean±s.e.m. (208.1±22.9 min versus 16.5±6.0 min; n=17 for interphase induction, n=19 for post-mitotic re-activation.) () mRNA production was 5 times more rapid during post-mitotic re-a! ctivation, as shown by quantitative analysis of mRNA productio! n at the locus during interphase induction and post-mitotic re-activation. The MS2–YFP signal was quantified across the imaging session for each cell and the average curve was generated (left) as described in the Methods. The rising time (right) of the MS2–YFP signal for interphase induction and post-mitotic re-activation was plotted as mean±s.e.m. (157.6±19.0 min versus 34.3±7.6 min; n=13 for interphase induction, n=25 for post-mitotic re-activation). * Figure 2: Global chromatin decondensation or a second interphase induction is not sufficient to provide a more rapid transcriptional induction. () Schematic diagram of induction during mitosis. Cells in which the locus was inactive were synchronized with nocodazole (Noco; 50 μg ml−1) and, after wash-out, doxycycline (Dox) was added and cells were allowed to progress through the cell cycle. Such cells will be referred to as mitosis-induced cells. () Rapid chromatin decondensation on exit from mitosis, in and of itself, is not sufficient for rapid transcriptional induction. The average curve of Pol II was generated (left) as previously described. The rising time (right) of the Pol II signal for interphase induction (taken from Fig. 1d, for ease of comparison) and post-mitotic induction was plotted as mean±s.e.m. (208.1±22.9 min versus 168±21.8 min; n=17 for interphase induction, n=7 for post-mitotic induction). () The average curve of MS2–YFP was generated (left) as previously described. The rising time (right) of the MS2–YFP signal for interphase induction (taken from Fig. 1e, for ease of comparison! ) and post-mitotic induction was plotted as mean±s.e.m. (157.6±19.0 min versus 185.2±45.5 min; n=13 for interphase induction, n=10 for post-mitotic induction). () Diagram of the experimental protocol for studying cells that have been transcriptionally induced in interphase twice. () Silencing of an active locus during interphase followed by a second transcriptional induction does not result in increased kinetics of transcriptional induction. The average curve of Pol II was generated (left) as previously described. The rising time (right) of the Pol II signal for initial (taken from Fig. 1d, for ease of comparison) and second interphase induction was plotted as mean±s.e.m. (208.1±22.9 min versus 227±41 min; n=17 for interphase induction, n=9 for interphase second induction). () The average curve of MS2–YFP was generated (left) as previously described. The rising time (right) of the MS2–YFP signal for initial (taken from Fig. 1e, for ease of comparison) and ! second interphase induction was plotted as mean±s.e.m. (157.6! ±19.0 min versus 169.2±49.6 min; n=13 for interphase induction, n=9 for interphase second induction). * Figure 3: H4K5ac is a bookmark for active transcription in interphase and is maintained during mitosis. () Schematic diagram of primer sets used for ChIP experiments. () H4K5ac showed the largest increase on the promoter region after interphase transcriptional activation, whereas the other active histone modifications (H3K4me3, H3K36me3, H4K8ac, H4K12ac and H4K16ac) did not show as large an increase. Cells stably expressing pTet-On were treated with or without doxycycline (Dox; 1 μg ml−1) for 24 h before being collected for ChIP experiments. Results were collected from three biologically independent experiments (mean±s.e.m.). () Increased H4K5ac after interphase transcriptional activation was preserved in mitotic cells. U2OS-2-6-3 cells, stably expressing pTet-On, were induced by doxycycline (Dox; 1 μg ml−1) for 24 h, followed by treatment with nocodazole (Noco; 50 μg ml−1) in the presence of doxycycline for another 16 h for synchronization, before being collected by mechanical shake-off for ChIP experiments. Results were collected from three biolog! ically independent experiments (mean±s.e.m.). () Association of acetylated H4 (TH4ac) at the locus during mitosis after doxycycline induction. Cells induced with doxycycline (1 μg ml−1, 24 h) were fixed and immunolabelled with anti-H4ac antibody (green). Association of TH4ac with the locus (enlarged, +Dox) can be detected in mitotic cells. Scale bars, 10 μm (main panels) and 2 μm (insets). * Figure 4: BRD4 regulates efficient post-mitotic re-activation of transcription. () BRD4 and Pol II were recruited to the same genetic locus, with different sequential dynamics on interphase induction versus post-mitotic re-activation (the daughter locus of the cell on the right is enlarged; white arrowheads indicate the presence of signal; yellow arrowheads indicate an absence of signal). Scale bar, 10 μm. () Pol II was recruited before BRD4 on interphase induction (n=7), whereas BRD4 was recruited before Pol II during post-mitotic re-activation (n=7; mean±s.e.m.). () siRNA knockdown of BRD4 delayed post-mitotic transcriptional re-activation. Arrowheads indicate mRNA production revealed by MS2–YFP signal. Scale bar, 10 μm. () BRD4 regulates efficient post-mitotic transcriptional re-activation (left), as shown by quantitative analysis of the rising time (right) of mRNA production at the locus in post-mitotic cells treated with either BRD4 siRNA 1 (258.75±38.0 min; n=5), BRD4 siRNA 2 (249.17±40.0 min; n=6) or a control siRNA (133.3±18.7 ! min; n=6; mean±s.e.m.). () Diagram of JQ1 experiments. Dox, doxycycline. () JQ1 treatment significantly slowed down the post-mitotic transcriptional activation (left), as shown by quantitative analysis of the rising time (right) of mRNA production at the locus in post-mitotic cells treated with either dimethylsulphoxide (DMSO) control (41±6.2 min; n=5) or JQ1 (100.91±8.1 min; n=11). The no-treatment control from Fig. 1e is re-plotted, for ease of comparison. NS, not significant. () Cells were transiently transfected with LacI–mCherry and were treated as indicated with Dox or Dox+JQ1 in interphase. Cells were fixed and immunolabelled for BRD4 and metaphase cells were examined. Loci are indicated by LacI–mCherry (arrowheads). DNA was also stained. Scale bars, 10 μm (main panels) and 2 μm (insets). () Loci are indicated by LacI–mCherry, and the area of the loci was determined (n=27, 40, 34 for −Dox, +Dox and +JQ1 groups, respectively). * Figure 5: BRD4 facilitates post-mitotic transcriptional re-activation through chromatin de-compaction. () Locus (arrowheads) size change under different treatments. The locus is compacted in the absence of doxycycline (Dox), and there was no CFP protein production (left). Following doxycycline addition the locus was de-compacted, and there was CFP protein production (middle). The locus was even more de-compacted with LacI–mCherry–BRD4 tethering in the absence of doxycycline, but no CFP was produced (right). (n=24, 27 and 36 for LacI−Dox, LacI+Dox and LacI–BRD4-Dox respectively.) Scale bar, 10 μm. () Tethering BRD4 protein, by fusing BRD4 with LacI–mCherry, to the locus in the absence of doxycycline de-compacted the locus without the recruitment of Pol II or mRNA production (arrowheads), in cells stably expressing YFP–Pol II or MS2–YFP. BRD4 tethering did not result in a significant increase of histone acetylation on the locus, nor did it result in decreased association of heterochromatin marker HP1α. Cells were transiently transfected with LacI–mCherry–B! RD4 and were fixed at interphase. Cells were immunostained for TH4 or HP1α (scale bar, 10 μm). () Tethering BRD4 protein to the locus significantly accelerated Pol II recruitment during interphase induction (75.8±10.6 min) when compared with control interphase induction (taken from Fig. 1d, for ease of comparison) without BRD4 tethering (208.1±22.9 min). However, Pol II kinetics was still significantly slower than that in post-mitotic re-activation. Post-mitosis re-plotted from Fig. 1d for ease of comparison (16.5±6.0 min; mean±s.e.m.). (n=17 for interphase induction, n=13for LacI–BRD4 interphase induction, n=19 for post-mitotic re-activation.) () Tethering BRD4 protein to the locus significantly accelerated mRNA synthesis during interphase induction (71.5±13.2 min) when compared with control interphase induction (re-plotted from Fig. 1e for ease of comparison) without BRD4 tethering (157.6±19.0 min). Tethering BRD4 results in kinetics that is closer t! o that observed in post-mitotic re-activation (34.3±7.6 min! ). Post-mitosis re-plotted from Fig. 1e for ease of comparison. (mean±s.e.m.; n=13 for interphase induction, n=17for LacI–BRD4 interphase induction, n=25 for post-mitotic re-activation.) * Figure 6: Molecular mapping of BRD4 function. () Diagram of the protein domains contained in different BRD4 deletion mutants. BD, bromodomain; ET, extra-terminal50; SEED, Ser/Glu/Asp-rich region51; CTD, carboxy-terminal domain50. () BD1, but not BD2, is critical for the de-compaction function of BRD4. Cells were transiently transfected with indicated LacI–mCherry-fused BRD4 mutants and localized (arrowheads). Among these mutants, 72% of the cells transfected with BRD4-722 (n=106 cells), 80% of those transfected with BRD4-Δ2 (n=108 cells) and 79% of those transfected with BRD4-NBD1 (n=104 cells) demonstrated de-compacted loci; no de-compacted locus could be identified in the cells transfected with the constructs indicated (−) in (>100 cells per construct). Tethering LacI–BRD4-NBD1 in the presence of JQ1 still resulted in de-compact loci (v, +JQ1) compared with control (v, −JQ1). Cells were transiently transfected with LacI–mCherry-fused BRD4-NBD1 and were treated with JQ1 for 6 h. () The minimum de-compactio! n mutant NBD1 was sufficient to accelerate interphase transcriptional induction. Analysis of the rising time of mRNA production in doxycycline-induced interphase cells expressing either LacI–mCherry–BRD4-Δ1 (141.25±19.4 min; n=12) or LacI–mCherry–NBD1 (85.77±4.7 min; n=13). () A model of transcriptional induction in interphase, and transcriptional re-activation in post-mitotic cells. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Tetsuya Nakamura & * Yu Fu Affiliations * Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA * Rui Zhao, * Yu Fu, * Zsolt Lazar & * David L. Spector * Graduate Program in Molecular and Cellular Biology, Stony Brook University, Stony Brook, New York 11794, USA * Rui Zhao * Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8519, Japan * Tetsuya Nakamura * Graduate Program in Neuroscience, Stony Brook University, Stony Brook, New York 11794, USA * Yu Fu Contributions R.Z., T.N. and D.L.S. designed the research; R.Z. and T.N. carried out the experiments and analysed data; Y.F. wrote the MatLab software for quantitative analysis and also analysed data; Z.L. carried out the super-resolution structured illumination microscopy and locus size analysis. R.Z. and D.L.S. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David L. Spector Author Details * Rui Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Tetsuya Nakamura Search for this author in: * NPG journals * PubMed * Google Scholar * Yu Fu Search for this author in: * NPG journals * PubMed * Google Scholar * Zsolt Lazar Search for this author in: * NPG journals * PubMed * Google Scholar * David L. Spector Contact David L. Spector Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2200K) Supplementary Information Movies * Supplementary Movie 1 (400K) Supplementary Information * Supplementary Movie 2 (2500K) Supplementary Information Additional data - BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol
- Nat Cell Biol 13(11):1305-1314 (2011)
Nature Cell Biology | Article BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol * Roger Geiger1 * Daniel Andritschke1 * Sarah Friebe1 * Fabian Herzog1 * Stefania Luisoni1 * Thomas Heger1 * Ari Helenius1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1305–1314Year published:(2011)DOI:doi:10.1038/ncb2339Received07 June 2011Accepted05 August 2011Published online25 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 How non-enveloped viruses overcome host cell membranes is poorly understood. Here, we show that after endocytosis and transport to the endoplasmic reticulum (ER), but before crossing the ER membrane to the cytosol, incoming simian virus 40 particles are structurally remodelled leading to exposure of the amino-terminal sequence of the minor viral protein VP2. These hydrophobic sequences anchor the virus to membranes. A negatively charged residue, Glu 17, in the α-helical, membrane-embedded peptide is essential for infection, most likely by introducing an 'irregularity' recognized by the ER-associated degradation (ERAD) system for membrane proteins. Using a siRNA-mediated screen, the lumenal chaperone BiP and the ER-membrane protein BAP31 (both involved in ERAD) were identified as being essential for infection. They co-localized with the virus in discrete foci and promoted its ER-to-cytosol dislocation. Virus-like particles devoid of VP2 failed to cross the membrane. The ! results demonstrated that ERAD-factors assist virus transport across the ER membrane. View full text Figures at a glance * Figure 1: SV40 undergoes a structural change in the ER. (–) Electron micrographs of CV-1 cells infected with SV40 for 2 h (), 6 h () and 19 h (). () Virions bind tightly to endosomal (arrows) or vesicular membranes (double arrowheads). () Virions in the ER appear smaller and more compact (red arrows), and exhibit electron-dense protrusions (yellow arrow), compared with virions bound to vesicular membranes (double arrowheads). () In the ER, the surface of the virus is not tightly associated with the membrane, and the membrane appears to form flattened surfaces around the virions (arrows). Scale bars, 100 nm. () Quantification of the diameter of virions in vesicles, endosomes and in the ER. n=50. Error bars represent the s.e.m. (,) Electron micrographs of SV40 treated with buffer () or with ER lumenal extract (). Scale bars, 100 nm. () Schematic representation of the iodixanol gradient used for liposome floating. () SV40 virions or VLPs (devoid of VP2 and VP3) were incubated with buffer or ER lumenal extract followed by! the addition of liposomes. Samples were overlaid with a step gradient followed by ultracentrifugation to separate membrane-bound virions (top fraction) from unbound virions (bottom). Fractions were precipitated with trichloroacetic acid and subjected to SDS–PAGE and western blotting with VP1 and VP3 antibodies. () Virions were treated as indicated and processed as in . Instead of precipitation with trichloroacetic acid, top fractions were subjected to phenol–chloroform extraction and the DNA content was determined by quantitative rtPCR. The DNA concentration measured in the top fraction after treatment of SV40 with buffer was set to 1. All other data points are relative to it. n=12 (for buffer and lumenal extract, LE), or n=6 (for heat-inactivated LE). Error bars represent the s.e.m. () Antibodies against the N- or C-terminal peptides of VP2 were added to the lumenal extract. N=12, error bars represent the s.e.m. Uncropped images of blots are shown in Supplementary Fig! . S7. * Figure 2: The N terminus of VP2 folds into an α-helix, integrates into the ER membrane and contains an essential residue, Glu 17. () Amino-acid sequence of VP2. Transmembrane segments as predicted by TMPred http://www.ch.embnet.org/software/TMPRED_form.html are in bold. The myristyl group at G2 is illustrated. Hydrophilic residues in the N-terminal peptide are coloured (red, potentially charged; green, polar). Green rectangles indicate the peptides used to raise VP2 antibodies used in Fig. 1j. () Helical wheel representation of the amphipathic N terminus of VP2 (residues 1–18) as generated at http://heliquest.ipmc.cnrs.fr. () CV-1 cells expressing the indicated constructs were homogenized and a microsomal fraction was prepared (pellet). Supernatants and pellets were analysed by SDS–PAGE followed by immunoblot analysis with antibodies against GFP. () Pellets of VP2(1–31)–GFP-expressing cells were treated as indicated for 20 min and subsequently centrifuged. Supernatants and pellets were analysed by immunoblot using antibodies against GFP, calnexin and p97. () CV-1 cells were transfected with w! ild-type or mutated NO-SV40 plasmids. Supernatants were collected 14 days post-transfection and 1 μl was used to infect 100,000 cells. The percentage of infected cells, measured by FACS analysis, is indicated. Bars represent the mean±s.e.m., n=6. () Reaction scheme. BG-labelled SV40 covalently reacts with SNAP. () CV-1 cells expressing ER-SNAP were inoculated with BG-labelled SV40. After 24 h, cells were lysed and analysed by SDS–PAGE followed by immunoblot analysis using antibodies against the Myc epitope of SNAP. Where indicated, cells were treated with 10 μg ml−1 BFA or 20 μM MG132. () ER-SNAP-expressing cells were inoculated with BG-labelled VLPs or BG–SV40 and treated as in . () Cytosol-SNAP-expressing cells were inoculated with BG-labelled VLPs or BG–SV40 and treated as in . Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 3: SV40 infection depends on BAP31, BAP29 and RMA1. (–) Top, HeLa cells (,–) and CV-1 cells () were transfected with 5 nM (,–) and 20 nM () of the indicated siRNAs. After 72h, cells were infected with SV40 for 24 h. After fixation, cells were imaged (,–) or analysed by FACS () to determine the numbers of infected cells (T-ag positive). The histogram shows infection indices relative to control siRNAs. Bars represent the mean±s.e.m., N=3, throughout. Bottom, HeLa cells (,–) or CV1 cells () were transfected with the indicated siRNAs as described above and lysed after 72 h. Cell lysate (20 μg) was analysed by SDS–PAGE and western blot analysis using antibodies against the target protein and a control protein (tubulin or actin). () The siRNA RMA1_A1 presumably induces an off-target effect, because infection is blocked but RMA1 expression was not affected. RMA1_A3 and RMA1_7 inhibit infection proportional to the RMA1 knockdown. () Owing to inefficient detection of endogenous HRD1 using HRD1 antibodies, cells! were additionally transfected with a HRD1–Myc plasmid and the ectopic protein was detected by a Myc antibody. () The siRNA p97_1 presumably induces an off-target effect, because two other siRNAs silence p97 expression without significantly affecting infection. () The asterisk indicates an unspecific band. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 4: Transport of SV40 to the ER is not affected following knockdown of BAP31. () Top, HeLa cells were transfected with 5 nM of the indicated siRNAs targeting the UTR of BAP31. After 72 h, cells were infected with SV40 for 24 h, fixed, stained and analysed by FACS. The histogram shows infection indices relative to control siRNAs. Bars represent the mean±s.e.m., n=3. Bottom, 20 μg of siRNA-transfected HeLa cell lysate was analysed by SDS–PAGE and western blot analysis using antibodies against BAP31 and tubulin. () HeLa cells were transfected with 5 nM control (All Star Negative) or UTR-BAP31_2 siRNA. After 72 h, cells were additionally transfected with either an EGFP or BAP31–Flag plasmid. After 24 h, cells were infected with SV40, and after a further 24 h cells were fixed, stained for viral T-ag and BAP31 expression and analysed by FACS. Bars represent the mean of infection indices ± s.e.m. relative to control siRNAs and plasmid transfection, n=4. () HeLa cells expressing BAP31–Flag were infected with SV40. Infection is inhib! ited with increasing overexpression of BAP31–Flag. Bars represent the mean±s.e.m., n=6. () CV-1 cells were transfected with 20 nM control or BAP31_8 siRNA and processed as in . BAP31_8 targets the coding region of BAP31. The expression of BAP31–Flag was therefore lower than in HeLa cells (not shown). Bars represent the mean±s.e.m., n=8. () CV-1 cells were transfected with 20 nM control (All Star Negative) or BAP31_8 siRNA for 72 h and then infected with purified virus for 2 or 8 h, lysed, separated by non-reducing SDS–PAGE and analysed by western blot analysis using VP1 and VP3 antibodies. Samples were additionally analysed by reducing SDS–PAGE and western blot analysis using tubulin and BAP31 antibodies. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 5: BAP31, BAP29 and SV40 accumulate in discrete spots in the ER. () CV-1 cells were fixed, stained with BAP31 and calnexin antibodies and analysed by confocal immunofluorescence microscopy. () Calnexin and BAP31 are located in different microdomains in the ER. Shown is a higher-magnification image of the area outlined in . The inset shows an area (outlined) where four tubules containing calnexin merge. The branch point is devoid of calnexin but contains BAP31. () CV-1 cells were infected with purified SV40 at a MOI of 50. At 18 h post-infection, cells were analysed as in . () CV-1 cells were infected with Alexa-Fluor-488-labelled SV40 (MOI=5). After 18 h, cells were fixed, stained with BAP31 antibodies and imaged. () BAP29–Flag-expressing CV-1 cells were infected with 488–SV40, fixed and stained with antibodies against Flag and BAP31. () 488–SV40 infected cells were fixed and stained with antibodies against calnexin and BAP31. Scale bars, 10 μm. () CV-1 cells were infected with SV40, VLPs or empty SV40 particles. After 18 h! , cells were fixed, stained with antibodies against BAP31 and imaged. Number of cells with BAP31-containing foci as in was determined. Bars represent the mean±s.e.m., n=3 (≥100 cells were analysed per experiment). * Figure 6: BAP31 interacts with the N-terminal peptide of VP2. () Predicted topology of BAP31. Residues mutated in and are indicated. (,) BAP31 depletion/add-back assay in HeLa cells followed by SV40 infection as in Fig. 4b. In addition, BAP31–Flag mutants were analysed. The infection of BAP31–Flag-expressing cells was set to 1. Infection indices are relative to wild-type BAP31–Flag throughout. Bars represent the mean±s.e.m., n=3 () or n=4 (). () BAP31 depletion/add-back assay in HeLa cells as in ,. Cells were infected with wild-type, VP2E17K or VP2E17R virus. Bars represent the mean±s.e.m., n=6. () BAP31 depletion/add-back assay in 3T6 cells with mPy. Bars represent the mean±s.e.m., n=6. () 3T6 cells were transfected with 20 nM mBAP31_3 siRNA. After 72 h, cells were lysed and 20 μg cell lysate was analysed by SDS–PAGE and western blot analysis using antibodies against BAP31 and tubulin. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 7: BiP is a critical factor in SV40 infection. (–) Top, HeLa cells (,,) and CV-1 cells () were transfected with 5 nM (,,) and 20 nM () of the indicated siRNAs. After 72 h, cells were infected with SV40 for 24 h. After fixation, cells were imaged (,,) or analysed by FACS () to determine the numbers of infected cells (T-ag positive). The histogram shows infection indices relative to control siRNAs. Bars represent the mean±s.e.m., n=3, throughout. Bottom, HeLa cells (,,) or CV1 cells () were transfected with the indicated siRNAs as described above and lysed after 72 h. Cell lysate (20 μg) was analysed by SDS–PAGE and western blot analysis using antibodies against the target protein and a control protein (tubulin or actin). () GRP94 is a weak hit because GRP94_5 siRNA decreases protein expression but barely affects infection. Geldanamycin, an inhibitor of GRP94, decreases infection indices by 25% to a level of 0.75 (ref. 6). (,) Cells were infected with 488–SV40 for 18 h, fixed and stained with antibodi! es against BAP31, and against BiP or ERp57. Scale bars, 10 μm. () CV-1 cells were treated for 72 h with 20 nM control siRNA or siRNA against BiP. Then, cells were infected with SV40 (MOI=50) and analysed as in Fig. 5g. Bars represent the mean±s.e.m., n=3 (≥100 cells were analysed per experiment). () CV-1 cells were treated for 72 h with 20 nM control siRNA or siRNA against BiP. Then cells were infected with purified SV40 for 2 h or 8 h, lysed, separated by non-reducing SDS–PAGE and analysed by western blot analysis using VP1 and VP3 antibodies. Samples were also analysed by reducing SDS–PAGE and western blot analysis using tubulin and BiP antibodies. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 8: BAP31 and BiP are essential for the transport of SV40 to the cytosol. (,) CV-1 cells were treated with indicated siRNAs, and transfected with ER-SNAP () or Cytosol-SNAP () plasmids. After 24 h, cells were exposed to BG–SV40 for 24 h. Cells were lysed, and analysed as in Fig. 2h. () CV-1 cells were treated for 10 min on ice with the indicated amounts of digitonin followed by centrifugation. Supernatants and pellets were analysed by SDS–PAGE and immunoblot analysis. When cells were treated with 40 μg ml−1 digitonin, the supernatant ('cytosolic fraction') contained cytosolic marker proteins but not ER markers (red rectangle). These conditions were used in ,. () CV-1 cells were mock-treated or infected with SV40 (MOI=50). In addition, cells were also infected in the presence of MG132 or BFA. After 8 h, cells were treated as in . Supernatants ('cytosolic fraction') and pellets were immuno-analysed using antibodies against VP1. () CV-1 cells were treated for 72 h with 20 nM of the indicated siRNAs. Then cells were infe! cted with SV40 (MOI=50). After 8 h, cells were treated as in . In addition, pellet fractions were immuno-analysed with antibodies against BAP31 and BiP to verify the siRNA-mediated knockdown. (,) The same as in , but cytosolic fractions were phenol–chloroform extracted and SV40 DNA was quantified by rtPCR. Data points are relative to negative controls (no virus). Error bars represent the s.e.m., N=3. () CV-1 cells were treated with 20 μM MG132 and infected with Alexa-488-labelled SV40. At 18 h post-inoculation, cells were fixed, stained with antibodies against BAP31 and calnexin and analysed by confocal microscopy. Scale bar, 10 μm. () A model for the changes that occur to an SV40 particle and its transfer through the ER membrane. See Discussion for description of steps involved. Uncropped images of blots are shown in Supplementary Fig. S8. Author information * Abstract * Author information * Supplementary information Affiliations * Institute of Biochemistry, ETH Zurich, Schafmattstrasse 18, CH-8093 Zurich, Switzerland * Roger Geiger, * Daniel Andritschke, * Sarah Friebe, * Fabian Herzog, * Stefania Luisoni, * Thomas Heger & * Ari Helenius Contributions R.G. designed and carried out experiments and analysed the data. D.A., S.F., F.H. and S.L. carried out experiments. T.H. wrote the MATLAB program. R.G. and A.H. wrote the manuscript. A.H. supervised the work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ari Helenius Author Details * Roger Geiger Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Andritschke Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah Friebe Search for this author in: * NPG journals * PubMed * Google Scholar * Fabian Herzog Search for this author in: * NPG journals * PubMed * Google Scholar * Stefania Luisoni Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Heger Search for this author in: * NPG journals * PubMed * Google Scholar * Ari Helenius Contact Ari Helenius Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (1800K) Supplementary Information Excel files * Supplementary Table 1 (100K) Supplementary Information * Supplementary Table 2 (39K) Supplementary Information * Supplementary Table 3 (30K) Supplementary Information Additional data - SHARPIN is an endogenous inhibitor of β1-integrin activation
- Nat Cell Biol 13(11):1315-1324 (2011)
Nature Cell Biology | Article SHARPIN is an endogenous inhibitor of β1-integrin activation * Juha K. Rantala1, 9 * Jeroen Pouwels1, 2, 9 * Teijo Pellinen1, 2 * Stefan Veltel1, 2 * Petra Laasola1 * Elina Mattila1, 2 * Christopher S. Potter3 * Ted Duffy3 * John P. Sundberg3 * Olli Kallioniemi1, 4 * Janet A. Askari5 * Martin J. Humphries5 * Maddy Parsons6 * Marko Salmi7 * Johanna Ivaska1, 2, 8 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1315–1324Year published:(2011)DOI:doi:10.1038/ncb2340Received18 May 2011Accepted09 August 2011Published online25 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 Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of β1-integrins in an RNAi screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations. View full text Figures at a glance * Figure 1: SHARPIN is an inhibitor of β1-integrin activity. () siRNA screen for endogenous integrin inhibitors in prostate cancer cells (PC3) using a cell spot microarray technique. Shown are representative images of array spots stained as indicated (scale bar 0.5 mm). On the right, control and SHARPIN siRNA positions are shown at higher magnification. () Z-score plot for active integrin labelling (12G10 normalized against DNA label 4,6-diamidino-2-phenylindole, DAPI) of the 44 highest-scoring siRNAs. Red indicates siRNAs for those genes in which both individual siRNAs significantly increased integrin activity. () PC3 cells were transfected as indicated and analysed with western blotting (uncropped blots are shown in Supplementary Fig. S5). () ScanR microscopy analysis of PC3 cells transfected as in and stained with two β1-integrin active epitope antibodies (9EG7 and 12G10) and one total β1-integrin antibody (K20; shown in Supplementary Fig. S2a). Shown are mean fluorescence intensities (MFIs) of 12G10 and 9EG7 relative to K20 (n! =3). Value 1.0 is assigned to control-siRNA-treated cells. () FACS analysis of PC3 cells transfected as indicated and stained for surface levels of active (9EG7 and 12G10) or inactive (Mab13 and 4B4) β1-integrins. Shown are representative histograms and the MFI. The MFI in the control transfected cells is set to 1.0 for each antibody, n=4. () FACS analysis of 9EG7 labelling from PC3 cells double transfected with siRNAs and plasmids expressing siRNA-resistant GFP–SHARPIN or GFP alone. Shown are the MFIs, n=3. () The binding of fibronectin (FN7–10 fragment) to control and SHARPIN-silenced cells was analysed using FACS. The staining intensities were normalized against total β1-integrin levels (n=3). () The adhesion of PC3 cells, transfected as indicated, to fibronectin for the indicated times was scored using propidium iodide stain (n=3). () Control and SHARPIN siRNA1 transfected PC3 cells adhering to the indicated concentrations of collagen I were scratch-wounded and an! alysed using time-lapse microscopy for 15 h. Shown are repre! sentative cell-tracks and quantitation of the migration speed (n=100 cells). () Migration on plastic of PC3 cells transfected with plasmids expressing GFP or GFP–SHARPIN, n=174 cells. All numerical data are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001, n.s., not significant. * Figure 2: SHARPIN interacts with the conserved membrane proximal segment of integrin α-tails. () Alignment of several C-terminal cytoplasmic domains of α-integrins. The conserved membrane-proximal (cons) and the C-terminal (cterm) peptides of α2-integrin used in this study are also indicated. (–) Streptavidin-bead pulldown assays with the indicated biotinylated integrin cytoplasmic-tail peptides or beads alone (no peptide) from MYC–SHARPIN transfected and non-transfected PC3 cell extracts () or with recombinant GST–SHARPIN and recombinant talin 1–400 fragment (). () Fluorescence polarization-based titration of GST–SHARPIN binding to the integrin peptides. Representative binding curves to wild-type (WT) and alanine-mutated α2-tails and the dissociation constant (Kd) values (mean±s.e.m., n=4) are shown. () Streptavidin-bead pulldown assay with recombinant GST–SHARPIN and the indicated biotinylated peptides in the presence or absence of competing peptides (tenfold excess soluble, unlabelled peptides). The uncropped blot is shown in Supplementary Fig. S5.! () Lysates from GD25 cells (β1-integrin null), co-transfected with plasmids expressing GFP–SHARPIN and ILR2α TAC subunit fused to the indicated integrin cytoplasmic tails, were immunoprecipitated with anti-TAC antibody and blotted as indicated. Uncropped blots are shown in Supplementary Fig. S5. * Figure 3: SHARPIN co-localizes with inactive β1-integrins in membrane ruffles and associates with them in cells. () Non-transfected NCI-H460 cells stained as indicated. Shown are confocal microscopy slices from the middle of the cell and from the bottom surface. Scale bar, 10 μm. Insets: Higher magnifications. The graph shows analysis of SHARPIN and Mab13 co-localization (Pearson's correlation coefficient, n=12). () Control- and SHARPIN-siRNA-transfected NCI-H460 cells stained as indicated. The arrows indicate Mab13-positive membrane ruffles. Scale bar, 10 μm. The graphs show quantitation of the percentages of cells with Mab13-positive membrane ruffles (n=43 cells) and the cell area (n=48 cells) for control- and SHARPIN-siRNA-transfected cells. () Co-immunoprecipitations of α-integrins and SHARPIN in non-transfected PC3 cells grown on plastic. (,) Co-immunoprecipitations of α2- () or α1-integrin () and SHARPIN in non-transfected PC3 cells kept in suspension or plated on the indicated substrates (collagen type I for α2-integrin and collagen type IV containing Matrigel for α! 1-integrin). () Co-immunoprecipitations (IP) of α5-integrin (wild type, WT, or 34AA mutant) and SHARPIN in PC3 cells transfected with plasmids expressing the indicated proteins (note the higher amount of SHARPIN in the α5–GFP 34AA lysates). Uncropped blots are shown in Supplementary Fig. S5. All numeric data are mean±s.e.m., ***P<0.001. * Figure 4: SHARPIN directly interacts with β1-integrins in cells and inhibits recruitment of talin and kindlin to β1-integrins. () PC3 cells transfected with GFP–SHARPIN with (lower row) or without (upper row) α5-integrin–mCherry subjected to FRET analysis