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In the United States, enrolment in graduate degree programmes in the biological sciences has risen sharply during the global economic downturn, but new graduates face an uncertain job market. What can prospective and current students do to ensure that a graduate degree remains a sound investment? - Working on a chain: E3s ganging up for ubiquitylation
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Myosin pulses all over Hippo restrains regeneration Coupling endocytosis to exocytosis Vasp: surfing on actin - Tissue elongation requires oscillating contractions of a basal actomyosin network
- ncb 12(12):1133-1142 (2010)
Nature Cell Biology | Article Tissue elongation requires oscillating contractions of a basal actomyosin network * Li He1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaobo Wang1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ho Lam Tang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Denise J. Montell1dmontell@jhmi.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1133–1142Year published:(2010)DOI:doi:10.1038/ncb2124Received15 July 2010Accepted28 October 2010Published online21 November 2010 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 Understanding how molecular dynamics leads to cellular behaviours that ultimately sculpt organs and tissues is a major challenge not only in basic developmental biology but also in tissue engineering and regenerative medicine. Here we use live imaging to show that the basal surfaces of Drosophila follicle cells undergo a series of directional, oscillating contractions driven by periodic myosin accumulation on a polarized actin network. Inhibition of the actomyosin contractions or their coupling to extracellular matrix (ECM) blocked elongation of the whole tissue, whereas enhancement of the contractions exaggerated it. Myosin accumulated in a periodic manner before each contraction and was regulated by the small GTPase Rho, its downstream kinase, ROCK, and cytosolic calcium. Disrupting the link between the actin cytoskeleton and the ECM decreased the amplitude and period of the contractions, whereas enhancing cell–ECM adhesion increased them. In contrast, disrupting cell–! cell adhesions resulted in loss of the actin network. Our findings reveal a mechanism controlling organ shape and an experimental model for the study of the effects of oscillatory actomyosin activity within a coherent cell sheet. View full text Figures at a glance * Figure 1: Stage-9 follicle cells undergo rapid periodic contractions and myosin accumulation. () Surface view of a live, late-stage-9 egg chamber expressing Sqh–mCherry (red) and cadherin–GFP (green). () Sagittal plane through the centre of the egg chamber. (, ). Sqh–mCherry label alone, in the same preparations as in and . Scale bar, 50 μm. () Schematic drawings of egg chambers in and , and the imaging of the apical and basal regions of the follicle cells. The red boxes in the left panel indicate a typical field of cells. The blue and red boxes on the right indicate the basal and apical focal planes, respectively. (–) Images of late-stage-9 egg chamber expressing Sqh–mCherry (red) and cadherin–GFP (green) at apical and basal focal planes. Scale bar, 20 μm. (–) Time-lapse series of one representative oscillating cell labelled with cadherin–GFP () and Sqh–mCherry (). The digitized cell contour is colour-coded on the basis of the percentage decrease in surface area relative to the maximum area captured during imaging, as indicated in the heat map ()! . Scale bar, 10 μm. The A–P and D–V orientations are shown at the right. * Figure 2: Quantification of basal periodic contraction and comparison with apical activity. () Dynamic change of basal area, D–V and A–P cell length from one representative cell. Peaks are indicated by arrowheads of the corresponding colour. () The distribution of periods observed over 375 oscillations with a mean of 6.3 min and s.d. of 1.2 min. () Change in apical area and apical myosin intensity from one representative cell over time. () Calculated surface area change for the indicated numbers (n) of cells over 30 min of imaging. () Calculated ratio of D–V to A–P length change over time; n is the number of individual cells analysed. () Changes in apical and basal myosin intensity compared with the average intensity for each, over time; n is the number of individual cells analysed. () Rate of change of myosin intensity (blue) and rate of decrease in basal area (purple) from the same sample. Peaks are marked with arrowheads. () Autocorrelation of time sequences of apical (red) and basal (blue) myosin intensity. All error bars indicate s.d. * Figure 3: Accumulation of basal myosin on stable actin filaments precedes the basal membrane contraction. (, ) Confocal micrographs of a clone of moesin–GFP in cells expressing Sqh–mCherry. Images in and were taken 3 min apart. Myosin intensity changes in two cells (left panel, arrows), whereas moesin-labelled F-actin does not change detectably. Scale bar, 10 μm. () Quantification of the dynamic change of moesin–GFP and Sqh–mCherry intensity in one oscillating cell. Intensity of each channel was normalized to its mean. () Correlation between the rate of decrease in basal area and change in myosin intensity. () Correlation between moesin and myosin intensity. Each row shows the correlation from a different cell as a function of various time offsets. () Average of all correlation coefficients with different time offsets. The blue line shows the average time-dependent correlation between rate of decrease in area and rate of change in myosin intensity from 43 individual cells. The red line shows the average time-dependent correlation between moesin and myosin intensity from! 37 individual cells. The correlation between the rate of basal area reduction and myosin accumulation reaches a maximum at −1 min, suggesting that myosin accumulation precedes area decrease. In contrast, the correlation between myosin intensity and F-actin intensity peaks at 0, indicating that they are virtually simultaneous. () The position of maximum correlation between contraction rate and intensity change rate (blue) is significantly different from zero with P < 0.001, whereas the moesin–myosin correlation (red) is not. Error bars indicate s.d. () The basal areas of cells (n = 45) were calculated and averaged. Even though the basal areas of individual cells fluctuated periodically, the average basal area did not change because the fluctuations were temporary and unsynchronized. () The ratio of A–P to D–V cell lengths during the time of imaging (n = 45). The periodic changes observed in individual cells were neither synchronous nor lasting; therefore no change w! as detected on average. () Diagram of different organizations ! and dynamics of the apical actomyosin for cells undergoing apical constriction versus the basal actomyosin for cells undergoing basal oscillations. * Figure 4: Global change in basal myosin during egg chamber development. () Schematic drawings of egg chambers at the indicated developmental stages. Red shading illustrates the overall distribution of cells with periodic myosin accumulation. Anterior is to the left. (–) Live egg chambers expressing Sqh–mCherry and cadherin–GFP at early stage 9 (), middle stage 9 (), stage 10A () and stage 10B (). Scale bar, 50 μm. () Basal myosin intensity relative to stage 8 and ratio of A–P to D–V egg chamber length at different stages. A–P or D–V length was defined as the maximum distance between two points of the tissue in the corresponding direction. (–) Side views of follicle cells from egg chambers of the indicated stages, showing the increase in basal myosin and comparatively constant apical myosin through development. Arrows indicate sites of basal myosin accumulation. Scale bar, 15 μm. () Apical myosin intensity normalized to stage 8, and the ratio of basal myosin intensity (in cells that exhibit basal myosin accumulation) to apical m! yosin in the indicated number (n) of cells from stages 8–10. * Figure 5: Basal actomyosin contractions control tissue shape. (–) Confocal micrographs of egg chambers treated with vehicle (DMSO, ), cytochalasin D (CytoD, ) or ionomycin () at 0 min (the beginning of imaging) and 20 min. Tissue contours at the two time points are shown at the right as red (0 min) and green (20 min) outlines. Scale bar, 50 μm. Control experiments and combination effects are shown in Supplementary Information, Fig. S3h–q. (–) Live images of basal F-actin (labelled with moesin–GFP) and myosin (Sqh–mCherry) after treatment for 30 min with DMSO (), CytoD () or ionomycin (). Scale bar, 10 μm. The more than 80% decrease in basal moesin–GFP signal after treatment with CytoD () suggests that most GFP intensity represents F-actin. Ionomycin showed little or no effect on basal F-actin (). Treatment with ROCK and calcium inhibitors decreased myosin intensity to near background but had little effect on actin (Supplementary Information, Fig. S3a–c), suggesting that the formation of basal actin and myosin contractil! e fibres is regulated independently. () Average basal myosin intensity and percentage change in egg chamber width during the 20-min imaging time after treatment with the indicated drugs; n is the number of samples analysed. () Quantification of basal moesin–GFP and myosin intensity after treatment with the indicated drugs; n is the number of cells analysed. All error bars indicate s.d. () An illustration of the proposed mechanism by which follicle cell relaxation or contraction leads to tissue rounding or elongation. As a result of curvature of the egg chamber, contractile force (F) generated at the basal side is exerted in part towards the centre. The red arrowheads indicate different magnitudes of contractile forces. * Figure 6: Rho, ROCK and cell adhesion regulate basal myosin accumulation and organ shape. (–) Basal view of follicle cell clones expressing the indicated transgenes, marked by co-expression of either nlsGFP (nuclear localization signal fused to GFP) or mCD8GFP (murine lymphocyte receptor CD8 transmembrane domain fused to GFP). Scale bar, 20 μm. () Quantification of relative basal and apical myosin intensities in the indicated number (n) of GFP-positive cells compared with wild-type cells in the same sample. Corresponding apical myosin and apical and basal actin images are shown in Supplementary Information, Figs S4 and S5. Verification of RNAi knockdown and overexpression is shown in Supplementary Information, Fig. S6. (–) Morphology of stage-10B (, , , ) and stage-14 (, , , ) egg chambers expressing the indicated transgene in all follicle cells. Egg chambers expressing hsGal4 served as controls. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 50 μm. () Quantification of the A–P to D–V length ratio in stage-10B and stage-14 egg chambers with the indic! ated genetic backgrounds; n is the number of samples analysed. In contrast, no significant difference was observed at late stage 8 (Supplementary Information, Fig. S7). All error bars are s.d. * Figure 7: Cell autonomy of myosin oscillation and pathways affecting its magnitude and period. (, ) Confocal micrographs of follicle cell clones in living egg chambers expressing Sqh–mCherry. () Wild-type (WT, GFP-negative) cells adjacent to cells expressing ROCK RNAi (GFP-positive) are indicated by white arrows. Wild-type cells without any contact with ROCK RNAi-expressing cells are indicated by blue arrows. Scale bar, 10 μm. () Wild-type (WT, GFP-negative) cells adjacent to cells expressing RhoV14 (GFP-positive) are indicated by white arrows. A wild-type cell without any contact with RhoV14 -expressing cells is identified by a blue arrow. (, ). Quantification of myosin intensity and oscillation period in wild-type cells adjacent to mutant cells showed no significant difference from those in wild-type cells not adjacent to mutant cells; n is the number of individual cells analysed. () Basal myosin intensity and oscillation period after addition of different concentrations of EGTA together with 2.5 μM ionomycin. A total of 48 individual cells were analysed, coveri! ng about 150 periods. () Basal myosin intensity and oscillation period after treatment with various concentrations of the ROCK inhibitor Y-27632. A total of 37 cells were analysed, covering about 110 periods. () Plot of oscillation period against basal myosin intensity in wild-type egg chambers. Samples were collected from 65 individual cells, covering more than 200 periods. () Dynamics of basal myosin intensity in a representative individual cell expressing UAS-talin RNAi, UAS-paxillin or no UAS transgene (WT). () Autocorrelation with different time offsets from data in . The first peak for each line provides the period for the corresponding genotype. () Quantification of the effect on period; n is the number of cells from three independent clones analysed. All error bars indicate s.d. * Figure 8: Model of tissue elongation controlled by basal actomyosin contraction. A schematic representation is shown of the distribution of molecules controlling oscillating basal contraction in an individual follicle cell and the organization of contractile forces into a super cellular band within the epithelium. Forces are indicated by red arrows. Local contraction force generated by basal myosin (red) transmitted through adhesions (blue) to the basal lamina (cyan) constrains tissue growth to the poles. Micrographs show a corresponding section through the middle of a stage-10 egg chamber labelled with cadherin–GFP and Sqh–mCherry. The Sqh–mCherry channel on its own is shown in black and white. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Li He & * Xiaobo Wang Affiliations * Department of Biological Chemistry, Center for Cell Dynamics, Johns Hopkins School of Medicine, 855 North Wolfe Street, Baltimore, Maryland 21205, USA. * Li He, * Xiaobo Wang, * Ho Lam Tang & * Denise J. Montell Contributions L.H. and X.W. performed the image acquisition and mutant analysis. L.H. processed and analysed images. H.L.T. conducted inhibitor treatments and calcium-related experiments. D.J.M. prepared the manuscript. All authors participated in the interpretation of the data and the production of the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Denise J. Montell (dmontell@jhmi.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (2M) Supplementary Information * Supplementary Movie 2 (5M) Supplementary Information * Supplementary Movie 3 (4M) Supplementary Information * Supplementary Movie 4 (4M) Supplementary Information * Supplementary Movie 5 (4M) Supplementary Information * Supplementary Movie 6 (9M) Supplementary Information * Supplementary Movie 7 (2M) Supplementary Information * Supplementary Movie 8 (3M) Supplementary Information * Supplementary Movie 9 (4M) Supplementary Information * Supplementary Movie 10 (2M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - A kinase cascade leading to Rab11-FIP5 controls transcytosis of the polymeric immunoglobulin receptor
- ncb 12(12):1143-1153 (2010)
Nature Cell Biology | Article A kinase cascade leading to Rab11-FIP5 controls transcytosis of the polymeric immunoglobulin receptor * Tao Su1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * David M. Bryant1, 2, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Frédéric Luton1, 2, 3, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Marcel Vergés1, 2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott M. Ulrich5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Kirk C. Hansen7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Anirban Datta1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dennis J. Eastburn1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alma L. Burlingame7 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevan M. Shokat5 Search for this author in: * NPG journals * PubMed * Google Scholar * Keith E. Mostov1, 2keith.mostov@ucsf.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1143–1153Year published:(2010)DOI:doi:10.1038/ncb2118Received20 January 2010Accepted06 October 2010Published online31 October 2010Corrected online12 November 2010 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 Polymeric immunoglobulin A (pIgA) transcytosis, mediated by the polymeric immunoglobulin receptor (pIgR), is a central component of mucosal immunity and a model for regulation of polarized epithelial membrane traffic. Binding of pIgA to pIgR stimulates transcytosis in a process requiring Yes, a Src family tyrosine kinase (SFK). We show that Yes directly phosphorylates EGF receptor (EGFR) on liver endosomes. Injection of pIgA into rats induced EGFR phosphorylation. Similarly, in MDCK cells, pIgA treatment significantly increased phosphorylation of EGFR on various sites, subsequently activating extracellular signal-regulated protein kinase (ERK). Furthermore, we find that the Rab11 effector Rab11-FIP5 is a substrate of ERK. Knocking down Yes or Rab11-FIP5, or inhibition of the Yes–EGFR–ERK cascade, decreased pIgA–pIgR transcytosis. Finally, we demonstrate that Rab11-FIP5 phosphorylation by ERK controls Rab11a endosome distribution and pIgA–pIgR transcytosis. Our result! s reveal a novel Yes–EGFR–ERK–FIP5 signalling network for regulation of pIgA–pIgR transcytosis. View full text Figures at a glance * Figure 1: Identification of EGFR as a Yes substrate in rat liver endosomes. () A Yes-GTM kinase reaction assay was performed in vitro on endosomal membranes for the indicated times. Each reaction mixture contained rat liver endosomes and [γ-32P]–N6(benzyl) ATP, with or without Yes-GTM as indicated. Proteins were separated by SDS–PAGE and 32P-labelled proteins were detected by a phosphorimager. Three major proteins of 170, 46 and 44 K are phosphorylated by Yes-GTM. Boxes indicate separate gels. () Quantification of the band intensities in . (, ) Kinase reaction assays were performed with or without Yes-GTM, and with PP2 or PP3 () or 1-NM-PP1 () at the indicated concentrations. Reaction mixtures contained rat liver endosomes and [γ-32P]–N6(benzyl) ATP. Proteins were separated by SDS–PAGE and 32P-labelled proteins were detected by a phosphorimager. PP3 is an inactive analogue of PP2. () Protein from endosomes was resolved by SDS–PAGE. Total (green) and phosphorylated EGFR (red) were identified by dual-colour infrared immunoblotting using an! tibodies specific to EGFR and phosphorylated tyrosine (pTyr). The phosphorylated tyrosine band occurs only in the presence of Yes-GTM, precisely co-migrating with EGFR. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 2: EGFR phosphorylation is induced in rat liver endosomes, and in pIgR-expressing MDCK cells, on pIgA stimulation. () EGFR phosphorylation in rats injected with pIgA. Top: endosome fractions, from rats treated with pIgA as indicated, were analysed by immunoprecipitation of EGFR, and immunoblotting with antibodies against phosphorylated tyrosine and EGFR. Antibody heavy chains from immunoprecipitation are indicated at the bottom of the blot. IP; immunoprecipitation, pEGFR; phosphorylated EGFR. Bottom: intensity of the phosphorylated tyrosine bands (normalized to EGFR bands), with or without pIgA treatment. Data are means ± s.e.m. Asterisk indicates P < 0.03, n = 3. () Top: MDCK cells expressing pIgR and stably expressing control, scrambled-sequence shRNA or Yes shRNA were treated basolaterally with pIgA for the indicated times. Cells were lysed and proteins were resolved by SDS–PAGE and immunoblotting with antibodies against phosphorylated tyrosine, EGFR and Yes at the indicated times after pIgA treatment (0 min represents control without pIgA treatment). Colour image represents overla! y of phosphorylated tyrosine/EGFR signals from infrared immunoblots. Note precise co-migration of phosphorylated tyrosine and the upper EGFR band in dual-colour immunoblots, representing phosphorylated tyrosine–EGFR. MLC; myosin light chain, loading control. Bottom: intensity of the phosphorylated EGFR bands (normalized to EGFR bands) at indicated times after pIgA treatment. Data are means ± s.e.m. Asterisks indicate P < 0.01, compared with control cells at 0 min, n = 4. () Top: pIgR-expressing MDCK cells were treated basolaterally with pIgA for the indicated times in the presence of SFK inhibitor (PP2). Cells were lysed at the indicated times after pIgA treatment and proteins were resolved by SDS–PAGE and immunoblotting. Bottom: intensity of the phosphorylated EGFR bands (normalized to EGFR bands), at indicated times after pIgA treatment. Data are means ± s.e.m., n = 4. () Top: MDCK cells stably expressing hEGFR and pIgR were treated basolaterally with pIgA for 5 min! , as indicated. Lysates were immunoblotted with antibodies spe! cific to EGFR proteins phosphorylated at the tyrosine residues indicated at the top, and antibodies against total EGFR and GAPDH (glyceraldehyde-3-phosphate dehydrogenase, as a control). Bottom: intensity of the phosphorylated EGFR bands (phosphorylated at the indicated residues and normalized to EGFR bands), with or without pIgA treatment. Data are means ± s.e.m. Asterisk indicates P < 0.03, compared with the respective cells not treated with pIgA (n = 4). Uncropped images of blot are shown in Supplementary Information, Fig. S7. * Figure 3: Interaction and co-localization of EGFR, pIgR and Yes. () Rat liver endosomes were solubilized and EGFR, pIgR or Yes were immunoprecipitated. The proteins were resolved by SDS–PAGE and co-immunoprecipitation was assessed by immunoblotting. Immunoprecipitations with non-specific rabbit serum (NSS) were performed as a negative control. In the RRC lane, 5 μg of protein was loaded as the input. The intensity of the co-immunoprecipitated protein bands are indicated, compared with the intensity of the immunoprecipitated protein bands (data are means ± s.e.m., n = 4). Boxes represent spliced regions from same gels, to maintain sample order format under all conditions. () pIgR, Yes or EGFR were immunoprecipitated from the lysates of MDCK cells expressing hEGFR and pIgR, and co-immunoprecipitation was detected by immunoblotting, as indicated. Immunoblotting of total cell lysates and immunoprecipitation and immunoblotting of non-specific serum (NSS) and was also performed. The intensity of the co-immunoprecipitated protein bands are i! ndicated as a percentage of the intensity of the protein bands in the input lysate (data are means ± s.e.m., n = 3). () Monolayers of MDCK cells expressing hEGFR and pIgR were fixed and stained with antibodies against the indicated proteins for immunofluorescence microscopy. Top: Representative images from the sub-apical and lateral (middle) regions of MDCK cell monolayers. Arrows indicate vesicular co-localization of pIgR, Yes and EGFR. White spots in the merged image indicate intracellular co-localization of EGFR, pIgR and Yes. Bottom: representative images from the sub-apical and lateral (middle) regions of MDCK cell monolayers on pIgA treatment. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 4: pIgA-stimulated pIgR transcytosis requires EGFR activity. () MDCK cells expressing pIgR were labelled with 35S-cysteine and basolaterally treated with pIgA or EGF, with or without EGFR kinase inhibitor (PD153035) for the indicated times. Apically released secretory component (Ap-SC) is represented as a percentage of total labelled pIgR. Data are means ± s.e.m. Asterisk indicates P < 0.05 and double asterisks indicate P < 0.001, compared with cells not treated with PD153035 for the indicated treatment. Control cells with PD153035 treatment, n = 8, all others, n = 6. () MDCK cells expressing hEGFR and pIgR, MDCK cells expressing pIgR with Yes knockdown, and their respective controls, were basolaterally treated with biotinylated pIgA for indicated times, and the apically transcytosed pIgA (Ap-pIgA) was measured by ELISA. Data are represented as a percentage of the total pIgA and are means ± s.e.m. Asterisk indicates P < 0.05, double asterisks indicate P < 0.001, compared with respective control cells. Control and hEGFR-expressing ce! lls, n = 8; control and Yes knockdown cells, n = 12. * Figure 5: ERK phosphorylation induced by pIgA treatment is required for pIgA–pIgR transcytosis in MDCK cells expressing pIgR. (–) MDCK cells expressing pIgR were treated basolaterally with pIgA for the indicated times. Cells were left untreated (, control), Yes was knocked down (), or cells were treated with SFK inhibitor (PP2; ), EGFR inhibitor (PD153035; ) or MEK inhibitor (U0126; ). Top: cell lysates were analysed by immunoblotting with specific phosphorylated ERK (pERK) and ERK antibodies. Bottom: quantification of the phosphorylated ERK band intensities (normalized to the ERK bands). Asterisk () indicates P < 0.03, compared with control cells at 0 min, n = 4). () Cells were pre-treated for 2 h, and throughout the experiment, with the indicated inhibitors. Control cells were treated with DMSO. Apical pIgA transcytosis of basolaterally applied pIgA was analysed at the indicated times after addition of inhibitor. Data are represented as a percentage of the total pIgA and are means ± s.e.m. Double asterisks indicate P < 0.001, compared with control cells, n = 4). * Figure 6: FIP5 phosphorylation is downstream of Yes–EGFR–ERK. () Comparison of the amino-acid sequences of FIP5 from the indicated species. A conserved ERK phosphorylation sequence at Ser 188 is indicated. Bottom: schematic representation of the FIP5 protein, indicating region compared at the top. N, amino-terminus; C, carboyxl terminus; RBD, Rab11-binding domain. () Immunoblotting of RRC fractions with antibodies against the indicated proteins. Box indicates splicing of bands from same gel. () Immunoblotting of the lysates from pIgR-expressing MDCK cells stably expressing FIP5 shRNA and control cells expressing scrambled-sequence shRNA. Duplicate samples are presented. GAPDH; loading control. () pIgA transcytosis in pIgR-expressing MDCK cells stably expressing scrambled-sequence shRNA, or FIP5 shRNA. Data are represented as a percentage of the total pIgA and are means ± s.e.m. Asterisks indicate P < 0.001, compared with control cells, n = 4. () Expression of FIP5 in MDCK cells. Levels of FIP5 were assessed in parental MDCK cells (lef! t) and in cells overexpressing wild-type human FIP5 or an S188A mutant, both tagged with GFP. GAPDH, loading control. Box indicates splicing of bands from same gel. () MDCK cells stably expressing pIgR and GFP–FIP5 (wild type or S188A) were treated with pIgA for the indicated times. Top: cell lysates were immunoprecipitated with antibodies against GFP–FIP5 and then immunoblotted with antibodies specific to GFP–FIP5 and phosphorylated serine (pS). Bottom: intensity of the phosphorylated serine bands (normalized to the intensity of the GFP–FIP5 bands). Data are means ± s.e.m. Single asterisks indicate P < 0.05, double asterisks indicate P < 0.001, n = 4. () MDCK cells expressing pIgR and GFP–FIP5 were treated with pIgA for indicated times and with the indicated inhibitors (untreated cells, control). Cell lysates were immunoprecipitated for GFP–FIP5, resolved by SDS–PAGE, and immunoblotted for GFP–FIP5 or phosphorylated serine. Intensity of bands for phosphory! lated serine were normalized to the intensity of GFP–FIP5 ba! nds. Data are means ± s.e.m. Asterisks indicate P < 0.05, n = 3. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 7: FIP5 Ser 188 phosphorylation regulates Rab11a localization and pIgA–pIgR transcytosis. () Filter-grown monolayers of MDCK cells expressing pIgR and stably expressing GFP–FIP5 (wild type or the S188A mutant, green) were immunostained for Rab11a (red) and E-cadherin (E-cad, blue) without (control) or with pIgA stimulation (15 min). Pairs of images on the right are higher-magnification images of boxed areas indicated in images on the left and demonstrate accumulation of Rab11a/FIP5-positive vesicles in the periphery of the sub-apical region of cells expressing GFP–FIP5S188A, as indicated by arrows. () pIgA transcytosis assays were performed on parental MDCK cells expressing pIgR or MDCK cells expressing pIgR and GFP–FIP5 (wild type or the S188A mutant). Cells were treated with biotinylated pIgA, which was allowed to accumulate intracellularly, followed by incubation. Transcytosed pIgA was measured at the indicated times. Data are means ± s.e.m. Asterisks indicate P < 0.001, n = 4. () MDCK cells expressing pIgR and GFP–FIP5 (green; wild type; top, or the ! S188A mutant; bottom) were treated basolaterally with biotinylated pIgA, and immunostained for pIgA (red) and F-actin (blue) after 60 min of transcytosis. Yellow, arrowheads indicate overlap of pIgA and wild-type GFP–FIP5 in the centre of the sub-apical region of cells. Images on the right are higher-magnification images of boxed areas indicated in images on the left. Scale bars, 20 μm. * Figure 8: A kinase cascade regulating pIgR transcytosis. () A Yes–EGFR–ERK–FIP5 signalling cascade controls pIgA–pIgR transcytosis in epithelial cells. pIgA stimulates pIgR activation, which associates with Yes, and directs phosphorylation of EGFR. Active phosphorylated EGFR, presumably through Ras/Raf, activates MEK/ERK, which in turn phosphorylates FIP5 on Ser 188 (pFIP5). FIP5, phosphorylated on Ser 188, functions with Rab11a to regulate transcytosis of pIgA–pIgR complexes. () FIP5 phosphorylation controls polarized distribution of Rab11a and pIgA transcytosis. A schematic representation of how pIgR–Yes–EGFR complexes are internalized and passaged through basolateral early endosomes (BEEs) to sub-apical endosomes, presumably the common recycling endosome (CREs). pIgA causes the disruption of the pIgR–Yes–EGFR complex in endosomes. The EGFR–MEK/ERK cassette phosphorylates FIP5 on Ser 188 (pS188), which controls re-distribution of Rab11a/FIP5 vesicles from the periphery to the centre of the apical region of ce! lls. This may represent the transition from the CRE to the apical recycling endosome (ARE). From here EGFR may be recycled to the basolateral plasma membrane. pIgA–pIgR complexes are delivered to the ARE, from where they are delivered to the apical plasma membrane. Thus the EGFR–MEK/ERK cassette represents an unappreciated regulator of transport through the transcytotic pathway. TJ; tight junctions. Change history * Abstract * Change history * Author information * Supplementary informationCorrigendum 12 November 2010In the version of this article initially published online, the amount of protein from cell lysate used in immunoprecipitation assays was incorrect. In addition the paper describing the FIP5 cDNA was not referenced. These errors have been corrected in both the HTML and PDF versions of the article. Author information * Abstract * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * David M. Bryant & * Frédéric Luton Affiliations * Department of Anatomy, University of California, San Francisco, CA 94158-2517, USA. * Tao Su, * David M. Bryant, * Frédéric Luton, * Marcel Vergés, * Anirban Datta, * Dennis J. Eastburn & * Keith E. Mostov * Biochemistry and Biophysics, University of California, San Francisco, CA 94158-2517, USA. * Tao Su, * David M. Bryant, * Frédéric Luton, * Marcel Vergés, * Anirban Datta, * Dennis J. Eastburn & * Keith E. Mostov * Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, CNRS-UMR6097, 06560 Sophia-Antipolis, France. * Frédéric Luton * Cardiovascular Genetics Centre, IdIBGi - University of Girona, 17003 Girona, Spain. * Marcel Vergés * Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158-2280, USA. * Scott M. Ulrich & * Kevan M. Shokat * Department of Chemistry, Ithaca College, Ithaca, NY 14850, USA. * Scott M. Ulrich * Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2517, USA. * Kirk C. Hansen & * Alma L. Burlingame * Proteomics Core, University of Colorado Health Sciences Centre, Aurora, CO 80045, USA. * Kirk C. Hansen Contributions T.S., D.M.B., F.L. and K.E.M. designed and analysed the experiments. T.S., D.M.B., M.V. and K.C.H. performed the experiments. A.D., D.J.E, S.M.U., K.M.S. and A.L.B. provided reagents. T.S., D.M.B. and K.E.M. wrote the manuscript. D.M.B. and K.E.M. supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Keith E. Mostov (keith.mostov@ucsf.edu.) Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data - Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria
- ncb 12(12):1154-1165 (2010)
Nature Cell Biology | Article Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria * Sudha B. Singh1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Wojciech Ornatowski1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Isabelle Vergne1 Search for this author in: * NPG journals * PubMed * Google Scholar * John Naylor1 Search for this author in: * NPG journals * PubMed * Google Scholar * Monica Delgado1 Search for this author in: * NPG journals * PubMed * Google Scholar * Esteban Roberts1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marisa Ponpuak1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sharon Master1 Search for this author in: * NPG journals * PubMed * Google Scholar * Manohar Pilli1 Search for this author in: * NPG journals * PubMed * Google Scholar * Eileen White3 Search for this author in: * NPG journals * PubMed * Google Scholar * Masaaki Komatsu4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Vojo Deretic1vderetic@salud.unm.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1154–1165Year published:(2010)DOI:doi:10.1038/ncb2119Received05 February 2010Accepted05 November 2010Published online21 November 2010 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 IRGM, a human immunity-related GTPase, confers autophagic defence against intracellular pathogens by an unknown mechanism. Here, we report an unexpected mode of IRGM action. IRGM demonstrated differential affinity for the mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd, but not IRGMb splice isoforms, caused mitochondrial depolarization and autophagy-independent, but Bax/Bak-dependent, cell death. By acting on mitochondria, IRGM confers autophagic protection or cell death, explaining IRGM action both in defence against tuberculosis and in the damaging inflammation caused by Crohn's disease. View full text Figures at a glance * Figure 1: IRGM localizes to mitochondria. () Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting. Numbers represent fractions from the gradient (1, top; 5, bottom). () Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit. () Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in . () Intracellular localization of IRGM analysed by confocal microscopy. Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red (MTR) where indicated. Images on the right are merged from images on the left. A–L; endogenous IRGM localization in HeLa cells relative to calnexin, MTR, cytochrome c and COX IV. M–O; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody. Scale bars, 5 μm.! () Analysis of IRGM (green) and COX IV (red) intensity as a function of distance along the line shown in the inset. Image in inset corresponds to panels J–L in . Uncropped images of blots are shown in Supplementary Information, Fig. S8. * Figure 2: IRGM co-fractionates with mitochondria and localizes to their inner membrane or matrix. () Purified mitochondria were left untreated, or were treated with Proteinase K (Pr.K) following osmotic shock (OS), or solubilization of the total protein using Triton X-100 (TX-100), as indicated. Samples were analysed by immunoblotting with antibodies against the indicated proteins. () Mitochondrial preparations were subjected to membrane disruption by freeze-thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting. (c) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density-gradient centrifugation. Numbers represent fractions from the gradient (1, top; 12, bottom). Uncropped images of blots are shown in Supplementary Information, Fig. S8. * Figure 3: IRGM affects mitochondrial fission. () Cells were transfected with control siRNA or with siRNA specific to IRGM (top) and DRP1 (bottom) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti-IRGM and anti-DRP1 antibodies. Actin was used as a loading control. () HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live-cell confocal microscopy. Scale bars, 5 μm. () Cells were treated with either control siRNA or with DRP1, IRGM, ATG7 or BECN1 (Beclin 1) siRNAs, labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information, Figure S2. () Cells were transfected with either control siRNA or with siRNA specific to MFN1/MFN2, IRGM or IRGM and MFN1/MFN2, and then labelled with MTR and analysed for mitochondrial mor! phologies. Data are means ± s.e.m. (, n = 3; , n = 4). Asterisk indicates P < 0.05, double asterisks indicate P < 0.01 and dagger indicates P > 0.05 (t-test). * Figure 4: Relationship between mitochondrial fission and autophagy and roles of IRGM, DRP1 and FIS1 in autophagic control of mycobacteria. () U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP–LC3. Autophagy was induced by treatment with hIFN-γ. GFP–LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm. () LC3 puncta per cell were quantified from cells treated as in . () LC3 puncta per cell quantified from cells treated as in , except that autophagy was induced by starvation rather than treatment with hIFN- γ. () After transfection with either control siRNA or siRNA specific to MFN1/MFN2, endogenous LC3 puncta per cell were quantified. () U937 cells were transfected with the indicated siRNA and infected with BCG. Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe (LTR) as a reporter of acidification8, 20. () U937 cells were treated with the indicated siRNA and infected with virulent M. tuberculosis H37Rv. Autophagy was induced by starvation, or ! cells were kept in full medium, as indicated. Colony forming units were counted, and expressed as a percentage of the number of plated bacterial colonies from untreated cells, to assess bacterial survival. Data are means ± s.e.m. (n = 6; two independent transfections, 6 independent infections). Asterisk indicates P < 0.05, and dagger indicates P > 0.05 (t-test) for the indicated data. * Figure 5: IRGMd binds to cardiolipin and causes loss of mitochondrial membrane potential. () Top: schematic representation of IRGM. The G1–G5 GTPase motifs and their sequences are shown. The change corresponding to the S47N mutation is indicated with an arrow. Bottom: splice variants of IRGM and Ras (p21). () HeLa cells were transfected with plasmids encoding fluorescent protein-tagged IRGMb, IRGMd WT (wild-type IRGMd) or IRGMS47N mutant (control cells; vector expressing GFP only), and after 48 h were labelled with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. () Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP–IRGMd positive and one GFP–IRGMd negative, as indicated in the inset. () Quantification of GFP+ MTR+ cells from an experiment performed as in . () Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST–IRGM. Binding was detected by immunoblotting. () Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were ! incubated with GST or GST–IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads. () Nitrocellulose filters spotted with the indicated lipids (defined in ) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in – were probed with anti-GST antibody. * Figure 6: IRGMd translocates to mitochondria, induces mitochondrial fragmentation and causes loss of mitochondrial ΔΨm independent of autophagic, but dependent on apoptotic, machinery. () HeLa cells transfected with plasmids encoding GFP or GFP–IRGMd were stained with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (–) Quantification of mitochondrial morphologies in cells transfected with IRGMd, IRGMdS47N or IRGMb GFP fusions (control cells express GFP only). () Cells were transfected with the indicated siRNAs and vector encoding GFP–IRGMd, and labelled with MTR. The percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. () Cells were transfected with the indicated siRNAs and vector encoding GFP–IRGMd (or vector encoding GFP only, as a control) and labelled with MTR. Percentages of GFP+ cells with MTR staining at 48 h were quantified. () Confocal microscopy images of HeLa cells transfected with vector encoding GFP–DRP1 (A) or GFP–IRGMd (B–G) and labelled with MTR (A–D) or antibodies against COX-IV (F). A, steady-state DRP1 distribution; B–D, translocati! on of IRGMd from the cytosol to mitochondria at indicated times post-transfection; E–G, co-localization of IRGMd and COX-IV (G is merged from E and F). Scale bars, 5 μm. () HeLa cells, co-transfected with GFP–IRGMd, and control, BECN1 or ATG7 siRNA, were labelled with MTR 48 h post-transfection, imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. () Cells from Atg7 wild-type (WT) or Atg7−/− MEFs transfected with vectors encoding GFP–IRGMd for 48 h were stained with MTR, imaged, and percentage of GFP+ cells that were also MTR+ cells was quantified. () Wild-type W2 (Bax/Bak+/+) or mutant D3 (Bax/Bak−/−) BMK cells were transfected with vectors encoding GFP–IRGMd and after 48 h were labelled with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. () HeLa cells were transfected with vectors encoding GFP–IRGMd, treated with z-VAD as indicated, and afte! r 48 h stained with MTR. Cells were imaged by live-cell micros! copy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P < 0.05 and double asterisks indicate P < 0.01 (t-test) for the indicated data. * Figure 7: IRGMd induces cell death. () Percentage of HeLa cells transfected with vectors encoding GFP or GFP–IRGMd that were rounded 48 h post-transfection. () HeLa cells transfected with plasmids encoding GFP or GFP–IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified. () HeLa cells transfected with vectors encoding GFP–IRGMd versus GFP–IRGMdS47N and YFP–IRGMb (vector encoding GFP only was transfected as a control) were stained with 7-AAD after 48 h, and percentages of GFP+ (for IRGMd and IRGMS47N) or YFP+ (for IRGMb) cells that were also 7-AAD+ were quantified. () HeLa cells were transfected with plamsids encoding GFP–IRGMd, GFP–IRGMDS47N or YFP–IRGMb (vector encoding GFP only was transfected as a control), and after 48 h were stained with propidium iodide (PI). Percentages of GFP+ or YFP+ cells that were also PI+ were quantified. () Images of HeLa cells transfected with vectors encoding GFP–IRGMd (bottom) were stained for active caspa! ses 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top). () HeLa cells were transfected with plasmids encoding GFP–IRGMd and were stained with antibodies against HMGB1 (absence of nuclear HMGB1 stain is a marker of HMGB1 release) or DAPI. Top: images indicate GFP–IRGMd (left), HMGB1 (middle) and DAPI (right) localization. Bottom: phase-contrast microscopy image of cells (left) and image merged from other panels (right). Scale bars, 5 μm. (–) Cells were transfected with plasmids encoding GFP or GFP–IRGMd, and control or DRP1 siRNA, as indicated. After 48 h, () cells were stained with 7-AAD and percentages of GFP+ cells that were also 7-AAD+ were quantified, () rounded cells were quantified, or () cells were stained with HMGB1 and HMGB1+ nuclei were quantified. Data are means ± s.e.m. (n = 3). Asterisk indicates P < 0.05 and double asterisk indicate P < 0! .01 (t-test). * Figure 8: Comparison of IRGMa, IRGMb and IRGMc effects. (, ) HeLa cells were transfected with plasmids encoding GFP, CFP–IRGMa, GFP–IRGMc or GFP–IRGMd, and after 24 h () or 48 h (), stained with MTR. Percentages of positively transfected cells that were also MTR+ were then quantified. (, ) Cells were transfected as in , , and after 24 h () or 48 h () the percentages of positively transfected cells that were also rounded by morphology were quantified. (, ) Cells were transfected as in , , and after 24 h () or 48 h (), stained with 7AAD. Percentages of positively transfected cells that were also 7AAD+ were then quantified. (, ) Cells were transfected as in , , and after 24 h () or 48 h (), stained with antibodies against HMGB1. Cells positively transfected with the indicated plasmids were assessed for retention of HMGB1 in the nucleus. Data are means ± s.e.m. (n = 3, ; n = 4, , ). Asterisk indicates P < 0.05 and double asterisks indicate P < 0.01 (t-test). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sudha B. Singh & * Wojciech Ornatowski Affiliations * Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, 915 Camino de Salud, NE, Albuquerque, NM 87131 USA. * Sudha B. Singh, * Wojciech Ornatowski, * Isabelle Vergne, * John Naylor, * Monica Delgado, * Esteban Roberts, * Marisa Ponpuak, * Sharon Master, * Manohar Pilli & * Vojo Deretic * Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. * Marisa Ponpuak * Cancer Institute of New Jersey, Department of Molecular Biology and Biochemistry, Rutgers University, CABM, 679 Hoes Lane, Piscataway, NJ 08854 USA. * Eileen White * Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8421, Japan. * Masaaki Komatsu * Department of Biochemistry, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan. * Masaaki Komatsu Contributions S.B.S., W.O., I.V., J.N., M.D., E.R., M.P. and S.M. carried out planning, experimental work and data analysis. E.W. and M.K. contributed MEFs and protocols for their use. V.D. carried out project planning, experimental work and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Vojo Deretic (vderetic@salud.unm.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (5M) Supplementary Information PDF files * Supplementary Information (1M) Supplementary Information Additional data - Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction
- ncb 12(12):1166-1176 (2010)
Nature Cell Biology | Article Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction * Balca R. Mardin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Cornelia Lange1 Search for this author in: * NPG journals * PubMed * Google Scholar * Joanne E. Baxter2 Search for this author in: * NPG journals * PubMed * Google Scholar * Tara Hardy2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sebastian R. Scholz1 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew M. Fry2 Search for this author in: * NPG journals * PubMed * Google Scholar * Elmar Schiebel1e.schiebel@zmbh.uni-heidelberg.de Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1166–1176Year published:(2010)DOI:doi:10.1038/ncb2120Received25 June 2010Accepted15 October 2010Published online14 November 2010 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 During interphase, centrosomes are held together by a proteinaceous linker that connects the proximal ends of the mother and daughter centriole. This linker is disassembled at the onset of mitosis in a process known as centrosome disjunction, thereby facilitating centrosome separation and bipolar spindle formation. The NIMA (never in mitosis A)-related kinase Nek2A is implicated in disconnecting the centrosomes through disjoining the linker proteins C-Nap1 and rootletin. However, the mechanisms controlling centrosome disjunction remain poorly understood. Here, we report that two Hippo pathway components, the mammalian sterile 20-like kinase 2 (Mst2) and the scaffold protein Salvador (hSav1), directly interact with Nek2A and regulate its ability to localize to centrosomes, and phosphorylate C-Nap1 and rootletin. Furthermore, we demonstrate that the hSav1–Mst2–Nek2A centrosome disjunction pathway becomes essential for bipolar spindle formation on partial inhibition of the ! kinesin-5 Eg5. We propose that hSav1–Mst2–Nek2A and Eg5 have distinct, but complementary functions, in centrosome disjunction. View full text Figures at a glance * Figure 1: Interactions between Nek2A, C-Nap1, hSav1 and Mst2. () Yeast cells were transformed with plasmids encoding the indicated proteins fused to the Gal4 DNA-binding domain (bait; right) and the Gal4 activator-domain (prey; left). Growth on YPD plates lacking leucine and tryptophan (–LW) indicates successful mating, and growth on YPD plates lacking leucine, tryptophan, histidine and adenine (–LWHA) indicates interaction of bait and prey proteins. Colonies from non-interactors (for example, empty plasmids) appear darker on –LW plates because cells did not express ADE2, and therefore accumulate a red pigment. () Lysates from HEK293 cells co-transfected with plasmids encoding HA-tagged hSav1 (left) or Mst2 (right) and the indicated Myc-tagged constructs (1–4) were immunoprecipitated with anti-HA. Immunoprecipitated proteins were analysed by SDS–PAGE and immunoblotting. () Extracts from cells expressing LAP–Nek2A were prepared and Nek2A was immunoprecipitated with GFP-binder protein (GBP) coupled (GBP IP) or not coupled (no! GBP) to NHS-activated Sepharose beads. Asterisk indicates LAP–Nek2A and arrowhead indicates endogenous Nek2. LAP–Nek2A also immunoprecipitates endogenous Nek2 owing to dimerization of Nek2 (ref. 43). Without enrichment by immunoprecipitation, LAP–Nek2A is difficult to detect in cell extracts with anti-Nek2 antibodies because of its low expression. () NusA- and 6His-tagged hSav, GST-tagged C-Nap1-CTD and Nek2ΔN were purified from E. coli, and wild-type (WT) and kinase-dead Mst2 (Mst2K56R) were purified from Sf21 insect cells. Recombinant proteins were resolved by SDS–PAGE and visualized by Simply Blue Safe staining. () Purified, recombinant GST–C-Nap1-CTD and GST–Nek2AΔN were incubated with recombinant His–hSav1 (top) or His–Mst2 (middle). C-Nap1-CTD and Nek2AΔN proteins were precipitated with glutathione–Sepharose beads and bound proteins were analysed by immunoblotting. Incubation with GST was used as a control; bottom gel indicates presence of GST-ta! gged proteins. () Sequence alignment of the SARAH domains of M! st1/Mst2 kinases and hSav1 with the C-terminal coiled-coil domain of Nek2A. Asterisks indicate fully conserved residues and arrowhead indicates the critical leucine residues for interactions. () Yeast two-hybrid analyses were carried out as in with the indicated protein constructs. () HA-tagged Mst2 or hSav1 constructs were co-expressed with Myc-tagged Nek2A constructs in HEK293 cells. HA-tagged proteins were immunoprecipitated with anti-HA antibodies. Co-immunoprecipitation of Myc-tagged proteins was determined by immunoblotting. IP; immunoprecipitate. Uncropped images of blots are shown in Supplementary Information, Fig. S8a. * Figure 2: hSav1 and Mst1/Mst2 are responsible for the centrosomal localization of Nek2. () Extracts of non-specific control- (NSC), hSav1- or Mst1/Mst2-siRNA-transfected RPE-1 cells were analysed by immunoblotting using anti-hSav1, anti-Mst2, anti-Nek2, anti-C-Nap1 and anti-tubulin antibodies. () RPE-1 cells were transfected with NSC, hSav1 or Mst1/Mst2 siRNA for 72 h. Cells were fixed and co-stained with anti-Nek2 and anti-γ-tubulin antibodies. DNA was stained with Hoechst 33342. Arrowheads indicate the centrosomes shown in insets. Scale bar, 10 μm. () Quantification of the Nek2 localized to centrosomes in cells treated as in . Results are from three independent experiments. n = 75 (NSC) and n = 50 (hSav1- and Mst1/Mst2-siRNA-transfected cells). Data are means ± s.e.m. () The DNA content of cells treated as in was analysed by flow cytometry. There is no significant difference in cell-cycle distribution between cells transfected with NSC, hSav1 or Mst1/Mst2 siRNAs. * Figure 3: Mst1/Mst2 and hSav1 regulate the centrosomal function and dynamics of Nek2A. () Top: schematic representation of protocol used to treat RPE-1 cells in –. Bottom: images of cells treated as indicated at the top using the indicated siRNA oligonucleotides, and stained with antibodies against γ-tubulin, and Hoechst 33342 to visualize DNA. Untransfected cells (bottom) were not transfected with plasmid encoding eGFP−NEK2a. Insets: magnifications of centrosomal signals indicated by arrowheads. Scale bar, 10 μm. () Extracts of cells, treated as in , were analysed by immunoblotting with the indicated antibodies. Untransfected; cells not transfected with siRNA or plasmid expressing eGFP–Nek2A. () Distance between two centrosomes in cells treated as in . Results are from three independent experiments; 20 cells were analysed for each condition. Data are means ± s.e.m. () Intensity of eGFP–Nek2A at centrosomes was quantified from cells treated as in . Results are from two independent experiments; 20 cells were analysed for each condition. Box-and-whisk! ers plots: boxes show upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 10 to the 90 percentile and dots correspond to outliers (asterisk indicates P = 0.032, compared with NSC-transfected cells, double-asterisks indicate P = 0.0022, compared with NSC-transfected cells). () Images of U2OS cells expressing Myc-tagged wild-type Nek2A or the Nek2AL413A mutant, stained with anti-Myc and anti-γ-tubulin antibodies and Hoechst 33342 to visualize DNA. Scale bar, 10 μm. () Quantification of cells, treated as in . Results are from three independent experiments; 20 cells were analysed for each condition. Data are means ± s.e.m. () Centrosomal dynamics of U2OS cells expressing Nek2–GFP, and transfected with the indicated siRNA, were measured by FRAP. Images were acquired at 65 ms intervals for 1 s before bleaching (pre-bleach), and after laser bleaching of the indicated areas, images were acquired every 65 ms for 20 s followed by an acquisiti! on of half a second for 60 s. Representative images are shown ! at indicated times. Scale bar, 10 μm. () Cells were treated as in and relative GFP intensity within a bleached area was recorded over time. Background intensities were subtracted and corrected for acquisition bleaching. The data were fitted using a single exponential equation and each data point represents the average intensity from 20 cells (P = 0.0002). Uncropped images of blots are shown in Supplementary Information, Fig. S8. * Figure 4: Mst2 phosphorylates Nek2A and regulates the ability of Nek2A to induce centrosome disjunction. () Recombinant C-Nap1-CTD and Nek2ΔN were incubated with wild-type Mst2 or Mst2K56R in the presence of [γ-32P]-ATP. Proteins were separated by SDS–PAGE, analysed by Coomassie Brilliant Blue (CBB; top) staining and autoradiography (bottom). () HEK293 cells were transfected with plasmids encoding HA-tagged wild-type Mst2 and either Myc-tagged wild-type Nek2A or Myc-tagged Nek2AS438A. Lysates were immunoprecipitated with anti-Myc and analysed by immunoblotting with indicated antibodies. Asterisk indicates the IgG heavy chain and arrowhead indicates Mst2 phosphorylated at Ser 438. () Lysates from untreated or Mst1/Mst2- or Nek2-depleted HeLa cells were immunoprecipitated with anti-Nek2. Samples were analysed by immunoblotting with the indicated antibodies. Asterisk indicates the IgG heavy chain and arrowhead indicates Mst2 phosphorylated at Ser 438. (–) U2OS cells were transfected with plasmids encoding Myc-tagged Nek2A (wild-type, -4A or -4D) constructs. () Cell lysates w! ere analysed by immunoblotting with the indicated antibodies. () Immunofluorescence microscopy of cells stained with anti-Myc and anti-γ-tubulin antibodies and Hoechst 33342 to visualize DNA. Arrowheads indicate centrosomes (shown in insets; for pairs of centrosomes yellow arrowheads indicate centrosomes in insets). Scale bar, 10 μm. () Quantification of cells, treated as in , with separated centrosomes. Results are from three independent experiments; 20 cells were analysed for each condition. Data are means ± s.e.m. () Intensity of Nek2A fluorescence at centrosomes was quantified from cells treated as in . Results are from three independent experiments. n = 20 cells analysed for each condition. Box-and-whiskers plots: boxes show the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 10 to the 90 percentile and dots correspond to outliers. () U2OS cells were transfected with NSC or Mst1/Mst2 siRNA oligonucleotides and transfected wit! h plasmids encoding the indicated Myc–Nek2A constructs. Cell! extracts were analysed with indicated antibodies. () Immunofluorescence microscopy images of cells treated as in and stained with anti-Myc and anti-γ-tubulin antibodies and Hoechst 33342 to visualize DNA. Arrowheads are as in . Scale bar, 10 μm. () Quantification of cells, treated as in . Results are from three independent experiments; 20 cells were analysed for each condition. Data are means ± s.e.m. () Extracts from RPE-1 cells transfected with plasmids encoding the indicated Myc–Nek2A constructs were analysed by immunoblotting using anti-Myc and antibody against C-Nap1 phosphorylated at Ser 2417 and Ser 2421. Bottom: band intensities, normalized to anti-Myc signal. * Figure 5: hSav1, Mst1/Mst2 and Nek2A regulate centrosome disjunction together with Eg5. () Top: schematic representation of protocol used to treat cells. Cells were incubated with nocodazole to block microtubule-dependent centrosome splitting. Bottom: representative images of RPE-1 cells treated as indicated at the top, using the indicated siRNA oligonucleotides. Cells were stained with antibodies against γ-tubulin and Hoechst 33342 to visualize DNA. Scale bar, 5 μm. () Distances between two centrosomes were analysed from cells treated as in . Results are from three independent experiments. S phase; distance was measured from cells treated with aphidicolin. NSC, S phase control; n = 50, cells transfected with Nek2, Mst1/Mst2 and hSav1 siRNA; n = 30. Data are means ± s.e.m. () Top: schematic representation of protocol used to treat cells. Thymidine block/release enriched cells in G2 phase. Monastrol treatment inhibited Eg5-dependent centrosome splitting. Bottom: representative images of RPE-1 cells treated as indicated at the top, using the indicated siRNA ol! igonucleotides. Cells were fixed and stained with α- and γ-tubulin antibodies and Hoechst 33342 to visualize DNA. Scale bar, 5 μm. () Distance between the two centrosomes was analysed from cells treated as in and with 100 μM monastrol. Results are from three independent experiments. NSC; n = 50, cells transfected with Nek2, Mst1/Mst2 and hSav1 siRNA; n = 30. Data are means ± s.e.m. () Percentage of cells with bipolar spindles was analysed from cells treated as in and with 50 μM monastrol. Results are from three independent experiments. NSC; n = 120, cells transfected with Nek2 siRNA; n = 98, cells transfected with Mst1/2 siRNA; n = 122, cells transfected with Sav1 siRNA; n = 110. Data are means ± s.e.m. * Figure 6: C-Nap1 phosphorylation and displacement is regulated by hSav1, Mst1/Mst2 and Nek2A. () Top: schematic representation of protocol used to treat RPE-1 cells expressing centrin–GFP. Bottom: representative images of RPE-1 cells treated as indicated at the top using the indicated siRNA oligonucleotides. Cells were stained with anti-C-Nap1 antibodies and Hoechst 33342 to visualize DNA. Scale bar, 5 μm. () Intensity of centrosomal C-Nap1 fluorescence at centrosomes was quantified from cells treated as in The average background intensity was subtracted and the intensities were normalized to corresponding centrin signal. Results are from three independent experiments. NSC; n = 35, Nek2, Mst1/Mst2 and hSav1 siRNA-treated cells; n = 30. Box-and-whiskers plots: boxes show the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 10 to the 90 percentile and dots correspond to outliers. Asterisk indicates P = 0.0002, double asterisk indicates P < 0.0001, triple asterisks indicate P = 0.0003, compared with NSC-siRNA-transfected cells.! () HeLa Kyoto cells were synchronized by treatment with a double thymidine block, before release into nocodazole. Samples were collected after 0, 5, 9 and 