Latest Articles Include:
- The scientist citizen
- ncb 13(4):339 (2011)
Nature Cell Biology | Editorial The scientist citizen Journal name:Nature Cell BiologyVolume: 13,Page:339Year published:(2011)DOI:doi:10.1038/ncb0411-339aPublished online01 April 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. As the US Congress debates the 2011 budget, US scientists must act to prevent damaging cuts to research funding. 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 - Protocol Exchange
- ncb 13(4):339 (2011)
Nature Cell Biology | Editorial Protocol Exchange Journal name:Nature Cell BiologyVolume: 13,Page:339Year published:(2011)DOI:doi:10.1038/ncb0411-339bPublished online01 April 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. Protocol Exchange, a new online repository to enable sharing of protocols. 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 - Do cilia put brakes on the cell cycle?
- ncb 13(4):340-342 (2011)
Nature Cell Biology | News and Views Do cilia put brakes on the cell cycle? * Peter K. Jackson1Journal name:Nature Cell BiologyVolume: 13,Pages:340–342Year published:(2011)DOI:doi:10.1038/ncb0411-340Published online01 April 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. Two papers in this issue show that dynein-binding proteins may regulate the G1–S transition through an effect on cilia. Nde1, a known partner of dynein light chain LC8, controls ciliary length in vitro and in zebrafish, and influences the G1–S progression. The phosphorylation of Tctex1, a dynein light chain, modulates cilia length and accelerates G1–S, thereby regulating proliferation–differentiation decisions in the developing mouse neocortex. 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 * Peter K. Jackson is at Genentech Inc, 1 DNA way, MC214, South San Francisco, California 94080, USA. Competing financial interests P.K.J. is an employee of Genentech, Inc. Corresponding author Correspondence to: * Peter K. Jackson Author Details * Peter K. Jackson Contact Peter K. Jackson Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Lysosome positioning coordinates mTORC1 activity and autophagy
- ncb 13(4):342-344 (2011)
Nature Cell Biology | News and Views Lysosome positioning coordinates mTORC1 activity and autophagy * Christian Poüs1, 2 * Patrice Codogno1, 3 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:342–344Year published:(2011)DOI:doi:10.1038/ncb0411-342Published online01 April 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. Under nutrient-rich conditions, the nutrient-sensitive kinase mTOR (mammalian target of rapamycin) is recruited to the surface of lysosomes where it becomes activated and can promote cell growth and inhibit autophagy. In contrast, mTOR is inhibited in nutrient-poor conditions, leading to the induction of autophagy. The intracellular positioning of lysosomes in response to nutrient availability is now shown to orchestrate mTOR activation and regulate autophagy. 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 * Christian Poüs and Patrice Codogno are at the Université Paris-Sud 11, IFR 141, Faculté de Pharmacie, 92296 Châtenay-Malabry, France. * Christian Poüs is also at the Université Paris-Sud 11, EA 4530, Faculté de Pharmacie, 92296 Châtenay-Malabry, France * Patrice Codogno is also at INSERM UMR984, 92296 Châtenay-Malabry, France. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Patrice Codogno Author Details * Christian Poüs Search for this author in: * NPG journals * PubMed * Google Scholar * Patrice Codogno Contact Patrice Codogno Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - The myosin-II-responsive focal adhesion proteome: a tour de force?
- ncb 13(4):344-346 (2011)
Nature Cell Biology | News and Views The myosin-II-responsive focal adhesion proteome: a tour de force? * Lisa Gallegos1 * Mei Rosa Ng1 * Joan S. Brugge1 * Affiliations * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:344–346Year published:(2011)DOI:doi:10.1038/ncb2230Published online20 March 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 formation and maturation of focal adhesions involves significant changes in protein composition and requires acto-myosin contractility. A mass spectrometry approach reveals changes to the focal adhesion proteome on myosin inhibition, providing a valuable resource for the cell adhesion field. 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 * Lisa Gallegos, Mei Rosa Ng and Joan S. Brugge are in the Department of Cell Biology, Harvard Medical School, Boston Massachusetts, USA Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Joan S. Brugge Author Details * Lisa Gallegos Search for this author in: * NPG journals * PubMed * Google Scholar * Mei Rosa Ng Search for this author in: * NPG journals * PubMed * Google Scholar * Joan S. Brugge Contact Joan S. Brugge Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Out of the shade and into the light
- ncb 13(4):347-349 (2011)
Nature Cell Biology | News and Views Out of the shade and into the light * Markus Grebe1Journal name:Nature Cell BiologyVolume: 13,Pages:347–349Year published:(2011)DOI:doi:10.1038/ncb0411-347Published online01 April 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. Plants reach for the sun by avoiding the shade and by directly growing towards the light. Two studies now suggest that the polar relocation of PIN3, a transporter directing the flow of the plant hormone auxin, drives both growth processes. PIN3 repolarization occurs downstream of shade perception through phytochrome photoreceptors, whereas blue light perceived by phototropin initiates polar recycling of PIN3 and growth towards the light. 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 * Markus Grebe is at the Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, SE-90187 Umeå, Sweden. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Markus Grebe Author Details * Markus Grebe Contact Markus Grebe Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Cell Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Research highlights
- ncb 13(4):350 (2011)
Nature Cell Biology | Research Highlights Research highlights Journal name:Nature Cell BiologyVolume: 13,Page:350Year published:(2011)DOI:doi:10.1038/ncb0411-350Published online01 April 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. Autophagy is induced in response to stress and allows cells to survive through recycling of cellular components. As such, autophagy enables cancer cell survival in oxygen-poor tumour regions. White and colleagues now show that oncogenic Ras upregulates basal autophagy, and that Ras-transformed cells require autophagy to maintain mitochondrial function (Genes Dev.25, 460–470; 2011). Expression of constitutively active Ras mutants induced autophagy in an mTOR-independent manner. Intriguingly, autophagy-defective Ras-transformed cells were not able to proliferate in nutrient-poor conditions and could not form tumours efficiently in vivo, suggesting a critical role for autophagy in Ras-mediated tumour maintenance. Indeed, basal autophagy was upregulated in human cancer cell lines expressing mutant Ras, and mutations in key autophagy genes suppressed proliferation in several of these cell lines. 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 - Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry
- ncb 13(4):351-360 (2011)
Nature Cell Biology | Article Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry * Sehyun Kim1 * Norann A. Zaghloul2, 4 * Ekaterina Bubenshchikova1 * Edwin C. Oh2, 5 * Susannah Rankin1, 3 * Nicholas Katsanis2, 5 * Tomoko Obara1 * Leonidas Tsiokas1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:351–360Year published:(2011)DOI:doi:10.1038/ncb2183Received03 October 2010Accepted20 January 2011Published online13 March 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 primary cilium is an antenna-like organelle that is dynamically regulated during the cell cycle. Ciliogenesis is initiated as cells enter quiescence, whereas resorption of the cilium precedes mitosis. The mechanisms coordinating ciliogenesis with the cell cycle are unknown. Here we identify the centrosomal protein Nde1 (nuclear distribution gene E homologue 1) as a negative regulator of ciliary length. Nde1 is expressed at high levels in mitosis, low levels in quiescence and localizes at the mother centriole, which nucleates the primary cilium. Cells depleted of Nde1 have longer cilia and a delay in cell cycle re-entry that correlates with ciliary length. Knockdown of Nde1 in zebrafish embryos results in increased ciliary length, suppression of cell division, reduction of the number of cells forming the Kupffer's vesicle and left–right patterning defects. These data suggest that Nde1 is an integral component of a network coordinating ciliary length with cell cycle prog! ression and have implications for understanding the transition from a quiescent to a proliferative state. View full text Figures at a glance * Figure 1: Depletion of Nde1 induces longer cilia. () Immunofluorescence microscopy staining of centrin2 (green) or Nde1 (red) in NIH-3T3WT cells. () Top: expression of endogenous Nde1 in asynchronous cultures of the indicated cell lines. Bottom: α-tubulin (loading control). NIH-3T3 cells stably transfected with a mouse Nde1-specific short hairpin RNA containing a three-nucleotide deletion in the sense strand were used as a negative control cell line (NIH-3T3KD-CON). () Cilia formation at indicated times during serum starvation in NIH-3T3WT and NIH-3T3Nde1–KD2 cells. Cilia or basal bodies were visualized by an antibody against acetylated α-tubulin (red) or γ-tubulin (green). () Ciliary length of NIH-3T3WT cells and NIH-3T3Nde1–KD2 cells at 0, 6, 12 and 24 h of serum starvation. Ciliary length was measured from ciliated cells per time-point from a representative experiment. () DAPI-, doublecortin (DCX)- and adenylyl cyclase 3 (ACIII)-labelled freshly dissociated, mouse E18.5 embryonic cortical neurons transiently trans! fected with GFP (top) or GFP and Nde1-specific shRNA (bottom). DCX-, ACIII-, or DAP1/GFP/ACIII/DCX-labeled neurons are shown in left. middle or right panels respectively () Ciliary length of GFP+/DCX+ embryonic neurons transiently transfected with GFP alone (WT) or GFP and Nde1-specific shRNA (NdeKD). () Expression of Nde1 (top) or α-tubulin (bottom) in RPE1-hTERT cells transfected with siRNA as indicated. () RPE1-hTERT cells transiently transfected with control siRNA (scrambled-sequence siRNA) or hNde1 siRNA (hNde1KD) were double-stained with antibodies against γ-tubulin (red) and acetylated α-tubulin (green) following 24 h serum starvation. () Ciliary length of cells transfected with control siRNA and hNde1KD RPE1-hTERT cells at 0, 12 and 24 h of serum starvation. Quantification was obtained from ciliated cells. () Ki-67 positive control or hNde1KD RPE1-hTERT cells were synchronized in mitosis by nocodazole treatment (M sync.), followed by a 6 h recovery in complete me! dia (0 h of serum starvation), and serum starvation for 12 h o! r 24 h (n = 3 independent experiments). Scale bars: , 2.5 μm; , 2.5 μm; , 5 μm; , 10 μm. Insets in images are higher-magnification images of indicated regions. , , , ; data are means ± s.e.m.; asterisks indicate P < 0.05, Student's t-test; values in bars indicate n values. * Figure 2: Expression of Flag-tagged human Nde1 rescues abnormally long cilia in NIH-3T3Nde1–KD2 cells. () Expression levels of Flag–hNde1 in NIH-3T3Nde1–KD2 cells. Lysates of NIH-3T3WT cells stably expressing GFP (NIH-3T3WT Mock), NIH-3T3Nde1–KD2 cells stably expressing GFP (NIH-3T3Nde1–KD2 Mock) or NIH-3T3Nde1–KD2 cells stably expressing Flag–hNde1 (NIH-3T3Nde1–KD2 Flag–hNde1) were immunoblotted for Nde1 and α-tubulin as loading control (top) or Flag (bottom). () Ciliary length of NIH-3T3WT cells stably expressing GFP (NIH-3T3WT Mock; n = 57), NIH-3T3Nde1–KD2 stably expressing GFP (NIH-3T3Nde1–KD2 Mock; n = 78), or NIH-3T3Nde1–KD2 stably expressing Flag–hNde1 (NIH-3T3Nde1–KD2 hNde1; n = 100) following serum starvation for 24 h. Data are means ± s.e.m. Asterisk indicates P < 0.05. () Cilia staining in mock-infected NIH-3T3WT (A) and NIH-3T3Nde1–KD2 cells (B), or NIH-3T3Nde1–KD2 cells infected with increasing amounts of Flag–hNde1 (C–L). Antibody against acetylated α-tubulin was used to visualize cilia (A–L), antibody against Flag was use! d to detect Flag–hNde1 (C–G), and antibody against γ-tubulin used to visualize the basal body (A, B and H–L). Note that ciliary morphology and length changed according to the amount of Flag–hNde1 expressed (C–G). Bottom; schematic representation of the dosage-dependent effect of Flag–hNde1 on ciliary length and morphology. Low levels of Flag–hNde1 converted abnormally long cilia back to cilia of normal size (compare A with C), whereas moderate or high levels of Flag–hNde1 resulted in bulged (D–F) or stumpy cilia (G), respectively. () RPE1-hTERT cells were transiently transfected with Flag–hNde1 and recovered for 48 h followed by an additional 24 h of serum starvation. Cells were double-stained with antibodies against Flag (red) or acetylated α-tubulin (green). () Schematic representation of full length Nde1. Coiled-coil domains are shown as grey boxes (residues 18–85 and 90–188). () Myc-tagged Nde1L135P,F138P was transiently transfected into NIH-3T! 3WT cells. Cells were serum starved for 24 h and double-staine! d with antibodies against acetylated α-tubulin (green) or the Myc epitope (red). () Summary of structure-function analysis. Scale bars: , 2.5 μm; , 7 μm; , 7 μm. * Figure 3: Nde1 suppresses ciliogenesis through LC8. () Immunofluorescence microscopy of NIH-3T3WT cells transiently co-transfected with wild-type Nde1–Myc and Flag–LC8 at plasmid ratios of 9:1 and 1:9. Co-transfection of Nde1–Myc and Flag-tagged bacterial alkaline phosphatase (Flag–BAP) in a plasmid ratio of 1:9 was used as a control. () Expression levels of LC8 (top) or α-tubulin (loading control, bottom) in NIH-3T3WT cells transiently transfected with Flag–LC8 (Flag–LC8OE; OE: overexpression), control siRNA or a mouse LC8-specific siRNA (LC8KD). () Immunofluorescence microscopy of NIH-3T3WT and NIH-3T3Nde1–KD2 cells transiently transfected with LC8-specific siRNA (LC8KD) and double-stained with antibodies against γ-tubulin (red) and acetylated α-tubulin (green), following 24 h of serum starvation. () NIH-3T3WT cells were transiently transfected with Flag–LC8OE and double stained with an antibody raised against the Flag epitope (Flag–LC8, red) or acetylated α-tubulin (green). (, ) Quantification of cili! ary length () or ciliation () of untransfected NIH-3T3WT cells (n = 35), or cells transiently transfected with Flag–LC8OE (n = 76), or LC8-specific siRNA (hNde1KD, n = 44). Ciliary length was measured from ciliated cells and percentage of ciliated cells was obtained from three independent experiments (n = 3). Data are means ± s.e.m. Asterisk indicates P < 0.05, Student's t test. (, ) GFP–PACT (green, ) or GFP–PACT–LC8/BS (green, ) was transiently expressed in NIH-3T3WT and NIH-3T3Nde1–KD2 cells, followed by immunofluorescence microscopy staining with γ-tubulin (yellow) or acetylated α-tubulin (red). Although both constructs were targeted specifically to the basal body, only GFP–PACT–LC8/BS caused the formation of bulged or stumpy cilia. One or two representative examples of bulged and stumpy cilia of NIH-3T3WT or NIH-3T3Nde1–KD2 cells, respectively, transfected with GFP-PACT-LC8/BS are depicted in boxed regions. Insets in images are higher-magnification i! mages of boxed regions. () Schematic representation summarizin! g the functional role of the Nde1–LC8 interaction in ciliogenesis. Sequestration of LC8 at the basal body suppresses cilia formation, whereas increase of unbound LC8 at the basal body promotes cilia formation. Scale bars: , 7 μm; , 10 μm; , 10 μm; , , 10 μm. * Figure 4: Nde1 expression inversely correlates with ciliogenesis. (, ) Centriolar expression of Nde1 decreases on ciliation. RPE1-hTERT () or NIH-3T3WT () cells were serum-starved for the indicated times. Nde1 (red) or acetylated α-tubulin (green) were visualized by indirect immunofluorescence microscopy. Arrows indicate Nde1 localization. (, ) Quantification of fluorescence intensity ratio of Nde1/γ-tubulin (red/green) signals at the centrosome at 0, 12 or 24 h of serum starvation. Fluorescence intensity of coinciding green or red pixels within the boxed area (inset) was measured in a projection of z series collected in 0.5 μm intervals. The range of fluorescence intensity per pixel in a box was from 0–255 (n is indicated on graph). Data are means ± s.e.m. () Cell-cycle-dependent regulation of Nde1 expression. Asynchronously proliferating NIH-3T3WT cells (Asyn.) were serum starved for 12h (12 h serum –), and 24h (24 h serum –), followed by serum re-stimulation for 6 h (6 h serum +), and 12 h (12 h serum +). Phosphorylation level! s of RB (pRBS807/811) and Cdc2 (pCdc2Y15) or levels of cyclin A, cyclin E and Nde1 were determined by immunoblotting. α-tubulin was used as a loading control. () Nde1 levels decrease as cells exit mitosis. Top: cell cycle analysis of NIH-3T3WT cells synchronized in mitosis by nocodazole treatment (600 ng ml−1) for 12 h (0 h), followed by wash and release into complete media (10% calf serum) for 0.5, 1, 2 or 4 h (top). Triangles indicate cells with 2n or 4n of DNA content. Black line represents the overall distribution of cells that fall into either 2n or 4n of DNA content. Red or orange peaks are cell populations that fall into 2n or 4n of DNA content, repectively. Bottom: lysates from cells arrested in mitosis (0 h), and cells released from mitosis for 0.5 h, 1 h, 2 h or 4 h were immunoblotted with antibodies against Nde1 or β-actin. () Schematic representation of Nde1 expression during the cell cycle and ciliogenesis. Scale bars: , 10 μm; , 2.5 μm. * Figure 5: Nde1 depletion causes a delay in cell cycle re-entry. () NIH-3T3WT (top) and NIH-3T3Nde1–KD2 (bottom) cells were arrested in G0 by 24 h serum starvation (0 h serum +) and induced to re-enter the cell cycle by serum re-stimulation for 12 h (12 h serum +). Cells were pulse-labelled with EdU and immunostained with antibodies against γ-tubulin (green), acetylated α-tubulin (red) and EdU (green, inset). For illustration purposes, EdU labeling is shown as 25% reduction in size of the main image. Arrows indicate EdU-labelled cells. () Percentage of EdU-positive NIH-3T3WT or NIH-3T3Nde1–KD2 cells following serum re-stimulation (n = 3 independent experiments). () Ciliary length of NIH-3T3WT cells and NIH-3T3Nde1–KD2 cells at 0 or 12 h of serum re-stimulation. () Percentage of ciliated NIH-3T3WT or NIH-3T3Nde1–KD2 cells following serum re-stimulation (n = 3 independent experiments). () Ciliary length of EdU-positive NIH-3T3WT or NIH-3T3Nde1–KD2 cells following serum re-stimulation. () RPE1-hTERT cells transiently transfected ! with control siRNA or Nde1 siRNA (hNde1KD) were immunostained with antibodies against γ-tubulin (green), acetylated α-tubulin (red) and EdU (green, inset). Arrows indicate EdU-labelled cells. () Percentage of control or hNde1KD RPE1-hTERT cells labelled with EdU at 24 h following serum starvation (0 h serum +) and at 12 h (12 h serum +), and 24 h (24 h serum +) following serum re-stimulation. n = 3 independent experiments. () Phosphorylated RB (pRBS807/811) and cyclin A levels in control (lanes 1, 3, 5 and 7) and hNde1KD RPE1-hTERT cells (lanes 2, 4, 6 and 8) at 6 h, 12 h, 18 h and 24 h following serum re-stimulation. α-tubulin was used as loading control. () Ciliary length of RPE1-hTERT control cells and hNde1KD cells at 0, 12 or 24 h of serum re-stimulation. () Percentage of ciliated control or hNde1KD RPE1-hTERT cells following serum re-stimulation (n = 3 independent experiments). Scale bars: , 2.5 μm; , 10 μm. –, , , ; data are means ± s.e.m. and asterisks indic! ate P < 0.05, Student's t test. , , ; n values are indicated i! n bars. * Figure 6: Knockdown of Nde1 causes a cilium-dependent delay in cell cycle re-entry. () Expression levels of Nde1 and IFT20 (top) or α-tubulin (loading control, bottom) in untransfected RPE1-hTERT cells (lane 1), RPE1-hTERT cells stably expressing an shRNAi construct targeting IFT20 (IFT20KD) and transiently transfected with an siRNA against hNde1 (lane 2), or IFT20KD cells (lane 3). () Expression levels of IFT88/polaris (top), Nde1 (middle) or α-tubulin (loading control, bottom) in untransfected RPE1-hTERT cells (lane 1), and in RPE1-hTERT cells transiently transfected with a siRNA against hNde1 (hNde1KD; lane 2), siRNA against iFT88/polaris (IFT88KD; lane 3), or siRNAs for both hNde1 and IFT88/polaris (IFT88/hNde1KD; lane 4). () RPE1-hTERT IFT20KD, IFT20/ hNde1KD, IFT88KD or IFT88/ hNde1KD cells were double stained with antibodies against γ-tubulin (green) and acetylated α-tubulin (red) along with DAPI (blue). () Cilia length distribution of RPE1-hTERT control, hNde1KD, IFT20KD, IFT20/ hNde1KD, IFT88KD and IFT88/hNde1KD cells. Overall percentile of cil! iation for each of the cell types is indicated. () RPE1-hTERT cells transfected with control siRNA or hNde1 siRNA (hNde1KD) were treated with Cytochalasin D (CD) and immunostained with antibodies against γ-tubulin (green) or acetylated α-tubulin (red). () Cilia length distribution the indicated cells. () Time course of cell cycle re-entry of the indicated RPE1-hTERT cells in response to serum re-stimulation. Data are means ± s.e.m. (n=3 independent experiments). () Time course of cell cycle re-entry of NIH-3T3WT cells transfected with control siRNA or IFT88/polaris-specific siRNA (NIH-3T3WTIFT88KD), and NIH-3T3Nde1–KD2 cells transiently transfected with control siRNA or IFT88/polaris-specific siRNA (NIH-3T3Nde1–KD2 IFT88KD). Data are means ± s.e.m. (n = 3 independent experiments). () Rab8aQ67L–GFP induces long cilia in NIH-3T3WT cells. GFP- or Rab8aQ67L–GFP-transfected NIH-3T3WT cells were stained for γ-tubulin (yellow or green) or acetylated α-tubulin (red). ! Scale bars, 10 μm. () Time course of cell cycle re-entry of N! IH-3T3WTGFP cells transfected with GFP or NIH-3T3WT cells transfected with Rab8aQ67L–GFP in response to serum re-stimulation. Data are means ± s.e.m. (n = 3 independent experiments). * Figure 7: Depletion of nde1 in zebrafish leads to longer cilia and a smaller Kupffer's vesicle. () Ciliary length at 10 somite stage (ss) of wild-type zebrafish embryos (WT) and embryos injected with nde1 morpholino oligonucleotides (nde1 MO), or co-injected with nde1 morpholino oligonucleotides and human NDE1 cap mRNA (rescue). () Whole-mount immunofluorescence microscopy staining of cilia at the Kupffer's vesicle in indicated embryos at 6 or 10 ss using antibodies against acetylated α-tubulin (green) and atypical PKC (aPKC, red). () Ciliary length in Kupffer's vesicle of indicated embryos at 6 or 10 ss. n represents ciliated cells from 6–10 embryos per group. () Number of cells in the Kupffer's vesicle (Kupffer's vesicle) in indicated embryos at 6 or 10 ss. () Percentage of ciliated cells in the Kupffer's vesicle of indicated embryos at 6 and 10 ss (n = 3 independent experiments). () Whole-mount immunofluorescence microscopy staining of the Kupffer's vesicle of 10 ss embryos. Antibodies against phosphorylated histone H3 (pH3, green) or atypical PKC (aPKC, red) wer! e used. () Percentage of pH3-positive cells in the Kupffer's vesicle of 10ss embryos (n = 3 independent experiments). () Left: percentage of the indicated embryos with no expression of southpaw (Absent), expression at the right side (Right), left side (Left), or expression on both sides (Bilateral) of the lateral plate mesoderm at 14 hpf. Right; example images of dorsal view of southpaw mRNA at 14 hpf. () Left: percentage of embryos in each group with no looping (No loop), leftward (Left), or rightward looping (Right) of the heart tube at 48 hpf determined by the expression pattern of myl-7 mRNA. Laterality defects are manifested as non-looping (No Loop) or leftward looping (Left), whereas wild-type embryos show rightward looping of the heart (Right). Right: example images of heart looping in embryos. Data in , , , ; means ± s.e.m.; asterisks indicate P < 0.001 (, ) and P < 0.05, Student's t test; n values are indicated in bars (, , ). Scale bars, 10 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Cell Biology, University of Oklahoma Health Sciences Center, 975 NE 10th Street, Oklahoma City, Oklahoma 73104, USA. * Sehyun Kim, * Ekaterina Bubenshchikova, * Susannah Rankin, * Tomoko Obara & * Leonidas Tsiokas * McKusick-Nathans Institute of Genetic Medicine, Departments of Ophthalmology, Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, USA. * Norann A. Zaghloul, * Edwin C. Oh & * Nicholas Katsanis * Department of Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma city, Oklahoma 73114, USA. * Susannah Rankin * Present address: University of Maryland School of Medicine, Department of Medicine, Division of Endocrinology, Diabetes and Nutrition, 660 W. Redwood Street, Baltimore, Maryland 21201, USA. * Norann A. Zaghloul * Department of Cell Biology and Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina 27710, USA. * Edwin C. Oh & * Nicholas Katsanis Contributions S.K., N.A.Z., E.B., E.C.O. and S.R. performed experiments. S.K., N.A.Z., E.B., S.R., N.K., T.O. and L.T. analysed data. S.K. and L.T. planned the project. S.K. and L.T. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Leonidas Tsiokas Author Details * Sehyun Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Norann A. Zaghloul Search for this author in: * NPG journals * PubMed * Google Scholar * Ekaterina Bubenshchikova Search for this author in: * NPG journals * PubMed * Google Scholar * Edwin C. Oh Search for this author in: * NPG journals * PubMed * Google Scholar * Susannah Rankin Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas Katsanis Search for this author in: * NPG journals * PubMed * Google Scholar * Tomoko Obara Search for this author in: * NPG journals * PubMed * Google Scholar * Leonidas Tsiokas Contact Leonidas Tsiokas Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration
- ncb 13(4):361-369 (2011)
Nature Cell Biology | Article Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration * Dong Zhang1 * Kyle D. Grode2, 5 * Shannon F. Stewman1, 5 * Juan Daniel Diaz-Valencia3, 5 * Emily Liebling1 * Uttama Rath1 * Tania Riera1 * Joshua D. Currie2 * Daniel W. Buster4 * Ana B. Asenjo1 * Hernando J. Sosa1 * Jennifer L. Ross3, 5 * Ao Ma1, 5 * Stephen L. Rogers2, 5 * David J. Sharp1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:361–369Year published:(2011)DOI:doi:10.1038/ncb2206Received16 November 2010Accepted07 January 2011Published online06 March 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 Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubul! e behaviours involved in cell migration. View full text Figures at a glance * Figure 1: Dm-Kat60 targets the cell cortex of interphase cells. () Immunofluorescence micrograph showing the localization of Dm-Kat60 in interphase S2 cells (antibody characterized in ref. 12). () Immunofluorescence micrograph of an interphase S2 cell double labelled for microtubules (anti-α-tubulin; red) and Dm-Kat60 (green). () High magnification of the region outlined in . () Immunofluorescence of an interphase S2 cell double labelled for actin (red) and Dm-Kat60 (green) and a higher magnification of the two regions outlined in the 'merge' panel. Scale bars, 10 μm (,,), 2 μm (). * Figure 2: Depletion of Dm-Kat60 causes significant microtubule curling and bundling beneath the cortex. () Confocal images of live GFP–α-tubulin-expressing S2 cells treated with control RNA. The cell border is marked with a dashed red line in the higher-magnification image shown on the bottom right. The bottom right panel is also shown in Supplementary Movie S1a. () Time series of a putative microtubule-severing event at the cortex. The green arrow marks the site of microtubule breakage and the dashed red line marks the cell border. See also Supplementary Movie S1c. () Images of live GFP–α-tubulin-expressing S2 cells treated with Dm-Kat60 RNAi. As in , the cell border is marked with a dashed red line in the higher-magnification image shown in the bottom right of this panel. This panel is also shown in Supplementary Movie S1b. () Time series of images obtained from a Dm-Kat60 RNAi-treated cell showing lateral growth of microtubule ends along the cortex and their subsequent incorporation into the underlying microtubule array. Individual ends are indicated by coloured circl! es and time (s) is labelled in each image. Scale bars, 10 μm (,), 1 μm (), 2 μm (). * Figure 3: Automated tracking and quantitative analysis of microtubule plus-end organization and dynamics in control and Dm-Kat60 RNAi-treated cells. () Ends of GFP-labelled microtubules were tracked with an automated tracking algorithm. Each tracked microtubule end is marked with a coloured dot: growing ends in blue, pausing ends in yellow and shrinking ends in pink. The stack of panels on the right is a time series of the outlined region. () Trajectories of microtubule ends tracked in a control RNA-treated cell. Individual microtubule trajectory paths are indicated by coloured lines. () Trajectories of microtubule ends tracked in a Dm-Kat60 RNAi cell. Again, individual trajectory paths are indicated by coloured lines. Relative to controls, significantly more microtubules continue to grow after reaching the cortex, and so microtubules are frequently observed to bend and grow parallel to the cortex. () The microtubule ends in Dm-Kat60 RNAi cells (five cells, 494 microtubules, 6,087 distances) spent significantly (P<0.0001) more time in the vicinity of the cortex, compared with control cells (seven cells, 257 microtubules,! 2,600 distances). Microtubule ends within the cell interior could not be identified and thus were not analysed. () Cortical microtubules spend more time growing after Dm-Kat60 knockdown. The change in dynamic behaviour is most prominent in microtubule ends very near the cell margin (<1.3 μm), but is still significant (P<0.0001) for ends further away (>1.3 μm). Numbers within bars indicate the number of frames in which microtubules grew parallel to the cortex/total number of frames observed. Error bars, s.e.m. Scale bars, 10 μm (all image panels). * Figure 4: Dm-Kat60 targets to the leading edge of motile D17 cells and negatively regulates their migration. () Immunolocalization of Dm-Kat60, tubulin and actin in polarized Drosophila D17 cells. The arrowheads indicate enrichment of Dm-Kat60 at the leading edge; the outlined areas are magnified in the far right panels. () Western blot showing the depletion of Dm-Kat60 in D17 cells using RNAi. () Representative phase-contrast images of scratch-wounds at 0 and 24 h after wounding. () Quantification of migration during wound closure for control and Dm-Kat60 RNAi-treated D17 cells. Migration area was calculated by subtracting the total wound area at 24 h from the total wound area at 0 h after wounding and then normalized to control RNA. P=8.5×10−5. Data represent mean values ± s.e.m. from four independent experiments. Numbers in bars are sample sizes. Scale bars, 10 μm (), 50 μm (). Uncropped images of blots are shown in Supplementary Fig. S8b. * Figure 5: Dm-Kat60 negatively regulates multiple parameters of D17-cell motility. () Representative migration tracks of 20 cells for each treatment group (control and Dm-Kat60 RNAi; also see Supplementary Movie S2). () Frequency distributions of instantaneous cell velocities from time-lapse imaging of individual D17-cell movements. P=8.2×10−40. () Quantification of the ratio of velocity frequencies between Dm-Kat60 and control RNAi treatments. The Dm-Kat60/control RNAi ratio was calculated by dividing the number of movements for Dm-Kat60 RNAi-treated cells by the number of movements for control RNA-treated cells for a given range of velocities from the graphs in . A ratio less than one (red) represents a decreased number of movements for Dm-Kat60 RNAi relative to control RNA, and a ratio greater than one (blue) represents an increased number of movements for Dm-Kat60 RNAi relative to control RNA. Relative to control, Dm-Kat60 depletion decreases the frequency of cells migrating at low rates while increasing the frequency of high migration rates. () Qua! ntification of total migration distance over a 3 h time period. P=1.5×10−5. () Quantification of intrinsic cell directionality. Directionally persistent migration (D/T) was calculated as a ratio of the direct distance between start and end points (D) to the total migration distance (T). P=3.6×10−2. () Quantification of persistent migration. The percentage of time that cells spent migrating was calculated by subtracting those movements between frames that were less than 2 μm (considered to be migratory pauses) from the total number of movements and then dividing by the total number of movements. P=3.7×10−5. Data represent mean values ± s.e.m. from four separate double-stranded RNA treatments. Numbers in bars are sample sizes. * Figure 6: Dm-Kat60 negatively regulates actin protrusions at the cell edge. () Time-lapse images showing the cortical dynamics of control and Dm-Kat60 RNAi-treated S2 cells expressing GFP–α-tubulin and mCherry–actin. Yellow lines outline the edges of the cortical actin arrays in each image. These lines are stacked vertically (and shown in red) on the far right to illustrate the time-dependent alterations in the morphology of the cell edge (protrusions) in each condition (T labels the time axis). () Representative kymographs of mCherry–actin-labelled cortical regions from a control and Dm-Kat60 RNAi-treated cell. () Dm-Kat60 RNAi significantly increases both the frequency (left) and average displacement (right) of actin-based membrane protrusions (P<0.0001 for both). Data represent mean ± s.e.m. and numbers in the columns indicate regions/cells analysed. Scale bars, 2 μm for and . * Figure 7: Dm-Kat60 severs and depolymerizes microtubules from their ends. () Coomassie-blue-stained SDS–polyacrylamide gel electrophoresis of the purified rDm-Kat60 protein used for analysis (see also Supplementary Fig. S8c). The arrow indicates the band corresponding to rDm-Kat60. () Time series of TIRF images showing the disassembly of a field of immobilized microtubules by 50 nM rDm-Kat60 and ATP. Green arrows mark the ends of an individual microtubule at the beginning of visualization; a red arrow marks a microtubule end that has shrunk from its initial position. Time (seconds) is indicated. () Left, time series of an individual microtubule (marked by arrows in ) before (pre) and after addition of rDm-Kat60. i–iii, Kymographs showing the depolymerization and severing (indicated by dotted yellow lines) of individual microtubules from other experiments. The time (t) axis is vertical and distance (x–y) axis horizontal in all kymographs shown in this figure. () Left, time series of an individual polarity-marked microtubule incubated with r! Dm-Kat60. The plus-end is dimly labelled and the minus-end is brightly labelled. i,ii, Kymographs showing the depolymerization and severing (dotted yellow lines) of further polarity-marked microtubules. () Upper, quantification of the frequency of microtubule severing by rDm-Kat60. All conditions included 2 mM ATP (or 2 mM AMPPNP). P=0.031. Lower, measured rates of microtubule plus- and minus-end depolymerization by rDm-Kat60. P=3.1×10−7. Again, all conditions included 2 mM ATP or AMPPNP. Data in both panels represent mean ± s.e.m. N= number of microtubules analysed. () Electron micrographs showing the ends of negatively stained microtubules after incubation with: no Dm-Kat60 (control), 50 nM rDm-Kat60 and 2 mM ATP (rDm-Kat60) or full-length KLP59D and ATP (KLP59D). () Quantification of microtubule ends per unit microtubule length. Compared with the no Dm-Kat60 and Dm-Kat60 (50 nM)+AMPPNP (2 mM) controls, Dm-Kat60+ATP (50 nM GFP–Dm-Kat60, 2 mM ATP)! induces three times more ends (P<0.0001). Numbers in the colu! mn indicate numbers of microtubules/electron microscopy fields. Data represent mean ± s.e.m. pooled from two independent experiments. Scale bars, 500 μm (), 10 μm (), 20 nm (). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Kyle D. Grode, * Shannon F. Stewman, * Juan Daniel Diaz-Valencia, * Jennifer L. Ross, * Ao Ma & * Stephen L. Rogers Affiliations * Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA * Dong Zhang, * Shannon F. Stewman, * Emily Liebling, * Uttama Rath, * Tania Riera, * Ana B. Asenjo, * Hernando J. Sosa, * Ao Ma & * David J. Sharp * Department of Biology and the Center for Carolina Genome Sciences, University of North Carolina, Chapel Hill, 421 Fordham Hall, CB#3280, Chapel Hill, North Carolina 27599, USA * Kyle D. Grode, * Joshua D. Currie & * Stephen L. Rogers * Department of Physics, University or Massachusetts, Amherst, 302 Hasbrouck Laboratory, Amherst, Massachusetts 01003, USA * Juan Daniel Diaz-Valencia & * Jennifer L. Ross * Department of Cell Biology and Anatomy and the Arizona Cancer Center, University of Arizona, Tuscon, 1515 N. Campbell Avenue, Tuscon, Arizona 85724, USA * Daniel W. Buster Contributions D.Z. carried out and analysed most experiments using Drosophila S2 and human cells. D.Z. also purified and carried out the initial in vitro characterization of rDm-Kat60. K.D.G. carried out most experiments with Drosophila D17 under the direction of S.L.R.; J.D.C. also carried out analyses in D17 cells. S.F.S. designed the automated tracking algorithm under the direction of A.M. and used it to track microtubules in live-cell movies provided by D.Z.; J.D.D-V. and J.L.R. carried out numerous in vitro assays to quantify the microtubule-severing and end-depolymerase activities of rDM-Kat-60. A.B.A., E.L. and T.R. carried out and analysed the electron microscopy under the direction of H.J.S. U.R. carried out KLP59D for the electron microscopy assay. D.W.B. helped in the design of many experiments in S2 cells and made the model figure shown in Supplementary Information. D.J.S. wrote the manuscript (with the help of all authors, but particularly S.L.R.) and coordinated the efforts ! of the multiple laboratories involved in this project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David J. Sharp Author Details * Dong Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Kyle D. Grode Search for this author in: * NPG journals * PubMed * Google Scholar * Shannon F. Stewman Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Daniel Diaz-Valencia Search for this author in: * NPG journals * PubMed * Google Scholar * Emily Liebling Search for this author in: * NPG journals * PubMed * Google Scholar * Uttama Rath Search for this author in: * NPG journals * PubMed * Google Scholar * Tania Riera Search for this author in: * NPG journals * PubMed * Google Scholar * Joshua D. Currie Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel W. Buster Search for this author in: * NPG journals * PubMed * Google Scholar * Ana B. Asenjo Search for this author in: * NPG journals * PubMed * Google Scholar * Hernando J. Sosa Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer L. Ross Search for this author in: * NPG journals * PubMed * Google Scholar * Ao Ma Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen L. Rogers Search for this author in: * NPG journals * PubMed * Google Scholar * David J. Sharp Contact David J. Sharp Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (4M) Supplementary Information * Supplementary Movie 2 (6M) Supplementary Information * Supplementary Movie 3 (2M) Supplementary Information * Supplementary Movie 4 (6M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - A role for actin arcs in the leading-edge advance of migrating cells
- ncb 13(4):371-382 (2011)
Nature Cell Biology | Article A role for actin arcs in the leading-edge advance of migrating cells * Dylan T. Burnette1 * Suliana Manley1 * Prabuddha Sengupta1 * Rachid Sougrat1 * Michael W. Davidson2 * Bechara Kachar3 * Jennifer Lippincott-Schwartz1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:371–382Year published:(2011)DOI:doi:10.1038/ncb2205Received17 August 2010Accepted06 January 2011Published online20 March 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 Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. How the two modules connect mechanistically to regulate directed edge motion is not understood. Using live-cell imaging and photoactivation approaches, we demonstrate that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. We propose that net edge extension occurs by nascent focal adhesions advancing the site at which new actin arcs slow down and form the base of the next protrusion event. The actin arc thereby serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion. View full text Figures at a glance * Figure 1: Retrograde-actin-flow rates change several times over a single edge-protrusion/retraction cycle. () Electron micrograph of a rotary-shadowed cell after live-cell extraction. Areas 1 and 2 show actin-filament organization in the lamellipodium and lamella, respectively. Scale bar, 1 μm. () Actin–tdEos speckle image of an entire PtK1 cell with a corresponding FSM flow map and a higher magnification of the leading-edge flow. LM, lamella; LP, lamellipodium. Vector colours reflect flow speed (colour bar), and arrows reflect direction. Scale bar, 10 μm. () Edge-motion rates relative to retrograde actin flow. Schematic of how the speckle-flow data were binned, and the resulting rearward-speckle-flow kymograph showing the change in retrograde-flow rates during protrusion (open arrows) and retraction (filled arrows) of the leading edge. Each data bin was 5 μm across and 1 μm high. () Schematics of how the edge-protrusion and retrograde-flow data were binned across the leading edge. Bins for edge protrusion were set at 500 nm parallel to the edge. Bins for retrogra! de flow were set at 1 μm parallel to the edge and 3 μm into the cell. () Edge-protrusion/retraction velocity and rearward actin velocity maps of the same cell used for the kymograph in . () Edge position, edge velocity and rearward actin flow of the region denoted by the dashed lines in plotted over time. Asterisks in edge-velocity and rearward-actin-flow graphs denote retractions and arrowheads denote protrusions corresponding to increases in rearward actin flow. Arrowheads denote slowing rearward actin flow immediately after edge retraction. * Figure 2: Differential actin-filament turnover during protrusion and retraction. () Montage of unconverted actin–tdEos (green) at the edge and actin–tdEos photoconverted (red) in the lamellipodium during edge protrusion. LM, lamella; LP, lamellipodium. () Montage of unconverted and converted actin–tdEos molecules during edge traction. Yellow line denotes region of photoconversion and arrowheads denote actin-bundle formation. () Quantification of fluorescence loss of converted actin–tdEos molecules in the lamellipodium during edge protrusion (green line) or retraction (red line). () Still frames showing that the actin bundle formed after retraction is arc shaped. Scale bar, 5 μm. () Montage showing unconverted and converted channels before, 0 min, and 6 min after photoconversion of actin–tdEos molecules incorporated into actin arcs in the lamella (dashed outline). Scale bar, 10 μm. () Time montage showing the recovery of fluorescence from the unconverted channel and loss of fluorescence from the converted channel of actin-tdEos in the ! lamella. * Figure 3: Actin-arc dynamics at the leading edge. () Three frames of a time-lapse recording of actin–mRFP showing the cytoskeletal organization at the leading edge during the transition from protrusion to retraction and back to protrusion. LP denotes actin network in the lamellipodium and arrow denotes actin arcs in the lamella (LM). Yellow arrowheads show actin filaments associated with focal adhesions. Red arrowheads show a newly forming actin arc. Scale bar, 10 μm. () Time-lapse montage of the area outlined in showing the formation of an actin arc (red arrowheads) between protrusion events (white lines). Green arrowhead shows the actin arc formed during the previous retraction event. White arrowheads show zone of actin depolymerization during edge protrusion. Yellow arrowheads show the removal of actin arcs. The change in intensity from frame 10 to 11 is due to focusing. () Kymograph of the line in showing multiple protrusion and retraction events over 1 h. Red arrows denote the first frame an actin arc was observ! able and yellow dashed line shows lamellar advance. () Zyxin–mCherry and monomeric GFP (mGFP)-tagged actin montage showing that an actin arc can form (white arrowheads) before coming in contact with focal adhesions (yellow arrow). Actin was psuedo-coloured red for consistency with other figures and zyxin was psuedo-coloured green. () Protrusion/retraction map showing edge-velocity (shown by colour bar) changes over time across the edge of the cell in . () Plot of the edge velocity from the dashed line in . () Fourier transforms of the velocity profiles represented by the coloured lines in . () Distribution of the period of the protrusion/retraction cycle among 41 cells. * Figure 4: Myosin II activity condenses the lamellipodium into an actin arc. () Organization of actin–mRFP, myosin IIA–GFP and overlay during edge protrusion. Myosin II localizes with older actin arcs in the lamella. Scale bar, 10 μm. () Time-lapse montages of actin–mRFP, myosin IIA–GFP and an overlay from the rectangle in showing the co-localization of myosin II with newly forming actin arcs. Arrowheads show co-translocation of myosin IIA and the newly formed actin arc. () Kymograph