by FLIM. Lifetime images mapping spatial FRET in cells are depicted using a pseudo-colour scale (blue, normal lifetime; red, FRET (reduced lifetime)). Scale bars, 10 μm. (,) β1−/− MEF cells transfected with plasmids expressing β1–GFP and mCherry–talin () or β1–GFP and mCherry–kindlin-2 (the predominant kindlin isoform expressed in fibroblasts; ) in combination with an empty control plasmid or MYC–SHARPIN and subjected to FRET analysis by FLIM (as in ), n=12–19 cells. Note the dose-dependent effect of MYC–SHARPIN in the two cells shown in . Scale bars, 10 μm. () PLA between β1-integrin and kindlin in SHARPIN or control-silenced PC3 cells. Scale bar, 10 μm. () Analysis by PCR with reverse transcription of Sharpin and actin mRNA levels in β1-null GD25 mouse cells transfected with Sharpin siRNA or control siRNA. Cel! ls double-transfected with the indicated siRNAs and plasmids expressing either β1–CFP (cyan fluorescent protein) wild type and α5–YFP (yellow fluorescent protein) wild type or legs together (LT) restrained mutants β1–CFP-LT and α5–YFP-LT were stained with an antibody recognizing active human α5β1-integrin (SNAKA51) and analysed with FACS. Shown are mean fluorescence intensities of SNAKA51 staining of CFP–YFP double-positive cells (n=3). All numeric data are mean±s.e.m., *P<0.05, ***P<0.001, n.s., not significant. * Figure 5: SHARPIN inhibits β1-integrin activity in primary human leukocytes. () SHARPIN and tubulin levels in freshly isolated PBL from two individuals and in PBL (no. 2) silenced with control or SHARPIN siRNA1 for 48 h. () Spreading of SHARPIN-siRNA1- or control-siRNA-transfected human PBL on 1 μg ml−1 fibronectin after 1 h. The cells were stained with DAPI (blue, nuclei) and phalloidin (green), and the cell areas (green) quantitated by microscopy (mean±s.e.m., n=1,671–3,681 cells). Scale bar, 10 μm. () Migration of SHARPIN-siRNA1- or control-siRNA-transfected human PBL on fibronectin (n=49 cells). Representative migration tracks and quantitation of the migration distance are shown. Scale bar, 10 μm. () SHARPIN- and control-silenced human PBLs were stained for α4- and α5-integrin and total β1-integrin (K20; n=3). () FACS staining of cell-surface 9EG7 levels in PBL (no. 2) transfected with control or SHARPIN siRNA1 together with plasmids expressing GFP or siRNA-resistant GFP–SHARPIN. The value 1.0 was assigned to cells transf! ected with control siRNA and plasmid expressing GFP (n=3). () FACS analysis of cell-surface levels of active (9EG7) and inactive (4B4) β1-integrin in human PBL transfected with plasmids expressing GFP or GFP–SHARPIN from three independent transfections. Binding in cells transfected with plasmids expressing GFP is assigned the value of 1.0. All numeric data are mean±s.e.m., *P<0.05, ***P<0.001, n.s., not significant. * Figure 6: Loss of SHARPIN correlates with increased β1-integrin activity in vivo. () Haematoxylin–eosin stainings and immunofluorescence co-stainings of active β1-integrin and IVL, KRT10 and KRT14 of skin samples from wild-type and SHARPIN-null (cpdm) mice. DAPI was used to stain nuclei. Scale bars, 50 μm. () Primary splenocytes isolated from wild-type (WT) and cpdm mice were analysed using FACS for binding to fibronectin (FN7–10 fragment) or 9EG7 antibody (n=6 mice per genotype). Shown are the mean fluorescence intensities relative to wild-type splenocytes, and representative histograms. () Binding of fibronectin (FN7–10 fragment) and 9EG7 to wild-type and cpdm primary leucocytes (bone marrow cells) was analysed using FACS (n=3 mice per genotype). Binding to wild-type cells is assigned the value of 1.0. () FACS analysis of total β1-integrin levels on the surface of wild-type and cpdm MEFs (left). Binding of fibronectin (FN7–10 fragment) to wild-type and cpdm MEFs (right) was analysed using FACS (n=4). Binding to wild-type cells is assigned t! he value of 1.0. () Binding of fibronectin (FN7–10 fragment) to MEFs from wild-type and cpdm mice transfected with plasmids expressing GFP or GFP–SHARPIN was analysed using FACS. The staining intensities are shown relative to wild-type MEFs transfected with plasmids expressing GFP (n=3). Analysis of migration speed of wild-type and cpdm MEFs transfected with plasmids expressing GFP or GFP–SHARPIN (n>25 cells). () MEFs from wild-type and cpdm mice were transfected with plasmids expressing full-length α5–GFP wild type or α5–GFP 34AA mutant and the α5β1-integrin activity was analysed with SNAKA51 antibody using FACS. Shown are mean fluorescence intensities of SNAKA51 stainings relative to GFP intensity (the intensity of wild-type MEFs expressing wild-type α5 is defined as 1.0; n=3). () Binding of fibronectin (FN7–10 fragment) to cpdm MEFs transfected with plasmids expressing GFP or GFP–SHARPIN in the presence or absence of membrane-permeable SHARPIN binding ! α-tail peptide (α1-TAT) or scramble peptide (ScrTAT) was ana! lysed using FACS (n=3). Control, without peptides. All numeric data are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001, n.s., not significant. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Juha K. Rantala & * Jeroen Pouwels Affiliations * Medical Biotechnology, VTT Technical Research Centre of Finland, 20521 Turku, Finland * Juha K. Rantala, * Jeroen Pouwels, * Teijo Pellinen, * Stefan Veltel, * Petra Laasola, * Elina Mattila, * Olli Kallioniemi & * Johanna Ivaska * Centre for Biotechnology, University of Turku, 20520 Turku, Finland * Jeroen Pouwels, * Teijo Pellinen, * Stefan Veltel, * Elina Mattila & * Johanna Ivaska * The Jackson Laboratory, Bar Harbor, Maine 04609, USA * Christopher S. Potter, * Ted Duffy & * John P. Sundberg * Institute for Molecular Medicine Finland (FIMM), Biomedicum 2U, University of Helsinki, 00014 University of Helsinki, Helsinki, Finland * Olli Kallioniemi * Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK * Janet A. Askari & * Martin J. Humphries * Randall Division of Cell and Molecular Biophysics, King's College London Guy's Campus, London SE1 1UL, UK * Maddy Parsons * MediCity Research Laboratory and Department of Medical Biochemistry and Genetics, University of Turku, and National Institute for Health and Welfare, FIN-20520 Turku, Finland * Marko Salmi * Department of Biochemistry and Food Chemistry, University of Turku, 20520 Turku, Finland * Johanna Ivaska Contributions J.K.R. and O.K. developed cell spot microarrays, J.K.R. and T.P. carried out the screen. J.K.R., J.P., P.L., S.V. and J.I. carried out the experiments. E.M. immortalized the MEFs. M.P. carried out the FRET-FLIM, C.S.P, T.D. and J.P.S. contributed to the mouse data, J.A.A. and M.J.H. contributed to the legs together integrin experiments and M.S. contributed to the leukocyte work. J.P., J.I. and M.S. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Johanna Ivaska Author Details * Juha K. Rantala Search for this author in: * NPG journals * PubMed * Google Scholar * Jeroen Pouwels Search for this author in: * NPG journals * PubMed * Google Scholar * Teijo Pellinen Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Veltel Search for this author in: * NPG journals * PubMed * Google Scholar * Petra Laasola Search for this author in: * NPG journals * PubMed * Google Scholar * Elina Mattila Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher S. Potter Search for this author in: * NPG journals * PubMed * Google Scholar * Ted Duffy Search for this author in: * NPG journals * PubMed * Google Scholar * John P. Sundberg Search for this author in: * NPG journals * PubMed * Google Scholar * Olli Kallioniemi Search for this author in: * NPG journals * PubMed * Google Scholar * Janet A. Askari Search for this author in: * NPG journals * PubMed * Google Scholar * Martin J. Humphries Search for this author in: * NPG journals * PubMed * Google Scholar * Maddy Parsons Search for this author in: * NPG journals * PubMed * Google Scholar * Marko Salmi Search for this author in: * NPG journals * PubMed * Google Scholar * Johanna Ivaska Contact Johanna Ivaska Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (700K) Supplementary Information Excel files * Supplementary Table 1 (32K) Supplementary Information * Supplementary Table 2 (32K) Supplementary Information Additional data - MCPH1 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry through the Chk1–Cdc25 pathway
- Nat Cell Biol 13(11):1325-1334 (2011)
Nature Cell Biology | Article MCPH1 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry through the Chk1–Cdc25 pathway * Ralph Gruber1, 4 * Zhongwei Zhou1, 5 * Mikhail Sukchev1, 5 * Tjard Joerss1 * Pierre-Olivier Frappart2 * Zhao-Qi Wang1, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1325–1334Year published:(2011)DOI:doi:10.1038/ncb2342Received27 January 2011Accepted11 August 2011Published online25 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 Primary microcephaly 1 is a neurodevelopmental disorder caused by mutations in the MCPH1 gene, whose product MCPH1 (also known as microcephalin and BRIT1) regulates DNA-damage response. Here we show that Mcph1 disruption in mice results in primary microcephaly, mimicking human MCPH1 symptoms, owing to a premature switching of neuroprogenitors from symmetric to asymmetric division. MCPH1-deficiency abrogates the localization of Chk1 to centrosomes, causing premature Cdk1 activation and early mitotic entry, which uncouples mitosis and the centrosome cycle. This misorients the mitotic spindle alignment and shifts the division plane of neuroprogenitors, to bias neurogenic cell fate. Silencing Cdc25b, a centrosome substrate of Chk1, corrects MCPH1-deficiency-induced spindle misalignment and rescues the premature neurogenic production in Mcph1-knockout neocortex. Thus, MCPH1, through its function in the Chk1–Cdc25–Cdk1 pathway to couple the centrosome cycle with mitosis, is re! quired for precise mitotic spindle orientation and thereby regulates the progenitor division mode to maintain brain size. View full text Figures at a glance * Figure 1: The microcephaly of Mcph1-del mice. () A dorsal view of newborn (P0) mouse brains of indicated genotypes. The cortex areas are marked for comparison. () Coronal sections of newborn mice stained with haematoxylin and eosin. ob, olfactory bulb; ctx, cerebral cortex; mb, midbrain; hp, hippocampus. The lower panels are the enlargements of the indicated region of the upper panel. () The Mcph1-del newborn cortex shows a decrease of radial thickness (left; solid line in ) and also in lateral dimension (right; dashed line in ). Mean±s.e.m. is shown. Student's t-test was carried out for statistical analysis. n, the number of mice. () E15.5 neocortex sagittal sections stained with haematoxylin and eosin. An enlarged view of the forebrain cortex from the rectangular areas is shown below. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. * Figure 2: Neocortical developmental analysis of Mcph1-del mice. () Immunofluorescence staining of E15.5 brains with BrdU antibody on sagittal sections. () The histogram shows BrdU-positive cells as a percentage of the total cell number in the VZ/SVZ. () Immunofluorescence staining of E18.5 brains with BrdU antibody on coronal sections. () The histogram shows quantification of BrdU-positive cells per optical section; 1,136 BrdU-positive cells from Mcph1-ctr embryos and 673 BrdU-positive cells from Mcph1-del embryos were scored. () E17.5 mouse coronal brain sections were stained with antibody against Ki67 to label cycling cells (red) and co-stained with DAPI (blue). () Quantification of Ki67-positive cells per optical section (0.377 mm2). () The TUNEL reaction on sagittal sections of E15.5 embryos. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. () The histogram shows TUNEL-positive cells as a percentage of the total cell number in the VZ/SVZ. n, the number of sections of at least two mice. Mean! ±s.e.m. is shown. Student's t-test was carried out for statistical analysis. NS, not significant. * Figure 3: Characterization of Mcph1-knockout neural progenitors in vitro and in vivo. () The images show representative primary (passage 0, p0), secondary (passage 1, p1) and tertiary (passage 2, p2) neurospheres formed after seven days in culture. At each passage, an equal number (4×104 cells ml−1) of dissociated neurosphere cells was replated. The number of neurospheres per millilitre and the number of cells per neurosphere were quantified. n, the number of embryos analysed. Scale bars, 200 μm. () The CCEI was measured after immunofluorescence staining of BrdU (red) and of the cell cycle marker Ki67 (green) in E15.5 brain sections. Doubly BrdU- and Ki67-positive staining indicates cells in the cell cycle, whereas BrdU-positive-only cells represent cells that have exited the cell cycle (G0). () The histogram shows the CCEI. The total number of each cell type (n) was scored from four sections of three mice of each genotype and is shown below in the histogram. Mean±s.e.m. of three embryos is shown. Student's t-test was carried out for statistical ! analysis. NS, not significant. * Figure 4: In vivo analyses of the cell division mode of neuroprogenitors. () The equal and unequal distribution of the apical plasma membrane in the anaphase neuroprogenitors of E15.5 brains. The sections were immunostained for pan-cadherin (Pan-cad) to mark the apical plasma membrane, also known as the 'cadherin hole' (yellow bars). The cleavage planes of the progenitors in anaphase (dashed line) that are relative to the ventricular surface (V) are shown. () Quantification of anaphase progenitors with an unequal distribution of the apical plasma membrane. Mean±s.e.m. of three embryos of each genotype is shown. Student's t-test was carried out for statistical analysis. () Examples of neuroprogenitor cells in E14.5 neocortex with equal or unequal distribution of the apical plasma membrane. The sections were immunostained for N-cadherin (N-cad; green) and the adherens junction protein ZO-1 (red), which indicates the apical plasma membrane, known as the 'cadherin hole' (yellow bar). DNA staining with DAPI was used to measure the cleavage p! lane (dashed line) of anaphase neuroprogenitor cells relative to the ventricular surface (V). () Quantification of neuroprogenitor cells in anaphase or telophase with an unequal distribution of the apical plasma membrane. Mean ± s.e.m. of three embryos is shown. Three Mcph1-ctr embryos (n=51cells) and three Mcph1-del embryos (n=53cells) were analysed. Student's t-test was carried out for statistical analysis. () Representative images of the cleavage planes of mitotic neuroprogenitors in vivo after immunostaining with α-tubulin antibody (green) and DAPI (blue). The angle of the cleavage plane (dashed thick line) to the ventricular surface (V, dashed thin line) was individually measured. () Quantification of the cleavage plane orientation of progenitors on the apical surface of the E15.5 neocortex. The dots represent each mitotic figure in a given angle range. The cleavage plane of Mcph1-del progenitors was significantly shifted when compared with Mcph1-ctr. Chi-squared t! est was carried out for statistical analysis. * Figure 5: Analysis of cell-cycle-dependent Chk1 centrosome localization, premature mitotic entry and centrosome maturation in primary neuroprogenitors. () The centrosome localization of Chk1 (arrows). The neuroprogenitor cells in G2 phase, prophase and metaphase were fixed and stained with antibodies against Chk1 (red) and γ-tubulin (green) and counterstained with DAPI (blue). () Quantification of the cells in with a co-localization of Chk1 and γ-tubulin at the indicated cell-cycle phase. Mean±s.e.m. of at least three embryos is shown. n, the total number of cells scored. Student's t-test was carried out for statistical analysis. NS, not significant. () Immunofluorescence microscopy analysis of the activation of Cdk1 in primary neuroprogenitors. Immunostaining of synchronized neuroprogenitors with antibodies against cyclin B1 and Cdk1-Y18p. Note the increase in the number of Mcph1-del cells with hypo-Cdk1-Y18p (dephosphorylation at Tyr 18). () Time course analysis of Cdk1 activation by immunoblotting. Primary neurospheres were synchronized by a double-thymidine block and proteins were analysed at indicated times after ! release from the block. The level of Cdk1-Y18p is presented as a percentage of endogenous actin. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 6: Analysis of premature mitotic entry and centrosome maturation in primary neuroprogenitors. () Left, p-H3 antibody staining of neuroprogenitor cells. The arrows point to p-H3-positive cells and insets are examples of the p-H3-positive cells. Right, quantification of mitotic cells (p-H3-positive) at 6 h after release from the double-thymidine block. n, the number of cells scored. Mean±s.e.m.of three embryos is shown. Student's t-test was carried out for statistical analysis. () Left, examples of mitotic EdU labelling. Embryonic brain sections were stained with p-H3-antibody (green) and EdU (magenta). The yellow arrows indicate the EdU+/p-H3+ double-positive cells in the ventricular zone. The red arrows indicate the single p-H3-positive cells. Mitotic cells in the SVZ were excluded from the analysis. Scale bar, 10 μm. Right, quantification of the mitotic EdU-labelling index of EdU+/p-H3+ double-positive cells as a percentage of p-H3+ cells at the apical surface. Mean±s.e.m. from at least three embryos. is shown. () Analysis of centrosome maturation. Top, 6�! �h after release from the double-thymidine block, primary neuroprogenitor cells were stained with antibodies against γ-tubulin and ODF2 (mature centrosome marker). Bottom, quantification of cells with the ODF2 marker in both centrosomes. The total number of neuroprogenitor cells (n) from three embryos was quantified for mature centrosomes (symmetric ODF2 staining; top panel). Student's t-test was carried out for statistical analysis. NS, not significant. () Quantitative analysis of centrosome maturation in metaphase neuroprogenitor cells after release from the double-thymidine block. Left, metaphase cells (10 h after release) were stained with γ-tubulin (green) and ODF2 (red). The insets show a higher magnification of centrosomes. Right, quantification of the ODF2 signal intensity (ODF2 channel) of presumably the daughter centrosome (weak signal) in relation to the signal intensity of the mother centrosome (strong signal). Mean±s.e.m. from at least three embryos is s! hown (Mcph1-ctr, n=29 cells; Mcph1-del, n=27 cells). Student�! �s t-test was carried out for statistical analysis. * Figure 7: Characterization of mitotic spindle defects in Mcph1-del neuroprogenitors and MEFs by Chk1 and/or Cdc25b knockdown. () Spindle alignment of mitotic Mcph1-del primary neuroprogenitor cells. The metaphases of neuroprogenitor cells were stained for α-tubulin (spindle), pericentrin (centrosome) and DAPI (DNA). A normal mitotic figure (top left image) and several abnormal mitotic figures are shown. () Quantification of the total mitotic abnormalities (see ). n, the number of cells scored. Mean±s.e.m. of four embryos of each genotype is shown. Student's t-test was carried out for statistical analysis. () Examples of mitotic spindle aberrations (multipolar and abnormal bipolar spindles) in Mcph1-del 3T3 MEFs transfected with a GFP-tagged Luc shRNA. GFP+ (green) anaphase cells were stained with antibodies against pericentrin (magenta), α-tubulin (red) and co-stained with DAPI (blue) at 24–28 h after release from contact inhibition. () Quantification of metaphase cells with spindle aberrations shown in . Number of metaphase cells analysed: Mcph1-ctr+untransfected (n=165); Mcph1-ctr+ Luc s! hRNA (n=142); Mcph1-ctr+ Chk1 shRNA (n=130); Mcph1-ctr+ Chk1 and Cdc25b shRNA (n=130); Mcph1-del+untransfected (n=107); Mcph1-del+ Luc shRNA (n=102); Mcph1-del+ Cdc25b shRNA (n=105). Mean±s.e.m. of at least three independent experiments is shown. A one-way analysis of variance test was carried out for statistical analysis. ***, P<0.001; NS, not significant. (,) Immunoblot analysis of knocking down Chk1 () and Cdc25b (). Wild-type 3T3 MEFs were untransfected (−) or transfected with GFP-tagged shRNA vector against control Luc, Chk1 () or Cdc25b (). GFP-positive cells were sorted 48 h after transfection using FACS. β-actin was used as a loading control. Uncropped images of blots are shown in Supplementary Fig. S9. * Figure 8: In vivo silencing of Chk1 and Cdc25b in the neocortex by in utero electroporation. () Analysis of the cleavage plane after Chk1 shRNA transfection in vivo. Left, the equal and unequal distribution of the apical plasma membrane of dividing neuroprogenitors of E15.5 brains that were electroporated at E13.5 with indicated EGFP-tagged shRNA vectors is shown. The sections were immunostained for N-cadherin (N-cad, red) to mark the apical plasma membrane (cadherin hole; yellow bars) of the transfected cells (green). The cleavage plane of mitotic neuroprogenitors is indicated with a white dashed line. Right, quantification. Mean±s.e.m. of three embryos of each genotype is shown. n, the number of mitotic figures scored. Student's t-test was carried out for statistical analysis. () Representative E15.5 brain sections two days after in utero electroporation with the indicated vector expressing EGFP–Luc shRNA, EGFP–Chk1 shRNA or EGFP–Chk1 shRNA together with Cdc25b shRNA. The staining channels from the dashed square are shown. () Quantification of the GFP-pos! itive cells in . The histogram shows the ratio of non-proliferating neuronal (Ki67−GFP+) cells versus all GFP-positive (Ki67+GFP+ and Ki67−GFP+) cells, which indicates the NPR of transfected progenitors. Mean±s.e.m. of three to five sections of at least three mice of each genotype is shown. A one-way analysis of variance test was carried out for statistical analysis. () Representative images of brain sections. The cell-cycle exit was measured in wild-type (Mcph1-ctr) or mutant (Mcph1-del) E15.5 brain sections following electroporation at E13.5 with vectors expressing control EGFP-tagged Luc or Cdc25b shRNA. The staining channels from the dashed square are shown below. () The NPR of transfected progenitors is quantified by scoring the number of GFP-positive-only (Ki67−GFP+) cells divided by the number of all GFP-positive (Ki67+GFP+ and Ki67−GFP+) cells. Mean±s.e.m. of at least three sections of three to four mice of each genotype is shown. A one-way analysis of var! iance test was carried out for statistical analysis. *, P<0.05! ; **, P<0.01; NS, not significant. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Zhongwei Zhou & * Mikhail Sukchev Affiliations * Leibniz Institute for Age Research—Fritz Lipmann Institute (FLI), Beurtenbergstrasse 11, 07745 Jena, Germany * Ralph Gruber, * Zhongwei Zhou, * Mikhail Sukchev, * Tjard Joerss & * Zhao-Qi Wang * Institute of Molecular Cell Biology, Centre for Molecular Biomedicine (CMB), Hans-Knöll-Strasse 2, 07745 Jena, Germany * Pierre-Olivier Frappart * Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, Beurtenbergstrasse 11, 07745 Jena, Germany * Zhao-Qi Wang * Present address: Cancer Research UK—London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK * Ralph Gruber Contributions R.G. carried out most of the experiments, analysed data and prepared the figures and the manuscript; Z.W.Z. carried out in situ hybridization, in utero electroporation, immunoblot analysis and analysed data; M.S. carried out gene targeting in embryonic stem cells and analysed gene expression; P-O.F. assisted with neurosphere experiments; T.J. contributed to mouse colony maintenance; Z-Q.W. designed experiments, analysed data and composed the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Zhao-Qi Wang Author Details * Ralph Gruber Search for this author in: * NPG journals * PubMed * Google Scholar * Zhongwei Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Mikhail Sukchev Search for this author in: * NPG journals * PubMed * Google Scholar * Tjard Joerss Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre-Olivier Frappart Search for this author in: * NPG journals * PubMed * Google Scholar * Zhao-Qi Wang Contact Zhao-Qi Wang Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (1200K) Supplementary Information Additional data - Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes
- Nat Cell Biol 13(11):1335-1343 (2011)
Nature Cell Biology | Article Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes * Oliver Florey1 * Sung Eun Kim1, 2 * Cynthia P. Sandoval3 * Cole M. Haynes1, 2 * Michael Overholtzer1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1335–1343Year published:(2011)DOI:doi:10.1038/ncb2363Received09 March 2011Accepted16 September 2011Published online16 October 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 Autophagy normally involves the formation of double-membrane autophagosomes that mediate bulk cytoplasmic and organelle degradation. Here we report the modification of single-membrane vacuoles in cells by autophagy proteins. LC3 (Light chain 3) a component of autophagosomes, is recruited to single-membrane entotic vacuoles, macropinosomes and phagosomes harbouring apoptotic cells, in a manner dependent on the lipidation machinery including ATG5 and ATG7, and the class III phosphatidylinositol-3-kinase VPS34. These downstream components of the autophagy machinery, but not the upstream mammalian Tor (mTor)-regulated ULK–ATG13–FIP200 complex, facilitate lysosome fusion to single membranes and the degradation of internalized cargo. For entosis, a live-cell-engulfment program, the autophagy-protein-dependent fusion of lysosomes to vacuolar membranes leads to the death of internalized cells. As pathogen-containing phagosomes can be targeted in a similar manner, the death of ep! ithelial cells by this mechanism mimics pathogen destruction. These data demonstrate that proteins of the autophagy pathway can target single-membrane vacuoles in cells in the absence of pathogenic organisms. View full text Figures at a glance * Figure 1: Entotic cell death involves recruitment of LC3 to a single-membrane vacuole. () Confocal time-lapse images of an MCF10A cell-in-cell structure expressing GFP–LC3 and H2B–mCherry. The arrow marks recruitment of LC3 to the entotic vacuole (see Supplementary Movie S1). Scale bar, 15 μm. () Images of MCF10A cells expressing GFP–LC3G120A and mCherry–LC3; note no recruitment of GFP–LC3G120A to the entotic vacuole. Scale bar, 15 μm. () LC3 is recruited from the host-cell cytosol onto the entotic vacuole membrane. Data points are the mean fluorescence intensity of GFP–LC3 on vacuoles versus cytoplasm for two independent cell-in-cell structures (see Supplementary Movie S2). () Correlative video-light–electron microscopy of an MCF10A GFP–LC3 cell-in-cell structure. Cells were imaged by time-lapse microscopy, followed by fixation after LC3 recruitment (see Supplementary Movie S3). Top left, post-fixation images of phase-contrast and GFP-fluorescence signals were taken demonstrating LC3 recruitment. Bottom left and right, electron microscop! y (EM) of the same cell shows the entotic vacuole is a single membrane (arrow in right image, which is a higher magnification of the area outlined in the bottom left image). () Percentage of MCF10A cell-in-cell structures, with and without autophagy inhibition, that show LC3 recruitment associated with death of the internalized cell. Data represent mean±s.e.m. from at least three independent experiments; n, total number of death events; ***P<0.0001. (,) Time-lapse images of MCF10A cell-in-cell structures expressing either 2×FYVE–mCherry (red) and GFP–LC3 (green; ) or Lamp1–GFP (green) and mCherry–LC3 (red; ). The arrows indicate recruitment to the entotic vacuole (see Supplementary Movies S4 and S5). Times are indicated as h:min:s (,) or min (). Scale bars, 5 μm. () Representative images of cell-in-cell structures in which only host cells express Cathepsin-B–mCherry. Left, a live inner cell with no Cathepsin B in the vacuole (arrow); middle, a live internaliz! ed cell with Cathepsin B from the host inside of the vacuole (! arrowhead); right, a dead internalized cell with Cathepsin B from the host throughout the corpse (asterisk). Scale bar, 15 μm. () Five representative timings of GFP–LC3 recruitment to entotic vacuoles (green bar) and death of internalized cells (cross) from time-lapse microscopy. () Initiation and duration of 2×FYVE domain (red bar, n=11), LC3 (green bar, n=20) and Lamp1 (orange bar, n=7) recruitment to entotic vacuoles, determined using double-expressing cells shown in ,. Error bars represent s.d. * Figure 2: Autophagy machinery in host cells controls the fate of starving internalized cells. () Left, model depicting the multiple cell fates of internalized cells; the arrow thickness represents the relative frequency of events. Right, images of MCF10A cell-in-cell structures (1, host cell; 2, internalized cell) that show a cell death (top images) or release (bottom images) event. Scale bar, 15 μm. () Quantification of MCF10A internalized cell fate over 20 h following autophagy inhibition. Data represent the mean from at least three independent experiments; n, total number of structures analysed; ***P<0.007, **P<0.004, *P<0.01. (,) Quantification of LC3 recruitment (), and cell fate (), of mixed cell-in-cell structures for which host or internalized cells were treated with ATG5 siRNA. Data represent mean±s.e.m.from three separate experiments; n, total number of structures analysed; *P<0.01, **P<0.004. () Left, quantification of type of internalized cell death after autophagy inhibition. Data represent the mean of three independent experiments; n, total death ! events analysed; **P<0.03. Right, representative images of non-apoptotic and apoptotic death of an internalized cell; the arrow points to fragmented apoptotic nuclei. Scale bars, 15 μm. () Quantification of mCherry/GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP–mCherry–LC3, with or without siRNA treatment as indicated. Data represent mean±s.d.; numbers of cells analysed for each data point (from left to right) 30, 30, 30, 25, 30, 25, 25, 25, 30, 27; *P<0.03, **P<0.003, ***P<0.0004. () Quantification of autophagosome and autolysosome area in control MCF10A (n=10) and internalized cell (n=10) cytoplasm imaged by electron microscopy; **P<0.005. () Representative electron micrographs of cell-in-cell structures (left) and internalized cell cytoplasm (middle and right) showing autophagosomes (arrow) and autolysosomes (arrowhead). * Figure 3: Inhibition of apoptosis and entotic cell death promotes anchorage-independent growth. () The fate of internalized cells (control MCF10A and MCF10A expressing E7 and Bcl2) was measured with and without autophagy inhibition. Data show the percentage of internalized cell release with autophagy inhibition, compared with each control without autophagy inhibition; mean±s.e.m. from at least three independent experiments (total cell numbers analysed: ATG5 siRNA, 261 cells; Bcl2 ATG5 siRNA, 97 cells; 3-MA, 170 cells; Bcl2 3-MA, 112 cells); *P<0.05, **P<0.02. () Effects of Y27632, 3-MA and VPS34 and ATG5 siRNA on MCF10A cell-in-cell formation in suspension for 7 h; >300 cells per condition were scored for cell-in-cell formation; ±s.e.m.from three independent experiments; **P<0.004. () Representative images of an MCF10A E7+Bcl2 cell-in-cell structure formed in soft agar. The arrow marks LC3 recruitment around internalized cells. Scale bar, 5 μm. () Quantification of MCF10A E7+Bcl2 cell-in-cell structures 48 h after seeding into soft agar (white bars), and colon! y formation after 2 weeks (grey bars). Data represent mean±s.e.m.from three independent experiments; *P<0.03, **P<0.004, ***P<0.001. () Representative wells for data in . The insets show higher magnifications. () Quantification of MCF10A E7+Bcl2 colony formation with Atg5 knockdown with two separate siRNAs. Data represent mean±s.e.m.from three independent experiments; *P<0.001. () Representative wells for data in . The insets show higher magnifications. See also Supplementary Movie S6. * Figure 4: LC3 recruits to single-membrane vacuoles containing apoptotic cells to facilitate corpse degradation. () Time-lapse images of MCF10A cell-in-cell structures with an internalized cell that undergoes apoptosis. The arrows mark the recruitment of LC3 around apoptotic fragments (see Supplementary Movie S7). Scale bar, 10 μm. () Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA; n, number of cells analysed; ***P<0.0001, chi-squared test. () Apoptotic U937 cells expressing H2B–mCherry phagocytized by J774 macrophages expressing GFP–LC3. Confocal images show LC3 recruitment to an engulfed corpse (arrow; see Supplementary Movie S8). Scale bar, 15 μm. () Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5-shRNA-expressing cells; ***P<0.0001, chi-squared test. () Correlative light–electron microscopy of an apoptotic phagosome with GFP–LC3 recruitment (top left image, white arrow). The LC3-loaded phagosome consists of a single membrane (black arrow in right image, which is a higher magnifica! tion of the area outlined in the bottom left image; EM, electron microscopy; AP, apoptotic cell nucleus). () Representative images of apoptotic cell phagocytosis and degradation in control (n=21) and Atg5 shRNA (n=21) J774 GFP–LC3 cells; ***P<0.0001, chi-squared test. Scale bar, 10 μm. See also Supplementary Fig. S3. * Figure 5: LC3 is recruited to macropinosomes independently of macroautophagy. () Representative images of a J774 macrophage expressing GFP–LC3 incubated in media containing red dextran. The arrow marks LC3 recruitment to a dextran-containing macropinosome. Scale bar, 2 μm. () Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages; n, number of macropinosomes analysed; ***P<0.0001, chi-squared test. See Supplementary Movie S9. () Representative image of LC3 recruitment to macropinosomes (arrows) in MCF10A cells expressing GFP–LC3 incubated with red dextran. Scale bar, 2 μm. () Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5; n, number of macropinosomes analysed; ***P<0.0001, chi-squared test. () J774 GFP–LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h, followed by the addition of LysoTracker red to mark acidified compartments. A macropinosome recruited LC3 (arrow) whereas engulfed! latex beads did not (arrowheads). Scale bar, 5 μm. See Supplementary Movie S10. * Figure 6: LGG-1 recruitment to apoptotic phagosomes during C. elegans embryonic development. () A C. elegans embryo expressing mCherry::RAB-5 and GFP::LGG-1. The arrows point to two apoptotic phagosomes. See Supplementary Movie S11. Scale bar, 10 μm. () Cropped time-lapse images showing RAB-5 and LGG-1 recruitment to an apoptotic phagosome (from , left arrow). () Representative images of apoptotic phagosomes in embryos from control-RNAi- or bec-1-RNAi-fed worms. The insets show higher magnifications of the areas outlined in the main panels. () Quantification of GFP::LGG-1- and mCherry::RAB-5-positive phagosomes in control- or bec-1-RNAi embryos determined by time-lapse imaging. Control, 21 embryos, 52 phagosomes; bec-1, 9 embryos, 25 phagosomes; ***P<0.0001, chi-squared test. () Representative DIC images from control- and bec-1-RNAi embryos at different stages of development (minutes after first division). The yellow arrowheads mark apoptotic corpses. Scale bar, 10 μm. () Quantification of apoptotic corpses in control- and bec-1-RNAi embryos; data show ± s.d.! ; ***P<0.0001. () Representative images of CED-1::GFP embryos with control or bec-1 RNAi. The yellow arrowheads mark engulfed corpses surrounded by CED-1::GFP. Scale bar, 5 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA * Oliver Florey, * Sung Eun Kim, * Cole M. Haynes & * Michael Overholtzer * BCMB Allied Program, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA * Sung Eun Kim, * Cole M. Haynes & * Michael Overholtzer * Department of Physiology, University of Arizona, Tucson, Arizona 85721, USA * Cynthia P. Sandoval Contributions O.F. and M.O. designed, carried out experiments and wrote the paper. S.E.K. and C.P.S. contributed experimental assistance and data. C.M.H. provided worm strains and carried out RNAi feeding of C. elegans. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael Overholtzer Author Details * Oliver Florey Search for this author in: * NPG journals * PubMed * Google Scholar * Sung Eun Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Cynthia P. Sandoval Search for this author in: * NPG journals * PubMed * Google Scholar * Cole M. Haynes Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Overholtzer Contact Michael Overholtzer Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (1500K) Supplementary Information Movies * Supplementary Movie 1 (900K) Supplementary Information * Supplementary Movie 2 (4M) Supplementary Information * Supplementary Movie 3 (360K) Supplementary Information * Supplementary Movie 4 (14M) Supplementary Information * Supplementary Movie 5 (7M) Supplementary Information * Supplementary Movie 6 (1700K) Supplementary Information * Supplementary Movie 7 (1200K) Supplementary Information * Supplementary Movie 8 (1100K) Supplementary Information * Supplementary Movie 9 (900K) Supplementary Information * Supplementary Movie 10 (760K) Supplementary Information * Supplementary Movie 11 (1M) Supplementary Information Additional data - Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins
- Nat Cell Biol 13(11):1344-1352 (2011)
Nature Cell Biology | Article Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins * Nancy N. Fang1 * Alex H. M. Ng1 * Vivien Measday2 * Thibault Mayor1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1344–1352Year published:(2011)DOI:doi:10.1038/ncb2343Received12 July 2011Accepted16 August 2011Published online09 October 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 Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with numerous aggregation diseases. Several protein quality control mechanisms degrade non-native proteins by the ubiquitin–proteasome system. Here, we use quantitative mass spectrometry to demonstrate that heat-shock triggers a large increase in the level of ubiquitylation associated with misfolding of cytosolic proteins. We discover that the Hul5 HECT ubiquitin ligase participates in this heat-shock stress response. Hul5 is required to maintain cell fitness after heat-shock and to degrade short-lived misfolded proteins. In addition, localization of Hul5 in the cytoplasm is important for its quality control function. We identify potential Hul5 substrates in heat-shock and physiological conditions to reveal that Hul5 is required for ubiquitylation of low-solubility cytosolic proteins including the Pin3 prion-like protein. These find! ings indicate that Hul5 is involved in a cytosolic protein quality control pathway that targets misfolded proteins for degradation. View full text Figures at a glance * Figure 1: Heat-shock stress induces protein misfolding and polyubiquitylation. () BY4741 cells were subjected to heat-shock (HS; 15 min at 45 °C) or not (No HS). Left, experimental triplicates were analysed by western (top) and dot blots (bottom) with anti-ubiquitin and anti-Pgk1 antibodies. Right, the region above Mr70K in the western blot and the whole spotted signal in the dot blot were quantified. Ubiquitylation signals were normalized to Pgk1 levels and standard deviations are shown. () Ubiquitylation levels in total cell extract (T), soluble (S) and pellet (P) fractions after 16,000g centrifugation from both unstressed and heat-shock-treated (15 min 45 °C) BY4741 cells shown by anti-ubiquitin western blot. () Schematic diagram of the proposed relationship between heat-shock and the increased ubiquitylation response. () Relative increases of Pgk1-normalized ubiquitylation levels after a 15 min 45 °C heat-shock in the indicated E2 double-deletion strains were quantified by dot blot and averaged values from three replicates are shown ! with standard deviations. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 2: Heat-shock mainly affects cytosolic proteins. () A schematic diagram of the workflow of the quantitative mass spectrometry analysis. HS: heat-shock; No HS: no heat-shock. () Percentage of proteins above the corresponding log2 values of the 14N heat-shock/15N no heat-shock ratios for three independent experiments (I: light; II: medium; and III: dark). Analysis of proteins in the whole-cell lysate (grey: I, 486; II, 730; and III, 399) and of IMAC-enriched ubiquitylated proteins (green: I, 302; II, 481; and III, 219) are shown. Proteins with a log2 ratio ≥0.5 are considered heat-shock affected. () Pin3–TAP solubility was assessed before and after a 15 min 45 °C heat-shock. An equal portion of each fraction (T: total; S: supernatant; P: pellet) was loaded on an SDS–PAGE gel for western-blot analysis with the anti-TAP antibody. () Subcellular localization of 155 proteins affected by heat-shock (green; identified as enriched in at least two of the three experiments in ), compared with the whole proteome (grey)—cy! tosol (C), nucleus (N), membrane (M) and mitochondria or peroxisome (M/P). Note that several proteins localize to more than one compartment. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 3: HUL5 is required for the full ubiquitylation response and cell fitness after heat-shock. () Ubiquitylation levels in unstressed (No HS) and heat-shocked (HS) cells are compared between wild-type and hul5Δ strains using the dot blot assay. HUL5 was deleted by two different cassettes (KanMX6 and NatMX4). Standard deviation is shown for three replicates and P values were calculated using an unpaired Student's t-test. () TAP-immunoprecipitation (TAP-IP) experiments with cells expressing or not Ubc4–TAP (left) or Ubc5–TAP (right) at endogenous levels with or without a plasmid expressing 13Myc–Hul5 were analysed by western blot using 9E10 or anti-TAP antibodies. Inputs (1%) are shown below. () The A600 of the first cell doubling following a 20 min incubation at 45 °C heat-shock (black) or at 25 °C (grey) of HUL5 (round) and hul5Δ::NAT (square) cells. hul5Δ::NAT cells carrying a centromeric plasmid (dashed lines) with HUL5 (round) or Hul5C878A (square) are also compared. Growth delay is defined by the difference of the first doubling time betwee! n the corresponding unstressed and stressed cells. Each data point is averaged from three replicates. () hul5Δ cells carrying either wild-type HUL5 or the catalytically inactive Hul5C878A were subjected to heat-shock (15 min at 42 °C). The increase in ubiquitylation levels (with standard deviations) was measured by dot blots with anti-ubiquitin and anti-Pgk1 antibodies in three replicates. An unpaired Student's t-test was used to assess the significance of the difference between the cell populations. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 4: Hul5 redistribution to the cytoplasm is important for its role in the heat-shock response. () Localization of Hul5–GFP was assessed in a strain that carries Nic96–mRFP as a nuclear periphery marker, in both unstressed and heat-shocked (30 min at 42 °C) cells. Images were taken with a ×40 objective and in focus z stacks were flattened with the wavelet extended function. The nuclear positions are outlined. Scale bar, 5 μm. () Histograms of the mean Hul5–GFP signal intensities (per pixel; with standard error) measured in a 0.7 μm2 area of the nuclei (N) and cytoplasm (C) of unstressed (No HS; 25 °C) and heat-shocked (HS; 30 min at 42 °C) cells (n=100), after subtraction of the average background signal from untagged cells. () Levels of Hul5–GFP in both nuclear and cytosolic fractions were analysed by western blot using anti-GFP, anti-Pgk1 and anti-histone H3 antibodies after subfractionation of both unstressed and heat-shocked (30 min at 42 °C) cells. () The localization of GFP–Hul5 (upper panels) and GFP-NLS–Hul5 (lower panels) e! xpressed from a plasmid was assessed in hul5Δ cells grown at 25 °C and subjected to heat-shock (30 min at 42 °C). DNA was stained with Hoechst (shown below GFP images); single-stack images were taken with a ×63 objective; arrowheads point to the nucleus. Scale bar, 5 μm. () The increase of ubiquitylation levels after heat-shock (15 min at 45 °C) is compared using the dot blot assay between HUL5 and hul5Δcells with an empty vector, and hul5Δ cells expressing GFP–Hul5 or GFP-NLS–Hul5 from a plasmid. Standard deviation is shown for three replicates and P values were calculated using an unpaired Student's t-test (in black or in grey when comparing with HUL5- or GFP–Hul5-expressing cells, respectively). () A600, following a 20 min heat-shock at 45 °C (black) or 20 min at 25 °C (grey), of hul5Δ cells carrying a plasmid with GFP–Hul5 (round) or GFP-NLS–Hul5 (square) are compared. Each data point is averaged (with standard deviations) from! three replicates. Uncropped images of blots are shown in Supp! lementary Fig. S7. * Figure 5: HUL5 is essential for the ubiquitylation of proteins misfolded in the absence of SSA-chaperone activity and for the degradation of pulse-labelled misfolded polypeptides. () Schematic diagram of the proposed model for the SSA-hsp70-chaperone inactivation. () The histogram shows Cdc28-normalized ubiquitylation levels in SSA1 and ssa1-45 cells (ssa2-4Δ) measured before and after shifting cells from 25 °C to 37 °C for 40 min. The ubiquitylation levels were quantified by dot blot assay from four replicates and are shown with standard errors. () The increased ubiquitylation levels in ssa1-45 HUL5 or ssa1-45 hul5Δ cells (two independent strains) were compared using five replicates for each assessed strain as described for . The Pvalues were determined by Student's t-test. () Wild-type (BY4741) and hul5Δ cells were subjected to 35S-pulse labelling (5 min) followed by a 3 h chase. The graph shows the averaged percentage (with standard errors) of pulse-labelled proteins (corresponding to newly synthesized protein) that were degraded at each time point from cells incubated at 25 °C or 38 °C from three replicates. () Histograms of! averaged ubiquitylation signals measured by dot blots in unstressed (no HS; light grey) and heat-shocked (HS; dark grey) cells in three replicates are shown with standard deviations. Cells were grown at 25 °C and pre-treated or not for 15 min with 100 μg ml−1 cycloheximide (CHX) before heat-shock (15 min at 45 °C). Student's t-test was used to assess the significance of the ubiquitylation level differences between the indicated strains. * Figure 6: HUL5 is required for ubiquitylation of low-solubility cytosolic proteins. () 14N and 15N metabolic labelling was carried out for wild-type and hul5Δ cells, respectively. Percentage of proteins above the corresponding log2 values of the 14N/15N ratios in three independent experiments (I: light; II: medium; and III: dark). Analysis of proteins in the whole-cell lysate (grey: I, 345; II, 346; and III, 240) and of IMAC-enriched ubiquitylated proteins (green: I, 661; II, 430; and III, 267) are shown. Proteins with an IMAC log2 ratio ≥1 are considered Hul5 candidate substrates. () Venn diagram representing all 95 proteins identified as more ubiquitylated in experiments I to III in . Bold protein names are confirmed Hul5 substrates in Fig. 7 and Supplementary Fig. S6. () Histogram showing the subcellular localization of the 99 (light green) and 95 (dark green) Hul5 candidate substrate proteins identified in heat-shock-stressed (HS) and unstressed cells (No HS), respectively: cytosol (C), nucleus (N), membrane (M) and mitochondria or peroxisome (M/P). * Figure 7: Hul5 targets proteins that are specifically ubiquitylated in the low-solubility cellular fraction. (–) Validation of the Hul5 substrate candidates Lsm7 and Pin3 using TAP-tagged strains expressing His8–Ubi. IMAC was carried out in denaturating conditions to pull down ubiquitylated proteins, and anti-TAP or anti-ubiquitin antibody was used for western-blot analysis. The asterisk denotes unspecific signal. Corresponding signal intensities for poly- and mono-ubiquitin were measured by subtracting the background signal in control cells (,). Solubility of ubiquitylated Lsm7–TAP () and Pin3–TAP () was assessed by comparing both soluble (S) and pellet (P) fractions subjected to IMAC and analysed by western blots with anti-TAP (,) and anti-ubiquitin (). A 20 min 45 °C heat-shock (HS) was also applied to Pin3–TAP-expressing cells (,). No HS, no heat-shock. (,) Turnover of Slh1 is dependent on cytosolic Hul5. Protein levels of Slh1–TAP were monitored by western-blot analysis after the addition of 100 μg ml−1 cycloheximide to both HUL5 and hul5Δ cells gro! wn at 25 °C (), and to hul5Δ cells expressing either GFP–Hul5 or GFP-NLS–Hul5 and shifted to 38 °C (). Relative averaged signal intensities (with standard deviations) were quantified and normalized to Pgk1 levels in three independent experiments. Uncropped images of blots are shown in Supplementary Fig. S7. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, University of British Columbia, 2125 East Mall, Vancouver, British Columbia V6T 1Z4, Canada * Nancy N. Fang, * Alex H. M. Ng & * Thibault Mayor * Wine Research Centre, University of British Columbia, 2205 East Mall, Vancouver, British Columbia V6T 1Z4, Canada * Vivien Measday Contributions N.N.F. and T.M. conceived the project and designed experiments; N.N.F. carried out experiments; A.H.M.N. designed and carried out the SSA1 analysis and developed reagents; V.M. designed and carried out part of the localization analysis; N.N.F., A.H.M.N., V.M. and T.M. analysed the data; N.N.F., V.M. and T.M. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thibault Mayor Author Details * Nancy N. Fang Search for this author in: * NPG journals * PubMed * Google Scholar * Alex H. M. Ng Search for this author in: * NPG journals * PubMed * Google Scholar * Vivien Measday Search for this author in: * NPG journals * PubMed * Google Scholar * Thibault Mayor Contact Thibault Mayor Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (1700K) Supplementary Information Excel files * Supplementary Table 1 (200K) Supplementary Information * Supplementary Table 2 (64K) Supplementary Information * Supplementary Table 3 (47K) Supplementary Information Additional data - miR-34 miRNAs provide a barrier for somatic cell reprogramming
- Nat Cell Biol 13(11):1353-1360 (2011)