14 h of release (indicated as G1, S, G2 and M fractions, respectively; according to DNA distribution in flow cytometry analysis). M-phase cells depleted of C-Nap1 were used as a control. Whole-cell extracts were analysed by immunoblotting using antibodies against C-Nap1 and C-Nap1 phosphorylated at Ser 2417 and Ser 2421. GAPDH was used as a loading control. () Extracts from RPE-1 cells transfected with the indicated siRNA oligonucleotides were arrested by nocodazole. Whole-cell extracts were analysed by immunoblotting using antibodies against C-Nap1 and C-Nap1 phosphorylated at Ser 2417 and Ser 2421. Bottom: relative intensity of the bands. Phosphorylated C-Nap1 was normalized to the anti-C-Nap1 signal. The result is a representative of three independent experiments. * Figure 7: Centrosomal localization of the linker protein rootletin is regulated by hSav1, Mst1/Mst2 and Nek2A. () Top: schematic representation of protocol used to treat RPE-1 cells expressing centrin–GFP. Bottom: representative fluorescence microscopy images of cells treated as indicated at the top using the indicated siRNA and immunostained with anti-rootletin antibodies and Hoechst 33342 to visualize DNA . Scale bar, 5 μm. () Intensity of centrosomal rootletin fluorescence at centrosomes was quantified from cells treated as in The average background intensity was subtracted and the intensities were normalized to corresponding centrin signal. Results are from three independent experiments. n = 30 cells for each condition. Box-and-whiskers plots: boxes show the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 10 to the 90 percentile and dots correspond to outliers. Asterisk indicates P = 0.001, double-asterisks indicate P = 0.0001, triple-asterisks indicate P = 0.03, compared with NSC-siRNA-transfected cells. () Top: schematic representatio! n of protocol used to treat RPE-1 cells expressing centrin–GFP. Bottom: representative fluorescence microscopy images of cells treated as indicated at the top, using the indicated siRNA and immunostained with anti-rootletin antibodies and Hoechst 33342 to visualize DNA. Scale bar, 5 μm. () Intensity of centrosomal rootletin fluorescence at centrosomes was quantified from cells treated as in . The average background intensity was subtracted and the intensities were normalized to corresponding centrin signal. Results are from three independent experiments. n = 20 cells were analysed for each condition. Box-and-whiskers plots: boxes show the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the 10 to the 90 percentile and dots correspond to outliers. Asterisk indicates P < 0.0001, double-asterisks indicate P = 0.0005, triple-asterisks indicate P < 0.0001, compared with NSC-siRNA-transfected cells. * Figure 8: Model for centrosome separation in mitotic entry. Centrosome disjunction in cells with hSav1–Mst1/Mst2–Nek2A and Eg5–microtubules. Step 1: Mst1/Mst2 aided by hSav1 phosphorylates Nek2A. This regulates the affinity and dynamics of Nek2A for centrosomes. Step 2: Nek2A phosphorylates the centrosomal linker proteins C-Nap1 and rootletin resulting in linker dissociation. Step 3: The Eg5–microtubule pathway cooperates with hSav1–Mst1/Mst2–Nek2A in centrosome disjunction and separation. The hSav1–Mst1/Mst2–Nek2A pathway becomes essential for spindle formation when Eg5 activity is reduced. Author information * Abstract * Author information * Supplementary information Affiliations * Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69117 Heidelberg, Germany. * Balca R. Mardin, * Cornelia Lange, * Sebastian R. Scholz & * Elmar Schiebel * Department of Biochemistry, University of Leicester, Leicester, LE1 9HN, UK. * Joanne E. Baxter, * Tara Hardy & * Andrew M. Fry Contributions B.R.M., A.M.F. and E.S. designed the experiments; B.R.M performed most of the experiments, C.L., J.E.B. and T.H. performed experiments with phospho-specific antibodies. S.R.S purified the GFP binder. B.R.M and E.S. wrote the manuscript with help from A.M.F. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Elmar Schiebel (e.schiebel@zmbh.uni-heidelberg.de) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases
- ncb 12(12):1177-1185 (2010)
Nature Cell Biology | Article The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases * Cheol-Sang Hwang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Shemorry1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Auerbach2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Varshavsky1avarsh@caltech.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1177–1185Year published:(2010)DOI:doi:10.1038/ncb2121Received14 July 2010Accepted18 October 2010Published online14 November 2010 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 Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1–Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1–Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that ! have been studied separately for two decades. View full text Figures at a glance * Figure 1: The Arg/N-end rule and UFD pathways. () The S. cerevisiae Arg/N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. () The S. cerevisiae UFD (Ub-fusion degradation) pathway38, 44, 45. One class of UFD substrates are engineered protein fusions that have in common a 'non-removable' N-terminal Ub moiety that acts as a degron38. Mgt1 is a physiological substrate of both the Arg/N-end rule and UFD pathways. A degron of Mgt1 is close to its N terminus but is distinct from an N-degron12. Polyubiquitylation and degradation of Cup9 are mediated by the Ubr1–Ufd4 complex. () Both the Arg/N-end rule pathway and a subset of the UFD pathway are mediated by the Ubr1/Rad6–Ufd4/Ubc4 complex discovered in the present work. Also cited are physiological substrates of these pathways in S. cerevisiae. Mgt1 and Cup9 contain internal degrons5, 12, 22. The separase-produced fragment of the Scc1 subunit of cohesin contains an Arg-bas! ed N-degron3, 6. * Figure 2: Ubiquitylation of Mgt1 by Ubr1–Ufd4. The in vitro ubiquitylation assay12 is described in Methods. Reaction mixtures were incubated at 30 °C for 15 min, followed by SDS–PAGE and autoradiography. 35S-Mgt1f3 and its polyubiquitylated derivatives are indicated on the right. Lane 1, Mgt1f3 in the complete reaction but without added Ub and with Ubr1 as the sole E3. Lane 2, as lane 1 but with wild-type Ub. Lane 3, as lane 1 but with UbK29R. Lane 4, as lane 1 but with UbK48R. Lane 5, as lane 1 but with UbK63R. Lane 6, 35S-Mgt1f3 in the complete reaction (containing both Ubr1 and Ufd4) but without added Ub. Lane 7, as lane 6 but with wild-type Ub. Lane 8, as lane 6 but with UbK29R. Lane 9, as lane 6 but with UbK48R. Lane 10, as lane 6 but with UbK63R. Lane 11, Mgt1f3 in the complete reaction but without added Ub and with Ufd4. Lane 12, as lane 11 but with wild-type Ub. Lane 13, as lane 11 but with UbK29R. Lane 14, as lane 1 but with UbK48R. Lane 15, as lane 11 but with UbK63R. * Figure 3: Physical interaction between Ubr1 and Ufd4. () Co-immunoprecipitation of fUbr1 and haUfd4 with anti-Flag antibody. fUbr1, either alone (lane 2) or together with haUfd4 (lanes 3 and 4) (see Methods). Cell extracts were immunoprecipitated (IP) with anti-Flag (lanes 2 and 4) or with antibody-free beads (lane 3). Immunoblotting (IB) with anti-Flag and anti-HA in the upper and lower panels, respectively. Lane 1, 1% input from cells expressing fUbr1 and haUfd4. Lane 2, extract containing fUbr1, with immunoprecipitation by anti-Flag. Lane 3, extract containing fUbr1 and haUfd4, with beads lacking antibody. Lane 4, same but with anti-Flag. () As in but immunoprecipitation with anti-HA (lanes 2 and 4) or beads alone (lane 3), with immunoblotting using anti-Flag and anti-HA. () Direct interaction of fUbr1 and fUfd4 (see Methods). Lane 1, 10% inputs of purified fUbr1 and fUfd4. Lanes 2 and 3, either no antibody or the previously characterized11 affinity-purified anti-Ubr1 antibody pre-bound to beads, respectively. () In vivo det! ection of Ubr1–Ufd4 interactions using split-Ub assay53. S. cerevisiae coexpressing bait and prey plasmids plated on either SC(–Leu, –Trp) or SC(–Leu, –Trp, –Ade, –His) medium (see Methods). () The UBR box, the BRR region, the RING domain and the AI (autoinhibitory) domain of the S. cerevisiae Ubr1 N-recognin11, 17, 23. Fragments of Ubr1 used to map its Ufd4-interacting region are shown below the diagram. () haUfd4 and either full-length fUbr1 or its Flag-tagged fragments were incubated with antibody-lacking beads (lanes 2, 5, 8, 11 and 14) or with anti-HA (lanes 3, 6, 9, 12 and 15), followed by immunoblotting of immunoprecipitates with anti-Flag. Input lanes, 1% of initial extracts. () Co-immunoprecipitation of fUbr1454–795 and haUfd4 with anti-HA. Lane 1, 1% input of the initial extract. Lanes 2 and 3, immunoprecipitation of fUbr1454–795 fragment and full-length haUfd4 beads alone and anti-HA, respectively, followed by immunoblotting with anti-Flag. () L! anes 1–3, as lanes 1–3 in , but immunoprecipitation was wi! th anti-Flag followed by immunoblotting with anti-HA. Uncropped images of blots are shown in Supplementary Information, Fig. S3. * Figure 4: Enhancement of ubiquitylation and degradation of Arg/N-end rule substrates by Ufd4. () X-eK-DHFRha (X = Met, Arg or Leu), denoted as X-DHFRha, are reporters54 produced from Ub–X-DHFRha through in vitro deubiquitylation55 (Supplementary Information, Fig. S1b). Purified X-DHFRha reporters were incubated in a ubiquitylation assay12 for 15 min at 30 °C, followed by immunoblotting with anti-HA. Lanes 1, 7 and 13, X-DHFRha in the absence of indicated assay's components. Lanes 2, 8 and 14, as lanes l, 7 and 13 but with Rad6. Lanes 3, 9 and 15, as lanes 1, 7 and 13 but with Ubr1 and Rad6. Lanes 4, 10 and 16, as lanes 1, 7 and 13 but with Ubc4. Lanes 5, 11 and 17, as lanes 1, 7 and 13 but with Ufd4 and Ubc4. Lanes 6, 12 and 18, as lane 1 but with Ubr1, Rad6, Ufd4 and Ubc4. Asterisk denotes an anti-HA-crossreacting protein. () As in but with Arg-DHFRha and the indicated Ub mutants (see Methods). Asterisks indicate two bands of proteins that crossreacted with anti-HA. Lanes 1, 4, 7, 10, 13 and 16, ubiquitylation of Arg-DHFRha with Ufd4/Ubc4 in the presence of UbK29! (lane 1), UbK48 (lane 4), a 50:50 mixture of UbK29 and UbK48 (lane 7), UbK29R (lane 10), UbK48R (lane 13), or a 50:50 mixture of UbK29R and UbK48R (lane 16). Lanes 2, 5, 8, 11, 14 and 17, as lanes 1, 4, 7, 10, 13 and 16, respectively, but with Ubr1/Rad6 instead of Ufd4/Ubc4. Lanes 3, 6, 9, 12, 15 and 18, as lanes 1, 4, 7, 10, 13 and 16, respectively, but with Ubr1/Rad6 plus Ufd4/Ubc4. () Lane 1, protein markers. Lane 2, Coomassie-stained preparation of the 26S proteasome. () Lanes 1–3, assay with 26S proteasome and polyubiquitylated Leu-DHFRha (prepared using Ubr1/Rad6), with chases for 10 and 20 min. Lanes 4–6, as lanes 1–3 but with polyubiquitylated Leu-DHFRha prepared with Ubr1/Rad6 plus Ufd4/Ubc4. () Quantification of data in , using ImageJ (http://rsb.info.nih.gov/ij/index.html). In plotting the levels of Leu-DHFRha for each data set (lanes 1–3 and 4–6 in ), the levels at time zero were taken as 100%. Open and filled circles, Leu-DHFRha that had been ubiquit! ylated by Ubr1/Rad6 and by Ubr1/Rad6 plus Ufd4/Ubc4, respectiv! ely. Uncropped images of blots are shown in Supplementary Information, Fig. S3. * Figure 5: Ufd4 augments the Arg/N-end rule pathway. () β-Gal activity in extracts from S. cerevisiae RJD347 (wild-type; white bars), AVY26 (ubr1Δ; grey bars), and CHY251 (ufd4Δ; black bars) that expressed His-β-gal or Tyr-β-gal. () S. cerevisiae expressing Ub–His-β-gal or Ub–Tyr-β-gal were labelled for 5 min with 35S-methionine/cysteine, followed by a chase for 20 and 60 min, immunoprecipitation with anti-β-gal, SDS–PAGE and autoradiography1, 54. Lanes 1–3 and 4–6, His-β-gal in wild-type (WT) and ufd4Δ cells, respectively. Lanes 7–9 and 10–12, Tyr-β-gal in wild-type and ufd4Δ cells, respectively. (, ) Quantification of the pulse-chase assay () for His-β-gal () and Tyr-β-gal (). Open and filled circles, wild-type and ufd4Δ cells, respectively. () Ubr1/Rad6-mediated polyubiquitylation of Cup9. In vitro ubiquitylation assay12 was performed with 35S-Cup9NSF (see Methods). Lane 1, 35S-Cup9 in the assay without E3s. Lanes 2–8, as lane 1 but with Ubr1/Rad6, in the presence of Arg-Ala and Leu-Ala (R-A/L! -A). Lane 9, as lane 1 but in a separate assay. Lanes 10–16, as lanes 2–8 but with Ubr1 plus Ufd4. () Maximal stimulation of Cup9 ubiquitylation by Ubr1–Ufd4 requires both type 1 and type 2 dipeptides. Lane 1, 35S-Cup9, with Ufd4 and wild-type Ub but in the absence of Ubr1 and type 1/2 dipeptides. Lane 2, as lane 1 but with Ubr1. Lane 3, as lane 2 but with 1 μM R-A. Lane 4, as lane 2 but in the presence of 1 μM L-A. Lane 5, as lane 2 but in the presence of R-A and L-A, each at 1 μM. Lane 6, as lane 2 but in the presence of A-R and A-L, each at 1 μM. Lane 7, as lane 6 but with UbK29R. Lane 8, as lane 7 but in the presence of R-A and L-A, each at 1 μM. () Dipeptide-mediated induction of PTR2 in the absence or presence of Ufd4. S. cerevisiae RJD347 (wild type; filled circles) and CHY251 (ufd4Δ; open circles) expressed Escherichia coli lacZ (β-galactosidase) from the PPTR2 promoter. Cells were grown to a A600 of about 0.8 in SHM medium at 30 °C in the presence of ! the indicated concentrations of R-A and L-A, followed by measu! rements in triplicate of β-gal activity in cell extracts. Standard deviations are shown. * Figure 6: Recognition and synergistic polyubiquitylation of UFD substrates by Ufd4 and Ubr1. () Ubiquitylation assay12 with Ub–ProtA (Supplementary Information, Fig. S1c), using immunoblotting with anti-ProtA. Lane 1, without E3s. Lane 2, as lane 1 but with Ubr1/Rad6. Lane 3, as lane 1 but with Ufd4/Ubc4. Lane 4, as lane 1 but with Ubr1/Rad6 plus Ufd4/Ubc4. Lane 6, as lane 1 but with Ufd2/Ubc4 plus Ubr1/Rad6. Lane 7, as lane 1 but with Ufd2/Ubc4 plus Ufd4/Ubc4. Lane 8, as lane 1 but with Ufd2/Ubc4, Ubr1/Rad6 and Ufd4/Ubc4. () Ubiquitylation assay with Ub–GST, as described in for Ub–ProtA. () A Ub-binding site in Ubr1. Equal amounts of purified fUbr1 (1 μg) were incubated (in the presence of indicated competitors) with GST alone or Ub–GST that had been linked to glutathione-Sepharose beads, with bound fUbr1 being detected by immunoblotting with anti-Flag (upper panel). Lower panel, Coomassie-stained membrane. () In vivo levels of endogenous Ubr1. Lanes 1–6, dilutions of purified fUbr1, with immunoblotting using affinity-purified anti-Ubr1 antibody11. Lanes! 7 and 8, extracts (50 μg) from wild-type and ubr1Δ cells. See also the main text. () Ubr1 and Ufd4 did not affect the Rsp5-mediated polyubiquitylation of T7-epitope-tagged Sic1PY. The PY motif is Pro-Pro-X-Tyr, which binds to the WW domain of Rsp5. Purified Sic1PY (Supplementary Information, Fig. S1c) was incubated in the ubiquitylation assay, followed by immunoblotting with anti-T7 antibody. Lane 1, Sic1PY without E3s. Lane 2, as lane 1 but with Ubr1/Rad6. Lane 3, as lane 1 but with Ufd4/Ubc4. Lane 4, as lane 1 but with Ubr1/Rad6 plus Ufd4/Ubc4. Lane 5, as lane 1 but with Rsp5/Ubc4. Lane 6, as lane 1 but with Rsp5/Ubc4 and Ubr1/Rad6. Lane 7, as lane 1 but with Rsp5/Ubc4 plus Ufd4/Ubc4. Lane 8, as lane 1 but with Rsp5/Ubc4, Ubr1/Rad6 and Ufd4/Ubc4. (, ) Equal amounts of cells from wild-type (JD52), ubr1Δ (JD55), ufd4Δ (CHY194) or ubr1Δ ufd4Δ (CHY195) strains were fivefold serially diluted, then plated on YPD plates containing 6% ethanol () or 0.4 mg ml−1 canavanine! () and incubated at 30 °C for 3 days and 1 day, respectively! . Uncropped images of blots are shown in Supplementary Information, Fig. S3. Author information * Abstract * Author information * Supplementary information Affiliations * Division of Biology, California Institute of Technology, Pasadena, California 91125, USA. * Cheol-Sang Hwang, * Anna Shemorry & * Alexander Varshavsky * Dualsystems Biotech AG, Grabenstrasse 11a, Schlieren 8952, Switzerland. * Daniel Auerbach Contributions C.-S.H, A.S., D.A. and A.V. designed experiments. C.-S.H. and A.S. performed the experiments. C.-S.H, A.S. and A.V. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alexander Varshavsky (avarsh@caltech.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (586K) Supplementary Information Additional data - A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A
- ncb 12(12):1186-1193 (2010)
Nature Cell Biology | Article A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A * Anaïck Lagana1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jonas F. Dorn1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Valérie De Rop1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anne-Marie Ladouceur1 Search for this author in: * NPG journals * PubMed * Google Scholar * Amy S. Maddox1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul S. Maddox1, 2paul.maddox@umontreal.ca Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1186–1193Year published:(2010)DOI:doi:10.1038/ncb2129Received07 May 2010Accepted20 October 2010Published online21 November 2010 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 Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. Here, we show that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, we found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, an! d the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A. View full text Figures at a glance * Figure 1: MgcRacGAP is required for CENP-A protein localization to centromeres. () Model of the licensing (1) and loading (2) steps of CENP-A into centromere chromatin shows the hypothetical replacement of H3 nucleosomes with CENP-A (see text for details). () Representative immunofluorescence images of HeLa cells transfected with shRNA specific to MgcRacGAP and expressing CENP-A–YFP (middle) and stained with antibodies specific to HsKNL2. Top image is merged from bottom two images. Depleted cells were identified by co-transfection with RFP–Histone H2B (red in merge); an untransfected control cell is also indicated. HsKNL2 localization (bottom panel) is missing from the control because HsKNL2 is normally lost after the end of G1. Thus, HsKNL2 localization is normal in both cases. Scale bar, 10 μm. () Intensity of CENP-A–YFP at centromeres in cells expressing CENP-A–YFP and transfected with the indicated shRNA plasmids, as assessed by high-resolution imaging of cells. Data are means ± s.e.m., n; number of cells analysed. () Western blot of lysat! es from control cells (diluted to the indicated percentages), compared with lysates from cells transfected with MgcRacGAP shRNA (right). The indicated antibodies were used for blotting. All depletions were confirmed by qPCR (Supplementary Information, Table S6). * Figure 2: MgcRacGAP localizes to centromeres transiently at the end of CENP-A loading. () Representative fluorescence microscopy images of endogenous MgcRacGAP and ACA (top; stained with indicated antibodies) and exogenous MgcRacGAP and Mis18 (MgcRacGAP–mCherry and Mis18–GFP; bottom panels) in HeLa cells in G1 phase. Arrows indicate co-localization of MgcRacGAP and centromeres. Images on the right are merged from images on the left, and insets are zooms of indicated nuclear regions; tubulin staining is overlaid in the top right panel in grayscale. Scale bar in nuclear insets, 2 μm and in merged image, 10 μm. Cells expressing MgcRacGAP–mCherry and CENP-A–YFP were imaged by time-lapse microscopy. Representative images are shown of MgcRacGAP–mCherry (left) and CENP-A–YFP localization (middle) at indicated times after initiation of G1 phase. Insets are ×2 zoom of indicated nuclear regions. Scale bar, 5 μm. () Quantification of CENP-A–YFP intensity (A.U.; arbitrary units) at centromeres and number of MgcRacGAP–mCherry spots that co-localize wit! h centromeres, from an experiment performed as in . () Schematic representation of CENP-A (green), HsKNL2 (blue) and