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• Reviewing refereeing
  - ncb 13(2):109 (2011)
  Nature Cell Biology | Editorial Reviewing refereeing Journal name:Nature Cell BiologyVolume: 13,Page:109Year published:(2011)DOI:doi:10.1038/ncb0211-109Published online01 February 2011 Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Considered and critical assessment of a manuscript is essential to peer review and the publication process, but what makes a good referee report? We highlight the central elements of the ideal referee report. View full text Read the full article * FREE access with registration Register now * Already have a Nature.com account? Login Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• RNA seeds nuclear bodies
  - ncb 13(2):110-112 (2011)
  Nature Cell Biology | News and Views RNA seeds nuclear bodies * Maria Carmo-Fonseca1 Contact Maria Carmo-Fonseca Search for this author in: * NPG journals * PubMed * Google Scholar * José Rino1 Contact José Rino Search for this author in: * NPG journals * PubMed * Google Scholar * AffiliationsJournal name:Nature Cell BiologyVolume: 13,Pages:110–112Year published:(2011)DOI:doi:10.1038/ncb0211-110Published online01 February 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. The interior of the eukaryotic cell nucleus is populated by a multitude of microscopic domains termed nuclear bodies. Despite having attracted much attention, how these compartments form and are maintained remained elusive. Now, two live-cell imaging studies provide compelling evidence that nascent RNAs can act as transiently immobilized scaffolds that recruit specific nuclear body proteins. View full text Figures at a glance * Figure 1: Tethering specific proteins and RNAs to a genomic locus. () A LacO array consisting of multiple copies of 256 repeats of the LacO binding sequence is stably integrated at a single site in the genome. The site of integration is visualized through binding of the Lac repressor protein (LacI; grey) fused to enhanced green fluorescence protein (EGFP). () A specific RNA tagged with a MS2 stem loop is immobilized through binding to MS2 coat protein fused to LacI and visualized by FISH. () A protein of interest (X) fused to LacI becomes immobilized at the LacO array. * Figure 2: Alternative models for nuclear body assembly. Left: according to the random self-organization model, a multi-component complex assembles from stochastic interactions between individual subunits. Right: in contrast, an ordered assembly model posits that specific subunits are recruited to the complex in a stepwise manner. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Maria Carmo-Fonseca and José Rino are in the Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal. carmo.fonseca@fm.ul.pt Competing financial interests The authors declare no competing financial interests. Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Proclaiming fate in the early mouse embryo
  - ncb 13(2):112-114 (2011)
  Nature Cell Biology | News and Views Proclaiming fate in the early mouse embryo * Magdalena Zernicka-Goetz1 Contact Magdalena Zernicka-Goetz Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature Cell BiologyVolume: 13,Pages:112–114Year published:(2011)DOI:doi:10.1038/ncb0211-112Published online01 February 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. In the mouse embryo, the first differences between cells that result in distinct lineages have long been thought to arise only as a consequence of differential cell positioning at relatively late preimplantation stages. Differences in Oct4 transcription factor kinetics between cells at the 4–8-cell stage are now shown to be predictive of future lineages, providing further evidence for much earlier initiation of cell fate decisions. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Magdalena Zernicka-Goetz is in the Gurdon Institute, The Henry Wellcome Building of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK. m.zernicka-goetz@gurdon.cam.ac.uk Competing financial interests The author declares no competing financial interests. Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Research highlights
  - ncb 13(2):115 (2011)
  Nature Cell Biology | Research Highlights Research highlights Journal name:Nature Cell BiologyVolume: 13,Page:115Year published:(2011)DOI:doi:10.1038/ncb0211-115Published online01 February 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Inhibitor of apoptosis (IAP) proteins act as ubiquitin-E3 ligases and regulate caspase activity through ubiquitin-mediated proteasomal degradation; deubiquitylating enzymes (DUBs) can reverse the activity of ubiquitin and ubiquitin-like (UBL) proteins, such as NEDD8. In transgenic Drosophila expressing IAP antagonists to induce apoptosis, Broemer et al. have now systemically knocked down individual DUBs using RNAi and identified three NEDD8-specific proteases that, when knocked down, suppress cell death in vivo (Mol. Cell40, 810–812; 2010). Apoptosis was reduced in the null mutants of one of these genes, Deneddylase 1 (DEN1), and the effector caspase drICE was found to be neddylated in vivo, which suggested a role for NEDD8 in apoptosis. Interestingly, UV-mediated reduction in Drosphila IAP 1 (DIAP1) protein levels reduced neddylation of drICE and induced apoptosis, indicating that DIAP1 is a NEDD8-E3 ligase for drICE. Further investigation showed that DIAP1 neddylates drI! CE in a RING- and binding-dependent manner leading to reduced drICE proteolytic activity, and further mass spectrometry analysis identified nine lysine residues in drICE as sites for neddylation. As XIAP, a mammalian IAP, also promoted neddylation of caspase 7 in a RING-dependent manner, IAP-mediated neddylation seems to be evolutionarily conserved. This study demonstrates that IAPs can function as E3 ligases for NEDD8 as well as for ubiquitin and extends the complexity of IAP-mediated signalling. IO View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Oct4 kinetics predict cell lineage patterning in the early mammalian embryo
  - ncb 13(2):117-123 (2011)
  Nature Cell Biology | Article Oct4 kinetics predict cell lineage patterning in the early mammalian embryo * Nicolas Plachta1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tobias Bollenbach2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Shirley Pease1 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott E. Fraser1 Search for this author in: * NPG journals * PubMed * Google Scholar * Periklis Pantazis1 Contact Periklis Pantazis Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:117–123Year published:(2011)DOI:doi:10.1038/ncb2154Received09 September 2010Accepted07 November 2010Published online23 January 2011Corrected online28 January 2011 Abstract * Abstract * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Transcription factors are central to sustaining pluripotency, yet little is known about transcription factor dynamics in defining pluripotency in the early mammalian embryo. Here, we establish a fluorescence decay after photoactivation (FDAP) assay to quantitatively study the kinetic behaviour of Oct4, a key transcription factor controlling pre-implantation development in the mouse embryo. FDAP measurements reveal that each cell in a developing embryo shows one of two distinct Oct4 kinetics, before there are any morphologically distinguishable differences or outward signs of lineage patterning. The differences revealed by FDAP are due to differences in the accessibility of Oct4 to its DNA binding sites in the nucleus. Lineage tracing of the cells in the two distinct sub-populations demonstrates that the Oct4 kinetics predict lineages of the early embryo. Cells with slower Oct4 kinetics are more likely to give rise to the pluripotent cell lineage that contributes to the inner! cell mass. Those with faster Oct4 kinetics contribute mostly to the extra-embryonic lineage. Our findings identify Oct4 kinetics, rather than differences in total transcription factor expression levels, as a predictive measure of developmental cell lineage patterning in the early mouse embryo. View full text Figures at a glance * Figure 1: Selective photoactivation in live mouse embryos allows imaging of Oct4–paGFP kinetic behaviours. () Schematic diagram of photoactivation of paGFP (green) within a defined volume (grey sphere) using 820 nm light. () Photoactivation of cytoplasmic (cytopl) paGFP within a small ROI (red circle) localized to one cell of a live 8-cell stage embryo. Following photoactivation paGFP fluorescence spreads throughout the cytoplasm of the photoactivated cell. () Photoactivation of Oct4–paGFP within a ROI localized to a single cell nucleus of the embryo. () Schematic representation of the FDAP assay. Following photoactivation within a single cell nucleus, Oct4–paGFP fluorescence (green) is tracked in four dimensions with confocal time-lapse imaging. () FDAP analysis. The photoactivated Oct4–paGFP (green in simplified model, left) is initially in the nucleus and then moves from the nucleus to the cytoplasm. The parameters in, out and deg are the rates of Oct4–paGFP import into the nucleus from the cytoplasm, export from the nucleus to the cytoplasm, and the rate of overall de! gradation within the entire cell, respectively. 1−μ denotes the immobile fraction of nuclear Oct4-paGFP. Theoretical time development of the average Oct4-paGFP fluorescence profile (I/Io; normalized to the initial level of nuclear Oct4–paGFP fluorescence) in a FDAP experiment. () Representative FDAP time-lapse images showing Oct4–paGFP fluorescence over time in the cell nucleus photoactivated in . Scale bar, 10 μm. * Figure 2: Oct4–paGFP kinetic behaviours identify two cell populations in the mouse embryo. () Examples of a pre-compaction embryo showing two photoactivated cell nuclei. Scale bar corresponds to 15 μm. () FDAP curves show average fluorescence intensity of Oct4–paGFP within the cell nuclei photoactivated in over time. Green lines are exponential fits to fluorescence data. () FDAP curves for Oct4–paGFP obtained from several cell nuclei of pre-compaction embryos combining 4-cell stage and 8-cell stage data. Cells show two distinct kinetic behaviours, divided into clusters 1 and 2. () FDAP curves for Oct4ΔHD–paGFP at pre-compaction stages. () FDAP curves for Oct4–paGFP in cells that overexpress Sox17 at pre-compaction stages. () Quantification of Oct4–paGFP nuclear export, import and immobile fraction. Cl 1, cluster 1; Cl 2, cluster 2; ΔHD, Oct4ΔHD–paGFP; Sox17, Sox17 overexpressed. In , Cl 1, n = 11; Cl 2, n = 5; ΔHD, n = 6; Sox 17, n = 15. Asterisks show statistically significant differences (export, P < 0.015; import, P < 0.006, immobile P < 1 × 1! 0−5). Error bars show standard deviations. * Figure 3: Oct4–paGFP kinetics are uncorrelated to total fluorescence in the cell nucleus. Relation between the initial level of Oct4–paGFP fluorescence within the cell nucleus and the export rate, import rate and immobile fraction values obtained using FDAP analysis for single cells from Fig. 2d. The initial values of Oct4–paGFP fluorescence are unrelated to any of the kinetic parameters. * Figure 4: Oct4–paGFP kinetics predict patterning of inside and outside cells. () Schematic representation of the experimental strategy used. Following FDAP analysis and photoactivation of a membrane-targeted paGFP, embryos undergo compaction and the pattern of cell division is determined. () Examples of two pre-compaction embryos (left panels) show Oct4–paGFP fluorescence following photoactivation of single cell nuclei, and mpaGFP fluorescence following photoactivation of part of the membrane. The FDAP curves show the distinct Oct4–paGFP kinetic behaviours in each cell. Following compaction and division (right panels), cell 1 (top row) divided asymmetrically, generating one inside and one outside cell. Cell 2 (lower row) divided symmetrically, generating two outside cells. Surface rendered three-dimensional views confirm that the inside cell is buried within the embryo whereas outside cells have part of their surface exposed to the outer region. A membrane-targeted RFP (pink) marks half of the cells in these embryos. The FDAP curves on the right s! how the distinct Oct4–paGFP kinetic behaviours in each daughter cell of the original photoactivated cell. Scale bar, 15 μm. () Quantification of symmetric and asymmetric divisions undertaken following compaction by cells that showed the FDAP profiles of clusters 1 and 2 at pre-compaction stages. () FDAP curves show Oct4–paGFP kinetic behaviours in several inside and outside cells at post-compaction stages. () Quantification of Oct4–paGFP nuclear export, import and immobile fraction. Asterisks show