showing myosin IIA dynamics over three protrusion/retraction cycles. White arrowheads denote the appearance and white arrows denote the movement of myosin IIA. Yellow arrowhead denotes the appearance and yellow arrow denotes the movement of myosin IIA associated with a small actin bundle left behind by the protruding lamellipodium. () Actin–mRFP before and after treatment with 25 μm blebbistatin. Kymograph shows the protrusion retraction cycle of the edge before and after blebbistatin treatment. The structure and movement of the actin arcs are diminished ! in the presence of blebbistatin. () Protrusion/retraction map showing edge motion before and after blebbistatin addition (arrow). Edge retractions denoted by arrowheads. * Figure 5: Oscillatory edge motion and net edge extension. () Edge-velocity map along the edge and a single region (bottom graph) along the edge from a crawling cell. () Edge-position map of the same cell as in . Edge-position map was created by colour-coding the lowest edge position as blue and the highest as red as in the colour bar. This allows for relative edge position along the same regions as in the velocity map in to be graphically shown over time. Bottom graph in shows the relative edge position at one point along the edge. Red dotted line and green dotted line graphically show the protrusion amplitude and retraction amplitude of one protrusion, respectively. () Protrusion amplitudes plotted against edge oscillation frequencies for individual cells. Correlation coefficient: −0.5129 (confidence interval: −0.7087,−0.2437). () Migration rate plotted against edge oscillation frequencies for individual cells. Correlation coefficient: 0.1966 (confidence interval: −0.1182,0.4755). () Migration rate plotted against protrusi! on amplitudes for individual cells. Correlation coefficient: 0.2650 (confidence interval: −0.0464, 0.5295). () Migration rate plotted against the ratio of protrusion and retraction amplitudes in individual cells. Correlation coefficient: 0.4254 (confidence interval: 0.1355, 0.6482). For –, n=41 cells. (,) Edge position and rearward-flow velocity plotted as in Fig. 1f for a cell that demonstrates net edge growth () and a cell that does not (). Note the pattern of rearward actin flow is similar in both cells. Pearson's correlation coefficient was used to quantify the correlation and the 95% confidence interval for each pair was computed using the Fisher transformation. * Figure 6: Differential slippage of focal adhesions in crawling versus non-crawling cells correlates with new-actin-arc movement. () Overlay of actin–mGFP (blue) and zyxin–mCherry at 0 min (purple) and 14 min (green). Arrows show focal adhesions that move during this time period. Scale bar, 10 μm. () Focal-adhesion movement, compared with pre-existing adhesions. New focal adhesion during protrusion (purple) and after edge retraction (green) in cells with different migrating rates (fast cell— 0.25 μm min−1; slow cell— 0.04 μm min−1). A pre-existing adhesion in each cell is shown in yellow. Total distance of the new adhesion from the previous adhesion during protrusion (purple double-headed arrows) and the net distance after new adhesion slippage (green double-headed arrows) are shown. () Net distance between new adhesions and pre-existing adhesions for the fast cell in (n=38 adhesions) and a slow cell in (n=29 adhesions). Distance was calculated for each new adhesion after the first and second edge-retraction event for which they are associated. () Net adhesion advance from ! focal adhesions labelled with vinculin. Average distance of a population of eight cells (n=132 adhesions), and for the fastest (0.31 μm min−1; n=17 adhesions) and slowest (0.01 μm min−1; n=39 adhesions) cell in the population are shown. Error bars in and , s.e.m. () Time-lapse montage of actin–mGFP and zyxin–mCherry in a crawling cell. First retraction-to-protrusion transition point (white asterisk) and second transition point (yellow asterisk) are denoted. White arrowheads show the pre-existing focal adhesion. Yellow arrowhead shows a nascent adhesion appearing and maturing (growing larger). () Similar time-lapse montage as in in a non-crawling cell. Pre-existing adhesion (white arrowheads) and new focal adhesions (yellow and green arrowheads) are shown. Adhesions move rearward (lines) during edge retraction. Brackets in , show the net movement of the base of the protrusion–retraction cycle/focal-adhesion advance. () Kymograph from a cell that increases! its rate of migration. There is no advance of the base protru! sion after two protrusion/retraction cycles (arrows), but there is advance after the third cycle (arrow). Yellow and green lines denote rapid and slow actin-arc translocation, respectively. * Figure 7: The advance of the lamella results from an actin-arc treadmill. () Actin–mGFP and (pseudo-coloured red) vinculin–mCherry (pseudo-coloured green) in a cell before and after net edge extension. Stars denote the lamellipodium and brackets denote the lamella. White arrowheads show focal adhesions at the boundary between the lamellipodium and lamella. Scale bar, 10 μm. () Edge-velocity map, edge-position map and kymograph showing dynamics and advance of the leading edge. Dotted yellow lines denote advance of the lamella. () Actin montage of area outlined in shows actin arcs are removed from the back of the lamella (arrowheads in actin montage) and new-focal-adhesion assembly (arrowheads in vinculin montage) leads to edge advance. Arrowhead colours denote distinct arcs or adhesions. The change in intensity in from frame 5 to 6 is due to focusing. * Figure 8: Model of the structural dynamics of the actin cytoskeleton underlying edge motion. Leading-edge advance is broken down into discrete steps. Step (1) shows the base of a previous retraction where a newly created actin arc is coupled to a focal adhesion. Hypothetically, the new lamellipodial protrusion could push off the arc to drive the membrane forward. During protrusion (2), actin-filament polymerization occurs behind the plasma membrane and depolymerization occurs a few micrometres away from the edge. Actin filaments treadmill through the lamellipodium during protrusion, and nascent adhesions form. At the peak of protrusion (2), myosin II filaments form in the lamellipodium and a local network contraction (similar to that proposed for keratocyte cell body translocation3) occurs that drives actin-arc formation and edge retraction (3). In cells that show net advance, the new actin arc slows at the nascent adhesion (4), most likely owing to strong coupling between the arc, adhesion and growth substrate. The base of the retraction in (4) is shifted forward w! hen compared with (1). As a consequence, the start of the new protrusion in (5) is also shifted forward and the edge protrudes farther than in (2). In cells that do not show net advance, the actin arc and adhesion slip rearward during edge retraction. This indicates that there is still strong coupling between the actin arc and the adhesion, and also indicates a weak coupling between the adhesion and the growth substrate. Actin-arc addition to the front of the lamella is balanced by actin-arc removal at the back of the lamella (5). Lamellipodial and arc actin filaments are yellow. Focal adhesions and associated actin filaments are green. Myosin II filaments are red. Relative actin-rearward-flow rates are represented by blue arrows. Author information * Abstract * Author information * Supplementary information Affiliations * National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA * Dylan T. Burnette, * Suliana Manley, * Prabuddha Sengupta, * Rachid Sougrat & * Jennifer Lippincott-Schwartz * National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida 32310, USA * Michael W. Davidson * National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA * Bechara Kachar Contributions D.T.B., S.M. and J.L-S. designed experiments and wrote the paper. D.T.B. carried out the experiments. D.T.B., S.M. and P.S. analysed the data. M.W.D. contributed new fluorescence probes. R.S. and B.K. contributed expertise in electron microscopy. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jennifer Lippincott-Schwartz Author Details * Dylan T. Burnette Search for this author in: * NPG journals * PubMed * Google Scholar * Suliana Manley Search for this author in: * NPG journals * PubMed * Google Scholar * Prabuddha Sengupta Search for this author in: * NPG journals * PubMed * Google Scholar * Rachid Sougrat Search for this author in: * NPG journals * PubMed * Google Scholar * Michael W. Davidson Search for this author in: * NPG journals * PubMed * Google Scholar * Bechara Kachar Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer Lippincott-Schwartz Contact Jennifer Lippincott-Schwartz Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (2M) Supplementary Information * Supplementary Movie 2 (4M) Supplementary Information * Supplementary Movie 3 (4M) Supplementary Information * Supplementary Movie 4 (4M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation
- ncb 13(4):383-393 (2011)
Nature Cell Biology | Article Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation * Jean-Cheng Kuo1, 4 * Xuemei Han2, 4 * Cheng-Te Hsiao3 * John R. Yates III2 * Clare M. Waterman1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:383–393Year published:(2011)DOI:doi:10.1038/ncb2216Received11 May 2010Accepted13 January 2011Published online20 March 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 Focal adhesions undergo myosin-II-mediated maturation wherein they grow and change composition to modulate integrin signalling for cell migration, growth and differentiation. To determine how focal adhesion composition is affected by myosin II activity, we performed proteomic analysis of isolated focal adhesions and compared protein abundance in focal adhesions from cells with and without myosin II inhibition. We identified 905 focal adhesion proteins, 459 of which changed in abundance with myosin II inhibition, defining the myosin-II-responsive focal adhesion proteome. The abundance of 73% of the proteins in the myosin-II-responsive focal adhesion proteome was enhanced by contractility, including proteins involved in Rho-mediated focal adhesion maturation and endocytosis- and calpain-dependent focal adhesion disassembly. During myosin II inhibition, 27% of proteins in the myosin-II-responsive focal adhesion proteome, including proteins involved in Rac-mediated lamellipodial! protrusion, were enriched in focal adhesions, establishing that focal adhesion protein recruitment is also negatively regulated by contractility. We focused on the Rac guanine nucleotide exchange factor β-Pix, documenting its role in the negative regulation of focal adhesion maturation and the promotion of lamellipodial protrusion and focal adhesion turnover to drive cell migration. View full text Figures at a glance * Figure 1: Development and validation of the focal adhesion isolation method. () Flow diagram of the protocol used to isolate and prepare focal adhesions (FAs) from HFF1 cells for compositional analysis by proteomic methods. Intact HFF1 cells (top) and isolated HFF1 focal adhesions (middle) were immunostained to localize paxillin (as a focal adhesion marker) and F-actin (phalloidin). Scale bars, 20 μm. () Size distribution of paxillin-containing focal adhesions quantified from images of intact cells (n = 7 cells, 1219 focal adhesions), hypotonically shocked cells (n = 7 cells, 944 focal adhesions) and isolated focal adhesions (n = 9 cells, 1217 focal adhesions). Data are means ± s.d. No significant differences were detected in the percentages of focal adhesions in each size range between the three samples (means: native, 2.91 ± 0.29 μm2; hypotonic shock, 2.90 ± 0.82 μm2; isolated, 2.94 ± 0.57 μm2). () Western blot comparison of protein concentration in total cell lysate (T), isolated focal adhesion fractions (before actin and fibrin immunodepl! etion; F) and cell body fractions (C). Equal total protein was loaded in each lane. The ratio shown on the right (F/C) indicates the relative concentration of protein between the isolated focal adhesion and cell body fractions quantified from the intensities of bands from western blots; n = 4 (paxillin, tubulin and GAPDH); n = 3 (vinculin, talin, actin, VASP, Akt, phosphorylated paxillin 118, phosphorylated FAK 397 and FGFR); data are means ± s.d. Note that this does not reflect the absolute amount of protein in isolated focal adhesion fractions and cell body fractions. pPaxillin118 and pFAK397; paxillin and FAK phosphorylated at residues 118 and 397, respectively. Uncropped image of blot is shown in Supplementary Fig. S8. * Figure 2: Proteome of isolated focal adhesions. () Criteria applied to data from MudPIT MS analysis for assembling the detectable and reproducible lists of proteins present in isolated focal adhesions. () Venn diagram and classification of proteins identified in isolated focal adhesions. Number of proteins in each class are shown in parentheses. () Pie diagram showing the percentage of proteins from the reproducible list categorized according to function. * Figure 3: Development of the MDR to characterize the effects of myosin II inhibition on protein abundance in isolated focal adhesions. () Effects of myosin II inhibition by blebbistatin treatment (50 μM, 2 h) on the organization and morphology of focal adhesions from intact cells or isolated focal adhesions, visualized by immunostaining for paxillin. Insets indicate higher-magnification images of indicated areas. Scale bars, 20 μm. () Size distribution of segmented paxillin-containing focal adhesions, quantified from images of intact blebbistatin-treated HFF1 cells (n = 3 cells, 526 focal adhesions) and isolated blebbistatin-treated focal adhesions (n = 4 cells, 252 focal adhesions). Data are means ± s.d. No significant differences were detected in the percentage of focal adhesions in each size range between focal adhesions isolated from intact cells and isolated focal adhesions. () Diagram of the procedure for calculating the MDR, which represents the change in protein abundance between focal adhesions isolated from control and blebbistatin-treated cells. Middle inset; western blot of paxillin abundance! in focal adhesions isolated from control (–) or blebbistatin (Blebb)-treated cells. Uncropped image of blot is shown in Supplementary Fig. S8. * Figure 4: Collective modulation by myosin II of the focal adhesion abundance of proteins in common biological pathways. () Number of proteins in different functional categories that exhibit change in abundance (relative to control) in isolated focal adhesions in response to blebbistatin treatment. Two-fold differences in abundance (MDR < 0.5 or > 2) were considered high-confidence significant changes. (–) Proteins are represented by boxes that are colour-coded according to the magnitude of their MDR (scale indicated at the bottom). () Myosin-II-mediated focal adhesion enrichment of proteins involved in focal adhesion maturation and stress fibres. () Myosin-II-mediated focal adhesion enrichment of proteins regulates integrin activation and actin linkage. () Myosin-II-mediated focal adhesion enrichment of proteins involved in calpain-dependent and endocytosis-dependent focal adhesion disassembly. () Myosin-II-mediated focal adhesion reduction of proteins involved in Rac1 activation and lamellipodial actin treadmilling. * Figure 5: Abundance of β-Pix in focal adhesions is negatively regulated by myosin-II-mediated maturation. () Focal adhesion fractions from control and blebbistatin-treated (50 μM) cells were analysed by western blotting using antibodies against β-Pix. Bottom: fold enrichment (fold) of β-Pix in isolated focal adhesions determined by western blotting (equal total protein loaded, n = 3 experiments, data are means ± s.d.) or MudPIT MS (fold in MS). () TIRF microscopy images of immunolocalized paxillin and β-Pix in control and blebbistatin-treated (50μM) cells. The 6.5 μm × 6.5 μm areas indicated in the left images are magnified on the right; scale bar, 1 μm. () Ratio of average density (intensity per μm2) of paxillin or β-Pix within segmented focal adhesions of blebbistatin-treated:control cells or small (< 2μm2):big (>2μm2) focal adhesions in control cells. Blebbistatin-treated cells, n = 9, control cells, n = 9; data are means ± s.d. **P < 0.05. () Fractions from focal adhesions isolated from cells plated on 1.5 kPa and 55 kPa substrates were analysed by western blo! tting using β-Pix antibodies (equal total protein loaded). Bottom: fold enrichment in isolated focal adhesions determined by western blotting (n = 3, data are means ± s.d.). () Immunolocalized paxillin and β-Pix in cells plated on 1.5 kPa and 55 kPa substrates. The 6.5 μm × 6.5 μm area indicated in the left images is magnified in right images; scale bar, 1 μm. () Ratio of average density of paxillin or β-Pix within segmented focal adhesions of cells plated on 1.5 kPa relative to 55 kPa substrates. 1.5 kPa, n = 6 cells; 55 kPa, n = 6 cells. Data are means ± s.d. **P < 0.05. () Left: timelapse TIRF microscopy of GFP–β-Pix and mApple–Paxillin accumulation in focal adhesions. Scale bar, 1 μm. Right: normalized integrated fluorescence intensity over time (mApple–paxillin, red; GFP–β-Pix, blue) and focal adhesion size (grey) in the focal adhesion marked with an arrow in images. () Left: timelapse TIRF microscopy of GFP–β-Pix and mApple–Paxillin accumulati! on in focal adhesions treated with Y27632 (10 μM; added at 0 ! s) Scale bar; 1 μm. Right: normalized integrated fluorescence intensity over time in the focal adhesions shown in the images. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 6: Effects of β-Pix on regulation of lamellipodia formation and Rac1 activation. () HFF1 cells overexpressing GFP–β-Pix (red) and treated with blebbistatin (50 μM, 2 h) were immunostained for paxillin, to localize nascent focal adhesions (green), and cortactin, to localize protruding lamellipodia (blue). The indicated 6 μm × 6 μm boxed regions in top left image is magnified in the merged and bottom three images; scale bar, 1 μm. () HFF1 cells expressing non-silencing or β-Pix-silencing shRNA (same as β-Pix#1 in ) were treated with 50μM blebbistatin or not (control) and immunostained for paxillin (green) and cortactin (purple). Scale bar, 20 μm. Values indicate ratio of cortactin-stained cell area relative to total cell area for the indicated treatments; n = 10 cells, data are means ± s.d.. **P < 0.05. () Effects of β-Pix-silencing on blebbistatin-induced Rac1 activation. HFF1 cells expressing non-silencing (non) or β-Pix-silencing shRNA (β-Pix#1 and β-Pix#2 indicate different sequence targets of β-Pix shRNA) alone or together with Myc-t! agged mouse β-Pix were treated with the indicated concentrations of blebbistatin for 2 h, and cell lysates prepared. The level of GTP-bound Rac1 isolated from lysates by GST–PAK–CRIB pulldown (top) and the protein level of β-Pix, Rac1 (Total Rac), and tubulin in the input lysate (bottom blots) were detected by western blot. Change relative to control in levels of GTP–Rac and β-Pix determined by western blot (Fold) and is indicated below the blots. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 7: Effects of β-Pix on focal adhesion dynamics and cell migration. () Paxillin immunostaining of focal adhesions in control cells and in cells overexpressing GFP–β-Pix or expressing non-silencing shRNA or shRNA against β-Pix (same as β-Pix#1 in Fig. 6c). Scale bar, 20 μm. () Size distribution of segmented focal adhesions in the periphery (within 15 μm of the cell edge) of cells treated as described in . n = number of focal adhesions. Data are means ± s.d. **P < 0.05 (compared with control). () Left: Timelapse TIRF microscopy of mApple–paxillin during focal adhesion turnover in a migrating cell that was expressing non-silencing or β-Pix shRNA (same as β-Pix#1 in Fig. 6c). Scale bar, 1 μm. Right: normalized fluorescent paxillin intensity over time in the focal adhesions highlighted with arrows in the images. () Focal adhesion lifetime in cells treated with β-Pix shRNA as indicated (β-Pix silenced: 40.19 ± 2.38min, control: 27.11 ± 3.00min). () Maximal total intensity of mApple–paxillin in focal adhesions in cells treated wi! th β-Pix shRNA as indicated. In and , non-silencing: n = 19 focal adhesions/5 cells; β-Pix-silencing: n = 15 focal adhesions/6 cells, data are means ± s.d. **P < 0.05. () Migration velocity of HFF1 cells expressing either non-silencing or β-Pix-silencing shRNAs with or without 50 μM blebbistatin treatment. n = number of control cells/number of blebbistatin-treated cells, data are means ± s.d. () Model of the effects of myosin II contractility on the composition and function of focal adhesions in the leading edge. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jean-Cheng Kuo & * Xuemei Han Affiliations * Cell Biology and Physiology Centre, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland 20892, USA. * Jean-Cheng Kuo & * Clare M. Waterman * Cell Biology, Scripps Research Institute, La Jolla California 92037, USA. * Xuemei Han & * John R. Yates III * Proteomics and Analytical Biochemistry Unit, Research Resources Branch, National Institute on Aging, NIH, Baltimore, Maryland 21224, USA. * Cheng-Te Hsiao Contributions J.C.K. and C.M.W designed experiments and wrote the paper, J.C.K. performed experiments, X.H. and J.R.Y performed MudPIT MS analysis and C.T.H. created the web site. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * John R. Yates III or * Clare M. Waterman Author Details * Jean-Cheng Kuo Search for this author in: * NPG journals * PubMed * Google Scholar * Xuemei Han Search for this author in: * NPG journals * PubMed * Google Scholar * Cheng-Te Hsiao Search for this author in: * NPG journals * PubMed * Google Scholar * John R. Yates III Contact John R. Yates III Search for this author in: * NPG journals * PubMed * Google Scholar * Clare M. Waterman Contact Clare M. Waterman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Information (6M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - Dynamics of ESCRT protein recruitment during retroviral assembly
- ncb 13(4):394-401 (2011)