MgcRacGAP (red) localization to centromeres with respect to the cell cycle. M is mitosis and S is S phase. * Figure 3: MgcRacGAP is required specifically to stabilize newly incorporated CENP-A. () Newly deposited CENP-A is specifically lost in the absence of MgcRacGAP. Cells stably expressing SNAP-labelled CENP-A were pulse-labelled with TMR. After pulse-labelling, total CENP-A was labelled by immunostaining (green; middle). Images indicate cells after treatment with (bottom) or without (control; top) MgcRacGAP shRNA. The average intensity of signal at centromeres in the cell, normalized with respect to the control cell, is reported in the lower left corner. Colour overlay and a colour scale of the relative ratio (right) indicate that MgcRacGAP is required for maintenance of newly incorporated centromere CENP-A. Scale bar, 5 μm. () Quantification of SNAP-tag-labelled CENP-A and total CENP-A in cells treated with MgcRacGAP shRNA, compared with control cells from experiment performed as in . Levels of SNAP-labelled CENP-A (old, red) were relatively unchanged (P = 0.5), but total CENP-A (green) was reduced to less than 50% (P < 0.001) after MgcRacGAP depletion, compa! red with controls. This indicates that the CENP-A lost was not at centromeres when the SNAP pulse label was administered in the previous cell cycle and was therefore newly incorporated CENP-A protein. Data are means ± s.e.m. * Figure 4: GAP-inactive MgcRagGAP mutant localizes persistently to centromeres. () Fluorescence microscopy images of a HeLa cell expressing GAP-inactive MgcRacGAP–mCherry and CENP-A–YFP. Scale bar, 10 μm. () Quantification of CENP-A–YFP intensity at centromeres and number of GAP-inactive MgcRacGAP–mCherry spots that co-localize with centromeres, as assessed from an automated time-lapse live-cell analysis. () The number of MgcRacGAP spots that co-localize with centromeres in cells expressing GAP-inactive MgcRacGAP–mCherry or wild-type MgcRacGAP–mCherry was compared by live-cell imaging. Co-localized spots were identified by visual inspection of dual-colour images. Blue, number of co-localizing spots per analysed cell; red, mean ± s.d. Control n = 22 cells; GAP dead n = 33 cells () Live-cell imaging was used to quantify time of co-localization in cells expressing CENP-A–YFP and either GAP-inactive MgcRacGAP–mCherry or wild-type MgcRacGAP–mCherry. Each row corresponds to a centromere that was tracked through time (horizontal axis). Blac! k dots indicate co-localization of MgcRacGAP–mCherry with the centromere track. * Figure 5: Automated analysis of CENP-A levels following shRNA depletion of various target proteins reveals differential defects in epigenetic regulation of centromeres. Representative images of cells at ×60 magnification from cells treated with indicated shRNA. Cells were stained with DAPI and the nuclei were segmented (Supplementary Information, Fig. S3). Images of DAPI (red) and CENP-A–YFP (green) localization are overlaid in the left column. The column labelled 'Auto' shows the CENP-A-YFP channel rescaled from 12 bits to 8 bits (required for display purposes) using an auto-scale where the highest pixel value is assigned 255 and the lowest 0 on the 8 bit scale. The column labelled 'Min' shows the same images as the Auto column, but is scaled from 12 bits to 8 bits using the dynamic range of the dimmest cell in the field of view (indicated by the white box in the Auto image). The resulting image reveals cells that have reduced CENP-A signal (affected cells) and saturates cells with 'normal' signal (unaffected cells). Comparing the Auto and Min columns gives a quick view of the level of CENP-A lost in various treatments; conditions resul! ting in greater loss of CENP-A have more bright cells in the Min column. In the last column, the Auto column has been subtracted from the Min column and pseudo-coloured to facilitate visualization. Redder colours indicate a greater difference between the Min and Auto columns. Therefore, fields with more red colours represent a greater effect on CENP-A levels. Numbers on the right of each image give the mean ± s.e.m. of CENP-A intensity per cell. n; number of cells measured. Scale bar, 20 μm. * Figure 6: Cdc42 localizes to centromeres and functions to maintain CENP-A levels independent of polymeric actin. () Representative fluorescence microscopy images of cells stained with antibodies against the indicated GTPases (left). Cells were expressing CENP-A–YFP (as shown in the middle images). Right: merge of images on the left. Co-localization of endogenous Cdc42 and CENP-A–YFP is indicated in the insets. All images are projections of 4–8 optical sections from deconvolved image stacks. Scale bar, 5 μm. () Representative fluorescence microscopy images of cells expressing CENP-A–YFP and stained with rhodamine-phalloidin to label polymeric actin. Cells were treated as indicated. Right: merge of images on the left, with DAPI used to stain nuclei. Scale bar, 20 μm. () Quantification of CENP-A–YFP intensity at centromeres in cells treated as indicated in , normalized to cells treated with DMSO. Data are means ± s.e.m.; n, number of cells analysed. * Figure 7: A small GTPase switch maintains newly incorporated CENP-A after loading. During G1, the centromere is specified in a three-step process. After licensing (1) and loading (2; see also Fig. 1), MgcRacGAP and ECT2 cycle Cdc42 GTPase activity to modify newly incorporated CENP-A nucleosomes to make them molecularly identical to pre-existing CENP-A nucleosomes (Step 3). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Anaïck Lagana & * Jonas F. Dorn Affiliations * Institute for Research in Immunology and Cancer (IRIC), Université de Montréal P.O. Box 6128, Station Centre-Ville Montréal QC, H3C 3J7 Canada. * Anaïck Lagana, * Jonas F. Dorn, * Valérie De Rop, * Anne-Marie Ladouceur, * Amy S. Maddox & * Paul S. Maddox * Department of Pathology and Cell Biology, Université de Montréal P.O. Box 6128, Station Centre-Ville Montréal QC, H3C 3J7 Canada. * Amy S. Maddox & * Paul S. Maddox Contributions A.L. conducted all immunoprecipitation and mass spectrometry experiments. A.L. and J.F.D. performed shRNA experiments and analysis, respectively. J.F.D. performed live-cell experiments. A.L. evaluated all shRNA experiments (qPCR, western blot). V.D.R. performed the small GTPases localization experiments. All authors performed essential tasks in generating figures. P.S.M., A.L., J.D., A.-M.L., and A.S.M. conceived the experiments. P.S.M. wrote the manuscript, assisted by all authors, in particular A.S.M. and J.F.D. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Paul S. Maddox (paul.maddox@umontreal.ca) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (2M) Supplementary Information * Supplementary Movie 2 (235K) Supplementary Information PDF files * Supplementary Information (945K) Supplementary Information Additional data - Competition amongst Eph receptors regulates contact inhibition of locomotion and invasiveness in prostate cancer cells
- ncb 12(12):1194-1204 (2010)
Nature Cell Biology | Article Competition amongst Eph receptors regulates contact inhibition of locomotion and invasiveness in prostate cancer cells * Jonathan W. Astin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer Batson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shereen Kadir1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica Charlet1 Search for this author in: * NPG journals * PubMed * Google Scholar * Raj A. Persad3 Search for this author in: * NPG journals * PubMed * Google Scholar * David Gillatt3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jon D. Oxley4 Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine D. Nobes1, 2Catherine.Nobes@bristol.ac.uk Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1194–1204Year published:(2010)DOI:doi:10.1038/ncb2122Received08 February 2010Accepted23 September 2010Published online14 November 2010Corrected online19 November 2010 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 Metastatic cancer cells typically fail to halt migration on contact with non-cancer cells. This invasiveness is in contrast to normal mesenchymal cells that retract on contact with another cell. Why cancer cells are defective in contact inhibition of locomotion is not understood. Here, we analyse the dynamics of prostate cancer cell lines co-cultured with fibroblasts, and demonstrate that a combinatorial code of Eph receptor activation dictates whether cell migration will be contact inhibited. The unimpeded migration of metastatic PC-3 cells towards fibroblasts is dependent on activation of EphB3 and EphB4 by ephrin-B2, which we show activates Cdc42 and cell migration. Knockdown of EphB3 and EphB4 restores contact inhibition of locomotion to PC-3 cells. Conversely, homotypic collisions between two cancer cells results in contact inhibition of locomotion, mediated by EphA–Rho–Rho kinase (ROCK) signalling. Thus, the migration of cancer cells can switch from restrained to i! nvasive, depending on the Eph-receptor profile of the cancer cell and the reciprocal ephrin ligands expressed by neighbouring cells. View full text Figures at a glance * Figure 1: Failure of CIL by PC-3 cells on contact with fibroblasts. () Representative time-lapse microscopy images, at the indicated times, of a PrEC/fibroblast collision (top and Supplementary Information, Movie S1) and a PC-3/fibroblast collision (middle and Supplementary Information, Movie S2). Asterisk indicates fibroblast cell and insets at the bottom indicate magnification of boxed area; false-colour indicates region of PC-3 cell lamella extending beneath the fibroblast. () CIL is quantified by comparing contact acceleration indices (Cx) of free-moving cells and colliding cells. Cells were tracked before (A) and after (B) a collision (free-moving cells were tracked for the same time periods). The component Cx of vector B–A represents the difference between how far the cell has progressed in the direction of A' and how far it would have gone had there been no collision. () Contact acceleration indices (Cx) of free-moving cells (F) versus colliding cells (C); PrEC/fibroblast (n = 36), DU-145/fibroblast (n = 32), PC-3/fibroblast (n = 29! ), PC-3/endothelial cell (n = 15). Triple-asterisks indicate P < 0.001, NS; not significant, determined by a Mann-Whitney test. () Scaled cell-displacement vector diagrams of free-moving cells and colliding cells, tracked during time-lapse microscopy. Thick red line denotes the scaled displacement of all cells before contact and thin black lines are those of each cell following contact. () Representative time-lapse microscopy images, at the indicated times, of collisions between two PrECs (top and Supplementary Information, Movie S3) and two PC-3 cells (bottom and Supplementary Information, Movie S4). Black arrows indicate direction of migration and white arrow indicates a new leading edge forming. () Contact acceleration indices (Cx) of free-moving cells (F) versus colliding cells (C); PrEC/PrEC (n = 31), DU-145/DU145 (n = 24), PC-3/PC-3 (n = 27). Triple asterisks indicate P < 0.001, determined by a Mann-Whitney test. () Scaled cell-displacement vector diagrams of homotypi! c collisions. Cells were tracked during time-lapse microscopy.! Thick red line denotes the scaled displacement of all cells before contact and thin black lines are those of each cell following contact. Scale bars: , 50 μm; , 25 μm. * Figure 2: Ephrin-A ligands are sufficient to induce CIL between PC-3 cells. () Immunofluorescence microscopy of cells treated with anti-Fc antibodies (green) to detect surface binding of ephrin-A1–Fc, ephrin-A5–Fc, ephrin-B2–Fc and control Fc, to PrEC, DU-145 and PC-3 cells. Hoechst (blue) stains nuclei. Scale bar, 50 μm. () Prostate cells were treated with clustered ephrin–Fc proteins before fixation and phalloidin staining. Data are expressed as percentage of cells with retraction of the cell periphery (rounded cells) for each treatment as indicated. Data are means ± s.d. Triple asterisks indicate P < 0.001 and double asterisks indicate P < 0.01, as determined by an unpaired Student's t-test (n = 4; 100 cells counted per experiment). () The underside of a transwell chamber was coated with ephrin–Fc proteins, as indicated, and the numbers of cells migrating through were scored. Data are expressed as fold-change with respect to control Fc-coated chambers (red dotted line). Data are means ± s.d. Asterisk indicates P < 0.05, as determined! by a paired Student's t-test (n = 5). A.U.; arbitrary units. () Representative time-lapse microscopy images, at the indicated times, of a PC-3 cell colliding with a silica protein-A bead (red pseudocolour) coated with ephrin-A5–Fc (top and Supplementary Information, Movie S5) or with Fc (bottom and Supplementary Information, Movie S6). Arrows indicate direction of migration. Scale bar, 25 μm. () Contact acceleration indices (Cx) of free-moving cells (F) versus cells colliding with beads (C), coated as indicated; Fc (n = 33), ephrin-A5–Fc (n = 25), EphA4–Fc (n = 28), ephrin-B2–Fc (n = 23). Double asterisks indicate P < 0.01, as determined by a Mann-Whitney test. * Figure 3: EphA2 and EphA4 are required for CIL between PC-3 cells. () Lysates of PC-3 cells mock transfected, transfected with a non-targeting siRNA oligonucleotide (control siRNA) or transfected with siRNA oligonucleotides specific to EPHA2 and EPHA4 (two different oligonucleotides for each) were immunoblotted using antibodies against the indicated proteins. Tubulin was used as a loading control. () PC-3 cells transfected with control siRNA or siRNA oligonucleotides specific to EPHA2 and EPHA4 were fixed, incubated with either ephrin-A1–Fc (top) or ephrin-A5–Fc (bottom) and stained with anti-Fc antibodies (green) and hoechst (blue). (, ) PC-3 cells, transfected with siRNA oligonucleotides as indicated, were treated with clustered ephrin-A1–Fc, ephrin-A5–Fc or Fc, and rounded cells were counted after fixation and staining with phalloidin. Data are means ± s.d. (siRNA 1, n = 4; siRNA 2 n = 3; 100 cells counted per experiment). Triple asterisks indicate P < 0.001, double asterisks indicate P < 0.01 and asterisk indicates P < 0.05, as! determined by an unpaired Student's t-test. () Representative time-lapse microscopy images, at the indicated times, of collisions between PC-3 cells transfected with siRNA oligonucleotides as indicated (Supplementary Information, Movies S7 and S8). Arrows indicate direction of migration. Following time-lapse microscopy, cells were fixed and EphA surface expression of recorded cells was determined by ephrin-A1–Fc binding detected with anti-Fc antibodies (right). () Contact acceleration indices (Cx) of free-moving (F) versus colliding (C) PC-3 cells transfected with siRNA oligonucleotides as indicated. Mock (n = 19), Control (n = 18), siRNA 1 (n = 22), siRNA 2 (n = 12). Triple asterisks indicate P < 0.001, NS; not significant, as determined by a Mann-Whitney test. () Scaled cell-displacement vector diagrams of colliding PC-3 cells treated as shown. Cells were tracked using time-lapse microscopy. Thick red line denotes the scaled displacement of all cells before contact and! thin black lines are those of each cell following contact. Sc! ale bars: , 50 μm; , 25 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S8. * Figure 4: Ephrin-B2 stimulates PC-3 cell migration. () Relative expression profiles of ephrin ligands by fibroblasts and endothelial cells versus PC-3 cells. Data are expressed as fold-change in mRNA with respect to PC-3 cell mRNA levels, as determined by real-time RT–PCR. Data are means ± s.d. (n = 3). () PC-3, fibroblast and endothelial cell lysates were immunoblotted with antibodies against the indicated proteins. () The underside of a transwell chamber was coated with Fc-coated ephrin-B-ligands as indicated, and numbers of cells migrating through the chamber were scored. Data are expressed as fold-change with respect to control Fc-coated chambers (grey dotted line). Data are means ± s.d. (n = 5). Asterisk indicates P < 0.05, as determined by a paired Student's t-test. () Left: EphB2 and EphB3 were immunoprecipitated from lysates of DU-145 or PC-3 cells and detected by immunoblotting. Right: lysates of DU-145 or PC-3 cells were immunoblotted with antibodies against EphB4. Tubulin was used as a loading control in both c! ases. Uncropped images of blots are shown in Supplementary Information, Fig. S8. * Figure 5: Ephrin-B2 induces filopodia by activating Cdc42 in PC-3 cells. () DU-145 or PC-3 cells were treated with clustered ephrin-B–Fc or Fc and analysed for the formation of filopodia. Data are expressed as mean number of filopodia per cell ± s.d. (n = 3, 50 cells counted per experiment). Triple asterisks indicate P < 0.001, as determined by an unpaired Students t-test. () Confocal microscopy images of DU-145 and PC-3 cells treated with clustered ephrin-B2–Fc or Fc and fixed and stained with phalloidin or with antibodies against fascin (PC-3 cell, bottom). () PC-3 cells were microinjected with expression vectors as indicated, and then treated with ephrin-B2–Fc or Fc, and analysed for the formation of filopodia. Data are expressed as mean number of filopodia per cell ± s.d. (pRK5 and Fc, n = 92, pRK5 and ephrin-B2–Fc, n = 82; N17Cdc42 and Fc, n = 87; N17Cdc42 and ephrin-B2–Fc, n = 101). Triple asterisks indicate P < 0.001, as determined by an unpaired Student's t-test. () Confocal microscopy images of phalloidin-stained (red) PC-3 c! ells after microinjection of indicated expression constructs, followed by treatment with ephrin-B2–Fc; injection marker (green). () PC-3 cells were treated with ephrin-B2–Fc. At the indicated times, cells were lysed, followed by pulldown of Cdc42–GTP, Rac–GTP (using PAK1-CRIB beads) and RhoA–GTP (using Rhotekin Rho-binding domain beads). Proteins were resolved by SDS–PAGE and detected by immunoblotting. () Quantification of Rho-GTPase activation in PC-3 cells following the addition of ephrin-B2–Fc. Experiments were carried out as in , band intensities were quantified, and normalised intensities were calculated relative to control untreated cells at 0 min. Data are means ± s.d. (n = 3). () DU-145 cells were treated with ephrin-B2–Fc. At the indicated times, cells were lysed, followed by pulldown of Cdc42–GTP using PAK1-CRIB beads. Proteins were resolved by SDS–PAGE and detected by immunoblotting. () Quantification of Cdc42 activation in DU-145 cells foll! owing the addition of ephrin-B2–Fc. Experiments were carried! out as in , band intensities were quantified, and normalized intensities were calculated relative to control untreated cells at 0 min. Data are means (n = 2). Scale bars: , , 20 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S8. * Figure 6: EphB3 and EphB4 knockdown restores CIL of PC-3 cells on contact with fibroblasts. () Contact acceleration indices (Cx) of free-moving cells (F) versus cells colliding (C) with beads coated with the indicated ephrin–Fc (1:1 molar ratios). DU-145 ephrin-A5–Fc + Fc (n = 20), DU-145 ephrin-A5–Fc + ephrin-B2–Fc (n = 40), PC-3 ephrin-A5–Fc + Fc (n = 28), PC-3 ephrin-A5–Fc + ephrin-B2–Fc (n = 24). Triple asterisks indicate P < 0.001, double asterisks indicate P < 0.01, NS; not significant, as determined by a Mann-Whitney test. () Immunoblot of lysates from PC-3 cells transfected with no vector (mock), pRK5 or pRK5 containing ephrin-B2 (at the indicated times after transfection), as indicated. Tubulin was used as a loading control.