Nature Cell Biology | Letter miR-34 miRNAs provide a barrier for somatic cell reprogramming * Yong Jin Choi1, 7 * Chao-Po Lin1, 7 * Jaclyn J. Ho1 * Xingyue He2 * Nobuhiro Okada1 * Pengcheng Bu1 * Yingchao Zhong1 * Sang Yong Kim3 * Margaux J. Bennett1 * Caifu Chen4 * Arzu Ozturk5 * Geoffrey G. Hicks5 * Greg J. Hannon6 * Lin He1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1353–1360Year published:(2011)DOI:doi:10.1038/ncb2366Received10 May 2011Accepted22 September 2011Published online23 October 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Somatic reprogramming induced by defined transcription factors is a low-efficiency process that is enhanced by p53 deficiency1, 2, 3, 4, 5. So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation1, 3, indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. Mir34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of Mir34a promoted iPSC generation without compromising self-renewal or differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (also known as N-My! c). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three miR-34 miRNAs acted cooperatively in this process. Taken together, our findings identified miR-34 miRNAs as p53 targets that play an essential role in restraining somatic reprogramming. View full text Figures at a glance * Figure 1: Generation of Mir34a and Mir34b/c knockout MEFs. () Diagrams of endogenous Mir34a and Mir34b/c gene structure (WT) and the knockout construct (Δ). Using recombineering, we engineered the Mir34a targeting vector with a ~6-kilobase (kb) homologous arm on both 5′ and 3′ ends, flanking a Kozak sequence, a lacZ complementary DNA and an FRT–Neo–FRT cassette. The Mir34b/c targeting vector (flNeo) contains a ~6-kb homologous arm at each end, with the Mir34b/c gene and a Neo selection cassette flanked by loxP sites. () Validating the germline transmission of the Mir34a and Mir34b/c targeted allele using Southern analysis. Putative wild-type, Mir34a+/− and Mir34a−/− animals derived from three independently targeted ESC lines were analysed by Southern blotting using probes either 5′ or 3′ to the homologous arms (left). Similar validation was carried out for wild-type, Mir34b/cfl/+ and Mir34b/c+/Δ animals (right). () Confirming loss of miR-34 expression in Mir34a and Mir34b/c knockout MEFs. Littermate-controlled wi! ld-type, Mir34a+/− and Mir34a−/− MEFs were analysed by real-time PCR to quantify the expression of Mir34a. Whereas wild-type MEFs showed robust miR-34a induction on culture stress, no miR-34a expression was detected in Mir34a−/−MEFs. The miR-34a level in Mir34a+/− MEFs was approximately half that of wild-type MEFs. Similar validation was carried out for Mir34b/c−/− MEFs. Error bar, s.d., n=3. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 2: Deficiency of miR-34 miRNAs increases reprogramming efficiency. () Four reprogramming factors triggered p53-dependent induction of miR-34 miRNAs. Three days after transduction, pri-miR-34a (the precursor transcript from the Mir34a locus), mature miR-34a and p21 were measured in uninfected and four-factor-induced wild-type (WT) and p53−/− MEFs. Induction of pri-miR-34a was dependent on the intact p53 response, and was comparable to that of p21. Induction of pri-miR-34b/c and mature miR-34b and miR-34c was determined in wild-type, Mir34a−/− and Mir34b/c−/− MEFs. Error bar, s.d., n=3. () Mir34a deficiency significantly enhanced three-factor-induced MEF reprogramming. 2,500 three-factor-infected wild-type, Mir34a−/− or p53−/− MEFs were plated to score reprogramming by alkaline phosphatase (AP)-positive colonies with characteristic ESC morphology. A representative image and quantitative analysis is shown out of five independent experiments, comparing littermate-controlled wild-type and Mir34a−/− MEFs (left, **P<0.01), ! as well as wild-type and p53−/− MEFs (right, **P<0.01). Error bar, s.d., n=4. () Single-sorted, four-factor-infected MEFs were cultured at a density of one cell per well. Four weeks post-plating, alkaline phosphatase-positive colonies with typical iPSC morphology were scored for wild-type, Mir34a−/− and p53−/− iPSCs. Four independent experiments confirmed this finding. *P<0.05for comparison between wild-type and Mir34a−/− MEFs. Error bar, s.e.m., n= experiments with independent MEF lines. () Mir34a deficiency significantly enhanced MEF reprogramming as measured by Oct4–Gfp reporter expression. Three- or four-factor-infected wild-type and Mir34a−/− MEFs that carry an Oct4–Gfp allele were sorted at the density of 2,500 cells per well and 1,000 cells per well, respectively. Reprogramming efficiency was quantified by GFP-positive clones. Images of Oct4–Gfp-positive iPSCs are shown on the left. A quantitative analysis for reprogramming efficiency trigge! red by four factors (left, **P<0.01) or three factors (right, ! *P<0.02) is shown. Scale bar, 100 μm. Error bar, s.d., n=3. OSKM, Oct4, Sox2, Klf4 and Myc; OSK, Oct4, Sox2 and Klf4. () miR-34a, b and c cooperatively regulate somatic reprogramming. Deficiency in Mir34a or Mir34b/c alone significantly promoted somatic reprogramming, yet deficiency in all Mir34 miRNAs exhibited further increase. Two independent experiments confirmed this finding. n, experiments with independent MEFs. All P-values were calculated on the basis of a two-tailed Student's t-test. * Figure 3: miR-34a and p21 cooperate to repress iPSC generation. () p21 was induced in Mir34a−/− MEFs during somatic reprogramming. Three days after retroviral transduction of four reprogramming factors, both p21 mRNA (left) and p21 protein (right) exhibited a significant increase in wild-type (WT) and Mir34a−/− MEFs. This increase correlated well with the elevated level of p53 proteins (right). α-tubulin (Tub) was used as a loading control. Error bar, s.d., n=3. () p21−/−MEFs proliferate more rapidly than Mir34a−/− MEFs. Cumulative population doublings were measured for six consecutive passages in littermate-controlled wild-type and Mir34a−/−MEFs (left), and in wild-type and p21−/− MEFs (right). Compared with the wild-type counterparts, p21−/− MEFs exhibited an enhanced cell proliferation rate, whereas Mir34a−/− MEFs showed little difference. Error bar, s.d., n=3 for triplicate measurements at each time. *P<0.05; **P<0.01 for comparisons between two lines of MEFs for each genotype. () miR-34a and p21 coop! erate to repress iPSC generation. The reprogramming efficiencies were compared among wild-type, Mir34a−/−, p21−/−, Mir34a−/−; p21−/− and p53−/− MEFs using either three (right) or four (left) reprogramming factors. Deficiency in Mir34a or p21 alone enhanced reprogramming efficiency to a comparable level. Deficiency in both Mir34a and p21 gave rise to an even greater reprogramming efficiency. Quantitative analyses of Oct4-positive colonies were carried out at 2 (four-factor-induced reprogramming) or 3 (three-factor-induced reprogramming) weeks post-plating using immunofluorescence analyses. Error bar, s.d., n=3. OSKM, Oct4, Sox2, Klf4 and Myc; OSK, Oct4, Sox2 and Klf4. All P-values were calculated on the basis of two-tailed Student's t-tests. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 4: Mir34a−/− iPSCs functionally resemble wild-type iPSCs. () iPSCs derived from both wild-type and Mir34a−/− MEFs exhibited ESC-like morphology in culture, with robust alkaline phosphatase expression. Scale bar, 20 μm for the left panel, 100 μm for the middle and right panels. () Both wild-type and Mir34a−/− iPSCs expressed pluripotency markers, including nucleus-localized Oct4 and membrane-localized SSEA1. Scale bar, 20 μm. (,) Wild-type and Mir34a−/− iPSCs both generated differentiated teratomas. Teratomas derived from four-factor-induced wild-type (left) and Mir34a−/− (right) iPSCs were collected from nude mice 4–6 weeks after subcutaneous injection. Haematoxylin and eosin staining (), as well as immunofluorescence staining (), revealed terminally differentiated cell types derived from all three germ layers. Scale bar in , 25 μm; in , 50 μm. () Four-factor-induced Mir34a−/− iPSCs efficiently contribute to adult chimaeric mice. We injected three independent lines of passage 7 Oct4–Gfp/+, Mir! 34a−/− iPSCs into albino-C57BL/6/cBrd/cBrd/cr blastocysts. The iPSC contribution to adult chimaeric mice was determined by coat colour pigmentation. DAPI, 4,6-diamidino-2-phenylindole. * Figure 5: Mir34a represses Nanog, Sox2 and N-Myc expression post-transcriptionally. () Schematic representation of the Nanog, Sox2 and N-Myc 3′UTR, and the predicted miR-34 binding sites. The mouse Nanog, Sox2 and N-Myc each contains one putative miR-34a-binding site within its 3′UTR. () Enforced expression of Mir34a, Mir34b and Mir34c in ESCs reduced the protein levels of Nanog, Sox2 and N-Myc, but not Oct4. Feeder-free ESCs were transfected with miRNA mimics for miR-34a, miR-34b and miR-34c, and a negative control, Gfp short interfering RNA (siRNA). At 48 h post transfection, western analysis indicated a significant reduction in the protein levels of Nanog, Sox2 and N-Myc, but not Oct4. The value of each band indicates the relative expression level normalized by the internal control, α-tubulin, averaged between two independent experiments, and presented as mean ± s.e.m. () Derepression of Nanog, Sox2 and N-Myc was observed in Mir34-deficient iPSCs. A significant increase of Nanog and Sox2, but not Oct4, was observed in four-factor-induced Mir34a�! �/− iPSCs, when compared with passage-matched, littermate-controlled wild-type iPSCs. A similar comparison was made for passage-matched, three-factor-induced wild-type and Mir34a−/−; Mir34b/c−/− double-knockout iPSCs, where derepression of Nanog, Sox2 and N-Myc was observed. For this western analysis, the quantitation of each band was carried out by Quantity One software, and was normalized against its own internal tubulin control. The s.e.m. of three independent iPSC lines are shown for each genotype. (,) Mir34a-deficient iPSCs exhibited slower kinetics during differentiation. Wild-type and Mir34a−/− iPSCs were both triggered to differentiate by withdrawal of LIF in the absence () or presence () of retinoic acid treatment. Images of a typical iPSC culture two days after each differentiation condition are shown on the left, and quantitative analyses of the decline of Nanog, Sox2 and Oct4 transcripts in response to these differentiating conditions are shown on ! the right. Error bar, s.e.m., n=3. *P<0.05, **P<0.01, ***P<0.0! 01. Scale bar, 100 μm. Uncropped images of blots are shown in Supplementary Fig. S6. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Yong Jin Choi & * Chao-Po Lin Affiliations * Division of Cellular and Developmental Biology, Molecular and Cell Biology Department, University of California at Berkeley, Berkeley, California 94705, USA * Yong Jin Choi, * Chao-Po Lin, * Jaclyn J. Ho, * Nobuhiro Okada, * Pengcheng Bu, * Yingchao Zhong, * Margaux J. Bennett & * Lin He * 40 Landsdowne Street, Cambridge, Massachusetts 02139, USA * Xingyue He * Transgenetic facility, 500 Sunnyside Blvd., Woodbury, New York 11797-2924, USA * Sang Yong Kim * Genomic Assays R&D, Life Technologies Corporation, 850 Lincoln Centre Dr., Foster City, California 94404, USA * Caifu Chen * Manitoba Institute of Cell Biology, 675 McDermot Ave. Rm. ON5029, Winnipeg, MB R3E 0V9, Canada * Arzu Ozturk & * Geoffrey G. Hicks * Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA * Greg J. Hannon Contributions Y.J.C., C-P.L. and L.H. designed all experiments and carried out the majority of the experiments shown in all figures and supplementary figures. J.J.H. and Y.Z. carried out immunofluorescence analyses and teratoma analyses to characterize the pluripotency of the iPSCs. L.H., X.H., P.B. and G.J.H. generated knockout constructs for Mir34a and Mir34b/c, and identified the correctly targeted ESC clones for Mir34a. N.O. and P.B. identified the correctly targeted ESC clone for Mir34b/c, validated Mir34a and Mir34b/c targeting in mice by Southern blotting and generated Mir34a−/−, Mir34b/c−/− and Mir34 triple knockout MEFs. S.Y.K. and G.J.H. carried out the blastocyst injection for Mir34a+/− and Mir34b/c+/− ESC clones. A.O., S.Y.K. and G.G.H. characterized the pluripotency of iPSCs using chimaera assays. M.J.B. and C.C. contributed to the identification of miR-34a targets. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Lin He Author Details * Yong Jin Choi Search for this author in: * NPG journals * PubMed * Google Scholar * Chao-Po Lin Search for this author in: * NPG journals * PubMed * Google Scholar * Jaclyn J. Ho Search for this author in: * NPG journals * PubMed * Google Scholar * Xingyue He Search for this author in: * NPG journals * PubMed * Google Scholar * Nobuhiro Okada Search for this author in: * NPG journals * PubMed * Google Scholar * Pengcheng Bu Search for this author in: * NPG journals * PubMed * Google Scholar * Yingchao Zhong Search for this author in: * NPG journals * PubMed * Google Scholar * Sang Yong Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Margaux J. Bennett Search for this author in: * NPG journals * PubMed * Google Scholar * Caifu Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Arzu Ozturk Search for this author in: * NPG journals * PubMed * Google Scholar * Geoffrey G. Hicks Search for this author in: * NPG journals * PubMed * Google Scholar * Greg J. Hannon Search for this author in: * NPG journals * PubMed * Google Scholar * Lin He Contact Lin He Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1300K) Supplementary Information Additional data - Microtubules induce self-organization of polarized PAR domains in Caenorhabditis elegans zygotes
- Nat Cell Biol 13(11):1361-1367 (2011)
Nature Cell Biology | Letter Microtubules induce self-organization of polarized PAR domains in Caenorhabditis elegans zygotes * Fumio Motegi1 * Seth Zonies1 * Yingsong Hao1 * Adrian A. Cuenca1 * Erik Griffin1 * Geraldine Seydoux1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1361–1367Year published:(2011)DOI:doi:10.1038/ncb2354Received11 May 2011Accepted05 September 2011Published online09 October 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A hallmark of polarized cells is the segregation of the PAR polarity regulators into asymmetric domains at the cell cortex1, 2. Antagonistic interactions involving two conserved kinases, atypical protein kinase C (aPKC) and PAR-1, have been implicated in polarity maintenance1, 2, but the mechanisms that initiate the formation of asymmetric PAR domains are not understood. Here, we describe one pathway used by the sperm-donated centrosome to polarize the PAR proteins in Caenorhabditis elegans zygotes. Before polarization, cortical aPKC excludes PAR-1 kinase and its binding partner PAR-2 by phosphorylation. During symmetry breaking, microtubules nucleated by the centrosome locally protect PAR-2 from phosphorylation by aPKC, allowing PAR-2 and PAR-1 to access the cortex nearest the centrosome. Cortical PAR-1 phosphorylates PAR-3, causing the PAR-3–aPKC complex to leave the cortex. Our findings illustrate how microtubules, independently of actin dynamics, stimulate the self-org! anization of PAR proteins by providing local protection against a global barrier imposed by aPKC. View full text Figures at a glance * Figure 1: PAR-2 dynamics at symmetry breaking. () Schematic representation of an embryo showing the distribution of PAR-1 and PAR-2 (green), anterior PARs (brown) and MTOC microtubules (magenta). Zygotes are oriented with the posterior to the right in this and all figures. (–) Confocal microscopy images of fixed mlc-4(RNAi) zygotes stained for tubulin (magenta) and PAR-2 (green). Note that shows a cross-section as in the schematic representations in , whereas and show superficial cortical sections. Scale bar, 10 μm. () The timing of GFP::PAR-2 appearance on the posterior cortex in live mlc-4(RNAi) zygotes relative to nuclear envelope breakdown (NEBD). Each dot represents an individual zygote. 'No PAR-2' refers to zygotes for which PAR-2 never loaded on the cortex. 'tbg-1(RNAi) nocodazole' refers to zygotes depleted for γ-tubulin and treated with nocodazole. Error bars represent s.d. from 10 control zygotes and 9 tbg-1(RNAi) nocodazole zygotes with a cortical GFP::PAR-2 domain. () Graph showing the size of t! he GFP::PAR-2 domain scored at nuclear envelope breakdown. Error bars represent s.d. in zygotes with a cortical GFP::PAR-2 domain as in . See Supplementary Fig. S1d for images of zygotes used to compile data in and . * Figure 2: Microtubule binding protects PAR-2 from aPKC phosphorylation and allows PAR-2 to interact with phospholipids in the presence of aPKC. () Schematic representation of PAR-2. The pink areas are regions that contribute to microtubule binding in vitro (see Supplementary Fig. S3a). The cortical-localization domain is the region sufficient for localization to the posterior cortex in the presence of endogenous PAR-2 (ref. 12 and F. Motegi, unpublished observation). The black bars indicate seven potential PKC-3 phosphorylation sites12. Ser 241 is required for maximal phosphorylation in vitro by aPKC () and for cortical exclusion in vivo (Fig. 3a). 