Nature Cell Biology | Article Dynamics of ESCRT protein recruitment during retroviral assembly * Nolwenn Jouvenet1, 2 * Maria Zhadina1 * Paul D. Bieniasz1, 3 * Sanford M. Simon4 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:394–401Year published:(2011)DOI:doi:10.1038/ncb2207Received14 October 2010Accepted07 January 2011Published online10 March 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 ESCRT (endosomal sorting complex required for transport) complexes and associated proteins mediate membrane scission reactions, such as multivesicular body formation, the terminal stages of cytokinesis and retroviral particle release. These proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the ATPase Vps4A. However, these events have never been observed in living cells, and their dynamics are unknown. By quantifying the recruitment of several ESCRT and associated proteins during the assembly of two retroviruses, we show that Alix progressively accumulated at viral assembly sites, coincident with the accumulation of the main viral structural protein, Gag, and was not recycled after assembly. In contrast, ESCRT-III and Vps4A were transiently recruited only when the accumulation of Gag was complete. These data indicate that the rapid and transient recruitment of proteins that act late in the ESCRT pat! hway and carry out membrane fission is triggered by prior and progressive accumulation of proteins that bridge viral proteins and the late-acting ESCRT proteins. View full text Figures at a glance * Figure 1: Characterization of the GFP–Chmp4b-expressing cell clone. HeLa cells stably expressing GFP–Chmp4b (green) were fixed and stained with anti-α-tubulin antibodies (red) and with DAPI (blue). Images show the distribution of GFP–Chmp4b in interphase cells (top panels) and in telophase cells (middle panels). Alternatively, cells were transfected with mCherry–Vps4-DN (bottom panels), fixed 18 h post-transfection and stained with DAPI (blue). Samples were observed with an epifluorescence microscope. Deconvolved optical sections acquired at the centre of the vertical dimension of the cell are shown. Expanded views are shown in insets. Scale bar, 10 μm. * Figure 2: Effect of stably expressed GFP-fused ESCRT proteins on cell proliferation, cytokinesis and virion assembly and release. () The stable expression of GFP-tagged ESCRT proteins does not affect cell proliferation. Cells (105) were plated into each well of a 24-well plate, collected and counted 48 h later. Error bars indicate s.d. from three independent experiments. () Stable expression of GFP-tagged ESCRT proteins does not disrupt cytokinesis. HeLa cells stably expressing GFP-tagged ESCRT proteins or transfected with GFP or GFP–Vps4K173Q were fixed, stained with both anti-α-tubulin antibodies and DAPI, and scored for multinucleated cells. Three-hundred cells from three independent experiments were analysed for the presence of more than one nucleus per cell for each factor. Error bars indicate s.d. () Kinetics of HIV-1 and EIAV assembly were not affected in the cell lines stably expressing GFP-tagged ESCRT proteins. The plots show the time to complete assembly for individual HIV-1 and EIAV VLPs, including wild-type and late-domain mutant (LD-) Gag proteins, in unmodified HeLa cells, or cell l! ines expressing the indicated GFP–ESCRT protein lines. Each symbol represents an individual VLP. The time to complete assembly was defined for each VLP as the interval between the points of inflection on plots of fluorescence intensity as a function of time. () The cell lines stably expressing GFP-tagged ESCRT proteins support the release of HIV-1 and EIAV Gag VLPs. Western blot analysis of HeLa cells and HeLa cells stably expressing GFP-tagged ESCRT transfected with HIV-1 (left panel) or EIAV (right panel) Gag. Samples were probed with an anti-p24 monoclonal antibody for HIV-1 or anti-EIAV horse serum. Supplementary Fig. S6 shows the corresponding unprocessed western blots. * Figure 3: Catalytically inactive Vps4A increases localization of stably expressed GFP-tagged ESCRT-III proteins at sites of HIV-1 assembly. () HeLa cells stably expressing GFP–Chmp4b (green) were transfected with HIV-1 Gag/Gag–mCherry (red), in the absence (top panel) or presence (bottom panel) of Vps4A-DN. Cells were fixed 24 h later and observed with a TIR-FM microscope. Expanded views are shown in insets. Scale bars, 10 μm. () Quantification of the co-localization between VLPs and puncta of ESCRT proteins. HeLa cells stably expressing GFP-fused ESCRT-III proteins were transfected with Gag/Gag–mCherry, in the absence (−) or presence (+) of Vps4A-DN, as indicated. Cells were observed under TIR-FM at 18 h post-transfection and the co-localization between puncta of Gag–mCherry and puncta of GFP was quantified by randomly selecting puncta of one marker (selected) and then enumerating the percentage of these puncta that were coincident with puncta of the other, non-selected marker. * Figure 4: Imaging Chmp1b, Chmp4b, Chmp4c and Vps4A recruitment during HIV-1 Gag assembly. (–) HeLa cells stably expressing Chmp1b–GFP (), GFP–Chmp4b (), GFP–Chmp4c () or GFP–Vps4A () were transfected with HIV-1 Gag/Gag–mCherry and observed under TIR-FM beginning at 6 h post-transfection. Each set of images illustrates the recruitment of GFP-labelled ESCRT proteins (green) during the genesis of an individual VLP (red). The time after the commencement of observation is given in minutes:seconds. Fields are 2.5×2.5 μm. Plots of fluorescence intensity in arbitrary units (a.u.) as a function of time for the GFP–ESCRT protein (green, right axis) and Gag–mCherry signals (red, left axis) associated with the assembly of three individual VLPs are shown. * Figure 5: Imaging Chmp1b, Chmp4b, Chmp4c and Vps4A recruitment during EIAV Gag assembly. (–) HeLa cells stably expressing Chmp1b–GFP (), GFP–Chmp4b (), GFP–Chmp4c () or GFP–Vps4A () were transfected with EIAV Gag/Gag–mCherry and observed under TIR-FM beginning at 6 h post-transfection. Each set of images illustrates the recruitment of GFP-labelled ESCRT proteins during the genesis of an individual VLP. The time after the commencement of observation is given in minutes:seconds. Fields are 2.5×2.5 μm. Plots of fluorescence intensity in arbitrary units (a.u.) as a function of time for the GFP–ESCRT protein (green, right axis) and Gag–mCherry signals (red, left axis) associated with the assembly of three individual VLPs are shown; the left graph in and corresponds to the microscopic images shown above. * Figure 6: Imaging Alix recruitment during EIAV Gag assembly. HeLa cells stably expressing GFP–Alix were transfected with EIAV Gag/Gag–mCherry and observed under TIR-FM beginning at 6 h post-transfection. Each set of images illustrates the recruitment of GFP-labelled Alix proteins during the genesis of an individual VLP. The time after the commencement of observation is given in minutes:seconds. Fields are 2.5×2.5 μm. Plots of fluorescence intensity in arbitrary units (a.u.) as a function of time for the GFP–Alix protein (green, right axis) and Gag–mCherry signals (red, left axis) associated with the assembly of three individual VLPs are shown; the left graph corresponds to the microscopic images shown above. * Figure 7: Dynamics and pattern of ESCRT protein recruitment during retroviral assembly. () The fraction of EIAV (left panel) and HIV-1 (right panel) VLPs at which GFP–ESCRT protein was detectable, as a function of time. For this analysis, T=0 was set as the point at which Gag recruitment to each VLP reached a plateau. () Percentage of VLP assembly events for which the recruitment of GFP-tagged ESCRT proteins was detected. HeLa cells stably expressing GFP-tagged ESCRT proteins were transfected with wild-type (WT) or late-domain mutant (LD−) HIV-1 (left panel) or EIAV (right panel) Gag/Gag–mCherry. Cells were observed live under TIR-FM beginning at 6 h post-transfection, for a period of 25–50 min. () Quantification of the number of pulses of ESCRT protein recruitment (percentage of VLPs for which each behaviour is observed) during HIV-1 and EIAV VLP assembly. () Removal of the GFP–ESCRT proteins from sites of HIV-1 and EIAV assembly. ESCRT-III and Vps4 proteins are generally completely recycled (as signified by the GFP signal at VLP assembly sites r! eturning to baseline levels following the pulse), but in some cases, the proteins seem to be only partially recycled (for example Fig. 4c, images and left panel). Alix is not recycled (that is, the GFP signal remains at a plateau after reaching its maximum, see Fig. 6). The percentage of VLPs showing each behaviour is plotted. Author information * Abstract * Author information * Supplementary information Affiliations * Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, New York, 10065 New York, USA * Nolwenn Jouvenet, * Maria Zhadina & * Paul D. Bieniasz * UMR 5236 CPBS CNRS, 1919 Route de Mende, 34293 Montpellier, France * Nolwenn Jouvenet * Howard Hughes Medical Institute, The Rockefeller University, New York, 10065 New York, USA * Paul D. Bieniasz * Laboratory of Cellular Biophysics, The Rockefeller University, New York, 10065 New York, USA * Sanford M. Simon Contributions N.J., S.M.S. and P.D.B. conceived and designed the experiments. N.J. carried out the experiments with help from M.Z. (Figs 2d and 5). N.J., S.M.S. and P.D.B. analysed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Paul D. Bieniasz or * Sanford M. Simon Author Details * Nolwenn Jouvenet Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Zhadina Search for this author in: * NPG journals * PubMed * Google Scholar * Paul D. Bieniasz Contact Paul D. Bieniasz Search for this author in: * NPG journals * PubMed * Google Scholar * Sanford M. Simon Contact Sanford M. Simon Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors
- ncb 13(4):402-411 (2011)
Nature Cell Biology | Article Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors * Aiqun Li1 * Masaki Saito1, 2, 3 * Jen-Zen Chuang1 * Yun-Yu Tseng1 * Carlos Dedesma1 * Kazuhito Tomizawa4 * Taku Kaitsuka4 * Ching-Hwa Sung1, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:402–411Year published:(2011)DOI:doi:10.1038/ncb2218Received17 September 2010Accepted27 January 2011Published online13 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Primary cilia are displayed during the G0/G1 phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression. However, the role of Tctex-1 in S-phase entry is dispensable in non-ciliated cells. Exogenously adding a phospho-mimic Tctex-1T94E mutant accelerates cilium disassembly and S-phase entry. These results support a model in which the cilia act as a brake to prevent cell cycle progression. Mechanistic studies show the involvement of actin dynamics in Tctex-1-regulated cilium resorption. Tctex-1 phosphorylated at Thr 94 is also selectively enriched at the ciliary transition zones of cortical neural progenitors, and has a key role in controlling G1 length, cell cycle entry and fate! determination of these cells during corticogenesis. View full text Figures at a glance * Figure 1: Tctex-1 is involved in cilium-dependent cell cycle re-entry. () Schematic representations of the timeline of transfection experiments (top) and the plasmids used for transfection (bottom). IRES; internal ribosome entry site. Grey and yellow boxes represent control and Tctex-1 shRNA sequences, respectively. () Representative immunoblots of lysates from control- or Tctex-1-sh-transfected RPE-1 cells probed with antibodies against the indicated proteins. Cells were treated as indicated in and the times at which cells were harvested after serum re-addition are shown. p-Rb and p-cdc2, phosphorylated Rb and cdc2, respectively; DIC, dynein intermediate chain. () Quantification of results from ; the signal levels were normalized to the DIC signal. Data are means ± s.e.m.; n = 3 experiments, **P < 0.01, t-test. (–) BrdU incorporation indices (fraction of BrdU-labelled GFP+ cells) of synchronized RPE-1 or IFT20KD-RPE-1 cells (), HeLa or COS-7 cells (), 3T3 cells (, ), and wild-type (WT) or Ift88 mutant MEF cells () after serum release. Cells! were treated as in and transfected with control vector or Tctex-1-sh plasmid; in cells were additionally transfected with plasmids encoding the indicated Tctex-1 proteins. Incorporation of BrdU in the control cells was taken to be 100%. Data are means ± s.e.m.; n = 5, 4, 7, 3 and 7 experiments for figures , , , and , respectively; an average of 400 cells was counted in each experiment; **P < 0.01, ***P < 0.001, t-test for –; *P < 0.05, one-way ANOVA test for . () Immunoblotting assays of lysates from the indicated cell types transfected with control vector or Tctex-1-sh plasmid. There was a reduction of endogenous Tctex-1 levels in various cell types transfected with Tctex-1-sh plasmid. Uncropped images of blots are shown in Supplementary Fig. S6. * Figure 2: Temporal activation of phospho(T94)Tctex-1 at the transition zone and its function in ciliary disassembly. () Immunofluorescence microscopy of phosphorylated Tctex-1 (p-Tctex-1; green), acetylated α-tubulin (Ac-Tub; red) and γ-tubulin (γ-Tub; cyan) in quiescent RPE-1 cells or cells treated with serum for 2 h or 24 h. DAPI; nuclei (blue). Insets are higher magnification images of indicated regions. Scale bar, 5 μm. () Left: representative immunoblots show the levels of (pan)Tctex-1 and phosphorylated Tctex-1 in cells harvested at the indicated times after serum re-addition. To improve the detection of phosphorylated Tctex-1, immunoprecipitation was first carried out using a saturated amount of anti-(pan)Tctex-1, the immunoprecipitates were resolved by electrophoresis and immunoblotted with antibody against phospho(T94)Tctex-1. Anti-HA (haemagglutinin) was used as an immunoprecipitation antibody control. Right: the relative expression level of phospho(T94)Tctex-1 level was normalized to the total amount of immunoprecipitated Tctex-1. () The relative intensity of phospho(T94)Tct! ex-1 in immunofluorescence microscopy images of RPE-1 cells at the indicated times after serum re-addition. The ratio of phospho(T94)Tctex-1:γ-tubulin intensity was quantified by MetaMorph software (data are means ± s.e.m.; n = 3 experiments). () Schematic representation of the timeline of cilium disassembly experiments used in –. () Fraction of transfected RPE-1 cells containing a cilium were scored in cells harvested at the indicated times after serum addition. An average of 500 cells were counted for each experiment (data are means ± s.e.m.; n = 3 experiments). () Quantification of the cilia lengths in transfected 3T3 cells harvested after serum addition (data are means ± s.e.m.; n = 3 experiments). () The fractions of transfected cells containing a cilium. Cells were harvested at indicated times after serum addition and scored and statistically analysed (data are means ± s.e.m.; n = 5 experiments; *P < 0.05, **P < 0.01; one-way ANOVA). Uncropped images of blots a! re shown in Supplementary Fig. S6. * Figure 3: Phospho(T94)Tctex-1 and actin dynamics participate in ciliary resorption. () Fractions of cells displaying cilia. RPE-1 cells were serum starved and left untreated (none) or treated with GFP-9R, Tctex-1T94E-9R or Tctex-1T94A-9R peptides, and harvested at indicated times after the treatments. Data are means ± s.e.m.; n = 4 experiments, except untreated control, where n = 3 experiments. () Cells in serum-free medium were treated with the indicated peptides followed by incubation with BrdU for 16 h. Fractions of BrdU-labelled cells are shown (data are means ± s.e.m.; n = 7 experiments; **P < 0.01; one-way ANOVA). () Cells were treated with DMSO (as a control) or Cyto D (0.5 μM) after serum addition and the fraction of ciliated cells was quantified (data are means ± s.e.m.; n = 3 experiments). () Representative image of cilia displayed in control cells versus Cyto D-treated cells 24 h after serum addition. Arrowheads and arrows indicate shortened and long cilia in the DMSO- and Cyto D-treated cells, respectively. Scale bar, 5 μm. () Fractions of ! cells that had cilia after the addition of GFP-9R or Tctex-1T94E-9R for indicated time in the presence of DMSO (n = 3 experiments) or Cyto D (n = 5 experiments). Data are means ± s.e.m. * Figure 4: Phospho(T94)Tctex-1 is expressed at the transition zone of radial glia in the developing neocortex. () Schematic representation of the radially elongated radial glia with their endfeet contacting both the ventricular and pial surfaces. The primary cilium, anchored on a basal body (BB), extends into ventricular spaces. Mitosis (M phase) typically occurs at the ventricular surfaces. Cells leave the cell cycle, migrate away from the ventricular zone (VZ), form multipolar post-mitotic neurons, and pause their migration in the intermediate zone (IZ) before reaching their cortical location. CP; cortical plate. () Left: anti-Tctex-1 mouse antibody recognized a single band of Mr ~12K on an immunoblot containing embryonic mouse brain lysates. Right: E13 mouse cortical slices were co-labelled for cilium marker Arl13b (ref. 42; green), Tctex-1 (red), and cell–cell junction marker ZO-1 (cy5). Dotted lines represent the ventral borders. Bottom images are enlarged views of the labelling of the ventricle surface. Scale bars, 20 μm (top); 2 μm (bottom). () Ventricle surfaces of E11–! E17 mouse neocortical slices were triple-labelled for Arl13b (green), phospho(T94)Tctex-1 (red) and γ-tubulin (cyan). () Higher-magnification images (right) of indicated area from an image of an E13 ventricle surface (left; as shown in ). Phospho(T94)Tctex-1 (red) was specifically located in the transition zone between the Arl13b-labelled cilia (green) and the γ-tubulin-labelled basal bodies (cyan) of the radial glia. (, ) Confocal microscopy images of the ventricular zone from mice brains, double labelled with antibodies against γ-tubulin and phospho(T94)Tctex-1 pre-absorbed with phosphopeptides corresponding to the antigen () or control peptides (). Arrows in indicate the γ-tubulin-labelled basal bodies and centrosomes, which lacked the phospho(T94)Tctex-1 signal. Arrowheads indicate the phospho(T94)Tctex-1 signals that remained at the ciliary base. Scale bar, 5 μm. () Co-labelling of phospho(T94)Tctex-1 and Arl13b in mouse cortical slices 24 h after transfection. No! te that cells transfected with GFP control plasmid (left, gree! n arrowhead) and neighbouring non-transfected cells (arrows) displayed similar levels of phospho(T94)Tctex-1 at the endfeet. However, cells transfected with Tctex-1-sh (right, green arrowhead) had reduced immunolabelling of phospho(T94)Tctex-1 (arrow indicates neighbouring non-transfected cell). Scale bar, 2 μm. () Post-mitotic mouse neurons located in the intermediate zone region had no detectable phospho(T94)Tctex-1 (red) between the Arl13-labelled cilia (green) and γ-tubulin-labelled basal bodies (cyan). Scale bar, 5 μm. * Figure 5: Suppression of Tctex-1 in radial glia induced premature neuronal differentiation. () Mouse cortical slices harvested 40 h after electroporation with the indicated plasmids. GFP was detected by direct green immunofluoresence microscopy. DAPI; blue. The dashed lines depict the borders of the cortexes. The intermediate zone (IZ) is defined by the presence of tangentially oriented cells. (, ) Representative images showing the labelling of Tuj1 () and brain lipid-binding protein (BLBP) () of vector- and Tctex-1-sh-transfected mouse brain slices. Bottom; higher-magnification images of cells transfected with Tctex-1-sh. Arrowheads point to the sites where the cell processes project from the cell bodies. () Percentage of total transfected GFP+ cells that were Tuj1+, Nestin+ or BLBP+, quantified from an experiment performed as in . Data are means ± s.e.m.; n = 4 experiments, except BLBP staining, where n = 5 experiments; an average of 600 cells were counted in each experiment; ***P < 0.001, one-way ANOVA. () Representative images depicting the cortical distributi! on of GFP cells in brains from mice at E18.5 (that is, 5-days post electroporation). Right; plots of GFP+ cell distribution. Top to bottom; the marginal zone (MZ), superficial (sCP), mid-, and deeper cortical plate (dCP) bins were defined as previously described43 and are as indicated on the images. () Quantification of the cortical location of transfected cells from experiment performed as in (data are means ± s.e.m.; n = 3 experiments; ***P < 0.001, t-test). * Figure 6: Phosphorylated Tctex-1 is required for cell cycling of radial glia. () Representative images of the ventricular zone of brains from mice transfected as indicated. Samples were immunolabelled with P-H3. Arrowheads indicate cells that were positive for both GFP and P-H3. () Representative confocal microscopy images of transfected mouse cortical slices subjected to cell cycle exit analysis. Left, GFP+ cells (green); middle, cells 24 h after BrdU treatment (red) and right, stained with antibodies against Ki67 (cyan). Arrows point to the cells that were positive for GFP and BrdU, but negative for Ki67. Arrowheads point to the cells that were positive for GFP, BrdU and Ki67. () Fractions of GFP+ and P-H3+ cells out of total GFP+ cells (the mitotic index). Data are means ± s.e.m.; n = 3, 6, 3, 5 and 6 experiments for vector, Tctex-sh, Tctex-1-sh/WT, Tctex-1-sh/T94E and Tctex-1-sh/T94A, respectively; ***P < 0.001, one-way ANOVA. () Fractions of GFP+, BrdU+, Ki67− cells out of total GFP+, BrdU+ cells (or cell cycle exit index). Data are means ± s! .e.m.; n = 3 experiments; total of 100 cells were scored; **P < 0.01, one-way ANOVA. * Figure 7: Phenotypic characterization of the developing neocortex of AurA and HDAC6 loss-of-function mutants, and Tctex-1 gain-of-function mutants. () Mouse cortical slices transfected with AurA-sh or HDAC6-sh plasmid and immunolabelled with P-H3 (red; middle panel). DAPI; blue. Bottom; schematic representation of the respective plasmids. Purple boxes indicate the shRNA-targeting sequences. () Cortical slices overexpressing the indicated Tctex-1 variants. Arrowheads point to P-H3-labelled, mitotic GFP+ cells at the ventricular zone. Scale bars, 100 μm (top), 20 μm (bottom). () Mitotic indices of GFP+ cells transfected with plasmids expressing the indicated Tctex-1 variants and GFP. Data are means ± s.e.m.; n = 3 experiments; total 1,200 cells were scored; **P < 0.01, one-way ANOVA. () The fractions of transfected mouse brains with BrdU incorporation (2 h incubation) that were GFP+ cells out of total GFP+ cells, 24 h and 40 h post-electroporation (data are means ± s.e.m.; n = 4 experiments; total 700 cells were scored; *P < 0.05, t-test). () Cumulative BrdU labelling curves of non-transfected cells and cells transfec! ted with vector alone or Tctex-1T94E/GFP. Data are means ± s.e.m.; n = 3 experiments; *P < 0.05, ***P < 0.001, one-way ANOVA. OE; overexpression. Author information * Abstract * Author information * Supplementary information Affiliations * Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10065, USA. * Aiqun Li, * Masaki Saito, * Jen-Zen Chuang, * Yun-Yu Tseng, * Carlos Dedesma & * Ching-Hwa Sung * Institute for International Advanced Interdisciplinary Research, Tohoku University International Advanced Research and Education Organization, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan. * Masaki Saito * Department of Cellular Signalling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan. * Masaki Saito * Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjyo, Kumamoto 860-8556, Japan * Kazuhito Tomizawa & * Taku Kaitsuka * Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10065, USA. * Ching-Hwa Sung Contributions A.L., J-Z.C. and C-H.S designed the overall study. A.L. and C.D. performed IUE experiments and phenotype characterization. J-Z. C. generated all constructs. A. L., Y-Y.T. and M.S. performed cell culture studies. K.T. generated the anti-phospho(T94)Tctex-1 antibody. K.T. and T.K. generated 9R peptides. A.L., J-Z.C. and C-H.S. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ching-Hwa Sung Author Details * Aiqun Li Search for this author in: * NPG journals * PubMed * Google Scholar * Masaki Saito Search for this author in: * NPG journals * PubMed * Google Scholar * Jen-Zen Chuang Search for this author in: * NPG journals * PubMed * Google Scholar * Yun-Yu Tseng Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos Dedesma Search for this author in: * NPG journals * PubMed * Google Scholar * Kazuhito Tomizawa Search for this author in: * NPG journals * PubMed * Google Scholar * Taku Kaitsuka Search for this author in: * NPG journals * PubMed * Google Scholar * Ching-Hwa Sung Contact Ching-Hwa Sung Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Control of PKA stability and signalling by the RING ligase praja2