() Contact acceleration indices (Cx) of free-moving cells (F) versus collisions (C) between PC-3 cells transfected with the indicated plasmids. Cells were imaged between 48–72 h post-transfection. pRK5-transfected cells, n = 23, pRK5-ephrin-B2-transfected cells, n = 23. Triple asterisks indicate P < 0.001, NS; not signif! icant, as determined by a Mann-Whitney test. () Lysates of PC-3 cells transfected with either control or CDC42 siRNA oligonucleotides were immunoblotted with antibodies against Cdc42. Tubulin was used as a loading control. () PC-3 cells transfected with the indicated siRNA oligonucleotides were added to transwell chambers coated with ephrin-B2–Fc, and numbers of cells migrating through were scored. Data are expressed as fold change with respect to control Fc-coated chambers (grey dotted line). Data are means ± s.d. (n = 5). Triple asterisks indicate P < 0.001, double asterisks indicate P < 0.01, as determined by an unpaired Student's t-test. () Representative time-lapse microscopy images, at the indicated times, of a PC-3 cell transfected with control siRNA colliding with a fibroblast (top and Supplementary Information, Movie S10), and a PC-3 cell transfected with EPHB3 and EPHB4 siRNA oligonucleotides colliding with a fibroblast (bottom panel and Supplementary Informati! on, Movie S11). White asterisk indicates the fibroblast, arrow! s indicate direction of migration. Scale bar, 50 μm. () Contact acceleration indices (Cx) of free-moving cells (F) versus collisions between PC-3 cells and fibroblasts (C). Cells are transfected with the indicated siRNA oligonucleotides. Control (n = 30), siRNA 1 (n = 18), siRNA 2 (n = 18). Triple asterisks indicate P < 0.001, NS; not significant, as determined by a Mann-Whitney test. Uncropped images of blots are shown in Supplementary Information, Fig. S8. * Figure 7: Immunohistochemical staining of EphB4 and ephrin-B2 in prostate cancer. () EphB4 expression in either benign prostate epithelium or in areas of prostate cancer (Gleason pattern 3+). Top and bottom panels represent sections from two different patients. ab-1 indicates staining with an antibody specific to the EphB4 C-terminal sequence and ab-2 indicates staining with an antibody specific to the EphB4 N-terminal sequence. The increased EphB4 staining was observed in 11 out of 15 cases. () Ephrin-B2 staining of stromal cells: smooth muscle cells of blood vessels (v) and smooth muscle within the stroma (s). A cohort of cancer cells (c) is indicated. H and E; haematoxylin and eosin. () An example of perineural invasion showing complementary staining of EphB4 in cancer cells (c) surrounding a nerve fibre (n), and ephrin-B2 in cells within the nerve fibre. () Complementary staining of EphB4 in cancer cells (nuclei indicated by arrows) and ephrin-B2 staining in the surrounding stromal cells (arrowheads) from serial sections. Similar complementary stainin! g was observed in 6 out of 6 cases. Scale bar, 50 μm. * Figure 8: Model of CIL regulation in PC-3 cells. There are two competing pathways that regulate CIL in PC-3 cells; repulsive EphA–RhoA signalling triggered by ephrin-A ligands and attractive EphB3/EphB4–Cdc42 signalling triggered by ephrin-B2 ligand. Thus, the ratio of ephrin-A/ephrin-B2 on a cell will dictate whether the PC-3 cell colliding with it will display CIL or not. () PC-3 cells have a high ephrin-A/ephrin-B2 ratio and therefore CIL is induced between PC-3 cells by EphA forward signalling, possibly by activation of RhoA. () Fibroblasts have a high ephrin-B2/ephrin-A ratio which activates EphB3/EphB4–Cdc42 signalling in PC-3 cells, stimulates migration and causes defective CIL. Change history * Abstract * Change history * Author information * Supplementary informationCorrigendum 19 November 2010In the version of this article initially published online, Fig. 4c was incorrectly labelled on the y axis. This error has been corrected in both the HTML and PDF versions of the article. Author information * Abstract * Change history * Author information * Supplementary information Affiliations * School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK. * Jonathan W. Astin, * Jennifer Batson, * Shereen Kadir, * Jessica Charlet & * Catherine D. Nobes * School of Physiology and Pharmacology, University of Bristol, Bristol, BS8 1TD, UK. * Catherine D. Nobes * Bristol Urological Institute, Southmead Hospital, Bristol, BS10 5NB, UK. * Raj A. Persad & * David Gillatt * Department of Cellular Pathology, Southmead Hospital, Bristol, BS10 5NB, UK. * Jon D. Oxley Contributions J.W.A., S.K. and C.D.N. designed experiments. J.W.A. and J.C. performed the RT–PCR, J.B. performed the Cdc42-knockdown experiments, C.D.N. performed microinjection experiments and immunohistochemistry, and J.W.A. carried out all other experiments. D.G. and R.P. provided prostate tissue and J.O., J.W.A. and C.D.N. prepared and analysed prostate immunohistochemistry. J.W.A. and C.D.N. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Catherine D. Nobes (Catherine.Nobes@bristol.ac.uk) Supplementary information * Abstract * Change history * Author information * Supplementary information Movies * Supplementary Movie 1 (4M) Supplementary Information * Supplementary Movie 2 (6M) Supplementary Information * Supplementary Movie 3 (4M) Supplementary Information * Supplementary Movie 4 (4M) Supplementary Information * Supplementary Movie 5 (5M) Supplementary Information * Supplementary Movie 6 (5M) Supplementary Information * Supplementary Movie 7 (5M) Supplementary Information * Supplementary Movie 8 (8M) Supplementary Information * Supplementary Movie 9 (4M) Supplementary Information * Supplementary Movie 10 (5M) Supplementary Information * Supplementary Movie 11 (5M) Supplementary Information PDF files * Supplementary Information (4M) Supplementary Information Additional data - Positive feedback between p53 and TRF2 during telomere-damage signalling and cellular senescence
- ncb 12(12):1205-1212 (2010)
Nature Cell Biology | Article Positive feedback between p53 and TRF2 during telomere-damage signalling and cellular senescence * Kaori Fujita1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Izumi Horikawa1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Abdul M. Mondal1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa M. Miller Jenkins2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ettore Appella2 Search for this author in: * NPG journals * PubMed * Google Scholar * Borivoj Vojtesek3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Christophe Bourdon4 Search for this author in: * NPG journals * PubMed * Google Scholar * David P. Lane4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Curtis C. Harris1curtis_harris@nih.gov Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1205–1212Year published:(2010)DOI:doi:10.1038/ncb2123Received01 February 2010Accepted24 September 2010Published online07 November 2010 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 telomere-capping complex shelterin protects functional telomeres and prevents the initiation of unwanted DNA-damage-response pathways. At the end of cellular replicative lifespan, uncapped telomeres lose this protective mechanism and DNA-damage signalling pathways are triggered that activate p53 and thereby induce replicative senescence. Here, we identify a signalling pathway involving p53, Siah1 (a p53-inducible E3 ubiquitin ligase) and TRF2 (telomere repeat binding factor 2; a component of the shelterin complex). Endogenous Siah1 and TRF2 were upregulated and downregulated, respectively, during replicative senescence with activated p53. Experimental manipulation of p53 expression demonstrated that p53 induces Siah1 and represses TRF2 protein levels. The p53-dependent ubiquitylation and proteasomal degradation of TRF2 are attributed to the E3 ligase activity of Siah1. Knockdown of Siah1 stabilized TRF2 and delayed the onset of cellular replicative senescence, suggesting! a role for Siah1 and TRF2 in p53-regulated senescence. This study reveals that p53, a downstream effector of telomere-initiated damage signalling, also functions upstream of the shelterin complex. View full text Figures at a glance * Figure 1: Replicative cellular senescence is associated with decreased TRF2 and increased Siah1. () Expression of TRF2, total p53, p21WAF1, p53 phosphorylated at serine 15 (pS15–p53) and Siah1 were examined by immunoblot in early-passage (Y) and senescent (S) human fibroblast strains MRC-5 and WI-38. The examined passage numbers were 30 (Y) and 65 (S) for MRC-5; and 30 (Y) and 58 (S) for WI-38. β-actin and histone H2B were loading controls. The top three panels used total protein lysates and the bottom three panels used nuclear extracts (NE). Three independent experiments gave reproducible results. () TRF2 mRNA levels are not changed during replicative senescence. The same set of cells as in were examined for TRF2 mRNA expression by real-time quantitative reverse-transcriptase PCR (qRT–PCR). β2-microglobulin mRNA expression was used as a control. Data are means ± s.d. from three independent experiments. () SIAH1 mRNA levels are increased during replicative senescence. The same set of cells as in and were examined for SIAH1 mRNA expression by qRT–PCR. β2-microg! lobulin mRNA expression was used as a control. Data are mean ± s.d. from three independent experiments. Asterisk indicates P < 0.01. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 2: p53 upregulates Siah1 and downregulates TRF2. () Loss of wild-type p53 results in increased TRF2 and decreased Siah1. Li-Fraumeni MDAH041 fibroblasts heterozygous and homozygous for p53 frame-shift mutation were examined by immunoblot for p53, TRF2 and Siah1. β-actin was used as a loading control. () shRNA knockdown of p53 results in increased TRF2 and decreased Siah1. hTERT-immortalized human fibroblasts (hTERT/NHF) were transduced with the p53 shRNA retroviral vector (+) or the control vector (−) and examined by immunoblot for expression of p53, TRF2, Siah1 and Siah2. NE, nuclear extracts. β-actin and histone H2B were loading controls. Three independent experiments gave reproducible results. () The same cells as in were examined for TRF2 mRNA expression by qRT–PCR, as in Fig. 1b. Data are means ± s.d. from three independent experiments. () Nutlin-3a activation of p53 results in decreased TRF2 and increased Siah1. hTERT/NHF cells with (+) or without (-) p53 shRNA were treated with 10 μM of Nutlin-3a for the ind! icated time period and examined by immunoblot for p53, TRF2 and Siah1 expression. NE, nuclear extracts. β-actin and histone H2B were loading controls. () The same cells as in and were examined for SIAH1 mRNA expression by qRT–PCR, as in Fig. 1c. Data are means ± s.d. from three independent experiments. Asterisk indicates P < 0.01. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 3: Siah1 knockdown stabilizes TRF2. () siRNA knockdown of Siah1 results in increased TRF2. MRC-5 fibroblasts were transfected with each of two independent siRNA oligonucleotides against Siah1 or a control oligonucleotide and examined by immunoblot for Siah1 and TRF2 expression. β-actin was a loading control. Relative expression levels of Siah1 and TRF2, assessed by signal intensities and indicated below each gel, were normalized to the intensity of β-actin. () MRC-5 fibroblasts transfected with control oligonucleotide or one of the SIAH1 siRNA oligonucleotides were treated with cycloheximide (CHX) at indicated time periods and examined for TRF2 expression by immunoblotting. () Quantitative analysis of TRF2 stability. Relative TRF2 expression levels were from densitometric analysis of . The value at 0 h was defined as 100% for each treatment. Two independent experiments gave reproducible results. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 4: TRF2 is subject to proteasomal degradation and ubiquitylated in vivo. () Proteasome inhibition increases TRF2. Early-passage (Y) and senescent (S) MRC-5 fibroblasts were incubated in the presence (+) or absence (−) of MG132 for 5 h and examined for TRF2 and p53 protein levels by immunoblotting. () Proteasome-mediated regulation of TRF2 depends on functional p53. hTERT/NHF cells, transduced with control vector, Δ133p53 overexpression vector23 or p53 shRNA vector, were treated with MG132 and examined for TRF2 and p53 protein levels, as in . () TRF2-containing protein complex is ubiquitylated in vivo. Protein lysates from WI-38 fibroblasts incubated with (+) or without (−) MG132 were used in immunoprecipitation (IP) with anti-TRF2 antibody or control IgG (immunoglobulin G). Immunoprecipitated proteins were analysed by immunoblot using anti-polyubiquitin antibody (Poly-Ub). Immunoblot with anti-TRF2 antibody confirmed the efficiency and specificity of the immunoprecipitation. () TRF2-associated ubiquitylation depends on Siah1 and p53. MRC-5 f! ibroblasts were either transfected with SIAH1 siRNA or control oligonucleotide, or transduced with p53 shRNA vector or control vector. Protein lysates prepared from cells after treatment with MG132, as well as those from untreated cells as negative controls, were used in immunoprecipitation with anti-TRF2 antibody, followed by immunoblotting with anti-Poly-Ub or anti-TRF2 antibody. Smear signals were quantified and expressed as relative values to cells without siRNA or shRNA (knockdown) after MG132 treatment (left lane; defined as 100%). The effectiveness of SIAH1 siRNA and p53 shRNA was confirmed by immunoblot using total protein lysates (before IP). The experiment was repeated three times with reproducible results. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 5: Siah1 interacts with, and ubiquitylates, TRF2. () Siah1 interacts with TRF2 in vitro. His6–TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST–Siah1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST–Siah1 was incubated with Ni-NTA magnetic agarose beads without His6-tagged TRF2. After extensive washing, the beads were boiled in SDS (sodium dodecyl sulfate) sample buffer and the eluted proteins were analysed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His6–TRF2, GST–Siah1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). () Siah1 ubiquitylates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah1, Siah1H59W or Siah1ΔRING) and a substrate (GST or GST–TRF2) were added to an in vitro ubiquitylation reaction as indicated. After reaction, glutathione–Sepharose 4FF-purified substrates were! analysed by immunoblot with anti-GST antibody. The position of non-ubiquitylated GST–TRF2 is indicated. Poly-ubiquitylated GST–TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitylated GST–TRF2. The experiment was repeated twice with reproducible results. () Siah1 is essential to TRF2 ubiquitylation in vivo. Myc-tagged TRF2, HA-tagged ubiquitin (HA–Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or two different SIAH1 siRNA (indicated as 1 and 2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analysed by immunoblot using anti-Myc antibody (top) and anti-HA antibody (bottom). The knockdown of Siah1 protein expression by SIAH1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation. β-actin was a loading control. White brackets indicate smear signals ind! icative of poly-ubiquitylation. The strong signals at the bott! om of the top image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which probably contained non-specific signals and TRF2-associated ubiquitylated proteins of smaller size. The open arrowhead indicates a ubiquitylated protein of currently unknown origin. The experiment was repeated twice with reproducible results. Uncropped images of blots are shown in Supplementary Information, Fig. S9. * Figure 6: Roles of Siah1 and TRF2 in cellular senescence in vitro and in vivo. () Siah1 knockdown extends cellular replicative lifespan in vitro. MRC-5 fibroblasts at passage number 51 were transfected with SIAH1 siRNA 1, SIAH1 siRNA 2 or control siRNA, every 4 days. The cumulative population doubling levels (PDL) were calculated and plotted to days after the first transfection. Data are means ± s.d from three independent experiments. () Immunoblot analysis of TRF2, Siah1, p53, p21WAF1 and p16INK4A. The same set of cells as in at days 20 and 88 were examined. () Cellular senescence in vivo is associated with decreased expression of TRF2. Eight pairs of matched colon adenoma (Ad) and non-adenoma (Non-ad) tissues were analysed in immunoblot (Supplementary Fig. S5g). These eight cases were previously described23 and the adenoma tissues had senescent phenotypes, such as positive staining for senescence-associated β-galactosidase and increased expression of p16INK4A and IL-8 (ref. 23). Normalized expression levels (TRF2/β-actin) were calculated from dens! itometric measurement and quantitative analysis of the immunoblot data. Paired Student's t-test was performed. () Cellular senescence in vivo is associated with increased expression of Siah1. RNA samples were isolated from the same eight cases as in and examined by qRT–PCR analysis for SIAH1 expression. SIAH1 mRNA levels were normalized to β2-microglobulin (β2MG) levels. Paired Student's t-test was performed. Uncropped images of blots are shown in Supplementary Information, Fig. S9. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Kaori Fujita & * Izumi Horikawa Affiliations * Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892-4258, USA. * Kaori Fujita, * Izumi Horikawa, * Abdul M. Mondal & * Curtis C. Harris * Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892-4256, USA. * Lisa M. Miller Jenkins & * Ettore Appella * Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech Republic. * Borivoj Vojtesek * University of Dundee, Ninewells Hospital, Department of Surgery and Molecular Oncology, Inserm-European Associated Laboratory, Dundee, DD1 9SY, UK. * Jean-Christophe Bourdon & * David P. Lane * Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore. * David P. Lane Contributions K.F., I.H., A.M.M. and L.M.M.J. performed experiments. B.V., J.-C.B. and D.P.L. provided essential reagents and suggestions. K.F., I.H., E.A., L.M.M.J. and C.C.H. coordinated the study and wrote the manuscript. C.C.H. was responsible for the overall project. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Curtis C. Harris (curtis_harris@nih.gov) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (1M) Supplementary Information Additional data - The orphan nuclear receptor Nurr1 restricts the proliferation of haematopoietic stem cells
- ncb 12(12):1213-1219 (2010)