statistical significant differences (export, P = 0.0012; import, P = 1.4737 × 10−4, immobile P = 1.4154 × 10−5). Error bars show standard deviations. In , , inside cells, n = 9; outside cells, n = 6. * Figure 5: Schematic illustration of Oct4–paGFP kinetics and cell lineage allocation in the early mouse embryo. Different accessibility of Oct4 DNA binding sites among cells, possibly due to a differential chromatin structure, the presence of an excess of another factor that blocks access or the absence of a cofactor required for high-affinity binding, results in segregation of Oct4–paGFP kinetic properties before lineage allocation. Cells with slower kinetics and a large immobile fraction divide more frequently in an asymmetric manner during the 8- to 16-cell transition, contributing more cells to the pluripotent cell lineage, whereas cells with faster kinetics and a small immobile fraction contribute more cells to the extra-embryonic lineage through symmetric divisions. Change history * Abstract * Change history * Author information * Supplementary informationErratum 28 January 2011In the version of this article initially published online and in print, the values for kout and kin in table 1 were incorrect. This error has been corrected in both the HTML and PDF versions of the article. Author information * Abstract * Change history * Author information * Supplementary information Affiliations * Division of Biology, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125, USA. * Nicolas Plachta, * Shirley Pease, * Scott E. Fraser & * Periklis Pantazis * Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. * Tobias Bollenbach * Present address: Institute of Science and Technology Austria, Am Campus 1, A-3400 Klosterneuburg, Austria. * Tobias Bollenbach Contributions T.B. performed the quantitative analysis. S.P. performed microinjections into mouse embryos. N.P., S.E.F. and P.P. designed and N.P. and P.P. carried out all other experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Periklis Pantazis Supplementary information * Abstract * Change history * Author information * Supplementary information Movies * Supplementary Movie 1 (41K) Supplementary Information * Supplementary Movie 2 (26K) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data
• PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1
  - ncb 13(2):124-131 (2011)
  Nature Cell Biology | Article PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1 * Wenqiang Tang1, 2, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Min Yuan1, 2, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Ruiju Wang2, 3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Yihong Yang1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Chunming Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Juan A. Oses-Prieto4 Search for this author in: * NPG journals * PubMed * Google Scholar * Tae-Wuk Kim2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hong-Wei Zhou5 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhiping Deng2 Search for this author in: * NPG journals * PubMed * Google Scholar * Srinivas S. Gampala2 Search for this author in: * NPG journals * PubMed * Google Scholar * Joshua M. Gendron2 Search for this author in: * NPG journals * PubMed * Google Scholar * Else M. Jonassen6 Search for this author in: * NPG journals * PubMed * Google Scholar * Cathrine Lillo6 Search for this author in: * NPG journals * PubMed * Google Scholar * Alison DeLong5 Search for this author in: * NPG journals * PubMed * Google Scholar * Alma L. Burlingame4 Search for this author in: * NPG journals * PubMed * Google Scholar * Ying Sun1, 2 Contact Ying Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Zhi-Yong Wang2 Contact Zhi-Yong Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:124–131Year published:(2011)DOI:doi:10.1038/ncb2151Received01 March 2010Accepted17 November 2010Published online23 January 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 When brassinosteroid levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. Brassinosteroid promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here, using tandem affinity purification, we identified protein phosphatase 2A (PP2A) as a BZR1-interacting protein. Genetic analyses demonstrated a positive role for PP2A in brassinosteroid signalling and BZR1 dephosphorylation. Members of the B' regulatory subunits of PP2A directly interact with BZR1's putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function for PP2A in dephosphorylating and activating BZR1 and completes the set of core components of t! he brassinosteroid-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus. View full text Figures at a glance * Figure 1: Identification of PP2A as a BZR1-interacting phosphatase. () Yeast two-hybrid assays showing the interaction between BZR1 and PP2A B′ family members. () Gel blots of phosphorylated (pBZR1) and unphosphorylated (BZR1) MBP–BZR1 were probed with GST–PP2AB′α (B′α) or GST–BIN2 (BIN2) followed by horseradish-peroxidase-labelled anti-GST antibody, or stained with Ponceau S (Stain) to show equal loading. () BiFC assays showing that BZR1 interacts with PP2A B′α, B′β and B′η, but not B′ε, in Nicotiana benthamiana leaf epidermal cells. Left column shows bright field and right column BiFC fluorescence. Scale bars, 50 μm. () Co-immunoprecipitation (IP) of BZR1 with PP2A. Wild-type (Col) Arabidopsis plants, or plants expressing BZR1::BZR1–CFP were treated with 100 nM brassinolide (BL) or mock solution for 15 min. BZR1–CFP was immunoprecipitated using anti-YFP antibody, and the immunoblots were probed with antibodies against YFP or the PP2A C subunits. Full scans of immunoblots are shown in Supplementary Fig. ! S9. * Figure 2: Overexpression of PP2AB′α and PP2AB′β activates brassinosteroid signalling and partially suppresses brassinosteroid signalling mutants. () Wild-type (Col), bzr1-1D, and seedlings overexpressing PP2AB′α–YFP (B′α-OX) or PP2AB′β–YFP (B′β-OX) fusion protein in the Col background were grown in the dark for 4 days on normal Murashige and Skoog medium (MS) or Murashige and Skoog medium containing 2 μM brassinazole (BRZ). Scale bar, 5 mm. () Overexpression of PP2AB′α–YFP and PP2AB′β–YFP partially suppresses the bri1-5 mutant. Wild type (WS), bri1-5, and bri1-5 transformed with35S:: PP2AB′α–YFP (B′α-OX) or PP2AB′β–YFP (B′β-OX) were grown in the dark for 4 days. Scale bar, 5 mm. Inset: Anti-YFP immunoblot (upper band) of the transgenic plants and Ponceau S staining as loading control (lower band). () Expression levels of the CPD gene in seedlings shown in were analysed by qRT-PCR. Values shown indicate fold-increase over normalized wild-type (WS) expression levels. The difference between bri1-5 and B′-OX or wild-type plants is statistically significant (P<0.05,n=3! ). Error bars represent s.e.m. () Four-week-old light-grown plants of wild type (WS), bri1-5, and bri1-5 overexpressing PP2AB′β (B′β OX/bri1-5). Scale bar, 10 mm. () Immunoblot analysis of BZR1 in transgenic plants overexpressing PP2AB′α or PP2AB′β in the bri1-5 mutant or wild-type background. Arrows mark the phosphorylated (+P) and unphosphorylated (−P) BZR1, and the asterisk marks a non-specific band that serves as loading reference. Full scan of immunoblot is shown in Supplementary Fig. S9. () Overexpression of PP2AB′β–YFP partially suppresses the homozygous bri1-116 null mutant. Scale bar, 10 mm. () Overexpression of PP2AB′β–YFP partially suppresses the homozygous bin2-1 mutant phenotype. The bin2-1 mutant was crossed with the bzr1-1D mutant and with transgenic plants overexpressing PP2AB′β, BSK3 and BSU1. Scale bar, 10 mm. * Figure 3: PP2A is essential for brassinosteroid signalling and BZR1 dephosphorylation. () A wild-type plant and two pp2ab′αβ double-mutant plants grown in the light for four weeks. Scale bar, 10 mm. () Immunoblotting analysis of phosphorylated (+P) and unphosphorylated (−P) BZR1 in plants shown in , using the anti-BZR1 antibody. St, Ponceau S staining of blot shows loading. The ratio between phosphorylated and unphosphorylated BZR1 bands (+P/−P) and the level of unphosphorylated BZR1 relative to that observed in the wild type (−P%) were calculated after normalization against the intensity of Ponceau S staining and are shown beneath the gel images. () Six-day-old dark-grown seedlings of wild type and the pp2ab′αβ double mutant with short hypocotyls. Scale bar, 5 mm. () Protein extracts were isolated from seven-day-old BZR1::BZR1–CFP transgenic Arabidopsis seedlings grown in the absence (−OA) or presence (+OA) of 250 nM okadaic acid and treated with 100 nM brassinolide (BL) for 30 min. After SDS–PAGE and transfer, the immunoblot was! probed with anti-YFP antibody. +P/−P, ratio between phosphorylated and unphosphorylated BZR1. () Nine-day-old light-grown seedlings of homozygous bin2-1 mutant were treated with mock solution or 1 μM okadaic acid for 1 h, then with 50 μM bikinin for 15 min. BZR1 phosphorylation was analysed by immunoblotting using the anti-BZR1 antibody. +P/−P, ratio between phosphorylated and unphosphorylated BZR1. Full scans of immunoblots are shown in Supplementary Fig. S9. * Figure 4: PP2A B′ subunit binds to the PEST domain of BZR1 to promote BZR1 dephosphorylation in vivo. () Sequence of the BZR1 region (aligned with the corresponding sequences of BZR2 and rice OsBZR1; conserved residues are highlighted by black background) that contains the PP2A-binding domain and the bzr1-1D, bzs247 and bzs248 mutations (amino-acid substitutions are indicated). () Yeast two-hybrid assay of PP2A binding by various fragments of BZR1 shown by box diagrams. Yeast growth on adenine and histidine dropout (−AH) medium indicates interaction between the BZR1 fragment and PP2AB′α. () Deletion of the PP2A-binding domain abolishes brassinosteroid-induced BZR1 dephosphorylation in plants. Two-week-old plants of a 35S::BZR1–YFP transgenic line expressing the wild-type BZR1–YFP, or three independent transgenic lines expressing the mutant BZR1–YFP containing deletion of amino acids 232–251 (ΔPEST-A, -B and -C lines), were treated with mock solution or 250 nM brassinolide (BL) for 1 h. The BZR1–YFP proteins were analysed by immunoblotting using anti-YFP a! ntibody (top) and the blot was stained with Ponceau S (Stain; bottom). () The bzr1-1D mutation increases dephosphorylation of BZR1 in planta. Top: immunoblot of BZR1/bzr1-1D protein extracted from wild-type (BZR1) and bzr1-1D mutant plants. In each sample, the upper band is phosphorylated (pBZR1) and the lower band is unphosphorylated (BZR1) protein. Bottom: Stain; Ponceau S staining shows equal loading. () PP2A binds more strongly to the bzr1-1D protein. Top: MBP–BZR1 and MBP–bzr1-1D proteins were gel blotted on a nitrocellulose membrane and probed sequentially with GST–PP2AB′α and anti-GST antibody. Bottom: Ponceau S staining of the gel blot. () From left to right are wild type, bzr1-1D, bzr1-1D bzs247 and bzr1-1D bzs248 seedlings grown in the dark on 2 μM BRZ for 5 days. Scale bar, 2.5 mm. () Anti-BZR1 immunoblot shows the phosphorylated BZR1 (pBZR1) and unphosphorylated BZR1 (BZR1) in the plants shown in . () The bzs mutations reduce PP2A binding. Equal am! ounts of MBP, MBP–BZR1, MBP–bzr1-1D, and MBP–bzr1-1D con! taining bzs247 and bzs248 mutations were gel blotted on nitrocellulose membrane and probed with GST–PP2AB′α or GST–BIN2 and anti-GST antibody, and the blots were subsequently stained with Ponceau S (Stain). Full scans of immunoblots are shown in Supplementary Fig. S9. * Figure 5: PP2A dephosphorylates BZR1 and abolishes its binding by the 14-3-3 proteins. () Top: MBP–BZR1 protein was phosphorylated by BIN2 using 32P-γATP, and then incubated at 30 °C for 3 h with PP2A (RCN1–YFP, PP2AA3–YFP) or BSU1–YFP, which were immunoprecipitated from brassinolide (BL)-treated (+, 1 h) or untreated (−) transgenic Arabidopsis plants. The control reaction used anti-YFP immunoprecipitate from non-transgenic plants. Bottom: Coomassie Blue staining of the precipitated YFP fusion proteins. () Recombinant MBP–BZR1 was phosphorylated by BIN2 in vitro (+BIN2) or left unphosphorylated (−BIN2), and then incubated with anti-YFP immunoprecipitate from wild-type control plants (Col) or from the 35S::PP2AB′β–YFP transgenic plants (B′β) for 3.5 h in the presence of 30 μM bikinin. The proteins were separated by SDS–PAGE, blotted, and incubated with GST–14-3-3 protein plus anti-GST antibody to detect binding by 14-3-3, or with anti-MBP antibody to detect phosphorylated (+P) and unphosphorylated (−P) MBP–BZR1. () Pre! -incubation of BIN2-phosphorylated BZR1 with the 14-3-3 protein did not interfere with its dephosphorylation by PP2A. BIN2-phosphorylated BZR1 was pre-incubated with GST or GST–14-3-3 for 1 h and then dephosphorylated by anti-YFP immunoprecipitation product from Col control (Col) or 35S::PP2A-B′β–YFP (B′β) for another 1 h. Phosphorylated (+P) and unphosphorylated (−P) BZR1 are marked by arrows. Full scans of immunoblots are shown in Supplementary Fig. S9. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Wenqiang Tang, * Min Yuan, * Ruiju Wang & * Yihong Yang Affiliations * Institute of Molecular Cell Biology, College of Life Science, Hebei Normal University, Shijiazhuang, Hebei, 050016, China * Wenqiang Tang, * Min Yuan, * Yihong Yang, * Chunming Wang & * Ying Sun * Department of Plant Biology, Carnegie Institution of Washington, Stanford, California 94305, USA * Wenqiang Tang, * Min Yuan, * Ruiju Wang, * Tae-Wuk Kim, * Zhiping Deng, * Srinivas S. Gampala, * Joshua M. Gendron, * Ying Sun & * Zhi-Yong Wang * Institute of Molecular Biology, College of Life Science, Nankai University, Tianjin 300071, China * Ruiju Wang * Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA * Juan A. Oses-Prieto & * Alma L. Burlingame * Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 02912, USA * Hong-Wei Zhou & * Alison DeLong * Centre for Organelle Research, University of Stavanger, N-4036, Norway * Else M. Jonassen & * Cathrine Lillo Contributions Z-Y.W. and Y.S. conceived the project and designed the experiments. C.W., M.Y. and Y.S. carried out BZR1 complex purification. Z.D. carried out in-gel digestion, and J.A.O-P. and A.L.B. did the mass spectrometry analysis (Supplementary Fig. S2). W.T. contributed data in Figs 1b,d, 2a–g, 3d, 4d,e,h and Supplementary Figs S4 and S5. M.Y. contributed data in Figs 1c, 2e, 3a–c,e, 4b,c,g, 5b,c and Supplementary Fig. S8. R.W. made contributions to Figs 1a,b, 2a–g, 3d, 4d,e,h and Supplementary Figs S4 and S5. Y.Y. contributed to Fig. 1a, Supplementary Fig. S3, Fig. 4b and Supplementary Fig. S7. T-W.K. contributed Fig. 5a. H-W.Z. and A.D.L. provided the YFP fusions to RCN1 and PP2AA3, the rcn1 pp2aa3 mutant and the anti-PP2AA antibodies. S.S.G. and J.M.G. identified the bzs247 and bzs248 mutants (Fig. 4f). E.M.J. and C.L. provided a pp2ab′αβ mutant and qRT-PCR data (Supplementary Fig. S6). Z-Y.W. and Y.S. wrote the paper together with W.T., M.Y. and R.W. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Ying Sun or * Zhi-Yong Wang Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (800K) Supplementary Information Additional data
• AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1
  - ncb 13(2):132-141 (2011)
  Nature Cell Biology | Article AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1 * Joungmok Kim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mondira Kundu2 Search for this author in: * NPG journals * PubMed * Google Scholar * Benoit Viollet3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kun-Liang Guan1 Contact Kun-Liang Guan Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:132–141Year published:(2011)DOI:doi:10.1038/ncb2152Received05 May 2010Accepted06 December 2010Published online23 January 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 is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction ! between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling. View full text Figures at a glance * Figure 1: Glucose starvation activates Ulk1 protein kinase through AMPK-dependent phosphorylation. () HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed. Proteins were resolved by SDS–PAGE and visualized with autoradiography (top) or western blotting (WB; bottom). () Cells were incubated in glucose-free medium (4 h) as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated. Endogenous Ulk1 mobility was examined by western blotting. () HA–Ulk1 was transfected into HEK293 cells together with wild-type (WT) AMPKα1 or a kinase-dead (DN) mutant. Cells were starved of glucose (4 h; Glu) or amino acids (–A.A) and treated with compound C (20 μM, C.C) as indicated. Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting. () HA–Ulk1 proteins were immunopurified from transfected HEK293 cells, which had undergone glucose starvation (4 h) as indicated. The HA–Ulk1 proteins were treated with λ PPase, and! in vitro kinase assays were performed in the presence of GST–ATG13. Proteins were resolved by SDS–PAGE; phosphorylated proteins were visualized with autoradiography , HA–Ulk1 by western blotting and GST–Atg13 by Coomassie staining. () HA–Ulk1 was immunopurified from transfected HEK293 cells under glucose-rich media and treated with AMPK in the presence of cold ATP for 15 min, followed by kinase assays as described in d. () AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEFs were incubated with or without glucose (4 h). Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured (mean ± s.d., n = 3). Autophosphorylation activity was normalized to Ulk1 protein level; relative activity is calculated by normalization to Ulk1 activity from AMPK wild-type MEFs in glucose-rich conditions. () HA–Ulk1 was transfected into HEK293 cells together with vector (Vec) or an AMPKα1 kinase-dead mutant (DN). The cells were starved of glucose (–Glu) or a! mino acids (–A.A), or treated with 50 nM rapamycin (Rapa) fo! r 3 h before lysis. Left: autophosphorylation activity was assessed and normalized as in (mean ± s.d., n = 3). Right: fold induction in Ulk1 autophosphorylation, compared with Ulk1 autophosphorylation from cells under nutrient-rich conditions. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 2: AMPK directly phosphorylates Ulk1 at Ser 317 and Ser 777. () AMPK phosphorylates the Ulk1 S/T domain in vitro. Top: schematic representation of Ulk1 domain structure and deletion constructs used to map phosphorylation sites. The mouse Ulk1 protein consists of an N-terminal kinase domain (KD; 1–278), serine/threonine-rich domain (S/T domain, 279–828), and C-terminal domain (CTD, 829–1051). Bottom: the indicated Flag–Ulk1 deletion mutants were immunopurified from transfected HEK293 cells and were used for in vitro AMPK assay as a substrate. Phosphorylation was examined by 32P-autoradiogram and protein level was determined by western blot. () Determination of AMPK phosphorylation sites in Ulk1. The indicated recombinant GST–Ulk1 mutants were expressed and purified from Escherichia coli, and used as substrates for in vitro phosphorylation by AMPK. Deletion analyses indicated that two Ulk1 fragments in the S/T domain, 279–425 and 769–782, were highly phosphorylated by AMPK in vitro. Mutation of Ser 317 abolished the majori! ty of phosphorylation in the Ulk1 fragment 279–425. Within the fragment 769–782, mutations of five serine residues (Ser 774, Ser 777, Ser 778, Ser 779 and Ser 780) to alanine, denoted as (769–782) 5SA, completely abolished the phosphorylation by AMPK. Reconstitution of Ser 777 in this mutation background, (769–782) 4SA-S777, but not any of the other four residues, restored the phosphorylation by AMPK. GST and GST–TSC2F (TSC2 fragment 1300–1367 containing AMPK phosphorylation site at Ser 1345) were used as negative and positive controls for AMPK reaction, respectively. Phosphorylation was determined by 32P-autoradiograph and the protein levels were detected by Coomassie staining. () Ser 317/Ser 777 are required for glucose-starvation induced Ulk1 phosphorylation in vivo. HA–Ulk1 and mutants were transfected into HEK293 cells. Cells were starved for glucose for 4 h as indicated. HA–Ulk1 was immunoprecipitated and examined by western blot for mobility. () Phosp! horylation of Ulk1 Ser 317 and Ser 777 are induced by AMPK. Wi! ld-type HA–Ulk1 or S317/777A mutant were co-transfected with AMPK into HEK293 cells as indicated. HA–Ulk1 was immunoprecipitated (IP) and phosphorylation of Ser 317 and Ser 777 were determined by western blotting. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 3: AMPK-dependent Ulk1 Ser 317 and Ser 777 phosphorylation is required for Ulk1 activation in response to glucose starvation. () AMPK wild-type or DKO MEFs were starved of glucose (4 h) as indicated. Total cell lysates were probed for Ulk1 protein and phosphorylation. () Time course of Ulk1 Ser 317 and Ser 777 phosphorylation in response to glucose starvation/re-addition. MEFs were starved of glucose (–Glu) for the indicated times. After 3 h starvation, the culture was switched to glucose-containing (25 mM) medium and samples were harvested (Re-Glu). In parallel, cells were treated with amino-acid-free (–A.A) medium or 50 nM rapamycin (Rapa) for 3 h. () Phosphorylation of Ulk1 Ser 317 and Ser 777 correlates with AMPK activity. MEFs were starved of glucose (4 h) as indicated in the presence or absence of 20 μM compound C (C.C). In parallel, cells were treated with 2 mM Metformin (Met, 2 h) in glucose-rich medium. Phosphorylation of ACC S79 was tested as a positive control for AMPK activation. () Ulk1 is highly phosphorylated at Ser 317 and Ser 777 by glucose starvation in vivo. To determine the! Ulk1 phosphorylation level in vivo, immunopurified HA–Ulk1 protein was phosphorylated by AMPK in vitro (100% represents full phosphorylation of Ulk1 by AMPK). In vitro phosphorylated HA–Ulk1 was diluted as indicated, and was immunoblotted along with the immunoprecipitated HA–Ulk1 from cells grown in either glucose-rich (+ Glu) or glucose-free (– Glu, 4 h) medium. The density of the bands was then quantified. By this measurement, approximately 50% of Ulk1 isolated from glucose-starved cells was phosphorylated on Ser 317 and Ser 777. () The indicated HA–Ulk1 proteins were immunopurified from transfected HEK293 cells grown in high-glucose medium, and then incubated with AMPK in the presence of cold ATP for 15 min in vitro. After the reaction, AMPK was removed by extensive washing, the resulting Ulk1 immuno-complexes were assayed for kinase activity in the presence of 32P-ATP. () HA–Ulk1 proteins (wild type or S317/777A mutant) were immunoprecipitated from the tran! sfected HEK293 cells, which were incubated with or without glu! cose (4 h) before lysis. An in vitro kinase reaction was performed in the presence of GST–ATG13 and FIP200. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 4: mTORC1 disrupts the Ulk1–AMPK interaction. () AMPK interacts with Ulk1. HEK293 cells were transfected with the various Flag–Ulk1 deletion mutants together with AMPK α/β/γ, Atg13 and FIP200. Flag–Ulk1 protein (indicated by white arrows) was immunoprecipitated and co-immunoprecipitation of AMPK α/β/γ, Atg13 and FIP200 were examined by western blots. () Deletion analysis of Ulk1 regions responsible for AMPK interaction. The indicated Flag–Ulk1 truncation mutants were immunoprecipitated from transfected HEK293 cells co-expressing AMPK complex (α/β/γ). Co-immunoprecipitation of AMPK subunits was determined by western blots. () Rheb inhibits the Ulk1–AMPK interaction. HA–AMPKα, Flag–Ulk1 and Myc–Rheb were co-transfected into HEK293 cells as indicated. Cells were treated with or without rapamycin (50 nM Rapa) for 1 h before lysis. Flag–Ulk1 was immunoprecipitated and co-immunoprecipitates of AMPKα were determined by western blot. () Rapamycin treatment enhances the interaction of endogenous Ulk1 a! nd AMPK. Endogenous Ulk1 proteins were immunoprecipitated from either Ulk1 or AMPK wild-type and knockout (single-knockout; KO or double-knockout; DKO) MEFs. Treatment with 50 nM rapamycin for 1 h is indicated (Rapa). Co-immunoprecipitation of endogenous AMPKα protein was determined by western blot. The arrow indicates AMPKα protein. () Phosphorylation by mTORC1 inhibits the ability of Ulk1 to bind AMPK in vitro. CBP/SBP–Ulk1 was purified from transfected HEK293 cells by streptavidin beads and the Ulk1–bead complex was incubated with mTORC1, which was prepared by Raptor immunoprecipitation, in the presence of cold ATP, as indicated. The resulting Ulk1 complex was incubated with the cell lysates containing AMPK, then extensively washed. The Ulk1 and associated AMPKα were detected by western blot. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 5: mTORC1 phosphorylates Ulk1 at Ser 757. () mTORC1 phosphorylates the Ulk1 S/T domain. Ulk1 deletion mutants were prepared from the transfected HEK293 cells and used for in vitro mTORC1 assay. Phosphorylation was examined by 32P-autoradiogram (top) and protein level was determined by western blot (bottom). () Ser 757 is phosphorylated by mTORC1. Left: the indicated recombinant GST–mUlk1 mutants were purified from E. coli and used for in vitro mTORC1 assay as substrates. Deletion analyses isolated the fragment (753–771) as a target for mTORC1. The Ulk1 (753–771) fragment contains five conserved serine/threonine residues, Thr 754, Ser 757, Ser 760, Thr 763 and Thr 770. Right: mutation of Ser 757 abolished Ulk1 phosphorylation by mTORC1 in vitro. GST was used as negative control for mTORC1 phosphorylation reaction. Phosphorylation was determined by 32P-autoradiograph (top), whereas protein levels were detected by Coomassie staining (bottom). () Rheb increases Ulk1 Ser 757 phosphorylation. HA–Ulk1 wild type and! the S757A mutant were immunoprecipitated from transfected HEK293 cells. Co-transfection with Rheb and rapamycin (Rapa) treatment are indicated. Ulk1 Ser 757 phosphorylation was determined by western blot. () Rheb induces a mobility shift in wild-type Ulk1, but not the Ulk1S757A mutant. HA–Ulk1 was transfected with or without Rheb into HEK293 cells. HA–Ulk1 was immunoprecipitated from the cells under nutrient-rich medium and Ulk1 mobility was examined by western blot. () Endogenous Ulk1 Ser 757 phosphorylation is elevated in Tsc1−/− MEFs. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 757 phosphorylation of endogenous Ulk1 was detected by a phospho-Ulk1 Ser 757 antibody. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 6: Phosphorylation of Ulk1 Ser 757 by mTORC1 inhibits the Ulk1–AMPK interaction. () Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA–AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitated HA–AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis). () Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1–AMPK interaction in vitro. CBP/SBP Ulk1 proteins (wild type or S757C) were prepared from transfected HEK293 cells, which were pre-incubated with 50 nM rapamycin (Rapa, 1h) as indicated. The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1–bead was incubated with the bacterial purified AMPKα/β/γ complex. AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody. L.E.; long exposure. () Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1−! /− MEFs. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777. () Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1. HA–Ulk1, AMPKα kinase-dead mutant (DN), and Myc–Rheb were co-transfected into HEK293 cells as indicated. The cells were incubated with glucose-free medium (–Glu, 4 h), in which either 20 μM compound C (C.C.) or 50 nM Rapamycin (Rapa) was added. Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317, Ser 777, Ser 757, and HA, as indicated. () Rheb inhibits glucose starvation-induced Ulk1 activation. HA–Ulk1 and Myc–Rheb was transfected into HEK293 cells, which were incubated with glucose-free (–Glu), amino-acid-free (–A.A) medium, or 50 nM rapamycin (Rapa) for 4! h before lysis. HA–Ulk1 was immunoprecipitated and kinase a! ssays were performed. Ulk1 activity was measured by 32P-autoradiogram and the protein level of HA–Ulk1 and GST–Atg13 used in the assay was determined by western blot and by Coomassie staining, respectively. Uncropped images of blots are shown in Supplementary Fig. S5. * Figure 7: AMPK phosphorylation is required for Ulk1 function in autophagy on glucose starvation. () Ser 317/Ser 777 is required for Ulk1 to protect cells from glucose starvation. Viability (24 h, mean ± s.d., n = 4; top) and PARP cleavage (8 h; western blot, middle; quantification, n = 2, bottom) was examined in Ulk1+/+ (WT), Ulk1−/− (KO), Ulk1−/− re-expressing wild-type Ulk1 (KO-WT), and Ulk1−/− re-expressing Ulk1 S317/777A mutant (KO-S317/777A) MEFs. Arrows in western blot indicate non-cleaved and cleaved PARP. () The Ulk1 S317/777A mutant is compromised in LC3 lipidation in response to glucose starvation. ULK1 MEFs were cultured in glucose-free medium for the indicated times. LC3-II level was determined by western blotting and the LC3-II accumulation was normalized by α-tubulin and quantified (bottom, n = 3, mean ± s.d.). A representative western blot was shown. The LC3 antibody used in this experiment seemed to preferentially recognise the lipid-modified form of LC3-II, which migrated faster on the gel. () The Ulk1 S317/777A mutant is defective in aut! ophagosome formation. The indicated MEFs were starved of glucose (4 h) and the formation of GFP–LC3-positive autophagosomes was examined by confocal microscopy. GFP–LC3; green and DAPI; blue. Scale bar, 20 μm. () Autophagy vacuole analysis by electron microscopy. Low-magnification images of Ulk1−/− (KO, upper left panel), Ulk1−/− reconstituted with wild-type Ulk1 (KO-WT, two middle panels with accompanying higher magnification images), and Ulk1−/− reconstituted with Ulk1 S317/777A (KO-S317/777A, lower left panel) are shown. High-magnification images of autophagosomes from KO-WT are shown in upper right and lower right panels. Autophagosome/autolysosome-like structures indicated by arrowheads on the lower-magnification images and arrows in higher-magnification images. Scale bars; lower-magnification, 1 μm; higher-magnification, 200 nm. * Figure 8: Model of Ulk1 regulation by AMPK and mTORC1 in response to glucose signals. Left: when glucose is sufficient, AMPK is inactive and mTORC1 is active. The active mTORC1 phosphorylates Ulk1 on Ser 757 to prevent Ulk1 interaction with and activation by AMPK. Right: when cellular energy level is limited, AMPK is activated and mTORC1 is inhibited by AMPK through the phosphorylation of TSC2 and Raptor. Phosphorylation of Ser 757 is decreased, and subsequently Ulk1 can interact with and be phosphorylated by AMPK on Ser 317 and Ser 777. The AMPK-phosphorylated Ulk1 is active and then initiates autophagy. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Pharmacology and Moores Cancer Center, University of California at San Diego, La Jolla, CA 92130, USA. * Joungmok Kim & * Kun-Liang Guan * Department of Pathology, St. Jude Children's Hospital, Memphis, TN 38105, USA. * Mondira Kundu * INSERM U1016, Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. * Benoit Viollet Contributions J.K. performed the experiments; M.K. and B.V. established the AMPK and Ulk1 knockout MEFs, respectively; J.K. and K.-L.G. designed the experiments, analysed data and wrote the paper. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kun-Liang Guan Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data
• Deubiquitylase HAUSP stabilizes REST and promotes maintenance of neural progenitor cells
  - ncb 13(2):142-152 (2011)
  Nature Cell Biology | Article Deubiquitylase HAUSP stabilizes REST and promotes maintenance of neural progenitor cells * Zhi Huang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Qiulian Wu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Olga A. Guryanova1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lin Cheng1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Weinian Shou3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeremy N. Rich1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shideng Bao1 Contact Shideng Bao Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:142–152Year published:(2011)DOI:doi:10.1038/ncb2153Received05 March 2010Accepted23 November 2010Published online23 January 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The repressor element 1-silencing transcription factor (REST) functions as a master regulator to maintain neural stem/progenitor cells (NPCs). REST undergoes proteasomal degradation through β-TrCP-mediated ubiquitylation during neuronal differentiation. However, reciprocal mechanisms that stabilize REST in NPCs are undefined. Here we show that the deubiquitylase HAUSP counterbalances REST ubiquitylation and prevents NPC differentiation. HAUSP expression declines concordantly with REST on neuronal differentiation and reciprocally with β-TrCP levels. HAUSP knockdown in NPCs decreases REST and induces differentiation. In contrast, HAUSP overexpression upregulates REST by overriding β-TrCP-mediated ubiquitylation. A consensus site (310-PYSS-313) in human REST is required for HAUSP-mediated REST deubiquitylation. Furthermore, REST overexpression in NPCs rescues the differentiation phenotype induced by HAUSP knockdown. These data demonstrate that HAUSP stabilizes REST through d! eubiquitylation and antagonizes β-TrCP in regulating REST at the post-translational level. Thus, HAUSP-mediated deubiquitylation represents a critical regulatory mechanism involved in the maintenance of NPCs. View full text Figures at a glance * Figure 1: HAUSP and REST protein levels decline coordinately during neuronal differentiation. () Immunoblotting of HAUSP, β-TrCP, REST and TUJ1 (a neuronal differentiation marker) during differentiation. 15167 NPCs (neural stem/progenitor cells derived from a fetal brain by Lonza) were induced by all-trans retinoic acid to undergo cellular differentiation for the indicated times. HAUSP and REST protein levels gradually decreased, whereas the β-TrCP E3 ubiquitin ligase and the TUJ1 (type III β-tubulin, a REST target gene) levels increased during NPC differentiation. () Immunofluorescent staining confirmed that both HAUSP (red) and REST (green) protein levels declined during NPC differentiation. 15167 NPCs were induced by retinoic acid to undergo differentiation for the indicated times, then fixed and immunostained with anti-HAUSP- and anti-REST-specific antibodies. Nuclei were counterstained with DAPI (blue). () Immunofluorescent staining showed that HAUSP (red) decreased, but the neuronal differentiation marker TUJ1 (green) increased, during differentiation. ENSte! mA NPCs (derived from a human ES cell line by Chemicon/Millipore) were differentiated by retinoic acid treatment for the indicated times, fixed and stained with anti-HAUSP- and anti-TUJ1-specific antibodies. Nuclei were counterstained with DAPI (blue). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 2: HAUSP knockdown reduces REST protein levels in NPCs. (,) Immunoblotting showed that HAUSP knockdown by two distinct shRNAs (B2 and B5) decreased protein levels of REST, but not CoREST, in ENStemA () and 15167 () NPCs. NPCs were infected with lentiviruses expressing HAUSP shRNA or non-targeting (NT) control shRNA for 48 h; whole cell lysates were collected for immunoblotting with the specific antibodies as indicated. (,) Immunofluorescent staining confirmed that HAUSP knockdown reduced REST levels in ENStemA () and 17231 () NPCs. Cells were cultured and attached on cover glasses coated with BD Matrigel hESC-qualified matrix, infected with lentiviruses expressing HAUSP shRNA or control non-targeting shRNA, treated without () or with () puromycin to select for infected cells, fixed and immunostained with anti-HAUSP- and anti-REST-specific antibodies. HAUSP was labelled in green, and REST was labelled in red. Nuclei were counterstained with DAPI (blue). Nuclei with reduced HAUSP and REST proteins are indicated by arrows in . All! puromycin-selected cells infected with lentiviruses expressing HAUSP-targeting shRNA showed reduced HAUSP and REST protein levels in . Lentiviral infection efficiency in NPCs with GFP-expressing lentiviruses is shown in Supplementary Fig. S7. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 3: HAUSP knockdown promotes neural differentiation and decreases NPC self-renewal, and REST overexpression rescues the differentiation phenotype induced by HAUSP knockdown. () Targeting HAUSP with shRNA promotes neuronal differentiation. 15167 NPCs were infected with lentiviruses expressing HAUSP shRNA (B5 clone) or non-targeting (NT) shRNA for 126 h and immunostained for Nestin (an NPC maker, in red) and TUJ1 (a neuronal differentiation marker, in green). () Quantified data from confirmed that HAUSP knockdown increased neuronal lineage specification. The fraction of cells expressing TUJ1 (green) significantly (P<0.001) increased and the fraction of cells expressing Nestin (red) decreased after HAUSP knockdown in the NPCs. Data are means±s.d. (n=3; 200  cells per experiment). () Immunofluorescent staining showed that ectopic REST expression rescued the differentiation phenotype induced by HAUSP knockdown. 17231 NPCs were transfected with Flag–REST or vector control, and infected with HAUSP shRNA (B5 clone) or non-targeting shRNA lentiviruses for 126 h and immunostained for Nestin (red) and TUJ1 (green). () Quantified data from indicate! d that ectopic expression of REST significantly (P<0.001) attenuated the increased fraction of cells expressing TUJ1 induced by HAUSP knockdown. Data are means±s.d. (n=3; 200  cells per experiment). () Immunoblotting confirmed that ectopic expression of REST repressed the TUJ1 expression induced by HAUSP knockdown. 17231 NPCs were transfected with Flag–REST or vector, and infected with HAUSP shRNA (B5 clone) or non-targeting shRNA lentiviruses for 96 h, and immunoblotted with specific antibodies against HAUSP, Flag, TUJ1 and α-tubulin. () Neurosphere formation assay showed that HAUSP knockdown reduced NPC self-renewal potential. 