- ncb 13(4):412-422 (2011)
Nature Cell Biology | Article Control of PKA stability and signalling by the RING ligase praja2 * Luca Lignitto1 * Annalisa Carlucci1 * Maria Sepe1 * Eduard Stefan2 * Ornella Cuomo3 * Robert Nisticò4, 5 * Antonella Scorziello3 * Claudia Savoia3 * Corrado Garbi1 * Lucio Annunziato3 * Antonio Feliciello1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:412–422Year published:(2011)DOI:doi:10.1038/ncb2209Received13 September 2010Accepted10 January 2011Published online20 March 2011Corrected online22 March 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 Activation of G-protein-coupled receptors (GPCRs) mobilizes compartmentalized pulses of cyclic AMP. The main cellular effector of cAMP is protein kinase A (PKA), which is assembled as an inactive holoenzyme consisting of two regulatory (R) and two catalytic (PKAc) subunits. cAMP binding to R subunits dissociates the holoenzyme and releases the catalytic moiety, which phosphorylates a wide array of cellular proteins. Reassociation of PKAc and R components terminates the signal. Here we report that the RING ligase praja2 controls the stability of mammalian R subunits. Praja2 forms a stable complex with, and is phosphorylated by, PKA. Rising cAMP levels promote praja2-mediated ubiquitylation and subsequent proteolysis of compartmentalized R subunits, leading to sustained substrate phosphorylation by the activated kinase. Praja2 is required for efficient nuclear cAMP signalling and for PKA-mediated long-term memory. Thus, praja2 regulates the total concentration of R subunits, t! uning the strength and duration of PKA signal output in response to cAMP. View full text Figures at a glance * Figure 1: Praja2 binds to PKA R subunits. () Schematic representation of human praja2 and its rat homologue neurodap1 (NDAP1, accession no. NP_620251). The cysteine-rich region (RING) and the C-terminal rat clone 33 isolated by the yeast two-hybrid system are shown. RBD, R-binding domain. The sequence of the RING-H2 domain of human praja2 along with the consensus sequence (outlined residues) are shown. Alanine-substituted Cys 634 and Cys 671 are indicated. () In vitro translated, [35S]-labelled RIIα and RIα subunits were subjected to pulldown assays with purified GST–praja2, GST–praja2Δ531–708 and GST–praja2531–631 fusions. *, proteolytic product. () Flag–praja2, the RING mutant (C634,671A; praja2rm) or deletion (Δ531–708) mutant were transiently transfected in HEK293 cells. Lysates were immunoprecipitated (IP) with rabbit polyclonal anti-RIIα/β antibody and immunoblotted (WB) with mouse monoclonal anti-Flag and anti-RIIα/β antibodies. () Flag–praja2 or its deletion mutants (Flag–praja2Δ63! 1–708, Flag–praja2Δ531–708 and Flag–praja2Δ431–708) were expressed, along with HA-tagged RIα, in HEK293 cells. Lysates were subjected to immunoprecipitation with rabbit polyclonal anti-RIα. The precipitates were immunoblotted with the indicated antibodies. () Lysates (2 mg) from HEK293 cells were subjected to immunoprecipitations using rabbit polyclonal anti-RIIα/β or non-immune IgG. Precipitates were immunoblotted with mouse monoclonal anti-RIIα/β and anti-praja2 antibody. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 2: Praja2 co-localizes with PKA R subunits. () Human neuroblastoma cells (SHSY) were subjected to double immunostaining with polyclonal anti-praja2 and monoclonal anti-RIIα/β antibodies. Images were collected and analysed by confocal microscopy. The fluorescence signals were quantified by correlation coefficient (Pearson's): R=0.51. Scale bar, 20 μm. Magnification of selected areas is shown (insets). () SHSY cells were transiently transfected with Flag–praja2 or Flag–praja2Δ531–708. At 24 h post-transfection, cells were fixed and doubly immunostained with anti-RIIα/β and anti-Flag antibodies. Before collecting, cells were treated with MG132 for 12 h. Images were collected and analysed by confocal microscopy. Flag–praja2/RII, Pearson's coefficient R=0.40; Flag–praja2Δ531–708/ RII, Pearson's coefficient R=0.01. Scale bar, 20 μm. () Primary hippocampal neurons were subjected to triple immunofluorescence with rabbit polyclonal anti-praja2, monoclonal mouse anti-CaMKIIα and goat polyclona! l anti-RIIβ antibodies. Right panels: profile plots refer to the cross-lines shown in the panels and express the intensity (a.u., arbitrary units) of fluorescence from left to right. Lower panels: Pearson's coefficients between praja2, RIIβ and CaMKIIα. Scale bar, 10 μm. * Figure 3: Praja2 triggers degradation of R subunits. (–) HEK293 cells were transiently co-transfected with HA–RIIα (), Myc–RIIβ () or HA–RIα () and Flag–praja2 expression vectors. CMV, cytomegalovirus. In (), two RING mutants were used: prajaC634A and Flag–praja2rm. At 24 h post-transfection, cells were collected and lysed. Lysates were immunoblotted with the indicated antibodies. () The same as in (), except the cells were treated for 8 h with MG132 (20 μM) as indicated before collecting. () Immunoblot analysis of endogenous R subunits on lysates from cells transiently transfected with Flag–praja2 vectors. Extracellular signal-regulated kinase 2 (ERK2) was used as a loading control. () Schematic representation of the PCA assay (AC, adenylate cyclase; Fsk, forskolin). () The effects of praja2 overexpression on PKA heterocomplex formation and dissociation under basal conditions and in response to Fsk (50 μM, 15 min; RLU, relative light units; data are means, ± s.d., n=3). HEK293 cells stably expr! essing RII–F[1]:PKAc–F[2] (ref. 36) were transiently transfected with Flag–praja2 (Wild type) or Flag–praja2rm (RM). At 24 h post-transfection, the cells were subjected to bioluminescence readout. Statistical significance was assessed using a paired Student t -test (* P<0.05; ** P<0.01 ). () Comparison of protein expression levels of endogenous and Rluc-PCA-tagged PKA subunits (PKAc, RII) and GAPDH by immunoblotting analysis. Shown is a representative experiment of n=3. Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 4: Praja2 ubiquitylates RIIα/β subunits. () HEK293 cells were transfected with HA–ubiquitin, RIIβ–Myc and Flag–praja2 or Flag–praja2rm. At 24 h post-transfection, cells were treated with MG132 (20 μM) for 8 h. Lysates were subjected to immunoprecipitations with anti-Myc and immunoblotted with anti-HA, anti-RIIα/β and anti-Flag antibodies. () HEK293 cells were transfected with HA-tagged ubiquitin. Where indicated, control (control siRNA) or SMARTpool praja2 siRNA was included in the transfection mix. At 24 h post-transfection, cells were either left untreated or stimulated with Fsk (40 μM) and IBMX (0.5 mM) for 3 h, in the presence of MG132. Lysates were subjected to immunoprecipitations with anti-RIIα/β and immunoblotted with anti-HA, anti-RIIα/β and anti-praja2 antibodies. () In vitro translated, [35S]-labelled RIIα was incubated with anti-Flag precipitates (Flag–praja2, Flag–praja2rm or Flag–praja2S342A,T389A) isolated from growing cells and 6×His-tagged ubiquitin, in the pr! esence of E1 and UbcH5c (E2). The reaction mix was denatured, size-fractionated by 7% SDS–polyacrylamide gel electrophoresis, and analysed by autoradiography. A fraction of the reaction mixture was immunoblotted with anti-Flag antibody (lower panel). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 5: PKAc binds to and phosphorylates praja2. () HEK293 cells transiently transfected with Flag–praja2 or Flag–praja2Δ531–708 vector were immunostained with anti-Flag and anti-PKAc antibodies. Pearson's coefficient: Flag–praja2/PKAc R=0.46 and Flag–praja2Δ531–708/ PKAc R=0.12. Scale bar, 20 μm. () Lysates from cells expressing Flag–praja2 and treated with MG132 were immunoprecipitated with anti-Flag antibody. The precipitates were immunoblotted with the indicated antibodies. Where indicated, cells were treated with Fsk (40 μM) and IBMX (0.5 mM) for 30 min ± H89 (10 μM). () Schematic illustration of the cAMP-precipitation strategy using 8-(6-Aminohexylamino)adenosine -3′,5′ -cyclic monophosphorothioate immobilized on agarose (Rp-8-AHA-cAMPS). () cAMP precipitation of endogenous PKA subunits and Flag–praja2 in response to 15-min treatment with either Fsk (100 μM) or isoproterenol (Iso, 10 μM). In the control, a molar excess of cAMP (5 mM) was added to the lysate. () Quantita! tive analysis of the experiments shown in (mean values ± s.e.m. of three independent experiments). () cAMP precipitation of endogenous PKA subunits and Flag–praja2rm. () Schematic representation of praja2, including putative PKA consensus sites (Ser 342 and Thr 389, underlined). Lower panels, cells transfected with vectors for Flag–praja2 or Flag–praja2S342A,T389A mutant were serum-deprived overnight and treated with Fsk and IBMX. Flag precipitates were immunoblotted with anti-phosphoSer/Thr PKA substrate antibody. CMV, cytomegalovirus. () Flag–praja2 and Flag–praja2S342A,T389A mutant were immunoprecipitated from lysates and subjected to an in vitro kinase assay. [32P]-labelled praja2 was visualized by autoradiography. An aliquot of the samples was immunoblotted with anti-Flag antibody. () cAMP precipitation of endogenous PKA subunits and Flag–praja2S342A,T389A mutant in response to isoproterenol treatment. () Complex formation between RII subunits and Flag–p! raja2S342A,T389A mutant (mean values ± s.e.m. from three inde! pendent experiments). () Co-immunoprecipitation of RIIα/β and Flag–praja2 or Flag–praja2S342A,T389A mutant from cell lysates. () Immunoblot analysis of cell lysates expressing HA–RIIα and Flag–praja2 or Flag–praja2S342A,T389A. () U2OS cells transfected with vectors for RII–F[1], PKAc–F[2] and Flag–praja2 versions were treated with ±50 μM Fsk for 30 min.The forskolin was washed out and cells were harvested 1 h later. Shown are the effects of praja2 on PKA holoenzyme dissociation and re-association. The untreated sample was set as 100% (RLU, relative light units; n=3 independent experiments; ± s.e.m.). Statistical significance was assessed using a paired Student t -test (* P<0.05; ** P<0.01). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 6: Praja2 controls PKA signalling in cells. () Double immunofluorescence for praja2 and RIIβ in human neuroblastoma cells (SHSY) transiently transfected with control siRNA or praja2 siRNA. Scale bar, 20 μm. () At 24 h post-transfection, cells were serum deprived overnight and stimulated with Fsk (40 μM) and IBMX (0.5 mM) for 30 and 60 min. Formalin-fixed cells were immunostained with anti-phosphoSer-133 antibody. Immunoblots (lower panels) show the expression of praja2 in control siRNA (lanes 1, 2) and praja2 -siRNA-transfected (lanes 3, 4) cells, left untreated (lanes 1, 3) or stimulated with Fsk for 60 min (lanes 2, 4). Scale bar, 30 μm. () Quantitative analysis of the experiments shown in . The data are expressed as the mean ± s.e.m. of three independent experiments carried out in triplicate. A total of 200–250 cells were scored in each set of experiments. * P<0.01 versus control (siRNAc). () Lysates from control siRNA- or praja2 -siRNA-transfected cells were serum deprived overnight. Before ! collecting, cells were left untreated or stimulated with Fsk and IBMX for the indicated times. Lysates were immunoblotted with the indicated antibodies. () Quantitative analysis of the experiment shown in . A mean of two independent experiments that gave similar results is shown. Squares, control siRNA; triangles, praja2 siRNA. () Immunoblot analysis of lysates from control (siRNAc) or praja2 -siRNA (3′UTR )-transfected cells. Before collecting, cells were serum deprived overnight and left untreated or stimulated with Fsk and IBMX for 60 min. Where indicated, Flag–praja2 and Flag–praja2Δ531–708 vectors were included in the transfection mixture. (,) Quantitative PCR with reverse transcription showing c-fos accumulation in cells transfected with control siRNA or praja2 siRNA and stimulated with isoproterenol (1 μM; ) or Fsk (40 μM) and IBMX (0.5 mM; ) for the indicated times. The data represent a mean value ± s.e.m. from three () or two () independent expe! riments carried out in triplicate. Where indicated, vectors fo! r Flag–praja2, Flag–praja2S342A,T389A mutant (double mutant, DM), Flag–praja2rm (RM) and PKAc were included in the transfection mixture. * P<0.01 versus control (siRNAc). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 7: Praja2 controls PKA signalling and LTP in rat brain. () Immunoblot analysis of lysates from hippocampal/striatal regions of rat brain intraventricularly perfused with siRNAs and subsequently treated with isoproterenol for the indicated times. () Quantitative analysis of phosphoCREB from the experiments shown in . The data are expressed as the mean ± s.e.m. of three experiments. * P<0.05 versus control. () Sections from hippocampal subregion CA1 of rat brain perfused with siRNAs and treated with isoproterenol as in were doubly immunostained with anti-phosphoCREB and anti-NeuN antibodies. The images were collected and analysed by confocal microscopy. Scale bar, 50 μm. () Quantitative analysis of the experiments shown in . The data are expressed as the means of two independent experiments that gave similar results. A total of 300–350 cells were scored in each set of experiments. () Quantitative PCR with reverse transcription showing c-fos accumulation in the striatum. The data represent a mean value from two independent e! xperiments made in duplicate that gave similar results. () E-LTP induced by one train of HFS is comparable between control siRNA (white squares, n=5) and praja2-siRNA rats (grey triangles, n=6). Field excitatory postsynaptic potential (fEPSP) amplitudes are expressed as percentages of the pretetanus baseline. The data are expressed as the mean ± s.e.m. () A TBS protocol elicits normal L-LTP in control siRNA rats (white squares, n=5), but deficient L-LTP in praja2-siRNA rats (grey triangles, n=6). Uncropped images of blots are shown in Supplementary Fig. S8. * Figure 8: Schematic representation of the role of praja2 in PKA signalling. Under resting conditions, the inactive PKA holoenzyme accumulates inside the cells as a consequence of the low ubiquitylation rate of R subunits. Elevation of intracellular cAMP levels by ligand (L) stimulation of the adenylate cyclase (AC) efficiently activates PKA, which in turn phosphorylates praja2. Phosphorylated praja2 ubiquitylates and degrades R subunits through the proteasome pathway. Accumulation of free, active PKAc (C) sustains substrate phosphorylation and positively impacts on the amplitude and duration of cAMP signalling. Change history * Abstract * Change history * Author information * Supplementary informationCorrigendum 22 March 2011In the version of this article initially published online Figures 2, 4 and 5 were mislabelled, and on page 5 line 21 a sentence has now been reworded for clarity. Author information * Abstract * Change history * Author information * Supplementary information Affiliations * Dipartimento di Biologia e Patologia Cellulare e Molecolare 'L. Califano', Universitá 'Federico II', 80131 Naples, Italy * Luca Lignitto, * Annalisa Carlucci, * Maria Sepe, * Corrado Garbi & * Antonio Feliciello * Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, 6020 Innsbruck, Austria * Eduard Stefan * Divisione di Farmacologia, Dipartimento di Neuroscienze, Università 'Federico II', 80131 Naples, Italy * Ornella Cuomo, * Antonella Scorziello, * Claudia Savoia & * Lucio Annunziato * Dipartimento Farmacobiologico, Università della Calabria, 87036 Rende, Italy * Robert Nisticò * Laboratorio di Neurologia Sperimentale, IRCCS Fondazione Santa Lucia, 00143 Rome, Italy * Robert Nisticò Contributions L.L., L.A., E.S. and A.F. designed the experiments. L.L. carried out most of the experiments except for the following: Figs 2c, 7c,d and Supplementary Figs S5 and S7b were carried out by A.S., C.S. and O.C. Figs 1d and 5g were carried out by M.S., who also generated praja2-deletion mutants. Figs 3f–h, 5c–f, i, j, m and Supplementary Fig. S6b were carried out by E.S. Figs 2a,b, 5a, 6a and Supplementary Figs S3 and S4 were carried out by C.G. Figs 6g,h, 7e and Supplementary Fig. S7a were carried out by A.C. Fig. 7f,g were carried out by R.N. L.L., E.S., L.A. and A.F. analysed the data. A.F. wrote the manuscript with contributions from L.A. and E.S. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Antonio Feliciello Author Details * Luca Lignitto Search for this author in: * NPG journals * PubMed * Google Scholar * Annalisa Carlucci Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Sepe Search for this author in: * NPG journals * PubMed * Google Scholar * Eduard Stefan Search for this author in: * NPG journals * PubMed * Google Scholar * Ornella Cuomo Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Nisticò Search for this author in: * NPG journals * PubMed * Google Scholar * Antonella Scorziello Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia Savoia Search for this author in: * NPG journals * PubMed * Google Scholar * Corrado Garbi Search for this author in: * NPG journals * PubMed * Google Scholar * Lucio Annunziato Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio Feliciello Contact Antonio Feliciello Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data - MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins
- ncb 13(4):423-433 (2011)
Nature Cell Biology | Article MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins * Kasey C. Vickers1 * Brian T. Palmisano1 * Bassem M. Shoucri1 * Robert D. Shamburek1 * Alan T. Remaley1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:423–433Year published:(2011)DOI:doi:10.1038/ncb2210Received20 July 2010Accepted11 January 2011Published online20 March 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 Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. Reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of messenger RNA reporters. Furthermore, HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be dependent on scavenger receptor class B type I. The human HDL–miRNA profile of normal subjects is significantly different from that of familial hypercholesterolemia subjects. Notably, HDL–miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets ! in cultured hepatocytes. Collectively, these observations indicate that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNAs. View full text Figures at a glance * Figure 1: HDL–RNA analysis. () Human plasma and HDL FPLC protein distribution (left and right axis, respectively; mAU, milli-absorbance unit). Red shading, HDL; green shading, LDL; blue shading, VLDL–exosome. Black line, human plasma; red line, purified HDL. () Human plasma and HDL FPLC total cholesterol distribution (mg dl−1). Black line, plasma; red line, purified HDL. () TEM of HDL and exosomes. Scale bars, 100 nm. () Quantification of ApoA-I (ng ml−1) protein. Exo, exosomes (n=3); Exo–Exo, FPLC exosome zone from total exosome preparations (n=3); Exo–HDL, FPLC HDL zone from total exosome preparations (n=4); HDL, purified from plasma (n=5). Data are means ± s.e.m. () Quantification of HSP70 protein (ng ml−1) in Huh7 cell lysates (n=4); Exo, exosomes (n=4); HDL, purified from plasma (n=4). Data are means ± s.e.m. () Western blot, anti-CD63. Purified exosomes; HDL-IP, HDL immunoprecipitation; HDL FPLC, HDL fast-protein liquid chromatography; HDL DGUC, HDL density-gradient ultracen! trifugation; Huh7 cell lysate. An uncropped image of the blot is shown in Supplementary Fig. S6. () Bioanalyzer Pico analysis of HDL total RNA (FU, fluorescence unit). () Bioanalyzer small-RNA analysis of HDL total RNA. () Digital gel electrophoresis of HDL small RNAs. Lane 1, miRNA standards 22 and 25 nucleotides (nt); Lane 2, HDL total RNA; Lane 3, miR-223 positive control. () Quantification of hsa-miR-223 levels (ng ml−1). n=4. * Figure 2: Human HDL carries distinct miRNA signatures in health and disease. () Hierarchical clustering heatmap of human HDL–miRNAs from normal (n=6) and familial hypercholesterolemia (FH; n=5) subjects. Blue to red, colour range gradient of mean abundance (−3 to 3). () StarGlyph distribution of each miRNA observed on HDL. Log values of the mean RQV. Red, normal; blue, familial hypercholesterolemia HDL. () Venn diagram of normal and familial hypercholesterolemia HDL–miRNA totals. Counted miRNAs were observed on ≥3 arrays within each group. () Histogram of frequency distribution of P values. Red line, P=0.05. Green line illustrates uniform distribution of P values. () Spearman non-parametric correlation between normal and familial hypercholesterolemia HDL–miRNA profiles. () Volcano plot of significantly differentially abundant miRNAs on familial hypercholesterolemia HDL, compared with normal HDL. Red marks, >2-fold change (Log2); P<0.05 (−Log10). (,) Expression (mean) ratios of miRNA pairs (miR/miR*, blue; miR-5p/miR-3p, red). Black line r! epresents expression ratio of 1 (x=y) and similar abundance of both strands of pairs. Log10 scale. () Human HDL–miRNA pairs from normal subjects. () Human HDL–miRNA pairs from familial hypercholesterolemia subjects. () Pie charts illustrating strand observations (percentages) for normal (left) and familial hypercholesterolemia (right) HDL. Blue, ≤1 strand of miRNA pair was observed; red, both strands of miRNA pair were observed but absolute fold change (AbFC) >2.0; green, both strands of pair were observed and had relatively equal abundance (AbFC<2.0). * Figure 3: The role of LDL and the LDLR in HDL–miRNA signatures. () miRNA abundance signatures of human