Nature Cell Biology | Letter The orphan nuclear receptor Nurr1 restricts the proliferation of haematopoietic stem cells * Olga Sirin1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Georgi L. Lukov2 Search for this author in: * NPG journals * PubMed * Google Scholar * Rui Mao2 Search for this author in: * NPG journals * PubMed * Google Scholar * Orla M. Conneely3 Search for this author in: * NPG journals * PubMed * Google Scholar * Margaret A. Goodell1, 2, 3goodell@bcm.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1213–1219Year published:(2010)DOI:doi:10.1038/ncb2125Received20 April 2010Accepted18 October 2010Published online14 November 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Successful haematopoiesis requires long-term retention of haematopoietic stem cells (HSCs) in a quiescent state. The transcriptional regulation of stem cell quiescence, especially by factors with specific functions in HSCs, is only beginning to be understood. Here, we demonstrate that Nurr1, a nuclear receptor transcription factor, has such a regulatory role. Overexpression of Nurr1 drives early haematopoietic progenitors into quiescence. When stem cells overexpressing Nurr1 are transplanted into lethally irradiated mice, they localize to the bone marrow, but do not contribute to regeneration of the blood system. Furthermore, the loss of only one allele of Nurr1 is sufficient to induce HSCs to enter the cell cycle and proliferate. Molecular analysis revealed an association between Nurr1 overexpression and upregulation of the cell-cycle inhibitor p18 (also known as INK4C), suggesting a mechanism by which Nurr1 could regulate HSC quiescence. Our findings provide critical insig! ht into the transcriptional control mechanisms that determine whether HSCs remain dormant or enter the cell cycle and begin to proliferate. View full text Figures at a glance * Figure 1: Nurr1 is highly expressed in quiescent HSCs (Hoechst 33342 side-population cells that are also c-Kit+, Sca1+ and Lin−; SP-KSL) and its overexpression in 32D cells results in a proliferative block. () Nurr1 expression in HSCs on the indicated days after injection with 5FU, as determined by microarray data extracted from ref. 13. Data was normalized to expression profile of Nurr1 from quiescent HSCs. () Verification of microarray data shown in by real-time PCR measuring Nurr1 levels in HSCs on the indicated days after injection with 5FU. Data were normalized relative to expression levels at day 0. Data are means ± s.e.m. from three experiments; P = 0.02 between day 0 and day 6. () Relative expression of Nurr1 in HSCs and the indicated differentiated cells, as assessed by microarray data extracted from ref. 14. () Nurr1 levels in the indicated cells, as assessed by real-time PCR. Expression levels are normalized with respect to B cells (data are means ± s.e.m. of 3 experiments; asterisks indicate P = 0.003). () Schematic representation of the vectors used for expression of Nurr1 in 32D cells. Both vectors were cloned into MSCV for subsequent infection of cells. The con! trol vector only expresses GFP. MSCV–Nurr1 expresses Nurr1 upstream of IRES–GFP. IRES, internal ribosome entry site; LTR, long terminal repeats. () Proliferation of 32D cells transduced with the indicated vectors, as represented in . Cells were transduced, cultured for 48 h and GFP-positive cells were identified by flow cytometry, before culture in 96-well plates. Viability was assessed by treatment of the cells with trypan blue on the indicated days. Data are means ± s.e.m.; n = 3; The curves were fitted using non-linear curve fitting, and Fisher's exact test indicated they were significantly different (P < 0.0001). () Left: representative flow-cytometry analysis of cells cultured as in and treated with propidium iodide and anti-annexin V to assess cell death and apoptosis. Percentages of cells that are dead (top), apoptotic (bottom right), or alive (bottom left), are indicated. Right: quantification of dead and apoptotic cells from flow cytometric analyses. Data are ! means ± s.d. from 3 experiments; asterisks indicate P = 0.004. * Figure 2: Overexpression of Nurr1 in bone-marrow cells reversibly blocks proliferation. () Schematic representation of overexpression experiments in bone-marrow cells using control and Nurr1 vectors shown in Fig. 1e. () CFU-C assay on cells cultured as indicated in (n = 4; asterisks indicate P < 0.0002). Data are colonies formed as a percentage of the total wells plated. () Cells were treated as indicated in , before treating with antibodies against annexin V and analysing staining by flow cytometry. Data are means ± s.e.m. of five experiments. () Lethally irradiated mice were transplanted with bone-marrow cells transduced with control or MSCV–Nurr1 vectors, as indicated in . Left: representative flow-cytometric analysis of peripheral blood 4 weeks after transplantation. Numbers indicate percentages of cells in each indicated quadrant. Right: quantification of GFP-positive cells from the flow cytometric analyses. Control; n = 7, Nurr1 n = 9; asterisks indicate P < 0.0001. () Quantification of GFP-positive cells in the bone marrow and spleen of lethally irrad! iated mice, transplanted with bone-marrow cells that were untransfected, or transfected with control or MSCV–Nurr1 vector. GFP-positive cells were identified by flow cytometric analysis 4 weeks after transplantation. Data are means ± s.e.m. of three experiments. Asterisk indicates P = 0.03 and double asterisks indicate P < 0.005. () Lethally irradiated mice were transplanted with bone-marrow cells transduced with control, MSCV–Nurr1 or the LXNurr1LX vector. The LXNurr1LX vector contains a loxP-flanked Nurr1 gene (schematic representation of vector is shown in Supplementary Information, Fig. 3), with Cre-mediated excision of the gene inducible by treatment of transduced cells with tamoxifen. Engraftment of transduced cells was assessed by flow cytometry, 8 weeks after the initial transplant (4 weeks after tamoxifen induction). Data are means ± s.e.m. Control, n = 4; Nurr1, n = 4; LXNurr1LX, n = 6; P = 0.3 for the comparison of TMX treated and non-treated LXNurr1LX. * Figure 3: Nurr1 overexpression leads to reduced cell-cycle proliferation. () Quantification of cells in G0. Sca-1-enriched bone-marrow cells were transduced with the indicated vectors . After transduction GFP-positive and Sca-1-positive cells were sorted by flow cytometry, and stained with Hoescht 33342 and pyronin Y to enable quantification of cells in G0 and G1 phases by flow cytometry. Data are means ± s.e.m. Control n = 5, Nurr1 n = 7. Asterisk indicates P = 0.05. () Quantification of Ki67-positive cells by flow cytometry. Haematopoietic progenitors (c-Kit+ Sca-1+ Lin−; KSL) were isolated from adult wild-type (WT) and Nurr+/− heterozygous mice by flow cytometry and Ki67-positive cells were then quantified by flow cytometry. Data are means ± s.e.m. of three experiments. Asterisks indicate P = 0.0007. () Colony forming assay of cells isolated from the bone marrow of wild-type and Nurr1+/− mice. Bone-marrow cells were sorted by flow cytometry into HSCs (SK; Sca-1+c-kit+), side population Lin− (SP-Lin−) and side population c-Kit+ Sca-1! + Lin− (SP-KSL) before the assay. () Survival curves of Nurr1+/+ mice (n = 5), compared with Nurr1+/− mice (n = 10) mice, over 55 days with weekly injections of 5FU (indicated by arrowheads). The survival curves were significantly different (P = 0.022), based on Gehan-Breslow-Wilcoxon test. * Figure 4: Dose effect of Nurr1 on expression of cell-cycle inhibitors and rescue of the Nurr1 phenotype by p18 expression. () Expression level of the indicated genes in SP-KSL cells isolated from the fetal liver of wild-type, Nurr1+/− and Nurr1−/− mice at E14.5. SP-KSL were isolated and sorted by flow cytometry and level of mRNA was assessed by real-time PCR. Data are means ± s.e.m. (n = 3). () Expression level of the indicated genes in HSCs (KSL, CD150+) transduced with the indicated vectors (GFP+) was assessed by real-time PCR. Cells were sorted by flow cytometry. Data are means ± s.e.m. (n = 4) and expression levels are relative to control. () Left: fold-change in p18 or p19 protein levels as quantified from the western blot shown to the right, after transfection of 32D cells with empty control vector, or vector containing construct shown in schematic representation at the bottom. Fold-change in expression is calculated relative to cells transfected with control vector. Data are means ± s.e.m. (n = 3; asterisk indicates P = 0.011; NS, not significant). Right: western blot of lysates ! from 32D cells transfected with the indicated vectors. Lysates were immunoblotted with antibodies against the indicated proteins. β-actin was used as a loading control. () KSL HSC progenitor cells were isolated from Nurr1+/− mice and transduced with either a control vector (GFP only) or a vector expressing p18. Proliferation of the cells was assessed by flow cytometry. Data are means ± s.e.m. and calculated relative to control cells (n = 3). () Colony formation assay of cells isolated from wild-type mice, and Nurr1+/− overexpressing p18 (compare with Fig. 3c). Data are means ± s.e.m. (n = 3). Uncropped image of blot is shown in Supplementary Information, Fig. S6. Author information * Author information * Supplementary information Affiliations * Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. * Olga Sirin & * Margaret A. Goodell * Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX 77030, USA. * Olga Sirin, * Georgi L. Lukov, * Rui Mao & * Margaret A. Goodell * Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA. * Orla M. Conneely & * Margaret A. Goodell Contributions This study was developed and designed by O.S., who also performed the experiments and co-wrote the manuscript. G.L.L. and R.M. helped carry out the experiments. O.M.C. provided Nurr1−/− mice and discussion. M.A.G. designed experiments and co-wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Margaret A. Goodell (goodell@bcm.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (636K) Supplementary Information Additional data - MicroRNA-199b targets the nuclear kinase Dyrk1a in an auto-amplification loop promoting calcineurin/NFAT signalling
- ncb 12(12):1220-1227 (2010)
Nature Cell Biology | Letter MicroRNA-199b targets the nuclear kinase Dyrk1a in an auto-amplification loop promoting calcineurin/NFAT signalling * Paula A. da Costa Martins1, 2, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Kanita Salic1, 2, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Monika M. Gladka1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anne-Sophie Armand3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stefanos Leptidis1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hamid el Azzouzi1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Arne Hansen4 Search for this author in: * NPG journals * PubMed * Google Scholar * Christina J. Coenen-de Roo5 Search for this author in: * NPG journals * PubMed * Google Scholar * Marti F. Bierhuizen6 Search for this author in: * NPG journals * PubMed * Google Scholar * Roel van der Nagel6 Search for this author in: * NPG journals * PubMed * Google Scholar * Joyce van Kuik7 Search for this author in: * NPG journals * PubMed * Google Scholar * Roel de Weger7 Search for this author in: * NPG journals * PubMed * Google Scholar * Alain de Bruin8 Search for this author in: * NPG journals * PubMed * Google Scholar * Gianluigi Condorelli9 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria L. Arbones10 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Eschenhagen4 Search for this author in: * NPG journals * PubMed * Google Scholar * Leon J. De Windt2l.dewindt@maastrichtuniversity.nl Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1220–1227Year published:(2010)DOI:doi:10.1038/ncb2126Received04 October 2010Accepted01 November 2010Published online21 November 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg MicroRNAs (miRs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length1, 2. Increasing evidence implicates miRs in myocardial disease processes3, 4, 5, 6, 7, 8, 9, 10, 11. Here we show that miR-199b is a direct calcineurin/NFAT target gene that increases in expression in mouse and human heart failure, and targets the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a), constituting a pathogenic feed forward mechanism that affects calcineurin-responsive gene expression. Mutant mice overexpressing miR-199b, or haploinsufficient for Dyrk1a, are sensitized to calcineurin/NFAT signalling or pressure overload and show stress-induced cardiomegaly through reduced Dyrk1a expression. In vivo inhibition of miR-199b by a specific antagomir normalized Dyrk1a expression, reduced nuclear NFAT activity and caused marked inhibition and even reversal of hypertrophy and fibrosis in mouse models of heart failure. Our results! reveal that microRNAs affect cardiac cellular signalling and gene expression, and implicate miR-199b as a therapeutic target in heart failure. View full text Figures at a glance * Figure 1: Increased expression of miR-199b in cardiac disease. () Northern blot analysis of miR-199b-5p expression in hearts from calcineurin transgenic mice (MHC-CnA), mice subjected to transverse aortic constriction (TAC; left panel), and quantification of corrected northern blot signal (right panel). Rnu6-2 was used as a loading control. () Northern blot analysis of miR-199b-5p expression in non-failing and failing human left ventricular myocardium. () Quantification of Rnu6-2-corrected northern blot miR-199b-5p signals from . () Northern blot analysis of miR-199b-5p expression in diverse mouse organs. () Schematic representation of genomic localization and precursor of hsa-miR-199b, located on the opposite strand in intron 15 of the dynamin-1 (DNM1) gene on chromosome 9 in the human genome. The mature miR-199b-5p strand is conserved among species. () Northern blot analysis of miR-199b-5p in left ventricular myocardium from Nfatc2+/+ (wild-type), MHC-CnA, Nfatc2−/−, and MHC-CnA mice harbouring a null mutation for Nfatc2. () Lucif! erase reporters with 1 kb upstream regions of the miR-199b gene were tested for calcineurin/NFAT responsiveness in transfection experiments. Luciferase reporters including site-directed mutations for the N2a (F2*), N2 (F2**) and N3 (F4*) NFAT binding sites were also tested. () Schematic representation of the NFAT binding site N3 showing its relative location to the start site of miR-199b and evolutionary conservation. () Electrophoretic mobility shift assays on an oligonucleotide probe of the NFAT-like site N3 from the mouse miR-199b gene (NFAT N3) or an oligonucleotide probe with a mutated N3 site (mut NFAT N3), in the presence or absence of an antibody against NFATc3 (NFAT) or control antibody (Ctrl). Unlabelled N3 oligonucleotide was added to determine specific binding. Complexes are indicated by an arrow, retarded complexes (antibody supershift) are indicated by an asterisk. () Chromatin immunoprecipitation demonstrates that endogenous NFATc3 forms a transcriptionally a! ctive complex on the N3-NFAT motif in the miR-199b gene in cal! cineurin transgenic hearts. NFAT, anti-NFATc3 antibody; H3, anti-acetyl histone H3 antibody. *P <.05, compared with the corresponding control group (mean ± s.e.m.). Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 2: miR-199b promotes hypertrophy and targets Dyrk1a in cardiomyocytes. () Northern blot analyses of miR-199b-5p and Rnu6-2 in neonatal rat cardiomyocytes infected with AdLacz, AdCnA, or AdmiR-199b, or transfected with synthetic precursor for miR-199b (pre-miR-199b) for 24 h. () Confocal microscopy images of neonatal rat cardiomyocytes infected with indicated adenoviruses or transfected with pre-miR-199b, nuclei visualized with DAPI and stained with antibodies against α-actinin or ANF. () Quantification of cell surface area from conditions in . () Northern blot analysis of miR-199b-5p and Rnu6-2 in neonatal rat cardiomyocytes transfected with scrambled antimir or antimir-199b, and infected with AdLacz, AdCnA, or treated with phenylephrine (PE, 10 μM) for 24 h. () Confocal microscopy images of AdLacz, AdCnA infected or PE-stimulated neonatal rat cardiomyocytes, pretreated with control antimir or antimir-199b and stained with DAPI or an antibody against α-actinin. () Quantification of cell surface area in conditions in . () Location of miR-199b! -5p binding region in human and mouse Dyrk1a 3′UTR. () Northern blot analyses of miR-199b-5p and Rnu6-2 in stably transfected clones expressing miR-199b in a doxycyclin (Dox)-inducible fashion. Western blots for Dyrk1a or GAPDH in corresponding clones. () Activity assay of luciferase reporter constructs harbouring intact or mutated Dyrk1a 3′UTR after transfection with pre-miR-199b. A scrambled precursor miR was used as a control. () Northern blot analysis for miR-199b-5p and Rnu6-2 and western blot analysis for Dyrk1a and GAPDH of neonatal rat cardiomyocytes unstimulated or stimulated for 24 h with PE (10 μM) or endothelin-1 (ET-1, 100 nM). () Confocal microscopy images of neonatal rat cardiomyocytes infected with an adenovirus expressing a NFATc1-GFP fusion and treated with PE (10 μM), scrambled antimir or antimir-199b, for assessment of nuclear translocation of NFAT. Nuclei were visualized with DAPI, cardiomyocytes visualized with an antibody against α-actinin. () ! Quantification of the percentage GFP-positive nuclei from cond! itions in . *P <0.05, compared with the corresponding control group; #P <0.05, compared with the experimental group (mean ± s.e.m.). Scale bars, 50 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 3: miR-199b overexpression sensitizes the myocardium to pathological cardiac hypertrophy. () Design of transgenic vector (upper panel). Northern blot analysis of miR-199b-5p and Rnu6-2 in hearts from nontransgenic littermates (nTg) or transgenic (Tg) lines overexpressing miR-199b (lower panel). () Gravimetric analysis of corrected heart weights in non-transgenic (nTg), single transgenic (MHC-199b or MHC-CnA) and double transgenic mice (DTg). () Representative image of whole hearts (top panels), Haematoxylin & eosin (H&E)-stained sections of the left ventricular free walls (second panels), high magnification H&E-stained sections (third panels), or Sirius Red stained (lower panels) histological sections of hearts from nTg, MHC-199b, MHC-CnA or DTg mice. () Western blot analysis of endogenous Dyrk1a, GAPDH, phospho-NFATc2 or total NFATc2 in hearts from nTg, MHC-199b, MHC-CnA or DTg mice. Phospho-NFATc2 or total NFATc2 were enriched by immunoprecipitation before blotting. () Real-time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts f! rom nTg, MHC-199b, MHC-CnA or DTg mice. () Gravimetric analysis of corrected heart weights in nTg or MHC-199b Tg mice subjected to sham or transverse aortic constriction (TAC) surgery for 1 week. () Representative image of whole hearts (top panels), H&E-stained (middle panels) or Sirius Red stained (lower panels) histological sections of hearts from nTg, or MHC-199b Tg mice subjected to sham or TAC. (), Real-time PCR analysis of transcript abundance for rcan1-4 in hearts from nTg or MHC-199b Tg mice subjected to sham or TAC. *P <0.05, compared with the corresponding control group; #P <0.05, compared with the experimental group (mean ± s.e.m.). Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 4: Dyrk1a haploinsufficiency exacerbates and miR-199b silencing prevents cardiac remodeling. () Confocal microscopy images of neonatal rat cardiomyocytes infected with indicated adenoviruses or transfected with an siRNA against Dyrk1a (si.Dyrk1a) or a scrambled sequence (si.scr). Nuclei were visualized with DAPI, cardiomyocytes visualized with an antibody against α-actinin. Scale bar, 50 μm. (), Quantification of cell surface area from conditions in . () Gravimetric analysis of corrected heart weights in Dyrk1A+/+ and Dyrk1a haploinsufficient (Dyrk1A+/−) mice subjected to sham or TAC for 1 week. () Representative image of whole hearts (top panels), H&E- (middle panels) or Sirius Red-stained (lower panels) histological sections of Dyrk1A+/+ and Dyrk1A+/− mice subjected to sham or TAC. () Real-time PCR analysis of transcript abundance for rcan1-4 in hearts from Dyrk1A+/+ and Dyrk1A+/− mice subjected to sham or TAC. () Design of antagomir study. () Northern blot analysis of miR-199b-5p and Rnu6-2 expression in hearts from mice treated with vehicle or antagomir-! 