15167 NPCs were infected with HAUSP shRNA or non-targeting shRNA lentiviruses and allowed to form neurospheres in serum-free suspension culture. HAUSP knockdown reduced the neurosphere size and induced the attachment of neurospheres on the uncoated dishes. () Quantified data from confirmed that HAUSP knockdown with two specific shRNAs (B2 an! d B5) significantly (P<0.001) decreased the number of neurosph! eres formed by 15167 NPCs. Data are means±s.d. (n=3). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 4: rtPCR analysis indicated that reduced HAUSP expression by shRNA did not significantly alter REST mRNA expression, but increased TUJ1 (a REST target gene) mRNA levels. ENStemA NPCs were targeted with HAUSP shRNA (B5 clone) or control non-targeting shRNA for 72 h through lentiviral infection. RNA samples were prepared for rtPCR analysis with specific primers for HAUSP, REST, CoREST and TUJ1. HAUSP mRNA was significantly downregulated, but REST mRNA levels were not significantly affected. Data are presented as means±s.d. (n=3). * Figure 5: HAUSP mediates REST deubiquitylation to regulate REST protein levels. (,) Immunoprecipitation (IP) showed that HAUSP and REST interact in NPCs. Cell lysates of ENStemA NPCs were immunoprecipitated with anti-REST (monocolonal antibody) or anti-HAUSP antibody or IgG control and then immunoblotted (IB) with anti-HAUSP and anti-REST (rabbit polyclonal) antibodies. () Ubiquitylation assays showed that HAUSP knockdown increased REST ubiquitylation in NPCs. 17231 NPCs were infected with lentiviruses expressing HAUSP shRNA or non-targeting shRNA for 48 h and then treated with the proteasome inhibitor MG132 for 6 h before collection for immunoprecipitation. Cell lysates were immunoprecipitated with an anti-REST- or anti-ubiquitin-specific antibody or control IgG, and then immunoblotted with anti-ubiquitin- or anti-REST-specific antibody. Both immunoprecipitation with anti-REST antibody and the reciprocal immunoprecipitation with anti-ubiquitin antibody confirmed that HAUSP knockdown increased REST polyubiquitylation. () Ectopic expression of wild-t! ype HAUSP (WT-HAUSP), but not the catalytic dead mutant HAUSPC223S (Mt-HAUSP), reduced REST ubiquitylation. 17231 NPCs were transfected with the Flag-tagged WT-HAUSP (WT), Mt-HAUSP (Mt) or vector (V) control through lentiviral infection for 36 h, then treated with the proteasome inhibitor MG132 for 6 h, and subjected to analysis of REST ubiquitylation. Ectopic expression of WT-HAUSP, but not Mt-HAUSP, reduced REST ubiquitylation in the NPCs. () In vitro deubiquitylation assay showed that the β-TrCP-mediated REST ubiquitylation was specifically inhibited by WT-HAUSP (lane 3), but not by the Mt-HAUSP (a catalytic dead mutant, lane 4) or control deubiquitylase USP1 (lane 5). Flag–WT-HAUSP, Flag–Mt-HAUSP, Myc–REST, HA–β-TrCP and Flag–USP1 were individually overexpressed in 293T cells, and then purified with the specific antibody or the corresponding tag antibody for this assay. () In vivo deubiquitylation assay confirmed that the β-TrCP-mediated REST ubiquityla! tion was specifically attenuated by the WT-HAUSP, but not the ! catalytic dead Mt-HAUSP. 293T cells were transfected with the indicated sets of plasmids for 48 h, treated with the proteasome inhibitor MG132 for 6 h and then subjected to analysis of REST ubiquitylation in the samples expressing different set of proteins as indicated. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 6: Neuronal differentiation induced by knockdown of endogenous HAUSP was rescued by ectopic expression of wild-type HAUSP, but not the catalytic dead HAUSP mutant. (–) As the B5 HAUSP shRNA clone targets the 3′-end non-coding region of endogenous HAUSP mRNA, ectopic expression of the wild-type and mutant HAUSP that lack the 3′-end non-coding region was not affected by the B5 HAUSP shRNA. () Immunofluorescent staining showed that ectopic expression of wild-type HAUSP (WT-HAUSP), but not the catalytic dead mutant HAUSP (Mt-HAUSP), rescued the neuronal differentiation phenotype induced by knockdown of endogenous HAUSP. 17231 NPCs were transfected with Flag-tagged WT-HAUSP or Mt-HAUSP, or vector control through lentiviral infection, and infected with lentiviruses expressing HAUSP shRNA (B5 clone) or control non-targeting (NT) shRNA for 96 h and immunostained with specific antibodies against Nestin (red) and TUJ1 (green). Ectopic expression of WT-HAUSP, but not the Mt-HAUSP, suppressed the expression of neuronal marker TUJ1 that was induced by knockdown of endogenous HAUSP. () Quantified data from indicated that ectopic expression o! f the WT-HAUSP, but not the Mt-HAUSP, in NPCs almost fully rescued the differentiation phenotype induced by knockdown of the endogenous HAUSP. Data are means±s.d. (n=4; 200 cells per experiment). () Immunoblotting validated that ectopic expression of WT-HAUSP, but not the Mt-HAUSP, resulted in suppression of TUJ1 expression. 17231 NPCs were transfected with Flag-tagged WT-HAUSP or Mt-HAUSP, or vector control through lentiviral infection, and infected with lentiviruses expressing HAUSP shRNA (B5 clone) or non-targeting shRNA for 72 h and immunostained with specific antibodies against REST, Flag, TUJ1 and α-tubulin (loading control). () Deubiquitylation assay showed that ectopic expression of WT-HAUSP, but not the catalytic dead Mt-HAUSP, attenuated the REST ubiquitylation induced by knockdown of endogenous HAUSP. 15167 NPCs were transfected with Flag-tagged WT-HAUSP or Mt-HAUSP, or vector control through lentiviral infection, and infected with lentiviruses expressing H! AUSP shRNA (B5 clone) or non-targeting shRNA for 48 h, treat! ed with the proteasome inhibitor MG132 for 6 h, immunoprecipitated with anti-REST-specific antibody or the IgG control, and immunoblotted with anti-ubiquitin-specific antibody. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 7: A consensus site of human REST (310-PYSS-313) is required for the HAUSP-mediated REST deubiquitylation and HAUSP counteracts β-TrCP-mediated REST ubiquitylation. () Schematic representation of human REST protein. A consensus sequence (310-PYSS-313) of human REST required for the HAUSP-mediated REST deubiquitylation and a critical mutation (S313A) on this site that disrupts the deubiquitylation are shown. NLS, nuclear localization signal. () In vivo deubiquitylation assay showed that a critical mutation (S313A) on the consensus sequence (310-PYSS-313) of human REST attenuated the HAUSP-mediated REST deubiquitylation (see lanes 2–4), whereas a similar mutation (S1042A) on another potential site (1039-PQES-1042) did not alter the HAUSP-mediated REST deubiquitylation (see lanes 3–5). S313A S1042A double mutations (Flag–RESTAA) and S313A single mutation showed a similar effect on the HAUSP-mediated REST deubiquitylation (see lanes 4–6). 293T cells were transfected with the indicated set of expression plasmids, and treated with the proteasome inhibitor MG132 for 6 h, and then subjected to analysis of REST ubiquitylation. () REST ! ubiquitylation increased during neuronal differentiation. 17231 NPCs differentiation was induced by all-trans retinoic acid for four days, and the cells were treated with MG132 for 6 h, and collected for a ubiquitylation assay. Cell lysates from NPCs or differentiated cells were subjected to immunoprecipitation with anti-REST antibody (Rabbit) and immunoblotted with anti-ubiquitin antibody. () Double-knockdown analysis confirmed that REST protein is controlled by both β-TrCP-mediated ubiquitylation and the HAUSP-mediated deubiquitylation in NPCs. 15167 NPCs were treated with retinoic acid for only 24 h to initiate differentiation, and transduced with HAUSP shRNA, β-TrCP shRNA, both HAUSP shRNA and β-TrCP shRNA, or non-targeting (NT) shRNA for 36 h through lentiviral infection, treated with MG132 for 6 h, and collected for ubiquitylation assessment. HAUSP knockdown alone increased REST ubiquitylation (lanes 1 and 2), whereas β-TrCP knockdown alone reduced REST ub! iquitylation (lanes 1 and 3). However, HAUSP and β-TrCP doubl! e knockdown restored REST ubiquitylation that was reduced by β-TrCP knockdown and abolished REST ubiquitylation induced by HAUSP knockdown (lanes 1–4). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 8: 'Ying–Yang' control model of REST protein at the post-translational level. Both HAUSP-mediated deubiquitylation ('Ying') and β-TrCP-mediated ubiquitylation ('Yang') regulate REST protein levels in NPCs. HAUSP deubiquitylase stabilizes REST protein to promote NPC maintenance. In contrast, the β-TrCP E3 ubiquitin ligase mediates REST ubiquitylation and degradation to promote neuronal differentiation. The net balance between the β-TrCP-mediated ubiquitylation and the HAUSP-mediated deubiquitylation controls REST protein levels and determines cellular fate. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, NE30, Cleveland, Ohio 44195, USA * Zhi Huang, * Qiulian Wu, * Olga A. Guryanova, * Lin Cheng, * Jeremy N. Rich & * Shideng Bao * Center for Experimental Research, The First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China * Lin Cheng * Riley Heart Research Center, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA * Weinian Shou Contributions Z.H., Q.W., O.A.G. and L.C. carried out and planned all experiments. S.B. developed the hypothesis, coordinated the study, oversaw the research and results, and wrote the manuscript. J.N.R. helped to write the manuscript and provided input into design and interpretation. W.S. provided reagents and helpful suggestions. The work was carried out in the laboratory of S.B. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Shideng Bao Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2000K) Supplementary Information Additional data
• Interpretation of the FGF8 morphogen gradient is regulated by endocytic trafficking
  - ncb 13(2):153-158 (2011)
  Nature Cell Biology | Letter Interpretation of the FGF8 morphogen gradient is regulated by endocytic trafficking * Matthias Nowak1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Anja Machate1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Shuizi Rachel Yu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mansi Gupta1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Brand1, 2 Contact Michael Brand Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:153–158Year published:(2011)DOI:doi:10.1038/ncb2155Received13 January 2010Accepted17 November 2010Published online23 January 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions1, 2. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated3. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphog! en signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns. View full text Figures at a glance * Figure 1: Cbl activity regulates Fgf signalling range without affecting the extracellular Fgf protein gradient. (–) Representative 60% epiboly whole-mount embryos stained for the expression of fgf8 (–), spry4 (–), spry2 (–) or pea3 (–) by in situ hybridizations; animal side of the embryo to the top, dorsal to the right. Injection of cbl-YF mRNA leads to an expansion of spry4, spry2 and pea3 expression (,,) when compared with control embryos (,,), whereas fgf8 expression remains unaffected (,). Target gene expression is strongly reduced on overexpression of Cbl-YF and a dominant-negative FgfR (XFD; ,,), indicating that it is still strictly Fgf signalling dependent. Scale bars, 100 μm. () Quantification of in situ results shown in –. Embryos were injected with wild-type c-cbl (green), cbl-YF (red) or cbl-YF together with XFD (purple) and the marginal expression domains of fgf8, spry4, spry2 and pea3 were measured. Blue represents un-injected controls. Values below bars give the respective n-number and error bars denote the standard deviation (s.d.). Statistical significan! ce was analysed by ANOVA tests (fgf8P=1.5×10−10; spry4P=6×10−16; spry2P=7.1×10−13; pea3P=3.2×10−8) followed by a post hoc Tukey test (values indicated on top of bars). Cbl-YF reduces the fgf8 expression domain, but still increases expression domain width of all tested target genes. This effect is counteracted by co-injection of XFD. Injection of wild-type Cbl leads to a mild reduction of spry4 and pea3 expression domains. () FCS autocorrelation measurements were taken in the extracellular space of live embryos at different distances from a GFP–Fgf8 source in un-injected control (blue; n=21) or cbl-YF-mRNA-injected embryos (red; n=14) to determine the extracellular Fgf8 concentration gradient. Data are binned in 20 μm intervals and fitted with a radial model10. Error bars denote standard deviation. Cbl-YF does not influence the Fgf8 gradient. () Fgf8–GFP fluorescence