exosomes, LDL and HDL from the same subject. Spearman non-parametric correlation between each profile. *P<0.0001. miRNAs are numerically ranked top-down and abundances are represented by horizontal band intensity. () Volcano plot of significant (P<0.05) differential (>2.0-fold) HDL–miRNA abundances in Ldlr−/− HFD mice (n=3), compared with wild-type controls (n=3). Red marks, >2-fold change (Log2); P<0.05 (−Log10). () Spearman non-parametric correlation between Ldlr−/− HFD mice and wild-type controls. R=0.43, P<0.0001. * Figure 4: HDL readily incorporates with miRNAs in vitro and in vivo. () TEM image of HDL + Nanogold-labelled miRNA (miR-223–Au) unenhanced (top); HDL + Nanogold-labelled miRNA (miR-223–Au) gold enhanced (Aue; 2 min; bottom). Scale bars, 100 nm. Bottom image inset for magnification. () FPLC separation of radiolabelled HDL ([3H]cholesterol)–miRNA ([32P]miR-125a) complexes. Red line, HDL + [32P]miR-125a (37 °C reaction); blue line, HDL + [32P]miR-125a (20 °C reaction); purple line, [32P]miR-125a alone; black dashed line, HDL ([3H]cholesterol) + cold miR-125a. Light shading indicates HDL complex zone, dark shading indicates uncomplexed free radiolabelled [32P]miR-125a and HDL ([3H]cholesterol) zone. Coloured arrows indicate peak associations, red (37 °C), blue (20 °C), purple (no HDL), black (HDL peak). S-200 column. () Quantification of HDL–miR-223 incorporation. hsa-miR-223 (ng ml−1) levels post-HDL immunoprecipitation, miR-223, positive control; native human HDL; native human HDL + miR-223; rHDL; rHDL + miR-223. n=2! . () Spearman non-parametric correlation between wild-type mouse and normal human HDL profiles. R=0.68, P<0.0001. () Hierarchal clustering heatmap of HDL–miRNA profiles (mean) in wellness, hyperlipidemia and atherosclerosis (mouse and human). n=7 conditions: FH, familial hypercholesterolemia (n=5); Apoe−/− HFD (n=4); wild-type hIP (n=3), human rHDL retrieved by immunoprecipitation from wild-type mice; Ldlr−/− HFD (n=3); wild-type chow diet (n=3); Apoe−/− chow diet (n=3); normal human HDL (n=6). * Figure 5: HDL transfers miRNAs to recipient cells with functional targeting. () nSMase2 regulates miRNA export to HDL. Quantification of HDL–miR-223 levels (qPCR) normalized to immunoprecipitated rHDL protein (ng μg−1). GW4869, chemical inhibitor of nSMase2; TO90131, LXRα agonist induces ABCA1 expression. n=3. Data are means ± s.e.m. () Quantification of intracellular miR-375 levels (qPCR) in hepatocytes (Huh7) treated with HDL alone (80 μg ml−1; n=4), HDL + miR-223 (80 μg ml−1; n=4) or HDL + miR-375 (80 μg ml−1; n=4). Data are means ± s.e.m. () Quantification of intracellular miR-223 levels (qPCR) in hepatocytes (Huh7) treated with HDL alone (80 μg ml−1; n=4), HDL + miR-223 (80 μg ml−1; n=4) or HDL + miR-375 (80 μg ml−1; n=4). Data are means ± s.e.m. () Quantification of RHOB mRNA levels (fold change) in hepatocytes (Huh7) treated with HDL + miR-375 (80 μg ml−1; n=4) or HDL + miR-223 (80 μg ml−1; n=4). Data are means ± s.e.m. () Quantification of EFNA1 mRNA levels (fold change) in h! epatocytes (Huh7) treated with HDL alone (80 μg ml−1; n=3) or HDL + miR-223 (80 μg ml−1; n=3). Data are means ± s.e.m. * Figure 6: HDL–miRNA delivery is SR-BI-dependent. () Quantification of intracellular hsa-miR-223 levels (fold change). Transfected BHK cells, pSwitch human SR-BI inducible (mifepristone 10 nM) expression system, treated with HDL alone (10 μg ml−1; n=3) or HDL + miR-223 (10 μg ml−1; n=3). Data are means ± s.e.m. () Quantification of HDL–miR-223 delivery, as determined by intracellular miR-223 levels (qPCR, miR-223 standard curve). Data reported as per cent control. Human hepatocytes (Huh7) transfected with SR-BI siRNA (100 nM, On-target Plus pool; n=3) or mock reagent (n=3), before HDL + miR-223 (10 μg ml−1) treatment. Data are means ± s.e.m. () Renilla luciferase activity normalized to Firefly (transfection control) luciferase activity. BHK cells transfected with pSwitch human SR-BI inducible (mifepristone 10 nM) expression system treated with HDL (80 μg ml−1; n=4) or HDL + miR-223 (80 μg ml−1; n=4). Renilla-SR-BI-3′UTR luciferase reporter activities reported as fold changes fr! om Renilla controls. Data are means ± s.e.m. * Figure 7: Atherosclerotic HDL induces differential gene expression through miRNA transfer. () Conceptualization of familial hypercholesterolemia HDL–miRNA mRNA target interactome. Putative targets of differentially abundant familial hypercholesterolemia HDL–miRNAs (unique colours) and their association to other miRNAs and targets. () Quantification of intracellular (Huh7) hsa-miR-105 (fold change) after human familial hypercholesterolemia (FH; n=4) or normal (n=4) HDL (80 μg ml−1) delivery. Data are means ± s.e.m. () Volcano plot illustrating significant differential gene expression (mRNA) changes (blue marks) attributed to familial hypercholesterolemia HDL–miRNA delivery, compared with normal HDL. −Log10(Benjamini–Hochberg-corrected P value) P<0.05; Log2 (fold change) >2-fold. n=3. () Genes that were downregulated as a result of familial hypercholesterolemia HDL–miRNA delivery with ≥3 predicted target sites of differential familial hypercholesterolemia HDL–miRNAs. Left, negative fold changes; right, number of familial hypercholesterolemia ! HDL–miRNA conserved target sites within mRNA 3′UTRs. () Genes that were downregulated as a result of familial hypercholesterolemia HDL–miRNA delivery that are putative targets of familial-hypercholesterolemia-HDL-specific hsa-miR-105. Predicted targeting score (TargetScan) range (−0.89 top to 0.04 bottom). n=3. Author information * Abstract * Author information * Supplementary information Affiliations * National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Dr. Building 10 8N222, Bethesda, Maryland 20892, USA * Kasey C. Vickers, * Brian T. Palmisano, * Bassem M. Shoucri, * Robert D. Shamburek & * Alan T. Remaley Contributions K.C.V., B.T.P. and A.T.R. designed the research plan and study. K.C.V., B.T.P. and B.M.S. carried out all experiments. R.D.S. provided human samples. K.C.V. and A.T.R. drafted and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kasey C. Vickers Author Details * Kasey C. Vickers Contact Kasey C. Vickers Search for this author in: * NPG journals * PubMed * Google Scholar * Brian T. Palmisano Search for this author in: * NPG journals * PubMed * Google Scholar * Bassem M. Shoucri Search for this author in: * NPG journals * PubMed * Google Scholar * Robert D. Shamburek Search for this author in: * NPG journals * PubMed * Google Scholar * Alan T. Remaley Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (500K) Supplementary Information * Supplementary Information (2M) Supplementary Information Additional data - Obesity-induced overexpression of miRNA-143 inhibits insulin-stimulated AKT activation and impairs glucose metabolism
- ncb 13(4):434-446 (2011)
- Light-mediated polarization of the PIN3 auxin transporter for the phototropic response in Arabidopsis
- ncb 13(4):447-452 (2011)
Nature Cell Biology | Letter Light-mediated polarization of the PIN3 auxin transporter for the phototropic response in Arabidopsis * Zhaojun Ding1, 2 * Carlos S. Galván-Ampudia3, 6 * Emilie Demarsy4 * Łukasz Łangowski1, 2 * Jürgen Kleine-Vehn1, 2 * Yuanwei Fan3 * Miyo T. Morita5 * Masao Tasaka5 * Christian Fankhauser4 * Remko Offringa3 * Jiří Friml1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:447–452Year published:(2011)DOI:doi:10.1038/ncb2208Received01 February 2010Accepted11 January 2011Published online13 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Phototropism is an adaptation response, through which plants grow towards the light1. It involves light perception and asymmetric distribution of the plant hormone auxin2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Here we identify a crucial part of the mechanism for phototropism, revealing how light perception initiates auxin redistribution that leads to directional growth. We show that light polarizes the cellular localization of the auxin efflux carrier PIN3 in hypocotyl endodermis cells, resulting in changes in auxin distribution and differential growth. In the dark, high expression and activity of the PINOID (PID) kinase correlates with apolar targeting of PIN3 to all cell sides. Following illumination, light represses PINOID transcription and PIN3 is polarized specifically to the inner cell sides by GNOM ARF GTPase GEF (guanine nucleotide exchange factor)-dependent trafficking. Thus, differential trafficking at the shaded and illuminated hypocotyl side aligns PIN3 polarity with ! the light direction, and presumably redirects auxin flow towards the shaded side, where auxin promotes growth, causing hypocotyls to bend towards the light. Our results imply that PID phosphorylation-dependent recruitment of PIN proteins into distinct trafficking pathways is a mechanism to polarize auxin fluxes in response to different environmental and endogenous cues. View full text Figures at a glance * Figure 1: PIN3-mediated asymmetric auxin response during hypocotyl phototropism. (–) Expression of the DR5rev::GFP auxin response reporter in hypocotyls in unilateral light (8 h). The fluorescence micrographs show increased DR5 activity at the shaded side in controls (24 out of 24 seedlings; ), no DR5 asymmetry in the phot1 mutant (26 out of 26 seedlings; ) and less-pronounced DR5 asymmetry in the pin3 mutant (14 out of 23 seedlings; ). Scale bars, 50 μm. In , the arrow indicates light direction; Ep, epidermis; V, vasculature. Arrowheads indicate increased DR5 auxin response. The histogram shows quantification of DR5rev::GFP fluorescence intensity in the wild-type, phot1 and pin3 seedlings (). The DR5 activity ratio is the fluorescent intensity at the shaded versus the illuminated side of the hypocotyl. Error bars represent standard deviation. Pairs for Student's t-test are indicated with brackets, **P<0.01. () Phototropic hypocotyl bending kinetics of pin3 mutants. Hypocotyl curvatures were measured under unilateral white-light illumination (2�! ��μmol m−2 s−1) and the average curvatures from three independent experiments were calculated (at least 15 seedlings for each). Error bars represent standard deviation. (Student's t-test between wild-type and pin3 mutant seedlings after 2 h of unilateral light treatment, *P<0.05.) () Redundant involvement of the PIN auxin carriers in hypocotyl phototropism. Hypocotyl curvatures were measured after 6 h of unilateral white-light illumination (2 μmol m−2 s−1) for different genotypes or NPA treatment. The average curvatures from three independent experiments were calculated (n=60). Error bars represent standard deviation (pairs for Student's t-test are indicated with brackets, **P<0.01, *P<0.05). * Figure 2: Light-mediated PIN3 polarization. (–) PIN3–GFP in endodermis cells of the upper hypocotyls. PIN3 is localized at both the outer and inner membranes in the dark (). There is gradual PIN3 polarization (weak outer and strong inner signal) only at the illuminated hypocotyl side after unilateral illumination with white light (2 μmol m−2s−1; ,). There is gradual PIN3 polarization (weak outer and strong inner signal) in all endodermis cells after illumination from all sides with white light (10 μmol m−2s−1; ,). Magnification of differences in signal intensity between inner and outer polar domains of the region marked by the rectangle in (). The asterisk depicts signal-free boundary between outer and inner lateral domains corresponding to the Casparian strip. Scale bars, 25 μm. Arrows indicate direction of illumination. V, vasculature. Open arrowheads depict outer side of endodermis cells. (–) Quantification of PIN3–GFP signal intensities of –. Relative PIN3–GFP signal at the outer (! 1) and inner (2) lateral cell sides of the left hypocotyl side or inner (3) and outer (4) lateral cell sides of the right hypocotyl side. The measurements were carried out in the same focal plane. Units are arbitrary fluorescence units (a.u.). Error bars represent standard deviation. * Figure 3: BFA-sensitive, GNOM-dependent PIN3 polarization by light. (–) PIN3–GFP in endodermis cells of hypocotyls after 4 h of unilateral illumination: PIN3–GFP polarization (weak outer and strong inner signal) at the illuminated hypocotyl side () is blocked in the presence of BFA (), but occurs normally in the BFA-resistant GNOMM696L line, even in the presence of BFA (). (,) Establishment of asymmetric DR5rev::GFP activity in unilateral illumination () is blocked by BFA (). Arrows indicate light direction. Open arrowheads depict outer side of endodermic cells. Ep, epidermis; V, vasculature. Scale bars, 25 μm (–), 50 μm (,). () Hypocotyl phototropism is sensitive to BFA treatment, defective in the mutant carrying the partial loss-of-function gnomR5 allele and BFA-insensitive in the BFA-resistant GNOMM696L line. Curvatures were measured after 6 h of unilateral white-light (70 μmol m−2 s−1) treatment. In each case, the average curvatures were calculated from three independent experiments (n=50). Error bars represe! nt standard deviations (Student's t test, **P<0.01, pairs for Student's t-test are indicated with brackets). Only around 9% of seedlings that were treated with BFA (n=43) or 10% of gnomR5 mutant seedlings (n=39) had very weak bending responses that could be measured. (,) The phot1pin3 double mutant had almost no bending response, similarly to the phot1 mutant, in low-intensity unilateral white light (2 μmol m−2 s−1) (), and the gnomR5phot1 double mutant had almost no bending response, similarly to the gnomR5 mutant, in both low- (2 μmol m−2 s−1) () and high- (70 μmol m−2 s−1) () intensity unilateral white light. Error bars represent standard deviation from three independent experiments (pairs for Student's t-test are indicated with brackets, **P<0.01). * Figure 4: PID involvement in PIN3 polarization and the phototropic response. () PID, but not phot1, phosphorylates GST–PIN3HL. GST and the dead version of PID* and phot1* kinases were used as negative controls. Arrowhead depicts unspecific signal present in all samples. () PID transcription is repressed by light. Quantitative real-time PCR measured relative to an internal tubulin control. RNA was isolated from 5-day-old seedlings that underwent treatment without and with white light for 6 h (10 μmol m−2 s−1). Error bars represent standard deviation from three independent repeats (Student's t test, **P<0.01). () Hypocotyl phototropism is strongly reduced in 35S::PID and wag1 wag2 pid lines. Curvatures were measured after 6 h of treatment with unilateral white light (2 μmol m−2 s−1). Average curvatures from three independent experiments (at least 15 seedlings were measured for each repeat) were calculated. Error bars represent standard deviations (Student's t test, **P<0.01). (–) Light does not change PIN3–GFP locali! zation in 35S::PID () and wag1 wag2 pid () hypocotyls when compared with dark-grown controls (). The dark-grown and light-treated controls are compared in Supplementary Fig. S2. Arrows indicate light direction. Open arrowheads depict outer side of endodermic cells. V, vasculature. Scale bars, 50 μm. * Figure 5: Model for the hypocotyl phototropic response. Hypocotyl in the unilateral light. At the shaded side (left), the PID activity is high and the presumably phosphorylated PIN3 auxin transporter is targeted to both inner and outer sides of the endodermis cells. At the illuminated side, light signalling leads to the repression of PID transcription, which results in the recruitment of presumably less phosphorylated PIN3 into the polar trafficking pathway, leading to preferential PIN3 targeting to the inner cell side. This differential PIN3 targeting at the shaded and illuminated sides results in PIN3 polarization aligned with the light direction. The auxin flow is thus diverted to the shaded side, where the accumulated auxin activates the auxin response (auxin gradient is visualized by the blue colour gradient in the background) and induces growth leading to bending of hypocotyls towards the light. Red tubes, PIN3; black dots, phosphorylation; yellow arrows, illumination; blue arrows, auxin flux. () Signal transduction cascade! : light perceived by phototropins and cryptochromes polarizes PIN3 subcellular distribution directly by repressing PID transcription; GNOM is also involved into the PIN3 polarization directly or by regulating phototropin and cryptochrome protein trafficking; PIN3-dependent auxin transport generates asymmetric auxin accumulation at the shaded side that drives differential growth and bending. Author information * Author information * Supplementary information Affiliations * Department of Plant Systems Biology, VIB, Universiteit Gent, Technologiepark 927, B-9052 Gent, Belgium * Zhaojun Ding, * Łukasz Łangowski, * Jürgen Kleine-Vehn & * Jiří Friml * Department of Plant Biotechnology and Genetics, Ghent University, B-9052 Gent, Belgium * Zhaojun Ding, * Łukasz Łangowski, * Jürgen Kleine-Vehn & * Jiří Friml * Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, The Netherlands * Carlos S. Galván-Ampudia, * Yuanwei Fan & * Remko Offringa * Center for Integrative Genomics, Genopode Building, University of Lausanne, CH-1015 Lausanne, Switzerland * Emilie Demarsy & * Christian Fankhauser * Graduate School of Biological Sciences, Nara Institute of Science and Technology, 630-0101 Ikoma, Japan * Miyo T. Morita & * Masao Tasaka * Present address: Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands * Carlos S. Galván-Ampudia Contributions Z.D., C.S.G-A., E.D., M.T., R.O., C.F. and J.F. conceived the study and designed the experiments. Z.D., E.D., C.S.G-A., J.K-V., Ł.Ł., Y.F. and M.T.M. carried out the experiments and analysed the data. Z.D. and J.F. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jiří Friml Author Details * Zhaojun Ding Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos S. Galván-Ampudia Search for this author in: * NPG journals * PubMed * Google Scholar * Emilie Demarsy Search for this author in: * NPG journals * PubMed * Google Scholar * Łukasz Łangowski Search for this author in: * NPG journals * PubMed * Google Scholar * Jürgen Kleine-Vehn Search for this author in: * NPG journals * PubMed * Google Scholar * Yuanwei Fan Search for this author in: * NPG journals * PubMed * Google Scholar * Miyo T. Morita Search for this author in: * NPG journals * PubMed * Google Scholar * Masao Tasaka Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Fankhauser Search for this author in: * NPG journals * PubMed * Google Scholar * Remko Offringa Search for this author in: * NPG journals * PubMed * Google Scholar * Jiří Friml Contact Jiří Friml Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (400K) Supplementary Information Additional data - Lysosomal positioning coordinates cellular nutrient responses
- ncb 13(4):453-460 (2011)
Nature Cell Biology | Letter Lysosomal positioning coordinates cellular nutrient responses * Viktor I. Korolchuk1, 5 * Shinji Saiki1, 5 * Maike Lichtenberg1, 6 * Farah H. Siddiqi1, 6 * Esteban A. Roberts2 * Sara Imarisio1, 3 * Luca Jahreiss1 * Sovan Sarkar1 * Marie Futter1 * Fiona M. Menzies1 * Cahir J. O'Kane3 * Vojo Deretic2, 4 * David C. Rubinsztein1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:453–460Year published:(2011)DOI:doi:10.1038/ncb2204Received07 May 2010Accepted07 January 2011Published online13 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg mTOR (mammalian target of rapamycin) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes, including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles1, 2 intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates3, 4, 5, 6, 7, 8. Here we report that lysosomal positioning coordinates anabolic and catabolic responses with changes in nutrient availability by orchestrating early plasma-membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, whereas starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH. Lysosomal positioning regulates mTORC1 si! gnalling, which in turn influences autophagosome formation. Lysosome positioning also influences autophagosome–lysosome fusion rates, and thus controls autophagic flux by acting at both the initiation and termination stages of the process. Our findings provide a physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux. View full text Figures at a glance * Figure 1: Changes in mTORC1 signalling in response to starvation correlate with lysosomal positioning. (–) HeLa cells were either left untreated, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunostained, or immunoblotted using antibodies as shown. Co-localization panels show an overlap between mTOR and LAMP1 signals. Note that changes in the positioning of lysosomal mTOR (quantified as the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles; ,) correlate with mTORC1 activity (levels of phosphorylated S6K relative to the total S6K; ). () Visualization of Akt activated in response to recovery after serum starvation. After nutrient recovery, LAMP1-positive vesicles localize to peripheral regions with higher concentrations of phospho-Akt. DAPI, 4,6−diamidino−2−phenylindole. For all panels, values are means ± s.e.m. of three independent experiments carried out in triplicate. *P<0.05, **P<0.01, ***P<0.005 Student's t-test; other comparisons are not significant (n.s.)! . Representative maximum-intensity projections of serial confocal optical sections are shown. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 2: Factors changing lysosomal positioning also affect mTORC1 signalling. (,) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability. Cells were treated as in Fig. 1a followed by incubation with dimethylsulphoxide (vehicle) or with nocodazole during the last 2 h before fixation/lysis. Samples were analysed by immunofluorescence () or immunoblotting (). Quantification of phopho-S6K levels is shown in (). (–) Changes in lysosomal positioning induced by kinesin- or small GTPase-family members (,) correlate with changes in mTORC1 activity (). HeLa cells were transfected with overexpression constructs (OE) or with siRNA as shown, followed by immunofluorescence (,) or by immunoblotting () analyses. Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P<0.05, ***P<0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplemen! tary Fig. S7. * Figure 3: Lysosomal positioning regulates recovery of mTOR signalling after starvation. (–) HeLa cells transfected with ARL8B or KIF2 siRNA, or with ARL8B overexpression construct (non-targeting siRNA and empty pcDNA vector used as transfection controls), were either left untreated, serum/amino-acid starved for 5 h, or starved and then recovered in amino-acid- and FBS-containing medium for 30 min. Cells were immunostained using LAMP1 antibody () and the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles was quantified () or subjected to immunoblotting () using antibodies as shown. Quantification of phospho-S6K levels relative to the total S6K is shown in (). Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P<0.05, **P<0.01, ***P<0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 4: Nutrients control lysosomal positioning by modulating pHi and lysosomal levels of KIF2 and ARL8B. () Starvation increases pHi in HeLa cells allowed to starve using three different protocols (see Methods) with or without subsequent recovery. (–) Changing pHi from 7.1 to 7.7 is sufficient to affect localization of lysosomes and mTORC1 activity. pHi was titrated in full tissue-culture medium containing nigericin, which enables changes in pHi to be forced by altering pH in the medium, followed by immunostaining (,) or western blotting () using antibodies as shown. () Changes in lysosomal localization have no effect on pHi. ARL8B and KIF2 were overexpressed or knocked down in HeLa cells followed by pHi measurement. (,) Nutrients and pHi affect levels of ARL8 and KIF2 in lysosomal fractions. Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation–recovery () or to 1 h changes of pHi in full tissue-culture medium containing nigericin () are shown. (,) Effect of nutrients a! nd pHi on binding of ARL8 and KIF2 to polymerized microtubules. HeLa cells treated as in () and (), followed by isolation of polymerized microtubule fraction and western blotting. The asterisk indicates a nonspecific band. For all panels values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P<0.05, **P<0.01, ***P<0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 5: Lysosomal positioning modulates autophagy. () Diagram illustrating how lysosomal positioning coordinates mTOR signalling and autophagy. Peripheral lysosomal localization (nutrient induced) increases mTOR activity (blocking autophagosome synthesis) and reduces autophagosome–lysosome fusion. Starvation-induced lysosomal clustering reduces mTOR activity (activating autophagosome synthesis) and facilitates autophagosome–lysosome fusion. () Serum and amino-acid starvation of HeLa cells for 5 h reduces LC3-II levels (above), but increases autolysosome numbers detected using tfLC3 (below). With tfLC3, GFP- and RFP-positive puncta represent autophagosomes before lysosomal fusion, whereas RFP-positive/GFP-negative puncta represent autolysosomes—GFP is more rapidly quenched by low lysosomal pH (see Methods). Increased autolysosomes indicate enhanced starvation-induced flux of LC3 to lysosomes. () ARL8B knockdown increases autophagosomal synthesis. siRNA-transfected HeLa cells were incubated for 48 h, then left untrea! ted or incubated with bafilomycin A1. LC3-II levels versus actin were quantified (bottom graphs). Asterisk: nonspecific band. () ARL8B overexpression inhibits autophagosome synthesis and degradation. HeLa cells overexpressing ARL8B or empty vector were analysed as in (). () HeLa cells were co-transfected with either ARL8B overexpression construct or siRNA (non-targeting siRNA and empty peGFP vector were transfection controls) together with mCherry–LC3 for 48 h. After fixation, cells were stained for endogenous LAMP1 and DNA (DAPI). Representative maximum-intensity projections of serial confocal optical sections are shown. Co-localization panels show overlapping mCherry–LC3 and LAMP1 signals. (–) Quantification of autophagosome–lysosome fusion in HeLa cells. Percentages of autolysosomes (positive for both mCherry–LC3 and LAMP1) to autophagosomes (positive for mCherry–LC3 and negative for LAMP1) were quantified. In (), we analysed cells treated for 2 h with no! codazole before fixation, which dispersed the perinuclear lyso! somal cluster (see Fig. 2a). () Autophagosomal and autolysosomal numbers in tfLC3-expressing cells after ARL8B overexpression and knockdown (Supplementary Fig. S5o shows representative cells.). In (–) we analysed 20 cells per group in three independent experiments. Values are means ± s.e.m of three independent experiments carried out in triplicate. *P<0.05, **P<0.01, ***P<0.005 Student's t-test; other comparisons not significant (n.s.). Uncropped images of blots are shown in Supplementary Fig. S7. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Maike Lichtenberg & * Farah H. Siddiqi Affiliations * Department of Medical Genetics, Cambridge Institute for Medical Genetics, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK * Viktor I. Korolchuk, * Shinji Saiki, * Maike Lichtenberg, * Farah H. Siddiqi, * Sara Imarisio, * Luca Jahreiss, * Sovan Sarkar, * Marie Futter, * Fiona M. Menzies & * David C. Rubinsztein * Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, 87131, USA * Esteban A. Roberts & * Vojo Deretic * Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK * Sara Imarisio & * Cahir J. O'Kane * Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, 87131, USA * Vojo Deretic * Joint first authors * Viktor I. Korolchuk & * Shinji Saiki Contributions All authors designed and analysed experiments. V.I.K., S. Saiki, M.L., F.H.S., E.A.R., S.I., L.J., S. Sarkar, M.F. and F.M.M. carried out experiments. V.I.K. and D.C.R. wrote the manuscript. D.C.R. supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David C. Rubinsztein Author Details * Viktor I. Korolchuk Search for this author in: * NPG journals * PubMed * Google Scholar * Shinji Saiki Search for this author in: * NPG journals * PubMed * Google Scholar * Maike Lichtenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Farah H. Siddiqi Search for this author in: * NPG journals * PubMed * Google Scholar * Esteban A. Roberts Search for this author in: * NPG journals * PubMed * Google Scholar * Sara Imarisio Search for this author in: * NPG journals * PubMed * Google Scholar * Luca Jahreiss Search for this author in: * NPG journals * PubMed * Google Scholar * Sovan Sarkar Search for this author in: * NPG journals * PubMed * Google Scholar * Marie Futter Search for this author in: * NPG journals * PubMed * Google Scholar * Fiona M. Menzies Search for this author in: * NPG journals * PubMed * Google Scholar * Cahir J. O'Kane Search for this author in: * NPG journals * PubMed * Google Scholar * Vojo Deretic Search for this author in: * NPG journals * PubMed * Google Scholar * David C. Rubinsztein Contact David C. Rubinsztein Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - The cilia protein IFT88 is required for spindle orientation in mitosis
- ncb 13(4):461-468 (2011)
Nature Cell Biology | Letter The cilia protein IFT88 is required for spindle orientation in mitosis * Benedicte Delaval1 * Alison Bright1 * Nathan D. Lawson2 * Stephen Doxsey1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:461–468Year published:(2011)DOI:doi:10.1038/ncb2202Received05 November 2010Accepted04 January 2011Published online27 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cilia dysfunction has long been associated with cyst formation and ciliopathies1. More recently, misoriented cell division has been observed in cystic kidneys2, but the molecular mechanism leading to this abnormality remains unclear. Proteins of the intraflagellar transport (IFT) machinery are linked to cystogenesis and are required for cilia formation in non-cycling cells3, 4. Several IFT proteins also localize to spindle poles in mitosis5, 6, 7, 8, indicating uncharacterized functions for these proteins in dividing cells. Here, we show that IFT88 depletion induces mitotic defects in human cultured cells, in kidney cells from the IFT88 mouse mutant Tg737orpk and in zebrafish embryos. In mitosis, IFT88 is part of a dynein1-driven complex that transports peripheral microtubule clusters containing microtubule-nucleating proteins to spindle poles to ensure proper formation of astral microtubule arrays and thus proper spindle orientation. This work identifies a mitotic mechanism! for a cilia protein in the orientation of cell division and has important implications for the etiology of ciliopathies. View full text Figures at a glance * Figure 1: IFT88 depletion leads to mitotic defects in HeLa cells, kidney cells from the Tg737orpk mouse mutant and zebrafish. () Immunofluorescence microscopy images of control (GFP) and IFT88-siRNA-treated mitotic HeLa cells. α-tubulin (microtubules) and γ -tubulin (spindle poles, arrow) staining show spindle pole defects. CREST (kinetochores) or DAPI (DNA) staining shows misaligned chromosomes. Scale bars, 5 μm. () Quantification of mitotic defects following IFT88- or control (GFP)-siRNA treatment in HeLa cells. Defects include disrupted poles (α - and γ -tubulin), misaligned chromosomes (DAPI staining) and spindle misorientation (spindle tilt, spindle poles in different focal planes). n=70 mitotic cells per experiment. () Side views of 3D reconstructed immunofluoresence images showing misoriented mitotic spindles in IFT88- versus control-siRNA-treated HeLa cells. Spindle (EB1), centrosomes (5051) and DNA (Phos-H3). () Histogram showing metaphase spindle-angle distribution in control- and IFT88-siRNA-treated cells. n=30 mitotic spindles. Schematic (top) showing spindle angle (α ) measurem! ent. H, hypotenuse. O, opposite. (,) Quantification () and time-lapse microscopy images () showing uneven timing of daughter-cell flattening onto the substrate after mitosis (misoriented cell division) in IFT88-siRNA-treated HeLa cells, compared with control. n=50 mitotic cells per experiment. Arrows, time when the first daughter cell begins flattening. Time, min. Scale bar, 10 μm. () Immunofluorescence microscopy images (left) showing a disrupted spindle pole (α -tubulin, arrow) in kidney cells derived from the IFT88 mouse mutant Tg737orpk (Tg737−/−), compared with wild type (Tg737+/+). Scale bars, 2 μm. Graph (right), quantification of mitotic defects in wild-type and Tg737orpk mutant cells. () Immunofluorescence microscopy images of mitotic spindles from the pronephric ducts of whole-mount zebrafish embryos. Control embryo, cell with aligned chromosomes and mitotic spindle oriented in the longitudinal plane of the duct. IFT88-depleted embryo, cell with non-ali! gned chromosomes and misoriented spindle. Lines, pronephric du! ct border. Dashed lines, spindle orientation. MO, morpholino. Right, enlargements of spindles outlined by dashed rectangles. Scale bar, 5 μm. * Figure 2: IFT88 depletion disrupts astral microtubules and the spindle pole localization of proteins involved in microtubule nucleation in HeLa cells. () Immunofluoresence microscopy images (left) of mitotic spindles showing disrupted astral microtubules (α -tubulin) at spindle poles of IFT88-depleted cells, compared with control. Pixel intensity range increased to visualize astral microtubules (arrow). Enlargements, spindle pole region. Graph (right), quantification of cells with long astral microtubules (>3 μm). n=70 mitotic spindles per experiment. () Side view of 3D reconstructed images (left) showing astral microtubules (EB1 staining) contacting the cortex in control cells (arrow, upper panel) and astral microtubules that fail to contact the cell cortex in IFT88-depleted cells (arrow, lower panel). Dotted lines, cell cortex. Graph (right), quantification of cells with both poles showing astral microtubules contacting cortex. n=50 mitotic spindles per experiment. (,) Immunofluorescence microscopy images () and quantification () of mitotic spindles showing loss of EB1 and γ -tubulin from spindle poles (arrow) in IF! T88-depleted cells, compared with control. Graph (), percentage of cells with disrupted pole localization of EB1 or γ -tubulin. n=50 mitotic spindles per experiment. Scale bar, 5 μm. () Immunoblots (WB) showing that IFT88 co-immunoprecipitates with EB1 (left) and that γ -tubulin co-immunoprecipitates with IFT88 (right) from lysates of mitotic HeLa cells, demonstrating a mitotic interaction between the proteins, either direct or indirect. Ig, rabbit antibody, negative immunoprecipitation (IP) control. Input, 5% of total lysate used for immunoprecipitation. For full scan of immunoblots, see Supplementary Fig. S8. () Quantification of γ -tubulin intensity at spindle poles of mitotic cells showing γ -tubulin recruitment to poles in a microtubule regrowth experiment. t, time after nocodazole washout (min). Bar, median. Experiment shown is representative of three independent experiments. a.u., arbitrary unit. () Immunofluoresence microscopy images showing microtubule regro! wth (α -tubulin) at mitotic spindle poles 0, 1 and 2 min af! ter nocodazole washout in IFT88- or GFP-depleted mitotic cells. t=0 min shows no nucleation in GFP- and IFT88-depleted cells, and t=1 min and 2 min show decreased nucleation in IFT88-depleted cells, compared with control cells. Scale bar, 2 μm. () Percentage of cells showing detectable nucleation (aster size ≥1 μm) 0, 1 and 2 min after nocodazole washout. n=50 mitotic cells per experiment; error bars, mean of at least three experiments ± s.d. * Figure 3: IFT88 is required for the movement of peripheral microtubule clusters containing microtubule-nucleating components towards spindle poles in LLC-PK1 cells stably expressing GFP– α -tubulin. () Immunofluoresence microscopy images showing IFT88 and dynein intermediate chain (Dyn) localizing to a peripheral microtubule cluster (GFP– α -tubulin) in a prometaphase cell. Pixel intensity range increased to visualize peripheral microtubule cluster. Scale bar, 5 μm. Inset, peripheral microtubule cluster. See Supplementary Fig. S4a for negative controls. () Quantification of GFP– α -tubulin LLC-PK1 metaphase cells with ectopic microtubule clusters following IFT88- or control- (lamin) siRNA treatment. n=50 mitotic cells per experiment. (,) Immunofluoresence microscopy images of GFP– α -tubulin LLC-PK1 control or IFT88-depleted metaphase cells. γ -tubulin (), EB1 (, left) and dynein (, right) localize to ectopic microtubule clusters. Insets, ectopic microtubule clusters. Scale bar, 5 μm. () Selected still images from time-lapse microscopy movies of GFP– α -tubulin LLC-PK1 cells. Control prometaphase, minus-end-directed motion of peripheral microtubule cl! usters towards spindle pole. In IFT88-depleted cells, peripheral clusters formed, but showed no movement towards spindle poles. Full cell (left); enlargement of spindle pole and microtubule cluster (right). Time (min); arrowhead, microtubule cluster; arrow, spindle pole. () Immunofluorescence microscopy images (left) and quantification (right) of the relocalization of microtubule clusters to spindle poles in a spindle reassembly assay (α -tubulin, microtubule regrowth following nocodazole washout). The decrease in cells with ectopic microtubule clusters over time correlates with their movement towards the poles. IFT88 depletion delays relocalization of microtubule clusters to poles. Arrows, spindle poles (localization confirmed with centrosome protein staining). Arrowheads, ectopic microtubule clusters. n=40 mitotic cells per experiment per time point. t, time after nocodazole washout (min). * Figure 4: IFT88 moves towards spindle poles and requires microtubules for its spindle pole localization. () Microtubule pulldown assay shows that IFT88 co-pelleted with taxol-stabilized microtubules in mitotic HeLa cell lysates. Nocodazole (Noc), inhibition of microtubule polymerization used as negative control. α -tubulin, microtubules. () Immunofluoresence microscopy images showing IFT88 foci formation (lower panel) after nocodazole washout (α -tubulin, microtubule regrowth; upper panel) in HeLa cells. t, time after nocodazole washout (min). Control without nocodazole (no Noc). Scale bar, 5 μm. (,) Immunofluorescence microscopy images showing the molecular composition of IFT88 foci in HeLa cells. Maximum projection of a cell with IFT88 foci 5 min after nocodazole washout () showing that IFT88 foci co-stain for α -tubulin (α -tub) and dynein intermediate chain (Dyn). Enlargements, single plane of the outlined foci. Enlargements of IFT88 foci () showing that microtubule clusters (α -tubulin; α -tub) can be observed extending from some foci, and that IFT88 foci co-sta! in with microtubule-nucleating components (5051, centrosome protein marker; γ -tubulin; EB1). Pixel intensity range increased to visualize foci. Scale bar, 1 μm. () Quantification of IFT88 intensity at spindle poles of mitotic HeLa cells showing IFT88 recruitment to poles following nocodazole washout. t, time after nocodazole washout (min). Experiment shown is representative of three independent experiments. Bar, median. a.u., arbitrary unit. No nocodazole (No Noc), untreated cells. () Still images from time-lapse microscopy imaging of a GFP–IFT88 LLC-PK1 cell line (left) showing one of the GFP–IFT88 foci (arrowhead) moving towards the GFP–IFT88-labelled spindle pole (arrow). Time elapsed is shown in seconds. Scale bar, 1 μm. Schematic representation (right) of several GFP–IFT88 foci moving towards (red arrow) or away from (black arrowhead) the spindle pole (grey dot). Time between points, 1 s. Arrows indicate the direction of the movement. * Figure 5: IFT88 is part of a dynein1-driven transport complex in mitosis. () Immunoblots (left) showing fractions of mitotic HeLa cell lysates obtained after gel-filtration fractionation and probed for IFT88, dynein intermediate chain (Dyn IC), dynactin p150/glued, p50 dynactin, IFT52 and IFT20. Input, total lysate before gel filtration chromatography. Arrowheads, peak elution fraction for calibration proteins: bovine serum albumin (Mr 66K), β -amylase (Mr 200K), thyroglobulin (Mr 669K). V, Void volume. Immunoprecipitation experiment (right) carried out on fractions 16–22 from gel filtration containing dynein. Immunoblots show that IFT88 co-immunoprecipitates with dynein (IC, intermediate chain) after gel filtration chromatography. For full scan of immunoblots, see Supplementary Fig. S8. () Immunofluorescence microscopy images of HeLa cells showing IFT88 redistribution from mitotic spindle poles to a more diffuse region surrounding the poles following dynein1 (D1) depletion, compared with control (GFP). α -tubulin; α -tub. Intensity pro! files, lower left panels; spindle pole enlargement, lower right panels. Scale bar, 5 μm. () Percentage of cells with focused IFT88 localization at poles following dynein1- (D1) or dynein2- (D2) siRNA treatment. n=70 mitotic spindles per experiment. () Schematic representation of IFT88 (green) redistribution in cilia when dynein2 (D2) is depleted, and around mitotic spindle poles when dynein1 (D1) is depleted. () Immunofluorescence microscopy images showing that D1 depletion in HeLa cells delays IFT88 (red) relocalization to spindle poles in a spindle reassembly assay (α -tubulin, green). The decrease of cytoplasmic foci over time, observed in control (GFP) cells correlates with the relocalization of IFT88 from foci to spindle poles. Despite the formation of microtubule clusters in D1-depleted cells, several IFT88 foci remain in the cytoplasm 30 min after nocodazole washout. Arrows, spindle poles; arrowheads, IFT88 foci. () Percentage of cells with more than ten cytopl! asmic foci. n=40 cells per experiment per time point. t, time ! after nocodazole washout (min). () Molecular model for IFT88 function in mitosis. IFT88 is depicted as a component of a minus-end-directed dynein1-driven transport complex. This complex is required for transport of microtubule clusters and their associated nucleating components (EB1 and γ -tubulin) to spindle poles. IFT88 thus contributes to the formation of astral microtubule arrays and consequently spindle orientation. Adapted from ref. 20. Author information * Author information * Supplementary information Affiliations * Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Suite 210, Worcester, Massachusetts 01605, USA * Benedicte Delaval, * Alison Bright & * Stephen Doxsey * Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA * Nathan D. Lawson Contributions B.D. and S.D. wrote the manuscript. B.D. conceived and planned the experimental work. B.D. and A.B. carried out the experimental work and analysed the data. N.D.L. provided the zebrafish facility and helped plan and guide the zebrafish experimental work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stephen Doxsey Author Details * Benedicte Delaval Search for this author in: * NPG journals * PubMed * Google Scholar * Alison Bright Search for this author in: * NPG journals * PubMed * Google Scholar * Nathan D. Lawson Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen Doxsey Contact Stephen Doxsey Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (500K) Supplementary Information * Supplementary Movie 2 (500K) Supplementary Information * Supplementary Movie 3 (700K) Supplementary Information * Supplementary Movie 4 (700K) Supplementary Information * Supplementary Movie 5 (50K) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component
- ncb 13(4):469-474 (2011)