162KRR164 is the basic cluster mutated in the single-substitution mutants K162A and R163A, and 183RRR185 is the basic cluster mutated in the triple-substitution mutant R183–5A. () Percentage of recombinant PAR-2 that co-sedimented with microtubules. Error bars represent s.d. of three independent experiments. () Photomicrographs of recombinant GFP::PAR-2 mixed with rhodamine-labelled microtubules and spread on slides. GFP::PAR-2R183–5A does not label microtubules as ef! ficiently as wild-type GFP::PAR-2. Scale bar, 5 μm. () Percentage of phosphorylated PAR-2 with respect to time from the start of incubation with aPKC kinase in the presence (dotted lines) or absence (solid lines) of microtubules. PAR-2 phosphorylation was monitored by [γ-32P]ATP incorporation. Error bars represent s.d. of three independent experiments. () Phosphorylation by aPKC inhibits PAR-2 binding to phospholipids. GST::PAR-2 fusions pre-treated with or without aPKC were incubated with lipid strips and detected using an anti-GST antibody. Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) 50-pmol spots are shown (see Supplementary Fig. S7b for the full dilution series). The numbers represent the percentage of binding normalized to wild type (100%). Ser 241 is one of seven predicted aPKC sites. '7 PKC sites SE' is a phosphomimic mutant for all seven sites. () Binding to microtubules is sufficient t! o protect PAR-2 from aPKC and retain binding to phospholipids.! The same as in , but GST::PAR-2 fusions were incubated with microtubules before incubation with aPKC. See Supplementary Fig. S7c for the full dilution series. * Figure 3: Microtubule binding is required for PAR-2 to localize to the cortex in the absence of cortical flows. () Live zygotes expressing the indicated GFP::PAR-2 fusions: wild type and K162A bind microtubules, whereas R163A and R183–5A do not. The percentages indicate zygotes with cortical PAR-2; numbers are presented in Supplementary Table S1. ECT-2 is the GEF for the small GTPase RHO-1 (ref. 29). ect-2(ax751) zygotes lack MTOC-induced cortical flows, but develop PAR-2-dependent cortical flows during mitosis4. MAT-1 is a subunit of the anaphase-promoting complex. mat-1(ax227) zygotes arrest in meiosis and become transiently polarized without cortical flows under the influence of the acentriolar meiotic spindle7. SPD-5 is a MTOC component required for PCM assembly8. spd-5(RNAi) zygotes localize GFP::PAR-2 to both the anterior and posterior cortex under the influence of the meiotic spindle remnant (anterior) and the slow-maturing MTOC (posterior)18. RNAi depletion of PKC-3 or mutations in the PKC phosphorylation sites (either 7 PKC sites SA or S241A) cause all fusions to localize u! niformly to the cortex. Scale bar, 10 μm. () FRAP was carried out on the cortex of pkc-3(RNAi) zygotes expressing the indicated GFP::PAR-2 fusions. The graph shows the average recovery half-time (t1/2) from five separate zygotes. Error bars represent s.d. Fluorescence recovery was faster at the boundary (Out) than at the centre (In) of the bleached area, indicating that at least some of the recovery is due to lateral diffusion of cortical GFP::PAR-2, as shown in ref. 26 (schematic representation of bleached area is shown on the left, with areas in which recovery was measured as indicated). See Supplementary Fig. S5b for representative recovery curves. () Cortical PAR-2 stimulates its own recruitment to the cortex. Live zygotes expressing the indicated GFP::PAR-2 fusions. The arrows point to the boundaries of the cortical GFP::PAR-2 domain. Scale bar, 10 μm. In mlc-4(RNAi);par-2(RNAi) zygotes, wild-type PAR-2 localizes to the posterior cortex, but the microtubule-bindi! ng mutant R183–5A and the RING mutant C56S do not. Endogenou! s PAR-2 (PAR-2(+)) rescues the localization of both mutants. Rescue is also observed in par-1(RNAi) and par-1 mutant zygotes, in which PAR-3 and PKC-3 are never excluded from the posterior cortex (see Fig. 4a). * Figure 4: PAR-2 recruits PAR-1 to the cortex, leading to exclusion of anterior PARs. () mlc-4(RNAi) zygotes with indicated mutations in PAR proteins stained for PAR-2, PAR-1, PAR-3 and PKC-3. par-1(it51) contains a mutation (R409K) that inhibits kinase activity14, and par-1(b274) contains a premature stop (Q814Stop) that eliminates the PAR-1 cortical-localization domain15. GFP::PAR-3S251A S950A contains mutations in the conserved PAR-1 phosphorylation sites and rescues par-3(it71) zygotes competent for cortical flows16. GFP::PAR-2 fusions were co-stained with PAR-1. PAR-2 and PKC-3 or PAR-1 and PAR-3 were co-stained in the other zygotes. The arrows indicate the boundary of the PAR domains. Scale bar, 10 μm. () Immunoprecipitation experiment showing that PAR-2 and PAR-1 interact in embryo extracts. Extracts from embryos expressing the indicated GFP fusions were immunoprecipitated with anti-GFP beads and the immunoprecipitates were blotted with the indicated antibodies. * Figure 5: Microtubule binding by PAR-2 is required for efficient polarity initiation in wild-type embryos. () Fluorescence micrographs of fixed zygotes expressing GFP::PAR-2 and depleted for endogenous PAR-2 by RNAi. Zygotes are stained for GFP::PAR-2 (green), PAR-3 (magenta) and DNA (white) and are shown at symmetry breaking (top two rows) or at nuclear envelope breakdown (NEBD; bottom row). Scale bar, 10 μm. () Top, kymographs from time-lapse movies of live zygotes expressing GFP::PAR-2 fusions and depleted for endogenous PAR-2 by RNAi. Times are with respect to the onset of cytokinesis. Wild-type GFP::PAR-2 appears on the posterior cortex earlier than the microtubule-binding mutant GFP::PAR-2R183–5A. Wild-type GFP::PAR-2 also accumulates transiently (asterisk) on the anterior cortex (owing to the transient influence of the meiotic spindle remnant18; 5 of 5 zygotes). GFP::PAR-2R183–5A does not show this localization (0 of 5), consistent with polarization by the meiotic spindle depending primarily on microtubules7. Bottom, the graph shows the fluorescence intensity at the! posterior-most cortex averaged from five zygotes. Accumulation of GFP::PAR-2R183–5A is delayed when compared with wild-type GFP::PAR-2 (29.0±11.2 s, P=0.03) but catches up by nuclear envelope breakdown. Error bars represent s.d. from five separate zygotes. () Model for polarization of the C. elegans zygote. 1, PKC-3 phosphorylates PAR-2 (ref. 12) and PAR-1, keeping them off the cortex. 2, MTOC breaks symmetry through two parallel mechanisms: 2a, microtubules at the MTOC protect PAR-2 from phosphorylation by PKC-3, allowing a few molecules of PAR-2 to load on the cortex close to MTOC; 2b, MTOC induces cortical flows by an unknown mechanism involving local inhibition of actomyosin3. Flows displace anterior PARs, allowing PAR-2 to accumulate in their place. 3, Cortical PAR-2 recruits additional PAR-2 molecules to expand the PAR-2 domain. The RING finger of PAR-2 stabilizes PAR-2 at the cortex. 4, PAR-2 recruits PAR-1 by binding to the C terminus of PAR-1. 5, PAR-1 phosph! orylates PAR-3 preventing its association with the cortex. 6, ! Anterior PARs stimulate their own displacement by recruiting myosin to the cortex and upregulating cortical flows3, 4. Not shown in this figure is LGL, a non-essential player in this process, which similarly to PAR-1 localizes to the posterior cortex and antagonizes the cortical association of anterior PARs (refs 24, 25). Author information * Author information * Supplementary information Affiliations * Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Center for Cell Dynamics, Johns Hopkins University School of Medicine, 725 N. Wolfe St., PCTB 706, Baltimore, Maryland 21205, USA * Fumio Motegi, * Seth Zonies, * Yingsong Hao, * Adrian A. Cuenca, * Erik Griffin & * Geraldine Seydoux Contributions F.M. and G.S. designed the study and wrote the manuscript. S.Z. carried out experiments shown in Fig. 3b and Supplementary Fig. S5b, Y.H. A.A.C. and E.G. carried out experiments shown in Supplementary Fig. S8c,d and F.M. carried out all other experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Geraldine Seydoux Author Details * Fumio Motegi Search for this author in: * NPG journals * PubMed * Google Scholar * Seth Zonies Search for this author in: * NPG journals * PubMed * Google Scholar * Yingsong Hao Search for this author in: * NPG journals * PubMed * Google Scholar * Adrian A. Cuenca Search for this author in: * NPG journals * PubMed * Google Scholar * Erik Griffin Search for this author in: * NPG journals * PubMed * Google Scholar * Geraldine Seydoux Contact Geraldine Seydoux Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1900K) Supplementary Information Excel files * Supplementary Table 1 (25K) Supplementary Information Additional data - USP15 is a deubiquitylating enzyme for receptor-activated SMADs
- Nat Cell Biol 13(11):1368-1375 (2011)
Nature Cell Biology | Letter USP15 is a deubiquitylating enzyme for receptor-activated SMADs * Masafumi Inui1 * Andrea Manfrin1 * Anant Mamidi1 * Graziano Martello1 * Leonardo Morsut1 * Sandra Soligo1 * Elena Enzo1 * Stefano Moro2 * Simona Polo3 * Sirio Dupont1 * Michelangelo Cordenonsi1 * Stefano Piccolo1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:1368–1375Year published:(2011)DOI:doi:10.1038/ncb2346Received16 June 2011Accepted17 August 2011Published online25 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 The TGFβ pathway is critical for embryonic development and adult tissue homeostasis. On ligand stimulation, TGFβ and BMP receptors phosphorylate receptor-activated SMADs (R-SMADs), which then associate with SMAD4 to form a transcriptional complex that regulates gene expression through specific DNA recognition1, 2. Several ubiquitin ligases serve as inhibitors of R-SMADs3, 4, yet no deubiquitylating enzyme (DUB) for these molecules has so far been identified. This has left unexplored the possibility that ubiquitylation of R-SMADs is reversible and engaged in regulating SMAD function, in addition to degradation5. Here we identify USP15 as a DUB for R-SMADs. USP15 is required for TGFβ and BMP responses in mammalian cells and Xenopus embryos. At the biochemical level, USP15 primarily opposes R-SMAD monoubiquitylation, which targets the DNA-binding domains of R-SMADs and prevents promoter recognition. As such, USP15 is critical for the occupancy of endogenous target promoters ! by the SMAD complex. These data identify an additional layer of control by which the ubiquitin system regulates TGFβ biology. View full text Figures at a glance * Figure 1: USP15 is a DUB required for TGFβ and BMP gene responses. () Diagram of the screening procedure to identify regulators of TGFβ and BMP signalling. () Left panels: immunoblots for PAI1 and p21Waf1, whose induction by TGFβ is inhibited by two independent USP15 siRNAs in MDA-MB-231 cells (lane 4 has been cropped from the same blot). Right panels: effect of USP15 depletion (USP15 siRNA no 1), compared with SMAD4 depletion (positive control) in HaCaT cells. LAMINB and β-catenin serve as loading controls. Similar results were obtained on stable expression of short hairpin RNA (shRNA) (Supplementary Fig. S1f). () Real-time PCR analysis with reverse transcription showing fold induction of SMAD7 and ID2 messenger RNAs in MDA-MB-231 cells transfected with the indicated siRNAs and treated with BMP2 or dorsomorphin (a BMP-receptor inhibitor, used to limit the background from autocrinally expressed BMP ligands). Data are normalized to the effect of dorsomorphin and represent the mean of two independent experiments each with duplicate biologi! cal replicates. See Supplementary Fig. S6 for uncropped images of blots. * Figure 2: USP15 is required for TGFβ and BMP biological effects. () Left: representative fields of hMSC cells (transfected with control siRNA or USP15 siRNA no 1). GM, growth medium. ODM, hMSC osteodifferentiation medium, as visualized by alkaline phosphatase staining. Right: bars show quantification of osteogenesis by measuring alkaline phosphatase activity over the total number of cells. Bars indicate the mean and s.d. of at least five independent fields counting a total of at least 500 cells for each point of a representative experiment. () USP15 is required for TGFβ-induced growth arrest in HaCaT cells as assayed by 5-bromodeoxyuridine (BrdU) incorporation (representative pictures on the left). DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 50 μm. Right: the number of cells in S phase in untreated control cultures was given an arbitrary value of one and all other values are depicted relative to this. SMAD4 siRNA is a positive control. Data are means of two independent experiments with two biological replicates. () Representative p! ictures of control, SMAD4- or USP15-depleted MDA-MB-231 cells migrating into a scratch introduced in confluent monolayers. Dots indicate the edges of the wound at the beginning of the experiment. Similar results were obtained with USP15 siRNA no 2 (data not shown). SB, SB505124 TGFβ receptor inhibitor. () Panels show in situ hybridization of Xenopus embryos for the Spemann Organizer marker Chordin (early gastrula), the pan-mesodermal marker Xbra (mid-gastrula) and the ventral marker Sizzled (late gastrula). Upper panels are embryos injected with control Morpholinos (Co MO, 80 ng); embryos in lower panels are injected with USP15/USP4 Morpholinos (40 ng each). Dorsal is up; embryos are shown as vegetal views. See Supplementary Fig. S2 and Table S1 for effectiveness of Morpholino injection, phenotypic rescue with overexpressed R-SMADs and quantifications of the observed phenotypes. * Figure 3: USP15 is a DUB for R-SMADs. () Loss of USP15 does not affect phospho-SMAD3 or total SMAD levels. Note that USP15 levels are not affected by TGFβ (third panel) or BMP treatments (Supplementary Fig. S3c). () Co-immunoprecipitation of overexpressed USP15 with SMADs. Immunoprecipitation (IP) and immunoblotting (IB) were carried out from transfected HEK293T cells untreated (−) or treated (+) with BMP2 (lane 3) or TGFβ1 (lanes 5, 7 and 9). Inp., input. () An endogenous protein complex between USP15 and SMAD2. The asterisk indicates an aspecific band. () USP15 directly interacts with the MH1-linker domain of SMAD3 as shown by GST-pulldown of recombinant proteins. S3FL, SMAD3 full length. () R-SMADs are mono–oligoubiquitylated. Monoubiquitylated (Ub1–SMAD1, Ub1–SMAD3), diubiquitylated (Ub2) or polyubiquitylated isoforms are indicated. The asterisk indicates the IgG band. Note that the same ubiquitylation pattern of R-SMADs was obtained when the same experiments were repeated under stringent immunopre! cipitation conditions; moreover, HA-positive bands were also positive after re-probing with anti-Flag antibody (R-SMAD; data not shown). () Gain of USP15 reverts SMAD2 mono–diubiquitylation. Expression of USP15 (WT, lane3), but not enzymatically inactive USP15C269A (CA) or USP15C269S (CS) (lanes 4 and 5), deubiquitylates SMAD2. The two anti-HA panels represent longer and shorter exposures of the same blot. See Supplementary Fig. S3d,e, for similar results on SMAD1 and SMAD3. () USP15 depletion by transfection of two independent siRNAs, USP15 siRNA no 1 and no 2, enhances SMAD3 mono- and diubiquitylation, but has no effect on SMAD4 monoubiquitylation (Supplementary Fig. S3h). Monoubiquitylation does not lead to enhanced degradation of R-SMADs (Supplementary Fig. S3i). The asterisk indicates IgG. Co, control. () USP15 depletion promotes SMAD1 monoubiquitylation as well as oligo- and polyubiquitylation triggered by Smurf1 (lane 1 has been cropped from the same blot). U15, US! P15. () USP15 deubiquitylates SMAD3 in vitro. Upper panel: the! input is a preparation of Flag-affinity-purified SMAD3 from HEK293T cell lysates that contains monoubiquitylated SMAD3 (as visualized by anti-HA western blotting and molecular weight). This preparation was used as substrate for an in vitro deubiquitylation reaction (see Methods). Lower panel: V5-affinity-purified USP15 (WT), but not C269A mutant (CA), deubiquitylates SMAD3. See Supplementary Fig. S6 for uncropped images of blots. * Figure 4: R-SMAD activation and nuclear entry promote monoubiquitylation. () On nuclear (N)–cytoplasmic (C) fractionation of HEK293T cell lysates, monoubiquitylated SMAD3 is enriched in the nuclear fraction (see Supplementary Fig. S4a for loading controls). IP, immunoprecipitation; IB, immunoblot. () Increased monoubiquitylation in NLS–SMAD3 when compared with wild-type (WT) SMAD3. All lanes were from the same exposure of the same blot. () Decreased monoubiquitylation in NES–SMAD3 when compared with wild-type SMAD3. See Supplementary Fig. S4b for positive controls of the effectiveness of these tags. All lanes were from the same exposure of the same blot. () TGFβ treatment promotes SMAD3 monoubiquitylation. SB, SB505124 TGFβ receptor inhibitor. () BMP treatment promotes SMAD1 monoubiquitylation. () Phosphorylated SMAD3 is monoubiquitylated. () TGFβ treatment promotes SMAD3 monoubiquitylation, but not in