intensity (count rate in kHz) at its source in control (blue) and cbl-YF-injected embryos ! (red). The intensities are indistinguishable, indicating that ! cbl-YF has no influence on the amplitude of the Fgf8–GFP gradient. Error bars denote the standard deviation. * Figure 2: Cbl-YF changes the tissue distribution profile of intracellular Fgf8. Cy5–Fgf8-coated beads were implanted into the animal pole of sphere-stage embryos. Embryos were incubated for 2 h and analysed. (,) Single confocal sections through the animal pole of embryos injected with wild-type c-cbl () or cbl-YF () mRNA. The Cy5–Fgf8 bead is partially visible as a half-circle at the left border of each panel. Grey lines demarcate the boundary of Fgf8 tissue penetration. Fgf8 endosomes are visible farther from the source in cbl-YF-injected embryos. Scale bars in ,, 10 μm. (,) Quantification of data shown in ,. Magnified view of the interval 60–180 μm (outlined in ). Endosome density (endosomes per square pixel, y axis) is plotted against distance to bead centre (μm, x axis). In cbl-YF-injected embryos (red; n=10), Fgf8 endosome density is lower close to the source (interval 1, 25–50 μm distance to centre of the bead), but higher when the distance is greater, compared with un-injected controls (interval 3, 100–170 μm to centre o! f the bead; blue, n=20). In contrast, wild-type c-cbl-injected embryos show an increase in endosome density close to the source (interval 1), but a reduction in intermediate distance (interval 2, 60–100 μm to centre of the bead; green; n=14) when compared with controls. Error bars denote the standard deviation. Statistical significance was analysed by non-parametric Mann–Whitney tests. * Figure 3: Cbl-YF induces a delay in lysosomal targeting of Fgf8 and FgfR1 and their enrichment in caveolae. Cy5–Fgf8-coated beads were implanted into the animal pole of sphere-stage embryos. Embryos were mounted and imaged from 10 min post-implantation (mpi) until 90 mpi in 10-min intervals. Quantification of Cy5–Fgf8 co-localization in embryos injected with CFP–Rab5c (), YFP–Rab7 (), mRFP–Lamp1 (), Cherry–Rab11 (), Caveolin1–GFP () or a marker for the plasma membrane (RFP–ras, PM; ) and wild-type c-cbl (green) or cbl-YF (red) and control embryos (blue). Percentage co-localization of Cy5–Fgf8-positive endosomes with indicated endosomal markers (y axis) is plotted against time (minutes post implantation, mpi, x axis). n-numbers are given at the right end of respective curves. Error bars denote the standard deviation. Statistical significance was analysed by ANOVA tests followed by post hoc Tukey tests. Post hoc statistical significance is indicated by filled circles for control cbl, open circles for control cbl-YF and asterisks for cbl/cbl-YF pairs (for detail! s, see Methods section). Cbl-YF causes a delay of lysosomal targeting of Cy5–Fgf8, but it does not influence co-localization of Cy5–Fgf8 with Rab5c () or Rab11 (). () mRNAs coding for FgfR1–GFP and tRFP–Rab5c, tRFP–Rab7, Cherry–Rab11, Caveolin1–tRFP or plasma-membrane RFP (RFP–ras, PM) were injected alone (Control, blue) or together with cbl (green) or cbl-YF (red) mRNA and quantitatively analysed for co-localization of FgfR1 with the respective markers. Statistical significance was analysed by an ANOVA test followed by post hoc Tukey tests. Results of the post hoc test are shown only for significant differences (for Rab7 PANOVA 0.000065; Caveolin PANOVA 0.00037; PM PANOVA 0.047). Cbl-YF causes a reduction in FgfR1 co-localization with late endosomal markers, but an increased co-localization in caveolae. * Figure 4: Cbl-YF causes an increase in co-localization of FgfR1 and Grb2, but a reduction in Grb2 localization to the degradative pathway. (–) mRNAs coding for FgfR1–GFP and Grb2–tRFP were injected alone (, Control) or together with cbl () or cbl-YF mRNA (). Scale bars, – are 5 μm. () Quantitative analysis of the co-localization of FgfR1 and Grb2. Numbers below bars represent n-value n-number. Statistical significance was analysed by an ANOVA test (P-value given in the middle of the panel) followed by post hoc Tukey tests (Pctr–cbl 0.77, Pctr–cbl−YF2.1×10−8, Pcbl–cbl−YF2.1×10−8). Cbl-YF causes an increase in FgfR1–Grb2 co-localization. () mRNAs coding for Myc–FgfR1, Grb2–tRFP and CFP–Rab5c, CFP–Rab7, GFP–Rab11 or Caveolin1–GFP, respectively, were injected alone or together with cbl or cbl-YF mRNA and were quantitatively analysed for co-localization of Grb2 with the respective markers. Statistical significance was analysed by an ANOVA test followed by post hoc Tukey tests. Results of the post hoc test are shown only for significant differences (for Rab7, PANOVA 0.0019). C! bl-YF causes a reduction in Grb2 co-localization with late endosomal markers. * Figure 5: Model for Cbl function during complex pattern formation. () On stimulation with Fgf8 (green dots), active receptor signalling complexes are formed (P) and are endocytosed into the early endosome (Rab5). At the same time, a fraction of the signalling complexes is ubiquitylated (Ub) by the Cbl ubiquitin ligase. Ubiquitylated receptors are efficiently sorted towards lysosomal degradation (Rab7, Lamp1), whereas non-ubiquitylated receptors are recycled (Rab11). () Loss of Cbl function leads to reduced sorting of receptors to the lysosome and a longer residence time of the signalling complexes on endosomes. () We propose a mechanism where regulation of signal intensity downstream of the extracellular morphogen gradient, but upstream of target gene induction, is used to facilitate morphogen gradient interpretation. Target cells cannot interpret an extracellular morphogen gradient (green dots) in a linear fashion, but only distinguish between areas of high (blue) and low (red) signalling (upper panel). Feedback regulation through an inact! ivation of Cbl (for example, phosphorylation of Cbl by Src) leads to an expansion of gradient interpretation and further refinement of the pattern: as interpretation of the gradient expands, a second pattern of high and low signalling areas is superimposed on top of the first one and new cell fates can be specified (that is, those that have encountered low signal before and high signal later, white). Author information * Author information * Supplementary information Affiliations * Developmental Genetics, Biotechnology Center, TUD, Tatzberg 47-49, 01307 Dresden, Germany * Matthias Nowak, * Anja Machate, * Shuizi Rachel Yu, * Mansi Gupta & * Michael Brand * Center for Regenerative Therapies, TUD, Tatzberg 47-49, 01307 Dresden, Germany * Matthias Nowak, * Anja Machate, * Shuizi Rachel Yu, * Mansi Gupta & * Michael Brand Contributions M.N., S.R.Y., M.G. and A.M. carried out experiments. M.N., S.R.Y. and M.B. analysed the data. M.N. and M.B. designed the project and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael Brand Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1200K) Supplementary Information Additional data
• Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis
  - ncb 13(2):159-166 (2011)
  Nature Cell Biology | Letter Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis * Stephen J. Terry1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ceniz Zihni1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ahmed Elbediwy1 Search for this author in: * NPG journals * PubMed * Google Scholar * Elisa Vitiello1 Search for this author in: * NPG journals * PubMed * Google Scholar * Isabelle V. Leefa Chong San1 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria S. Balda1, 2 Contact Maria S. Balda Search for this author in: * NPG journals * PubMed * Google Scholar * Karl Matter1, 2 Contact Karl Matter Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:159–166Year published:(2011)DOI:doi:10.1038/ncb2156Received14 September 2010Accepted24 November 2010Published online23 January 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading1, 2. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. Here we show that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight-junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell–cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment3, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a compl! ex containing myosin II, Rock II and the junctional adaptor cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis. View full text Figures at a glance * Figure 1: p114RhoGEF is a tight-junction-associated RhoA GEF that regulates junction formation and the actin cytoskeleton. () Caco-2 and HCE cells transfected with non-targeting (Control) siRNA or siRNAs directed against p114RhoGEF (p114RG). Lysates were analysed by immunoblotting p114RhoGEF and α-tubulin. Uncropped images are shown in Supplementary Fig. S7. (–) HCE and Caco-2 cells were processed for immunofluorescence microscopy using antibodies against the proteins indicated. In , cells were transfected with siRNAs targeting p114RhoGEF before immunofluorescence. and are epifluorescence images, whereas and are confocal x–y () and z () line scans. () Levels of active RhoGTPases in HCE cells were measured after transfection of either control or p114RhoGEF-targeting siRNAs. Shown are averages ±1 s.d., n=3. (,) Caco-2 and HCE cells were transfected with siRNAs (for p114RhoGEF, a pool containing siRNA1 and 2 was used) and then processed for immunofluorescence microscopy using antibodies against the indicated proteins () or fluorescent phalloidin to label F-actin (). For the columnar Caco-2 c! ells, two epifluorescence images are shown for the actin staining, one taken focusing on the apical region and one from the base of the cells. For the flat HCE cells, a single epifluorescence image is shown that includes lateral and basal actin staining. See Supplementary Fig. S1 for other tight-junction markers and experiments with individual siRNAs. Scale bars, 10 μm. * Figure 2: p114RhoGEF regulates epithelial barrier formation. (–) Caco-2 cells transfected with control or two different pools of p114RhoGEF-specific siRNAs (OnT refers to the On-Target pool) were trypsinized and plated onto filters in low-calcium media for 24 h to prevent the formation of cell–cell contacts. Expression of p114RhoGEF and other junctional proteins was assayed at the end of the experiment by immunoblotting (). Uncropped images are shown in Supplementary Fig. S7. Normal-calcium-containing medium was added and cell contact formation was monitored during the next 48 h by measuring transepithelial electrical resistance (TER; , shown are averages ±1 s.d., n=3). Paracellular tracer permeability was then determined using fluorescently labelled dextrans (, shown are averages ±1 s.d., n=3). (–) Cells transfected with siRNAs and plated in low-calcium media followed by incubations in normal-calcium media were processed for immunoblotting (, 24 h of calcium, uncropped images are shown in Supplementary Fig. S7) or fixed! at the indicated times and processed for immunofluorescence analysis using an epifluorescence microscope (, ZO-1; , F-actin; , p114RhoGEF and myosin IIA). See Supplementary Fig. S2 for additional junctional markers analysed. Focal planes for the epifluorescence images in – were chosen so that they include lateral and basal staining. Note, p114RhoGEF-depleted cells, in contrast to GEF-H1-depleted, spread normally but did not assemble morphologically normal junctions. Scale bars, 10 μm. * Figure 3: p114RhoGEF regulates epithelial morphogenesis in three-dimensional cultures. (–) p114RhoGEF was depleted by transfecting siRNAs into Caco-2 cells (–) or by generating MDCK cells expressing shRNAs (–). The cells were then cultured in three-dimensional gels. After 5 days, the cultures were analysed by phase-contrast microscopy (,) and quantified by counting cysts with a single lumen or structures with a disorganized appearance and multiple lumens (,; averages ±1 s.d.; , n=3; , n=5). The cultures were then fixed and processed for analysis by confocal microscopy using the indicated antibodies and fluorescent labels (,). Scale bars, 30 μm (,) and 10 μm (,). See Supplementary Fig. S2e for immunoblot analysis of p114RhoGEF expression. * Figure 4: p114RhoGEF regulates RhoA signalling at cell junctions. (,) Cells were transfected with siRNAs and, after 2 days, with a RhoA FRET biosensor. RhoA activity was then imaged by gain of cyan fluorescent protein fluorescence after acceptor bleaching. Shown are images of control, p114RhoGEF- and GEF-H1-depleted cells taken from the apical part of the cells that contains the junctional complex (; see Supplementary Fig. S3a for basal sections). Images to monitor Rho activity during junction formation were taken between 1 and 2 h after adding calcium (Ca-switch). Images were quantified by