Nature Cell Biology | Letter Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component * Viola Baumgärtel1 * Sergey Ivanchenko1 * Aurélie Dupont1 * Mikhail Sergeev2 * Paul W. Wiseman2 * Hans-Georg Kräusslich3 * Christoph Bräuchle1 * Barbara Müller3 * Don C. Lamb1, 4 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 13,Pages:469–474Year published:(2011)DOI:doi:10.1038/ncb2215Received18 October 2010Accepted20 January 2011Published online10 March 2011Corrected online15 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at lea! st two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ~35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release. View full text Figures at a glance * Figure 1: Recruitment of VPS4A to HIV assembly sites. () A wide-field image and time projections (5,926 s) from a TIRFM image series exemplify frequent co-localization of eGFP–VPS4A bursts (green) with nascent HIVmCherry particles (magenta). The zoomed-in area shown in the TIRFM images is marked in the wide-field image with a white rectangle. () TIRFM images of assembly and release of a HIVmCherry particle (top panel, arrows) and the corresponding eGFP–VPS4A channel (bottom panel, arrows). Numbers above the fields give the time in minutes:seconds. () Number of VPS4A bursts per 200 frames (528 s) in the presence of the indicated HIV derivatives (left) and number of HIV budding sites detected (right); mean and s.d. from six individual cells are shown. () A wide-field image and time-projected TIRFM image series (5,565 s) of cells co-expressing eGFP–VPS4A (green) and HIVmCherry(late−) (magenta). The zoomed-in area shown in the TIRFM images is marked in the wide-field image with a white rectangle. All scale bars, 800 n! m. * Figure 2: Duration of eGFP–VPS4A bursts. () Histogram of burst width at baseline level from all eGFP–VPS4A bursts detected at HIV assembly sites (bin=5 s). t; mean burst width ± s.e.m. () Shape of eGFP–VPS4A bursts averaged over 89 events from a single cell. The average shape is asymmetric with a rapid assembly process, peak and shoulder with slower disassembly. The mean intensity is shown in black and the standard deviation of the data as a grey halo. () Background-corrected fluorescence intensity of a representative eGFP–VPS4A burst at an HIV assembly site showing distinct steps during the dissociation process. * Figure 3: Number of eGFP–VPS4A molecules per burst. () Exemplary TIRFM images of fixed HeLa cells expressing eGFP (left), GPI–eGFP (middle) or co-expressing eGFP–VPS4A and HIVmCherry (right) used for image correlation analysis. Insets show a 7.5-fold higher magnification of a region of the respective cell. The cells expressing eGFP–VPS4A showed VPS4A bursts captured by fixation. Scale bars, 5 μm. () Mean fluorescence brightness (normalized to monomeric GFP) assuming a single population for columns 1–4 and two populations, a dim one (light grey bars) and a bright one (dark grey bars), in columns 5–7. Columns 5 and 6 are from different data sets collected on different days. Mean values and s.e.m. are shown. () Histogram showing brightness distribution of eGFP–VPS4A bursts in cells co-expressing HIV. GEU, GFP equivalent unit; ROI, region of interest. * Figure 4: Correlation of VPS4A wild-type bursts with Gag assembly phases. () Number of VPS4A bursts per manually tracked HIV assembly site from five individual cells (nT, nB: number of traces, bursts in statistics). () Background-corrected intensity trace of an individual HIVmCherry particle and corresponding intensity in the eGFP–VPS4A channel. Vertical lines indicate the endpoint of each phase. () Top, averaged Gag–mCherry intensity traces (nT=78) normalized to the average kinetic rate and smoothed over three data points. Bottom, histogram of VPS4A burst position (nB=108, bin=2.5 min) relative to the start of manual tracking for all bursts (open bars) or for traces co-localizing with a single burst event (filled bars). Change history * Change history * Author information * Supplementary informationCorrigendum 15 March 2011In the version of this letter initially published online, the first sentence was erroneously truncated and eGFP–VPS4A was misspelled in the second paragraph. Author information * Change history * Author information * Supplementary information Affiliations * Physical Chemistry, Department of Chemistry and Biochemistry, Munich Center for Integrated Protein Science (CiPSM) and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstraße 11, 81377 Munich, Germany * Viola Baumgärtel, * Sergey Ivanchenko, * Aurélie Dupont, * Christoph Bräuchle & * Don C. Lamb * Physics Department, McGill University, Montreal, Quebec H3A 2T8, Canada * Mikhail Sergeev & * Paul W. Wiseman * Department of Infectious Diseases, Virology, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany * Hans-Georg Kräusslich & * Barbara Müller * Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA * Don C. Lamb Contributions V.B. and S.I. carried out experiments. M.S. and P.W.W. carried out the ICS analysis. V.B., S.I. and A.D. analysed data. B.M., D.C.L., H-G.K., S.I., A.D., M.S., P.W.W., C.B. and V.B. wrote the manuscript. B.M., D.C.L., H-G.K., C.B. designed and guided the project. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Barbara Müller or * Don C. Lamb Author Details * Viola Baumgärtel Search for this author in: * NPG journals * PubMed * Google Scholar * Sergey Ivanchenko Search for this author in: * NPG journals * PubMed * Google Scholar * Aurélie Dupont Search for this author in: * NPG journals * PubMed * Google Scholar * Mikhail Sergeev Search for this author in: * NPG journals * PubMed * Google Scholar * Paul W. Wiseman Search for this author in: * NPG journals * PubMed * Google Scholar * Hans-Georg Kräusslich Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph Bräuchle Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara Müller Contact Barbara Müller Search for this author in: * NPG journals * PubMed * Google Scholar * Don C. Lamb Contact Don C. Lamb Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Change history * Author information * Supplementary information Movies * Supplementary Movie 1 (7M) Supplementary Information * Supplementary Movie 2 (4M) Supplementary Information * Supplementary Movie 3 (8M) Supplementary Information * Supplementary Movie 4 (8M) Supplementary Information * Supplementary Movie 5 (8M) Supplementary Information PDF files * Supplementary Information (500K) Supplementary Information Additional data - Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation
- ncb 13(4):475-482 (2011)
Nature Cell Biology | Letter Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation * Maria Maldonado1 * Tarun M. Kapoor1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:475–482Year published:(2011)DOI:doi:10.1038/ncb2223Received05 November 2010Accepted08 February 2011Published online13 March 2011Corrected online24 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Accurate chromosome segregation depends on biorientation, whereby sister chromatids attach to microtubules from opposite spindle poles. The spindle-assembly checkpoint is a surveillance mechanism in eukaryotes that inhibits anaphase until all chromosomes have bioriented1, 2, 3. In present models, the recruitment of the spindle-assembly checkpoint protein Mad2, through Mad1, to non-bioriented kinetochores is needed to stop cell-cycle progression3, 4, 5, 6. However, it is unknown whether Mad1–Mad2 targeting to kinetochores is sufficient to block anaphase. Furthermore, it is unclear whether regulators of biorientation (for example, Aurora kinases7) have checkpoint functions downstream of Mad1–Mad2 recruitment or whether they act upstream to quench the primary error signal8. Here, we engineered a Mad1 construct that localizes to bioriented kinetochores. We show that the kinetochore localization of Mad1 is sufficient for a metaphase arrest that depends on Mad1–Mad2 binding.! By uncoupling the checkpoint from its primary error signal, we show that Aurora, Mps1 and BubR1 kinases, but not Polo-like kinase, are needed to maintain checkpoint arrest when Mad1 is present on kinetochores. Together, our data suggest a model in which the biorientation errors, which recruit Mad1–Mad2 to kinetochores, may be signalled not only through Mad2 template dynamics9, but also through the activity of widely conserved kinases, to ensure the fidelity of cell division. View full text Figures at a glance * Figure 1: An mCherry–Mis12–Mad1 fusion can recruit Mad2 to kinetochores independently of microtubule attachment. () Schematic showing experiment design. Endogenous Mad1 (grey) localizes to kinetochores that are not attached to microtubules and recruits Mad2 (dark green), forming a Mad1–Mad2 core tetramer. The Mad1-bound Mad2 catalytically converts open-Mad2 molecules (dark green square) to closed-Mad2 molecules (dark green circle). Microtubule (light green) binding displaces Mad1, and therefore Mad2, from kinetochores. We fused Mad1 to Mis12 (red), a protein whose kinetochore localization is microtubule-binding independent. This construct could retain Mad1, and Mad2, to microtubule-attached kinetochores. () Analysis of mCherry–Mis12–Mad1 localization in live cells. Differential interference contrast (DIC) and mCherry-fluorescence micrographs of a metaphase cell transfected with mCherry–Mis12–Mad1. (–) mCherry–Mis12–Mad1 localizes at kinetochores, even when they are attached to microtubules. Immunofluorescence images of HeLa cells (control; ) and cells transfected with m! Cherry–Mis12–Mad1 (), stained for DNA, tubulin, CREST and mCherry. Overlay shows tubulin (green), CREST (blue) and mCherry (red). Insets (selected optical sections; ) show individual microtubule-attached kinetochores from , fivefold magnification. () Analysis of Mad2 recruitment by mCherry–Mis12–Mad1. DIC, mCherry- and GFP-fluorescence micrographs of a HeLa cell stably expressing GFP–Mad2, transfected with mCherry–Mis12–Mad1, are shown. MG132 (10 μM, 1 h) was used to accumulate live cells at metaphase with many microtubule-attached chromosomes. Scale bars, 5 μm. * Figure 2: Constitutive kinetochore localization of Mad1 causes a persistent, Mad2-dependent metaphase arrest. () Analysis of mitotic index and phenotypes in HeLa cells (control), and cells transfected with mCherry–Mis12–Mad1 and mCherry–Mis12–Mad1 AA. Cells were fixed 24 h and 30 h after transfection. mCherry, tubulin and DNA staining was used to determine mitotic index and fraction of cells in metaphase, anaphase and all other mitotic states (other mitotic; n=3 independent experiments, >400 cells counted per condition per time). () The increase in mitotic index induced by mCherry–Mis12–Mad1 is Mad2 dependent. HeLa cells were transfected with small-interfering RNA (siRNA) against Mad2 or GFP (control), 24 h before transient transfection with mCherry–Mis12–Mad2 or mCherry–Mis12–Mad1 AA, or no transfection (control). Mitotic indices were determined after another 24 h (n=3 independent experiments, >250 cells counted per condition per time). (,) mCherry–Mis12–Mad1 AA localizes at kinetochores, but does not recruit GFP–Mad2. mCherry- () and GFP- () fluor! escence micrographs of a metaphase cell transfected with mCherry–Mis12–Mad1 AA. MG132 (10 μM, 1 h) was used to accumulate live cells at metaphase with many microtubule-attached chromosomes. (–) Forced kinetochore localization of Mad1 does not disrupt cold-stability of kinetochore microtubules. HeLa cells () and cells transfected with mCherry–Mis12–Mad1 () or mCherry–Mis12–Mad1 AA () were incubated in MG132 (10 μM, 1 h) to accumulate cells at metaphase and were then placed on ice (10 min) before fixation. Cells were stained for DNA, CREST (blue), mCherry (red) and tubulin (green). Individual channels and an overlay are shown. Insets (selected optical sections; ,,) show individual cold-stable microtubule-attached kinetochores (fourfold magnification). Scale bars, 5 μm. Average ± s.e.m. shown. * Figure 3: Analysis of kinetochore protein localization in cells expressing mCherry–Mis12–Mad1. HeLa cells and cells transfected with mCherry–Mis12–Mad1 were incubated in nocodazole (1 μg ml−1, 45 min; columns 1–3) or left unperturbed (columns 4 and 5). Cells were stained for mCherry and p150Glued, CENP-E, Bub1, ROD or Zw10. Scale bars, 5 μm. * Figure 4: Forced localization of Mad1 to chromosomes by fusion to H2B recruits Mad2, but does not affect the mitotic index. (–) HeLa cells (–) or HeLa cells stably expressing GFP–Mad2 (–) were transfected with mCherry–H2B–Mad1 and processed 48 h later. DIC (,,,), mCherry-(,,,) and GFP-fluorescence (,) micrographs of an interphase cell (,), a cell undergoing anaphase (,,–) and a prometaphase cell (–) are shown. () Mitotic indices were calculated by analysing mCherry, tubulin and DNA staining (n=3 independent experiments, >350 cells counted per condition per time). Scale bars, 5 μm. Average ± s.e.m. shown. * Figure 5: Inhibition of BubR1, Mps1 or Aurora B, but not Plk1, is sufficient for entry into anaphase, even when Mad1 persists at kinetochores. (–) Analysis of mitotic index and phenotypes in HeLa cells (control) and cells transfected with mCherry–Mis12–Mad1 and mCherry–Mis12–Mad1 AA on BubR1 depletion () or Polo-like kinase (), Mps1 () or Aurora B () inhibition. Cells were transfected with siRNA against GFP (control) or BubR1 (), or incubated in dimethylsulphoxide (DMSO), BI2536 (80 nM, 90 min before fixation), Mps1-IN-1 (10 μM, 80 min) or ZM447439 (2 μM, 60 min) (–). mCherry, tubulin and DNA staining was used to calculate mitotic index and fraction of cells in metaphase, anaphase and all other mitotic states (,,) or with monopolar spindles, in anaphase or all other mitotic states (; n=3 independent experiments, >350 cells counted per condition per time). (–) Analysis of the effects of inhibition of Mps1 (–,) or Aurora B (–,) in live cells transfected with mCherry–Mis12–Mad1. Metaphase mCherry-positive cells were selected before the medium was changed to one containing DMSO, Mps1! -IN-1 (10 μM) or ZM447439 (2 μM; n=3 independent experiments, >10 cells per condition per experiment). Each of those cells was imaged by multi-point re-visiting using microscope software. DIC (,,,) and mCherry-fluorescence (,,,) micrographs at the indicated times before and after Mps1-IN-1 or ZM447439 wash-in are shown. (,) Cumulative frequency of the imaged cells entering anaphase after Mps–IN-1 () or ZM447439 () wash-in. Scale bars, 5 μm. Average ± s.e.m. shown. Change history * Change history * Author information * Supplementary informationErratum 24 March 2011In the version of this article initially published online and in print, Figure 5b was incorrectly labelled. This error has been corrected in the HTML and PDF versions of the article. Author information * Change history * Author information * Supplementary information Affiliations * Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, New York 10065, USA * Maria Maldonado & * Tarun M. Kapoor Contributions M.M. and T.M.K. designed the experiments and wrote the paper. M.M. carried out the experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tarun M. Kapoor Author Details * Maria Maldonado Search for this author in: * NPG journals * PubMed * Google Scholar * Tarun M. Kapoor Contact Tarun M. Kapoor Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Change history * Author information * Supplementary information PDF files * Supplementary Information (3M) Supplementary Information Additional data - A tetrapyrrole-regulated ubiquitin ligase controls algal nuclear DNA replication
- ncb 13(4):483-487 (2011)
Nature Cell Biology | Letter A tetrapyrrole-regulated ubiquitin ligase controls algal nuclear DNA replication * Yuki Kobayashi1 * Sousuke Imamura2 * Mitsumasa Hanaoka1 * Kan Tanaka1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 13,Pages:483–487Year published:(2011)DOI:doi:10.1038/ncb2203Received16 March 2010Accepted06 January 2011Published online06 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In plant cells, organelle DNA replication (ODR) is coordinated with nuclear DNA replication (NDR), with ODR preceding NDR during cell cycle progression. We previously showed that the occurrence of ODR is signalled by a tetrapyrrole compound, most likely Mg-protoporphyrin IX (Mg-ProtoIX), resulting in the activation of cyclin-dependent kinase A (CDKA) and consequent initiation of NDR (refs 1, 2, 3). Here we identify an F-box protein of SCF-type E3 ubiquitin ligase (Fbx3) in the red alga Cyanidioschyzon merolae, which inhibits CDKA by ubiquitylating the relevant cyclin and inducing its degradation. Mg-ProtoIX binds to Fbx3 and inhibits cyclin ubiquitylation. Thus, these observations indicate that Fbx3 serves as the receptor for the plastid-to-nucleus retrograde signal Mg-ProtoIX and thereby contributes to a checkpoint mechanism ensuring coordination of ODR and NDR. View full text Figures at a glance * Figure 1: Role of ubiquitin and proteasome-dependent protein degradation in NDR. () Protocol for synchronization of the cell cycle in C. merolae by exposure to light–dark cycles. When the absorbance at 750 nm (A750 nm) of cultures reached 10, the cells were diluted (arrow) to an A750 nm of 0.4. The arrowhead indicates reagent addition to culture medium and the initiation time point for cell sampling. () Cells were incubated in the dark for the indicated times after the end of the second dark period and the addition of Mg-ProtoIX (20 μM, left) or epoxomicin (100 nM, right), and changes in DNA copy number for the nucleus (Nu), plastid (Pt) and mitochondrion (Mt) were determined by microscopic measurement of the fluorescence intensity of 4,6-diamidino-2-phenylindole (DAPI)-stained DNA. Data are expressed as relative copy number normalized to the G1 state and are means ± s.d. (n=30 cells). () Cells were either illuminated or exposed to Mg-ProtoIX or epoxomicin in the dark and were sampled at the indicated times thereafter. Total protein samples! were then subjected to SDS–polyacrylamide gel electrophoresis, and the gel was either stained with Coomassie brilliant blue (lower panel of each pair) or subjected to immunoblot analysis with antibodies against Cyclin 1 (upper panels). () Cells were illuminated or exposed to Mg-ProtoIX or epoxomicin in the dark and were sampled after 1 h. Total ubiquitylated protein was affinity-purified and subjected to immunoblot analysis with antibodies against Cyclin 1. Arrowheads indicate ubiquitylated Cyclin 1. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 2: Interaction of Fbx proteins with Cyclin 1 and tetrapyrroles. () Schematic representation of the structures of Fbx1 to Fbx5. Rectangles indicate Pfam domains: F, F-box domain; L, leucine-rich repeat; SPOC, SPOC domain; TyrKc, tyrosine kinase catalytic domain. N and C denote amino and carboxy termini, respectively. () GST-tagged Cyclin 1 (or GST) and TF-6×His-tagged Fbx1 to Fbx4 were pulled-down by glutathione–Sepharose. The precipitates were subjected to immunoblot analysis with antibodies against 6×His or GST. () TF-6×His-tagged Fbx proteins were mixed with the indicated concentrations of Mg-ProtoIX and then isolated with Ni-agar beads. After elution from the beads with imidazole, protein-bound Mg-ProtoIX was quantified by spectrofluorometry. Eluted proteins were quantified by immunoblot analysis with antibodies against 6×His (upper panel). Mg-ProtoIX fluorescence intensity was then normalized by 6×His–Fbx abundance determined by immunoblot analysis (lower panel); data are expressed in arbitrary units (a.u.) and are means ± ! s.d. (n=3 independent experiments). Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 3: Effects of tetrapyrroles on the Fbx3–Cyclin 1 interaction. (–) GST-pulldown assay with GST-tagged Cyclin 1 and either TF–6×His-tagged Fbx3 (–) or TF–6×His–Fbx4 () in the presence of the indicated concentrations of Mg-ProtoIX (,), ProtoIX () or haemin (). The precipitates were subjected to immunoblot analysis with antibodies against 6×His or GST. () GST-tagged Fbx3 protein was expressed in C. merolae cells, and pulled down by glutathione–Sepharose. GST–Fbx3-expressing cells (GF3) or wild-type cells were illuminated (L) or incubated in the absence (D) or presence (Mg) of Mg-ProtoIX (20 μM) in the dark after the end of the second dark period, and were sampled after 1 h. Total proteins were subjected to GST-pulldown analysis and detected using antibodies against GST (top) or Cyclin 1 (bottom). Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 4: Analysis of an Fbx3-null mutant. () Protocol for induction of G1 arrest by dark incubation. When the A750 nm of cultures reached 10, the cells were diluted to an A750 nm of 0.4 (arrow). The arrowhead indicates the addition of nalidixic acid to the culture medium and the initiation time point for cell sampling. (,) Wild-type (left panel) or DS19 (right panel) cells were incubated in the absence () or presence of nalidixic acid () for the indicated times, and changes in DNA copy number for the nucleus (Nu), plastid (Pt) and mitochondrion (Mt) were determined by microscopic measurement of the fluorescence intensity of DAPI-stained DNA. Data are expressed as relative copy number normalized to the G1 state and are means ± s.d. (n=30 cells). () Total protein samples of wild-type (left panel) or DS19 (right panel) cells at the indicated times relative to the onset of illumination were subjected to SDS–polyacrylamide gel electrophoresis, and the gel was either stained with Coomassie brilliant blue (CBB) or s! ubjected to immunoblot analysis with antibodies against Cyclin 1. () Wild-type or DS19 cells maintained in the dark for 48 h were sampled at 1 h after epoxomicin addition. Total ubiquitylated protein was affinity-purified and subjected to immunoblot analysis with antibodies against Cyclin 1. Arrowheads indicate ubiquitylated Cyclin 1. Uncropped images of blots are shown in Supplementary Fig. S7. * Figure 5: Model for Mg-ProtoIX signalling in C. merolae. During dark-arrested G1 phase or conditions of ODR inhibition, an SCF-type E3 ubiquitin (Ub) ligase containing Fbx3 ubiquitylates Cyclin 1 and thereby triggers its rapid degradation by the proteasome. Mg-ProtoIX accumulates in the cell during or on completion of ODR, and its interaction with Fbx3 results in the release of both Cyclin 1 and Fbx3 from the SCF complex and consequent inhibition of Cyclin 1 ubiquitylation. The stabilized Cyclin 1 forms a complex with and activates CDKA, thereby triggering NDR. Author information * Author information * Supplementary information Affiliations * Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan * Yuki Kobayashi, * Mitsumasa Hanaoka & * Kan Tanaka * Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan * Sousuke Imamura Contributions K.T. designed the research; Y.K., S.I. and M.H. carried out the research; and Y.K., M.H. and K.T. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Kan Tanaka Author Details * Yuki Kobayashi Search for this author in: * NPG journals * PubMed * Google Scholar * Sousuke Imamura Search for this author in: * NPG journals * PubMed * Google Scholar * Mitsumasa Hanaoka Search for this author in: * NPG journals * PubMed * Google Scholar * Kan Tanaka Contact Kan Tanaka Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Klotho suppresses RIG-I-mediated senescence-associated inammation
- ncb 13(4):487 (2011)
Nature Cell Biology | Erratum Klotho suppresses RIG-I-mediated senescence-associated inammation * Feng Liu * Su Wu * Hongwei Ren * Jun GuJournal name:Nature Cell BiologyVolume: 13,Page:487Year published:(2011)DOI:doi:10.1038/ncb0411-487Published online01 April 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, 254–262 (2011); published online 20 February 2011; corrected after print 16 March 2011 In the version of this article initially published online and in print, the middle graph in Figure 3b was incorrectly labelled. Additionally two sentences on page 257 have been altered to; 'Interestingly, knockdown of RIG-I in senescent HUVECs led to extension of the lifespan of the senescent cells (Fig. 2g)...' and 'This is consistent with observations that inhibition of IL-6 and IL-8 extends the proliferative capacity of cultured cells passages4,5,7'. In the legend for figure 4c, wording has been changed to: '(c) Growth curves of control HUVECs and klotho-overexpressing HUVECs. Senescent HUVECs were transfected with control plasmid or klotho-expression plasmid'. On page 258, il-8 should have been IL-8. These errors have been corrected in both the HTML and PDF versions of the article. Additional data Author Details * Feng Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Su Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Hongwei Ren Search for this author in: * NPG journals * PubMed * Google Scholar * Jun Gu Search for this author in: * NPG journals * PubMed * Google Scholar
No comments:
Post a Comment