SMAD4-depleted cells. Co, control; Sm4, SMAD4. () Localization of SMAD2/3 was visualized by immunofluorescence in HaCaT cells. In control ! cells, SMAD2/3 protein (red signal) localizes in both cytoplasm and nuclei, and accumulates into nuclei on TGFβ1 treatment. DAPI staining (blue) indicates nuclei. After a 1 h pulse of TGFβ treatment, cells were washed, incubated with the SB505124 TGFβ receptor inhibitor and fixed at the indicated times. Scale bar, 20 μm. See Supplementary Fig. S6 for uncropped images of blots. * Figure 5: USP15-dependent regulative SMAD3 ubiquitylation inhibits DNA binding. () DNA pulldown and immunoblot with anti-SMAD2/3 and anti-SMAD4 antibodies of the endogenous SMAD complex bound to DNA after TGFβ stimulation. SMAD–DNA association is detected in control cells but not in USP15-depleted cells. See Supplementary Fig. S5b for similar results obtained in the presence of the proteasome inhibitor MG132. WB, western blot. () Diagram of SMAD3 lysine mutants and their monoubiquitylation pattern. () Upper: structure of SMAD3 MH1 domain bound to DNA, as previously described19. The location of the three lysines found positive in the ubiquitylation mapping in is shown in yellow. Lower: in silico modelling of Lys 81-ubiquitylated SMAD3 (ubiquitin in green). For modelling of Lys 33, see Supplementary Fig. S5d. The functional significance of Lys 53 ubiquitylation remains to be determined. () The input is a purified preparation of monoubiquitylated SMAD3/non-ubiquitylated SMAD3 eluted as a 1:2 mix. Right: after DNA pulldown of this preparation, unmodified! SMAD3 binds to DNA, but monoubiquitylated SMAD3 does not. () Affinity-purified SMAD3 was subjected to in vitro ubiquitylation assay with purified Smurf2. Similar results were obtained with recombinant NEDD4 (not shown). () SMAD3 bound on SBE DNA (on streptavidin beads) was subjected to in vitro ubiquitylation reaction (see Methods). Lane 1: SMAD3 remains bound to the DNA beads after incubation in ubiquitylation buffer (without E3). Lane 2: SMAD3 dissociates from DNA on addition of a 1:1 mix of Smurf2 and NEDD4; similar results were obtained using individual enzymes (not shown). Lanes 3 and 4: western blots of the supernatants of DNA beads. Lane 3: unmodified SMAD3 passively diffusing from DNA during incubation. Lane 4: ubiquitylated SMAD3 dissociated from DNA. () GAL4–SMAD3 is insensitive to USP15 depletion but remains dependent on FAM (ref. 7). All error bars are s.d. Experiments were repeated three times with at least duplicate biological replicates. SB, SB505124 TGFβ! receptor inhibitor. () ChIP assays to measure the occupancy o! f the active SMAD complexes on their endogenous targets (two independent SBEs for the p21Waf1 promoter). Corresponding inputs are shown in Supplementary Fig. S5g. () A model for R-SMAD regulation by USP15. See Discussion. See Supplementary Fig. S6 for uncropped images of blots. Author information * Author information * Supplementary information Affiliations * Department of Medical Biotechnologies, Section of Histology and Embryology, University of Padua, viale G. Colombo 3, 35100 Padua, Italy * Masafumi Inui, * Andrea Manfrin, * Anant Mamidi, * Graziano Martello, * Leonardo Morsut, * Sandra Soligo, * Elena Enzo, * Sirio Dupont, * Michelangelo Cordenonsi & * Stefano Piccolo * Department of Pharmaceutical Sciences, Molecular Modelling Section, University of Padua, 35100 Padua, Italy * Stefano Moro * IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Istituto Europeo di Oncologia, 20139 Milan, Italy * Simona Polo Contributions M.I. and S.P. designed research; M.I. and A. Manfrin carried out all the biochemical and functional assays in cells and Xenopus embryos. A. Mamidi and S.D. purified SMADs and carried out in vitro binding assays; G.M., S.S. and M.C. helped with Xenopus assays; L.M. and E.E. helped with mesenchymal stem cells and immunofluorescence; S.M. carried out the modelling analysis; S.P. prepared recombinant E3 ligases; S.P. coordinated the work; M.I. and S.P. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stefano Piccolo Author Details * Masafumi Inui Search for this author in: * NPG journals * PubMed * Google Scholar * Andrea Manfrin Search for this author in: * NPG journals * PubMed * Google Scholar * Anant Mamidi Search for this author in: * NPG journals * PubMed * Google Scholar * Graziano Martello Search for this author in: * NPG journals * PubMed * Google Scholar * Leonardo Morsut Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra Soligo Search for this author in: * NPG journals * PubMed * Google Scholar * Elena Enzo Search for this author in: * NPG journals * PubMed * Google Scholar * Stefano Moro Search for this author in: * NPG journals * PubMed * Google Scholar * Simona Polo Search for this author in: * NPG journals * PubMed * Google Scholar * Sirio Dupont Search for this author in: * NPG journals * PubMed * Google Scholar * Michelangelo Cordenonsi Search for this author in: * NPG journals * PubMed * Google Scholar * Stefano Piccolo Contact Stefano Piccolo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1100K) Supplementary Information Additional data - The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks
- Nat Cell Biol 13(11):1376-1382 (2011)
Nature Cell Biology | Letter The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks * Mayura Meerang1, 2 * Danilo Ritz3, 4 * Shreya Paliwal5 * Zuzana Garajova1 * Matthias Bosshard2 * Niels Mailand6 * Pavel Janscak5 * Ulrich Hübscher2 * Hemmo Meyer3, 4 * Kristijan Ramadan1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:1376–1382Year published:(2011)DOI:doi:10.1038/ncb2367Received15 August 2011Accepted23 September 2011Published online23 October 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death1. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion2, 3. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood4, 5, 6, 7. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1–NPL4 and Lys-48-linked ubiquitin (K48–Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48–Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors ! critical for DNA repair and genome surveillance mechanisms3, 7, 8. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97–UFD1–NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability. View full text Figures at a glance * Figure 1: The p97–UFD1–NPL4 ATPase complex is involved in the response to DNA DSBs. () U2OS cells were depleted of indicated proteins by siRNA for 2 days or treated with non-silencing (NS) siRNA, exposed to 3 Gy of ionizing radiation (IR), fixed after 1 h or 8 h (recovery time) and stained with DAPI and antibodies against γ-H2AX. Note the persistence of γ-H2AX foci specifically in p97-, NPL4- and MMS21-depleted cells (Supplementary Fig. S1a shows the efficacy of depletion). () Quantification of the results in , showing the percentage of nuclei with an amount of γ-H2AX foci higher than the background. n=4 experiments with >500 nuclei per condition. (,) Stable U2OS cells were sensitized with BrdU (+BrdU) or not (−BrdU). Mild p97–myc expression was induced with doxycycline (+DOX) or left non-induced (−DOX), and confirmed by western blotting to be at a ratio of about 1:1 to endogenous p97 (). Cells were micro-irradiated with a moderate laser dose (55% energy output) to generate a discrete line of DNA DSBs (γ-H2AX positive), pre-extracted and fix! ed after 30 min. p97–myc and γ-H2AX were immunodetected by epifluorescence microscopy (). () Quantification of the results in ; n=4; >100 cells per condition. Error bars, s.e.m. Scale bars, 10 μm. An uncropped image of the blot is shown in Supplementary Fig. S8. * Figure 2: p97–UFD1–NPL4 is essential for DSB repair and survival after exposure to ionizing radiation. (,) Stable HEK293 cells were induced (+DOX) to express wild-type p97 () or dominant-negative p97EQ (), or left non-induced (−DOX) for 24 h, and exposed to 30 Gy of ionizing radiation (IR). Genomic DNA was analysed by mPFGE at the indicated times after exposure to ionizing radiation. l.c., loading control (short exposure of intact DNA). () U2OS cells were treated with NPL4 or non-silencing (NS) siRNA, irradiated and analysed as in ,. Note the persistent DNA fragments at late times following p97EQ expression or NPL4 depletion. () Readout of NHEJ (left) and homologous recombination (HR; right) reporter assays after treatment with indicated siRNAs; XRCC4 or RAD51 served as positive controls. (,) Colonigenic survival assay; sensitivity to ionizing radiation after p97, NPL4, UFD1 or MMS21 depletion for 2 days () or wild-type p97 or p97EQ induction for 24 h (). For –, n=3 experiments with triplicates for each condition; error bars, s.e.m. *P<0.05, **P<0.001 and ***P<0.000! 1. * Figure 3: p97 is recruited to DSB sites in an RNF8-dependent manner. (–) U2OS cells were treated with siRNA targeting indicated proteins before laser micro-irradiation (55% energy output) as in Fig. 1c, pre-extracted and fixed after 30 min. NS, non-silencing. γ-H2AX and K63–Ub (,) or myc–p97 (,) were immunolocalized with specific antibodies. The percentage of cells with K63–Ub chain or myc–p97 localizing to γ-H2AX lines was determined (,). n=3 experiments, >100 nuclei per condition. () Stable, inducible RNF8-shRNA-expressing U2OS cells were transiently co-transfected with various RNF8 shRNA-resistant Flag–RNF8 constructs and induced (+DOX) or left non-induced (−DOX). Cells were exposed to laser micro-irradiation and left to recover for 30 min, pre-extracted, fixed and stained with p97 specific antibody (Supplementary Fig. S4i). () Top, the amount of endogenous p97, presented here in arbitrary units (AU) of fluorescent intensity, on DSB lines was quantified in two independent experiments as described in Methods. n>70 cel! ls per condition and experiment. Bottom, western blotting shows the efficiency of RNF8 depletion and complementation with wild-type RNF8, RNF8 RING* (C403S, E3 ligase deficient) and RNF8 FHA* (R42A, deficient in binding to DSBs). Error bars, s.e.m. Scale bars, 10 μm. **P<0.001 and ***P<0.0001. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 4: p97 turns over RNF8-dependent K48–Ub conjugates at damage sites. () U2OS cells were treated with siRNA targeting the indicated proteins, before being laser micro-irradiated (55% energy output) as in Fig. 1c, pre-extracted and fixed after 30 min. NS, non-silencing. K48–Ub chains and γ-H2AX were immunolocalized with specific antibodies. () The accumulation of K48–Ub was quantified as fluorescence intensity (AU) on the DSB line in two independent experiments. n>70 cells per condition and experiment. (,) Stable U2OS cells were induced (+DOX, lower panel in ) to express p97EQ–myc or left non-induced (−DOX, upper panel in ) and analysed for the amount of K48–Ub on DSB sites () as described above in three independent experiments. (,) Stable HEK293 cells were induced (+DOX) to express Strep-tagged p97EQ or left uninduced (−DOX), treated with siRNA as indicated and exposed to 20 Gy of ionizing radiation (IR) or left untreated. Finally p97EQ, combined with endogenous p97 in about 1:1 ratio to form p97 hexamer (see double band), was! isolated over Strep-Tactin Sepharose beads with co-purified K48–Ub conjugates () and quantified in three independent experiments (). (,) p97EQ HEK293 cells were co-transfected with Flag–RNF8 wild type, induced and irradiated (20 Gy) as indicated. p97EQ complexes were isolated and associated RNF8 was analysed (). Complex formation of RNF8 with p97 was quantified in three independent experiments (). Error bars, s.e.m. Scale bars, 10 μm. *P<0.05, **P<0.001 and ***P<0.0001. # indicates that the difference between two groups is not significant. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 5: p97–UFD1–NPL4 governs BRCA1, 53BP1 and RAD51 localization to DSB sites. (–) U2OS cells were treated with the indicated siRNAs for 2 days and afterwards irradiated with 3 Gy of ionizing radiation (IR). NS, non-silencing. Cells were fixed after 1 h in the case of 53BP1 () and BRCA1 () or after 8 h in the case of RAD51 (). Recruitment of indicated proteins to γ-H2AX foci was analysed by confocal microscopy (,,). BRCA1 was assayed only in S/G2, as confirmed by cyclin A staining (red pan-nuclear signal). Note the aberrant 53BP1 and BRCA1 relocalization after exposure to ionizing radiation, classified in two categories: a complete pan-nuclear distribution without foci localization in some p97-depleted cells comparable to RNF8-depleted cells, and a partial pan-nuclear localization with irregular foci formation in NPL4- and some p97-depleted cells (Supplementary Fig. S6e). RAD51 foci formation was completely abolished in p97- or NPL4-depleted cells. The percentage of cells with proper 53BP1 or BRCA1 distribution or formation of RAD51 foci was d! etermined (,,). In addition, the effect on recruitment was compared with MG132 treatment for 90 min before exposure to ionizing radiation. n=3 experiments, >100 nuclei per condition. () Readout of homologous recombination (HR) and NHEJ reporter assays after treatment with indicated siRNAs. XRCC4 or RAD51 served as positive controls. n=3 experiments with triplicates for each condition; error bars, s.e.m. *P<0.05, **P<0.001 and ***P<0.0001. Scale bars, 10 μm. Author information * Author information * Supplementary information Affiliations * Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Winterthurerstrasse 260, 8057 Zürich, Switzerland * Mayura Meerang, * Zuzana Garajova & * Kristijan Ramadan * Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland * Mayura Meerang, * Matthias Bosshard & * Ulrich Hübscher * Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany * Danilo Ritz & * Hemmo Meyer * Institute of Biochemistry, ETH Zürich, Schafmattstrasse 18, 8093, Zürich, Switzerland * Danilo Ritz & * Hemmo Meyer * Institute of Molecular Cancer Research, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland * Shreya Paliwal & * Pavel Janscak * Ubiquitin Signaling Group, Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark * Niels Mailand Contributions M.M. carried out most of the experiments in the manuscript. D.R. created inducible stable HEK293 cell lines expressing wild-type p97 or p97EQ. S.P. carried out NHEJ and homologous recombination reporter assays. Z.G. carried out the K48–Ub immunofluorescence microscopy study. M.B. carried out FACS analysis. N.M. provided the stable transfected RNF8-shRNA-expressing U2OS cell line. K.R. initiated the project, carried out mPFGE experiments and conceived the study. K.R. and H.M. designed the experiments and wrote the manuscript. All authors discussed the experiments and gave suggestions for the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Hemmo Meyer or * Kristijan Ramadan Author Details * Mayura Meerang Search for this author in: * NPG journals * PubMed * Google Scholar * Danilo Ritz Search for this author in: * NPG journals * PubMed * Google Scholar * Shreya Paliwal Search for this author in: * NPG journals * PubMed * Google Scholar * Zuzana Garajova Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Bosshard Search for this author in: * NPG journals * PubMed * Google Scholar * Niels Mailand Search for this author in: * NPG journals * PubMed * Google Scholar * Pavel Janscak Search for this author in: * NPG journals * PubMed * Google Scholar * Ulrich Hübscher Search for this author in: * NPG journals * PubMed * Google Scholar * Hemmo Meyer Contact Hemmo Meyer Search for this author in: * NPG journals * PubMed * Google Scholar * Kristijan Ramadan Contact Kristijan Ramadan Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase
- Nat Cell Biol 13(11):1383 (2011)
Nature Cell Biology | Corrigendum Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase * Kambiz N. Alavian * Hongmei Li * Leon Collis * Laura Bonanni * Lu Zeng * Silvio Sacchetti * Emma Lazrove * Panah Nabili * Benjamin Flaherty * Morven Graham * Yingbei Chen * Shanta M. Messerli * Maria A. Mariggio * Christoph Rahner * Ewan McNay * Gordon C. Shore * Peter J. S. Smith * J. Marie Hardwick * Elizabeth A. JonasJournal name:Nature Cell BiologyVolume: 13,Page:1383Year published:(2011)DOI:doi:10.1038/ncb2369Published online02 November 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, 1224–1233 (2011); published online 18 September 2011; corrected after print 27 September 2011 In the version of this article initially published online and in print, the affiliation denoted by number 4 was incorrect. The correct affiliation is: 4Department of Neuroscience and Imaging, and Ce.S.I. Aging Research Center, Università G.D'Annunzio of Chieti-Pescara, I-66013 Chieti, Italy. This error has been corrected online in both the HTML and PDF versions of the article. Additional data 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 Search for this author in: * NPG journals * PubMed * Google Scholar
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