normalizing FRET signals in specific regions (cell–cell contacts or interior cytoplasm) with the total FRET signal in the quantified fields (; averages ±1 s.d.; n: Caco-2, 30 for control and p114RhoGEF and 12 for GEF-H1; HCE, 20 for control, 14 for p114RhoGEF, 12 for GEF-H1; Ca-switch, 10). (,) Caco-2 cells () and HCE cells (), transfected with the specified siRNAs, were processed for immunofluorescence as indicated and analysed by epifluorescenc! e microscopy. For Caco-2 cells, images taken from the apical and the basal regions are shown, whereas a single image is shown for HCE cells that includes lateral and basal staining. () Levels of active RhoA were measured after tetracycline-induced expression of p114RhoGEF–VSV, labelled as WT, or p114RhoGEFY260A–VSV, labelled as Y260A, in MDCK cells. Shown are averages ±1 s.d., n=3. () HCE cells were transfected with the indicated constructs for p114RhoGEF and GEF-H1 expression and then processed for immunofluorescence and confocal microscopy. Shown are z line scans and x–y sections. Note, the contracted appearance of cells expressing active p114RhoGEF. () Images from samples such as those in panel for HCE cells and analogous samples generated with Caco-2 cells (Supplementary Fig. S5a) were quantified by counting transfected cells with a rounded/contracted appearance and cells that remained normal/flat. Shown are averages ±1 s.d., n=3. () HCE cells transfected as in ! panel were stained for ZO-1 and β-catenin, and imaged by epif! luorescence microscopy (the antibody against ZO-1 crossreacts with a nuclear antigen in human cells). The overlay shows ZO-1 in green, β-catenin in blue and VSV in red. Scale bars, 10 μm. * Figure 5: p114RhoGEF forms a complex with myosin II, cingulin and Rock II. () p114RhoGEF was immunoprecipitated from cell extracts of normally cultured Caco-2 cells or cells kept in low calcium to prevent junction formation. Beads conjugated with a non-related goat IgG were used as negative control. Co-precipitating proteins were analysed by immunoblotting as indicated. Uncropped images are shown in Supplementary Fig. S7. () GST fusion proteins containing different domains of p114RhoGEF (left) were bound to beads and used for pulldown experiments with Caco-2 cell extract (DH, Dbl (Diffuse B-cell lymphoma) homology domain; PH, pleckstrin homology domain; CC, coiled-coil domain; P, proline-rich region; FL, full length; NTD, N-terminal domain; CTD, C-terminal domain). Pulldowns were analysed by immunoblotting (right). () Cells were transfected with control or cingulin-targeting siRNAs and expression of the indicated proteins was analysed by immunoblotting. (,) Caco-2 and HCE cells were transfected with siRNAs as in and then fixed and processed for imm! unofluorescence to localize p114RhoGEF and junctional proteins. Shown are fields from depleted samples that still express some cingulin (labelled with asterisks). () HCE cells were transfected with siRNAs as indicated and then processed for immunofluorescence. All immunofluorescence images shown were acquired by epifluorescence microscopy and include lateral and basal structures. Scale bars, 10 μm. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Maria S. Balda & * Karl Matter Affiliations * Department of Cell Biology, UCL Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK * Stephen J. Terry, * Ceniz Zihni, * Ahmed Elbediwy, * Elisa Vitiello, * Isabelle V. Leefa Chong San, * Maria S. Balda & * Karl Matter Contributions S.J.T. carried out most of the experiments. All other authors carried out particular subsets of experiments. S.J.T., M.S.B. and K.M. designed the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Maria S. Balda or * Karl Matter Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (1400K) Supplementary Information Additional data
• Nucleation of nuclear bodies by RNA
  - ncb 13(2):167-173 (2011)
  Nature Cell Biology | Letter Nucleation of nuclear bodies by RNA * Sergey P. Shevtsov1 Search for this author in: * NPG journals * PubMed * Google Scholar * Miroslav Dundr1 Contact Miroslav Dundr Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:167–173Year published:(2011)DOI:doi:10.1038/ncb2157Received05 May 2010Accepted06 December 2010Published online16 January 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 biogenesis of the many functional compartments contained in the mammalian cell nucleus is poorly understood. More specifically, little is known regarding the initial nucleation step required for nuclear body formation. Here we show that RNA can function as a structural element and a nucleator of nuclear bodies. We find that several types of coding and noncoding RNAs are sufficient to de novo assemble, and are physiologically enriched in, histone locus bodies (with associated Cajal bodies), nuclear speckles, paraspeckles and nuclear stress bodies. Formation of nuclear bodies occurs through recruitment and accumulation of proteins resident in the nuclear bodies by nucleating RNA. These results demonstrate that transcription is a driving force in nuclear body formation and RNA transcripts can function as a scaffold in the formation of major nuclear bodies. Together, these data suggest that RNA-primed biogenesis of nuclear bodies is a general principle of nuclear organizatio! n. View full text Figures at a glance * Figure 1: Immobilization of histone pre-mRNA to chromatin leads to formation of a HLB with associated Cajal body. () Schematic representation of RNA-tethering system used to analyse the role of specific RNAs (the human H2b-MS2 transcript is shown here) in the formation of subnuclear bodies in cells. Green arrow indicates the cleavage of 3′-end of H2b–MS2 () Histone H2b transcripts tagged with the MS2 loop were transiently co-expressed with the monomeric GFP–LacI–NLS-MS2 coat protein in HeLa cells containing the LacO array. Tethering to the LacO array was detected by specific RNA FISH (arrow indicates higher magnification shown in inset). () Fluorescence microscopy on HeLa cells transiently transfected with wild-type H2b-MS2 pre-mRNA and expressing the monomeric GFP–LacI–NLS-MS2 coat protein (green), and incubated with antibodies against NPAT (red) and coilin (white). Arrows indicate HLB with physically associated de novo Cajal body (shown in inset). () Imaging performed as in , but cells were transfected with empty vector rather than H2b-MS2 pre-mRNA. () Microscopy analysis ! of endogenous HLBs and Cajal bodies formed around clusters of active histone genes in HeLa cells, detected by RNA FISH using a fluorescently labelled probe against the conserved 3′ end of histone pre-mRNAs (red) and combined with immunofluorescence microscopy using anti-FLASH (green) and anti-coilin (white), and co-stained with DAPI. () Northern blot using a specific oligonucleotide probe against the MS2 loop sequence indicates processing defects of mutant H2b-MS2 RNAs. (, ) Deletion of the conservative stem loop structure () and mutation of HDE at the 3′ end of histone H2b-MS2 () abolish HLB and Cajal body formation. (, ) Mutation of the 3′-end cleavage site () and adding the polyadenylation site to the mutant with the blocked 3′-end cleavage site of histone H2b-MS2 () leads to the formation of de novo HLB and Cajal body. Insets; high-magnification images of HLBs and Cajal bodies. Arrows indicate the location of tethered H2b-MS2 RNAs. () Quantitative analysis of de! novo formation of HLB and Cajal body efficiency by tethered H! 2b-MS2 transcripts. Values represent averages (n = 65–80 cells) from two independent experiments. Scale bars, 2 μm. * Figure 2: Live-cell 4D imaging of de novo Cajal body formation nucleated by non-cleavable histone H2b–MS2 transcripts and de novo formation of HLB and Cajal body by factors involved in histone gene expression and histone 3′ end processing. () Time-lapse microscopy images of Cajal body formation at indicated times after IPTG washout. Images are maximum intensity projections of z stacks. Cajal bodies (marked with GFP–coilin; green) formed de novo on the LacO array, nucleated by non-cleavable mutant histone H2b–MS2 transcripts tethered by Cherry–LacI–NLS-MS2 coat protein (red). After IPTG washout, Cherry–LacI–NLS-MS2 coat protein binding on the LacO array was restored after approximately 30 min (arrow; high magnification insets) and de novo Cajal body was formed on the array after 1 h, 45 min–2 h (arrow, high magnification insets). (–) Tethering of factors involved in histone gene expression and histone 3′-end processing on chromatin leads to de novo formation of HLBs with associated Cajal bodies. Immunofluorescence microscopy on HeLa cells transiently transfected with factors involved in histone gene synthesis and 3′-end processing, fused with GFP–LacI. Immobilization of NPAT (), FLASH () a! nd Lsm11 (a unique component of U7 snRNP; ; all shown in green), which are present in de novo formed HLBs, leads to de novo Cajal body formation (detected by anti-coilin or anti-SMN antibody; white). To distinguish the position of the LacO array from endogenous HLBs and Cajal bodies, cells were co-transfected with Cherry–LacI (red; –). Tethering of SLBP () and CPSF30 () on chromatin (green) nucleates the de novo formation of HLB and Cajal body (detected by anti-NPAT and anti-coilin antibodies; white) in contrast to CtsF77, a factor exclusively required for proper 3′-end cleavage of polyadenylated pre-mRNAs (). Arrows indicate LacO arrays with de novo formed HLB's and Cajal bodies, which are shown in higher magnification in insets. Scale bars, 2 μm. * Figure 3: Tethering spliced β-globin–MS2 pre-mRNA on chromatin leads to association with splicing speckle or de novo formation of nuclear speckle. () Schematic representation of the β-globin–MS2 minigene system and unspliced and intron-less mutants. () Wild-type and the unspliced and intronless mutants of the β-globin–MS2 pre-mRNA were resolved on an agarose gel. Schematic representation indicates composition of bands. (–) RNA FISH using a probe against the β-globin minigene (red) combined with immunofluorescence microscopy using anti-SC35 antibody (white) on HeLa cells transiently co-transfected with the indicated β-globin–MS2 constructs and GFP–LacI–NLS-MS2 coat protein (visualized in green). Immobilized spliced β-globin–MS2 pre-mRNA on chromatin leads to protrusion towards an existing speckle () or to de novo speckle formation () in contrast to tethered unspliced and intron-less β-globin–MS2 RNAs (, ). () Quantitative analysis of association of tethered β-globin–MS2 constructs with existing speckle plus de novo speckle formation. Values represent averages (n = 65–80 cells) from two indepen! dent experiments. Scale bars, 2 μm. * Figure 4: Tethering noncoding NEAT1 RNA leads to formation of a de novo paraspeckle. (–) RNA FISH using a probe against NEAT1 (red) combined with immunofluorescence microscopy using indicated antibodies against paraspeckle components (white) on HeLa cells transiently co-transfected with NEAT1–MS2 and GFP–LacI–NLS-MS2 coat protein. () Cells were transfected with GFP–LacI–PSP1 (and GFP–LacI to distinguish the position of the LacO array from endogenous HLBs and Cajal bodies). RNA FISH and immunofluorescence microscopy were performed as in –. Immobilization of paraspeckle protein PSP1 on chromatin also leads to paraspeckle formation. () Quantitative analysis of de novo paraspeckle formation efficiency. Values represent averages (n = 65–75 cells) from two independent experiments. Scale bars, 2 μm. * Figure 5: Immobilization of the noncoding satIII transcripts on chromatin, without heat-shock induction, leads to formation of nSB. (–) RNA FISH using a probe against the satIII transcript (red) combined with immunofluorescence microscopy using antibodies against the indicated proteins (white) on HeLa cells transiently co-transfected with satIII–MS2 and GFP–LacI–NLS-MS2 coat protein (green), with (, ) or without (, ) heat shock. On heat shock nSB form around hyperactivated tandem satIII repeats (, ). Tethered satIII–MS2 transcripts on chromatin and episomal satIII–MS2 transcription sites form de novo nSB without heat shock induction (, ). Scale bars, 2 μm. Author information * Author information * Supplementary information Affiliations * Department of Cell Biology, Rosalind Franklin University of Medicine & Science, North Chicago, IL 60064, USA. * Sergey P. Shevtsov & * Miroslav Dundr Contributions M.D. and S.P.S. conceived and designed the experiments. S.P.S. and M.D. performed the experiments and analysed the data. M.D. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Miroslav Dundr Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (487 KB) Supplementary Information * Supplementary Video 2 (125 KB) Supplementary Information PDF files * Supplementary Information (1 MB) Supplementary Information Additional data
• Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation negatively modulates EGFR-mediated ERK activation
  - ncb 13(2):174-181 (2011)
  Nature Cell Biology | Letter Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation negatively modulates EGFR-mediated ERK activation * Jung-Mao Hsu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Chun-Te Chen1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Chao-Kai Chou1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hsu-Ping Kuo1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Long-Yuan Li3, 4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Chun-Yi Lin3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Hong-Jen Lee1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ying-Nai Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mo Liu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hsin-Wei Liao1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Bin Shi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Chien-Chen Lai6 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark T. Bedford7 Search for this author in: * NPG journals * PubMed * Google Scholar * Chang-Hai Tsai5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Mien-Chie Hung1, 2, 3, 4, 5 Contact Mien-Chie Hung Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:174–181Year published:(2011)DOI:doi:10.1038/ncb2158Received15 October 2010Accepted23 November 2010Published online23 January 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Epidermal growth factor receptor (EGFR) can undergo post-translational modifications, including phosphorylation, glycosylation and ubiquitylation, leading to diverse physiological consequences and modulation of its biological activity. There is increasing evidence that methylation may parallel other post-translational modifications in the regulation of various biological processes. It is still not known, however, whether EGFR is regulated by this post-translational event. Here, we show that EGFR Arg 1175 is methylated by an arginine methyltransferase, PRMT5. Arg 1175 methylation positively modulates EGF-induced EGFR trans-autophosphorylation at Tyr 1173, which governs ERK activation. Abolishment of Arg 1175 methylation enhances EGF-stimulated ERK activation by reducing SHP1 recruitment to EGFR, resulting in augmented cell proliferation, migration and invasion of EGFR-expressing cells. Therefore, we propose a model in which the regulatory crosstalk between PRMT5-mediated Arg ! 1175 methylation and EGF-induced Tyr 1173 phosphorylation attenuates EGFR-mediated ERK activation. View full text Figures at a glance * Figure 1: EGFR Arg 1175 is monomethylated. () In vivo methylation of EGFR. A431 cells were metabolically labelled with L-[methyl-3H]methionine (left) or L-[35S]methionine (right) in the presence or absence of protein synthesis inhibitors, as indicated. Immunoprecipitates (IP) of EGFR or control antibodies from L-[methyl-3H]methionine-labelled cells were analysed by fluorography (lanes 1 and 2), Coomassie Blue staining (lanes 3 and 4) or western blotting with EGFR antibody (lanes 5 and 6). Whole-cell lysates of L-[35S]methionine-labelled cells were analysed by Coomassie Blue staining (lanes 7 and 8) or autoradiography (lanes 9 and 10). () Mass spectrometry analysis of endogenous EGFR immunopurified from A431 cells. () Amino acid sequence of peptides corresponding to the EGFR 1171–1182 region, in which Arg 1175 is unmodified, monomethylated or dimethylated. Different amounts of peptides were spotted on PVDF membranes and detected by anti-EGFR or anti-EGFR methylated-Arg 1175 (me-Arg 1175) antibodies. () Western blot ! analysis of exogenous EGFR in HEK293 cells transfected with control vector, EGFR (WT) or EGFR (R1175K). () Western blot analysis of exogenous EGFR in HEK293 cells transfected with empty vector, EGFR (WT) or EGFR (R1175K). Anti-EGFR methylated-Arg 1175 antibody was pre-incubated with peptides, as indicated before use. () Western blot analysis of endogenous EGFR in MDA-MB-468 cells transfected with control or EGFR siRNAs. () Confocal microscopy analysis of MDA-MB-468 cells stained with total endogenous EGFR (red), methylated-Arg 1175 (green) and DAPI (blue). The third columns shows higher-magnification images of the areas outlined in the second column. * Figure 2: PRMT5 interacts with EGFR and methylates Arg 1175. () Western blot analysis of exogenous EGFR and PRMTs in the input and anti-EGFR immunoprecipitates from HEK293 cells transfected with EGFR and GFP–PRMTs, as indicated. () In vitro methylation assay of unmodified EGFR peptide by immunopurified HA–PRMT3, 5 or 8. Methylation of peptides was detected by western blotting (top) and scintillation counting (bottom). Error bars represent s.d. (n=3). () Confocal microscopy analysis of MDA-MB-468 cells stained with endogenous EGFR (red), PRMT5 (green) and DAPI (blue). () Western blot analysis of endogenous EGFR and total PRMT5 of the MDA-MB-468 cells in which endogenous PRMT5 was knocked down by three PRMT5 siRNAs (lanes 1–4) and then rescued with an siRNA-resistant PRMT5 mutant (RR-PRMT5; lane 5). * Figure 3: Suppression of Arg 1175 methylation promotes EGFR-mediated cell proliferation, migration and invasion. () Left: western blot analysis of MCF7 stable transfectants expressing EGFR (WT), EGFR (R1175K) or empty vector. Right: in vitro cell proliferation rates were assayed using the MTT colorimetric method. Error bars represent s.d. (n=5). () Left: in vivo cell proliferation was measured using an orthotopic breast cancer mouse model. Error bars represent s.d. (n=10). Right: two representative tumours from each group in the sixth week after inoculation. () Migration assay of these stable transfectants. Statistical analysis was carried out using Student's t-test. Error bars represent s.d. (n=3). () Invasion assay of these stable transfectants. Statistical analysis was carried out using Student's t-test. Error bars represent s.d. (n=3). * Figure 4: Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation. () Top: western blot analysis of exogenous EGFR in EGF-stimulated MCF7-EGFR (WT) and MCF7-EGFR (R1175K) stable transfectants. Bottom: densitometry of phospho-EGFR Tyr 1173 (p-Tyr 1173) blot. Error bars represent s.d. (n=3). () Top: western blot analysis of endogenous EGFR in EGF-stimulated MDA-MB-468 cells transfected with control or PRMT5 siRNA #1. Bottom: densitometry of phospho-EGFR Tyr 1173 (p-Tyr 1173) blot. Error bars represent s.d. (n=3). () In vitro kinase assay of unmodified (open symbols) and monomethylated (filled symbols) EGFR peptides by immunopurified EGFR proteins. Phosphorylation of peptides was detected by western blotting using anti-EGFR phospho-Tyr 1173 antibody (left) and scintillation counting (right). Error bars represent s.d. (n=3). * Figure 5: Suppression of Arg 1175 methylation inhibits SHP1 recruitment and prolongs ERK activation. () Top: western blot analysis of EGFR, SHP1, Grb2 and SHC in the input and anti-EGFR immunoprecipitates from EGF-stimulated MCF7-EGFR (WT) and MCF7-EGFR (R1175K) stable transfectants. Bottom: densitometry of EGFR-bound SHP1 blot. Error bars represent s.d. (n=3). () Top: western blot analysis of endogenous EGFR, PRMT5, SHP1, Grb2 and SHC in the input and anti-EGFR immunoprecipitates from EGF-stimulated MDA-MB-468 cells transfected with control or PRMT5 siRNA #1. Bottom: densitometry of EGFR-bound SHP1 blot. Error bars represent s.d. (n=3). () Top: western blot analysis of endogenous ERK, PLC-γ, STAT3 and AKT in EGF-stimulated MCF7-EGFR (WT) and MCF7-EGFR (R1175K) stable transfectants. Bottom: densitometry of phospho-ERK (p-ERK) blot. Error bars represent s.d. (n=3). () Top: western blot analysis of endogenous EGFR, PRMT5, ERK, PLC-γ, STAT3 and AKT in EGF-stimulated MDA-MB-468 cells transfected with control or PRMT5 siRNA #1. Bottom: densitometry of phospho-ERK (p-ERK) blot.! Error bars represent s.d. (n=3). Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Chun-Te Chen, * Chao-Kai Chou & * Hsu-Ping Kuo Affiliations * Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA * Jung-Mao Hsu, * Chun-Te Chen, * Chao-Kai Chou, * Hsu-Ping Kuo, * Hong-Jen Lee, * Ying-Nai Wang, * Mo Liu, * Hsin-Wei Liao, * Bin Shi & * Mien-Chie Hung * The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030, USA * Jung-Mao Hsu, * Chun-Te Chen, * Chao-Kai Chou, * Hsu-Ping Kuo, * Hong-Jen Lee, * Mo Liu, * Hsin-Wei Liao & * Mien-Chie Hung * Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan * Long-Yuan Li, * Chun-Yi Lin & * Mien-Chie Hung * Graduate Institute of Cancer Biology, China Medical University, Taichung 404, Taiwan * Long-Yuan Li, * Chun-Yi Lin & * Mien-Chie Hung * Department of Biotechnology, Asia University, Taichung 413, Taiwan * Long-Yuan Li, * Chang-Hai Tsai & * Mien-Chie Hung * Department of Medical Research, China Medical University Hospital, Taichung 404, Taiwan * Chien-Chen Lai & * Chang-Hai Tsai * Science Park-Research Division, The University of Texas MD Anderson Cancer Center, Smithville, Texas 78957, USA * Mark T. Bedford Contributions J-M.H. carried out experimental design and most of the experimental work. J-M.H. and M-C.H. wrote the manuscript. C-K.C. conducted cell migration and invasion experiments. H-P.K. generated stable transfectants and carried out cell proliferation assays. C-T.C. conducted animal experiments. L-Y.L. and C-Y.L. generated antibodies. H-J.L., Y-N.W. and H-W.L. carried out sucrose gradient centrifugation and confocal microscopy analyses. M.L. and B.S. conducted immunoprecipitation assays. C-C.L. carried out mass spectrometry analyses. M.T.B. contributed to PRMT plasmids and reagents. C-H.T. and M-C.H. supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Chun-Te Chen or * Chao-Kai Chou or * Hsu-Ping Kuo or * Mien-Chie Hung Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (700K) Supplementary Information Additional data
• CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity
  - ncb 13(2):182 (2011)
  Nature Cell Biology | Corrigendum CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity * Manu Sharma Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline Burré Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas C. Südhof Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature Cell BiologyVolume: 13,Page:182Year published:(2011)DOI:doi:10.1038/ncb0211-182aPublished online01 February 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, 30–39 (2011); published online 12 December 2010; corrected after print 21 December 2010; In the version of this article initially published online and in print, grant information was missing from the acknowledgements. The acknowledgements have been updated as follows: "We thank S. Chandra for discussions, and J. Mitchell, A. Roth and I. Kornblum for technical support. This work was supported by the National Institute on Aging (NIH grant RC2AG036614 to T.C.S.), and fellowships from the Human Frontiers Program (LT00527/2006-L to M.S.) and the 'Deutsche Akademie der Naturforscher Leopoldina' (BMBF-LPD 9901/8-161 to J.B.)." Additional data
• Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans
  - ncb 13(2):182 (2011)
  Nature Cell Biology | Corrigendum Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans * Lukas J. Neukomm Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas P. Frei Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Cabello Search for this author in: * NPG journals * PubMed * Google Scholar * Jason M. Kinchen Search for this author in: * NPG journals * PubMed * Google Scholar * Ronen Zaidel-Bar Search for this author in: * NPG journals * PubMed * Google Scholar * Zhong Ma Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa B. Haney Search for this author in: * NPG journals * PubMed * Google Scholar * Jeff Hardin Search for this author in: * NPG journals * PubMed * Google Scholar * Kodi S. Ravichandran Search for this author in: * NPG journals * PubMed * Google Scholar * Sergio Moreno Search for this author in: * NPG journals * PubMed * Google Scholar * Michael O. Hengartner Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature Cell BiologyVolume: 13,Page:182Year published:(2011)DOI:doi:10.1038/ncb0211-182bPublished online01 February 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, 79–86 (2011); published online 19 December 2010; corrected after print 07 January 2011; In the version of this letter initially published online and in print, supplementary information figures S3, S4 and S5 were presented in the incorrect order. This error has been corrected in the HTML version of the letter. Additional data