199b. () Gravimetric analysis of corrected heart weights in vehicle and antagomir-199b treated mice. () Representative image of whole hearts (top panels), H&E- (middle panels) or Sirius Red-stained (lower panels) histological sections of hearts from vehicle or antagomir-199b-treated mice. () Western blot analysis of Dyrk1a, GAPDH as loading control, phospho-NFATc2 or total NFATc2 in hearts from antagomir-199b or vehicle treated mice. Phospho-NFATc2 or total NFATc2 were enriched by immunoprecipitation before blotting. () Real-time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from antagomir-199b or vehicle-treated mice. *P <0.05, compared with the corresponding control group; #P <0.05, compared with the experimental group (mean ± s.e.m.). Uncropped images of blots are shown in Supplementary Information, Fig. S6. * Figure 5: miR-199b silencing prevents and reverses cardiac remodelling and dysfunction. () Design of antagomir prevention and reversal transverse aortic constriction (TAC) studies. () Representative image of whole hearts (top panels), H&E- (middle panels) or Sirius Red-stained (lower panels) histological sections from hearts of vehicle or antagomir-199b-treated, pressure overloaded mice. () Gravimetric analysis of corrected heart weights in vehicle and antagomir-199b treated mice from the prevention (TACp) and reversal (TACr) study. () Quantification of fractional shortening (FS) by echocardiography in antagomir-199b or vehicle-treated, pressure overloaded mice. () Measurements of the left ventricular internal diameters in systole (LVIDs) by M-mode echocardiography in antagomir-199b or vehicle-treated, pressure overloaded mice. () Representative images of western blot analysis of Dyrk1a and calsequestrin (CSQ) in human non-failing or failing myocardium. () Quantification of calsequestrin corrected Dyrk1a western blot signals from (). () Pearson's correlation re! flecting a linear relationship between miR-199b and Dyrk1A abundance in human hearts. () Model depicting the activation of calcineurin/NFAT signalling under cardiac stress, resulting in a gene program that induces cardiac hypertrophy, remodelling and heart failure. The antagonistic function of Dyrk1a on cardiac NFAT signalling is reduced by NFAT-mediated induction of miR-199b, constituting a pathological feed forward mechanism. An antagomir against miR-199b silences miR-199b, derepresses Dyrk1a, and antagonizes calcineurin/NFAT signalling, cardiac remodelling and heart failure. *P <0.05, compared with the control group; #P <0.05, compared with the experimental group (mean ± s.e.m.). Uncropped images of blots are shown in Supplementary Information, Fig. S6. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work * Paula A. da Costa Martins & * Kanita Salic Affiliations * Hubrecht Institute and Interuniversity Cardiology Institute Netherlands, Royal Netherlands Academy of Sciences, PO Box 85164, Utrecht, The Netherlands. * Paula A. da Costa Martins, * Kanita Salic, * Monika M. Gladka, * Stefanos Leptidis & * Hamid el Azzouzi * Department of Cardiology, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, Maastricht, The Netherlands. * Paula A. da Costa Martins, * Kanita Salic, * Stefanos Leptidis, * Hamid el Azzouzi & * Leon J. De Windt * Centre d'Etude de la Sensori-Motricité, UMR 8194 CNRS, Université Paris Descartes, 75270 Paris Cedex 06, Paris, France. * Anne-Sophie Armand * Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany. * Arne Hansen & * Thomas Eschenhagen * Harlan Laboratories, Horst, PO Box 553, The Netherlands. * Christina J. Coenen-de Roo * Departments of Medical Physiology, University Medical Center Utrecht, PO Box 85500, Utrecht, The Netherlands. * Marti F. Bierhuizen & * Roel van der Nagel * Departments of Pathology, University Medical Center Utrecht, PO Box 85500, Utrecht, The Netherlands. * Joyce van Kuik & * Roel de Weger * Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, PO Box 80125, Utrecht, The Netherlands. * Alain de Bruin * Institute of Biomedical Technologies, Consiglio Nazionale delle Ricerche, 20090 Segrate (MI), Milan, Italy. * Gianluigi Condorelli * Center for Genomic Regulation (CRG) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Centre de Regulació Genòmica, 08003 Barcelona, Spain. * Maria L. Arbones Contributions P.D.C.M, K.S., M.G., A.S., S.L., H.A., A.H., M.B., R.N., J.K and L.D.W. performed the experiments; P.D.C.M, K.S., M.G., A.S., C.C., M.A., T.E. and L.D.W. analysed the data; P.D.C.M., K.S. and L.D.W designed the study; P.D.C.M, K.S. and L.D.W. wrote the manuscript; P.D.C.M. and K.S. contributed equally as joint first authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Leon J. De Windt (l.dewindt@maastrichtuniversity.nl) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - A cytoplasmic dynein tail mutation impairs motor processivity
- ncb 12(12):1228-1234 (2010)
Nature Cell Biology | Letter A cytoplasmic dynein tail mutation impairs motor processivity * Kassandra M. Ori-McKenney1, 2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jing Xu3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Steven P. Gross3, 5sgross@uci.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Richard B. Vallee1, 2, 5rv2025@columbia.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 12,Pages:1228–1234Year published:(2010)DOI:doi:10.1038/ncb2127Received14 July 2010Accepted14 October 2010Published online21 November 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mutations in the tail of the cytoplasmic dynein molecule have been reported to cause neurodegenerative disease in mice. The mutant mouse strain Legs at odd angles (Loa) has impaired retrograde axonal transport, but the molecular deficiencies in the mutant dynein molecule, and how they contribute to neurodegeneration, are unknown. To address these questions, we purified dynein from wild-type mice and the Legs at odd angles mutant mice. Using biochemical, single-molecule, and live-cell-imaging techniques, we find a marked inhibition of motor run-length in vitro and in vivo, and significantly altered motor domain coordination in the dynein from mutant mice. These results suggest a potential role for the dynein tail in motor function, and provide direct evidence for a link between single-motor processivity and disease. View full text Figures at a glance * Figure 1: Purification and biochemical analysis of wild-type and mutant cytoplasmic dynein. () Pellets containing vesicles isolated from the brains of the indicated mice strains were fractionated by flotation through a 2M, 1.5M and 0.6M sucrose step gradient. Vesicles floated to the 0.6–1.5 M interface. Left: immunoblot of samples taken from the pellet (P), 0.6 M sucrose step (0.6), vesicles at the sucrose interface (V) and the 1.5 M sucrose step (1.5 M), using antibodies against the heavy and intermediate chains of dynein (synaptotagmin was used as a marker for vesicles). Right: percentage of heavy chain in vesicles was quantified from band intensities in the immunoblot (n = 3 experiments, data are means ± s.d.). WT; wild-type mice. () Coomassie-stained gel of cytoplasmic dynein purified from the brains of wild-type and Loa+/− mice. () ATPase activity of dynein purified from wild-type and Loa+/− mice as a function of microtubule concentration, at low- and high-ionic strength. Activity data are means ± s.d., determined from n = 3 experiments (10 mM KCl), or! from n = 6 experiments (50 mM KCl) and fitted with Michaelis-Menten kinetics. Left: dynein purified from wild-type (top) or Loa+/− (bottom) mice were incubated with microtubules (MTs) and ATP, as indicated. After centrifugation, binding of dynein was assessed by immunoblotting for heavy chain, intermediate chain and tubulin in the supernatant (S) or microtubule-containing pellet (P) fractions. Input is 20% of total protein. Right: amount of dynein in the microtubule pellet in the absence and presence of ATP. Data are means ± s.d. from n = 3 different experiments per genotype, per ATP condition (Asterisk indicates P < 0.001). Uncropped images of blots are shown in Supplementary Information, Fig. S1. * Figure 2: Single-molecule dynamics of cytoplasmic dynein isolated from wild-type and mutant mice. Dynein was linked to quantum dots using an antibody to the intermediate chain, and then applied to microtubules in the absence of ATP, and monitored by fluorescence microscopy in the presence of 500 μM ATP. () Velocities for quantum dot runs > 200 nm for dynein isolated from wild-type, Loa+/−, and Loa-/- mice (n > 111 quantum dots). () Average run-length at molar ratios of dynein:quantum dots of 1:35 and 1:50. Data are means ± s.e.m, P < 0.001 for dynein isolated from mutant mice, compared with wild-type mice (n = 175, 168 and 166 for wild-type, Loa+/− and Loa−/− mice, respectively). () Left: representative kymographs of dynein-bound quantum dots on microtubules. Top images show initial position of dynein-bound quantum dot on a microtubule (purple). The percentage of quantum dots that exhibited unidirectional and bidirectional dynamics is indicated below the kymographs (n = 175, 168 and 166 for wild-type, Loa+/− and Loa−/− mice, respectively). Scale bars, 1 �! �m (x axis) and 5 s (y axis). Right: distribution of net run lengths. n values and mean square displacement (± s.e.m.) are indicated. * Figure 3: Optical-trap analysis of wild-type and mutant cytoplasmic dynein dynamics in single- and multi-motor regions. The fraction of beads bound to microtubules was used as an indicator for the average number of available motors per bead: ≤ 30% corresponds to single-motor levels and > 50% to multiple-motor levels (Supplementary Information). Dynein-coated beads were positioned on a microtubule using an optical trap. () Distribution of run velocities for beads attached to dynein isolated from wild-type or Loa+/− mice. The mean velocity ± s.e.m. is indicated for dynein isolated from each mouse strain (n = 110 and 135 runs for dynein isolated from wild-type and Loa+/− mice, respectively; P = 0.97). () Representative single-motor stall force traces for dynein purified from mice with the indicated genotypes. After positioning of the dynein-bound beads on a microtubule, a trap stiffness of 2.2 pN per 100 nm was applied. A stall was scored if the bead proceeded away from the trap centre and held its plateau position for > 200 ms before detachment. The mean stalling force ± s.d. is indicat! ed for dynein isolated from each mouse strain (n = 29, 34 and 51 stalls for wild type, Loa+/− and Loa−/− dynein, respectively). The standard deviations from the mean for the stall forces are consistent with the systematic noise of the optical trap, 0.3–0.4 pN. () Distribution of axial step sizes for beads bound to dynein, purified from wild-type and Loa+/− mice, under 2.2 pN per 100 nm applied load (n = 7269 and 1979 axial steps for wild-type and Loa+/− dynein, respectively, P = 0.89). The two peaks represent both forward and backward stepping for dynein isolated from wild-type and Loa+/− mice. The bulk of steps larger than 8 nm in both directions represent unresolved, consecutive 8 nm steps (see Supplemental Information). () Run-lengths of beads were measured at the indicated bead–microtubule binding fractions. Data are means ± s.e.m. Wild-type: n = 38, 64 and 127 runs for binding fraction of 50, 75, and 100%, respectively; Loa+/−: n = 39 and 296 runs fo! r binding fraction of 75 and 100%, respectively. Asterisks ind! icate P < 0.03). Loa+/− run-length at 50% binding fraction was below measurement limit under assay conditions used here (Supplementary Information, Fig. S4c). * Figure 4: Analysis of retrograde transport of lysosomes in wild-type and mutant axons, with theoretical comparison. () Kymographs of retrograde lysosome transport in axons isolated from mice with the indicated genotype. Images used to generate kymographs were representative of processive lysosome runs. Scale bars, 5 mm (x axis) and 5 s (y axis). () Top: simulations of how motor number affects run length. The simulation was constrained by the experimental measurements of single-motor processivity from this study. Simulated data was used to predict number of motors (N; indicated by dotted line) involved in axonal transport of lysosomes, based on in vivo measurements of run-length in wild-type mice (5.27 ± 0.34 μm). Bottom: comparison of in vivo measurements of lysosome run-length (solid bars) and run-lengths calculated from simulations (hatched bars), using an average of 7.7 dynein motors per cargo as calculated from simulation shown at the top. Data are means ± s.e.m., n > 400, 300 and 600 simulated runs for wild type, Loa+/− and Loa−/−, respectively and n = 68, 78 and 55 uninterr! upted retrograde runs in neurons isolated from wild type, Loa+/− and Loa−/− mice, respectively. () Instantaneous and average lysosome velocities ± s.e.m. for each genotype (n = 102, 98 and 111 analysed lysosomes from wild type, Loa+/− and Loa−/− mice, respectively. P < 0.001 for lysosome average velocity in axons from mutant mice, compared with wild-type mouse neurons). * Figure 5: Biophysical and biochemical evidence of altered motor coordination in mutant dynein. () Representative lateral-position traces of beads carried by a single kinesin, or a dynein isolated from wild-type or Loa+/− mouse. () Top: distribution of the changes in lateral position of beads attached to dynein purified from wild-type and Loa+/− mice. Bottom: difference in counts shown at the top demonstrates a significant deviation of mutant dynein from wild-type (n = 32 and 18 runs for wild-type and Loa+/− dynein, respectively; P < 0.05). Error bars represent s.d. () Effect of ATP concentration on the ATPase activity of dynein purified from wild-type and Loa+/− mice, incubated with 10 μM microtubules. Low ATP concentration range indicated in graph on left is expanded in graph on the right. Activities ± s.d. were determined from three different experiments per genotype and fitted with Michaelis-Menten kinetics. () Proposed effect of Loa mutation on motor coordination. In wild-type dynein, the stepping head (green) inhibits the tightly bound head (red) from b! inding ATP. In Loa dynein, the stepping head (green) does not adequately inhibit the tightly bound head (red), which binds ATP prematurely and causes release from the microtubule. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Kassandra M. Ori-McKenney & * Jing Xu Affiliations * Department of Pathology and Cell Biology, Columbia University. New York, NY 10032, USA. * Kassandra M. Ori-McKenney & * Richard B. Vallee * Department of Biological Sciences, Columbia University, New York, NY 10027, USA. * Kassandra M. Ori-McKenney & * Richard B. Vallee * Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA. * Jing Xu & * Steven P. Gross * Co-senior authors. * Steven P. Gross & * Richard B. Vallee Contributions K.M.O.M., J.X., S.P.G. and R.B.V. designed the research. K.M.O.M. and J.X. performed experiments and analysed data. K.M.O.M., J.X., S.P.G. and R.B.V. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Richard B. Vallee (rv2025@columbia.edu) or * Steven P. Gross (sgross@uci.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (2M) Supplementary Information Additional data - Caenorhabditis elegans EFA-6 limits microtubule growth at the cell cortex
- ncb 12(12):1235-1241 (2010)
Nature Cell Biology | Letter Caenorhabditis elegans EFA-6 limits microtubule growth at the cell cortex * Sean M. O'Rourke1seanor@molbio.uoregon.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Sara N. Christensen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce Bowerman1bbowerman@molbio.uoregon.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Cell BiologyVolume: 12,Pages:1235–1241Year published:(2010)DOI:doi:10.1038/ncb2128Received20 April 2010Accepted18 October 2010Published online14 November 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Microtubules are polymers of tubulin heterodimers that exhibit dynamic instability: periods of growth followed by periods of shrinkage1. However, the molecular regulation of dynamic instability remains elusive. Here, we show that EFA-6, a cortically-localized protein2, limits the growth of microtubules near the cell cortex of early embryonic cells from Caenorhabidits elegans, possibly by inducing microtubule catastrophes. Compared with wild type, embryos lacking EFA-6 had abnormally long and dense microtubules at the cell cortex, and growing microtubule plus ends resided at the cortex for up to five-fold longer. Loss of EFA-6 also caused excess centrosome separation and displacement towards the cell cortex early in mitosis, and subsequently a loss of anaphase spindle-pole oscillations and increased rates of spindle elongation. The centrosome separation phenotype was dependent on the motor protein dynein, suggesting a possible link between the modulation of microtubule dynami! cs at the cortex and dynein-dependent force production. EFA-6 orthologues activate ARF6-type GTPases to regulate vesicle trafficking3. However, we show that only the C. elegans EFA-6 amino-terminus is both necessary and sufficient to limit microtubule growth along the cortex, and that this function is independent of ARF-6. View full text Figures at a glance * Figure 1: Loss of EFA-6 affects early microtubule-dependent processes. () Images from one-cell embryos expressing GFP–γ-tubulin and GFP–histone reporter fusions to mark centrosomes and chromosomes, respectively. Times indicated are relative to pronuclear meeting, or to paternal pronuclear envelope breakdown in the dhc-1(RNAi); efa-6(RNAi) and tba-1(or346dm,ts); efa-6(RNAi) embryos in which pronuclei did not meet. Multiple z sections were captured every 4–10 s and merged to give the images. Red arrowheads indicate localization of centrosomes that have detached from the male pronucleus. () Intercentrosomal distance over time during pronuclear migration for representative embryos. Spindle poles were tracked in three dimensions over time and the intercentrosomal distances were plotted. dhc-1(or195ts) and tba-1(or346dm,ts) mutant embryos were imaged for 30–120 min after shifting worms from 15 °C to 26 °C. The wild-type and dhc-1(or195ts) strains were grown on control RNAi-expressing bacteria. () Images (left) and plots of spindle pole tra! nsverse positions (right) of GFP–β-tubulin-expressing embryos. Left: three z sections 1 μm apart were captured every 2 s and merged. The two images of each embryo represent the extreme transverse distances travelled by the spindle poles during one oscillation. Right: spindle pole transverse positions were determined every 2 s for 152 s during anaphase. The x axes represent time. () Distances between spindle poles were plotted over the course of 6 min, starting before anaphase onset (corresponding to nuclear–centrosomal rotation and nuclear envelope breakdown) in a strain expressing GFP–γ-tubulin and GFP–histone. Each of the efa-6(RNAi) embryos had normal centrosome attachment to the pronuclei by this time. The time series were aligned at anaphase onset (dashed line). Images were captured every 2 s. The bottom plot indicates the rate of spindle elongation with respect to time. Data are means and the shaded regions represent the s.e.m. with a confidence interval of! 0.95. Scale bars, 10 μm. * Figure 2: EFA-6 prevents long cortical microtubules in one- and two-cell embryos. () Images from wild-type and efa-6(tm3124) embryos expressing GFP–β-tubulin. Three z sections with 0.5 μm spacing were captured and merged to generate the images. Shown for each time point are images captured at the cortical focal plane (left) and the central focal plane (right). Top row: one-cell-stage embryos, middle row: two-cell-stage embryos, Bottom row: 12–16-cell-stage embryos. Arrowheads indicate examples of enhanced cortical GFP–β-tubulin in central focal planes. () Left: representative images of wild-type and efa-6(tm3124) embryos, expressing GFP–β-tubulin, at the cortical focal plane (left) and central focal plane (right). Right: cortical microtubule lengths were determined 100–105 s after pronuclear meeting; 107 microtubules were measured from 9 embryos for the wild type, and 118 microtubules were measured from 10 embryos for efa-6(tm3124). Data are means ± s.e.m. with a confidence interval of 0.95. Scale bars, 10 μm. * Figure 3: EFA-6 limits microtubule growth at the cortex. () Images of embryos, expressing EBP2–GFP and mCherry–histone, at the cortical focal plane after pronuclear meeting. Top, single-frame images; bottom, images merged from five frames, captured at 1 s intervals. At the cortical plane, wild-type embryos have EBP-2–GFP puncta that tracked for short times as seen in the merged time points. Embryos lacking EFA-6 demonstrated EBP-2–GFP puncta tracking along the cortex for extended periods, which resulted in lines of puncta in the projected image. See Supplementary Information, Movie S6 for the entire time series. Scale bar, 10 μm. ( Single cortical EBP-2–GFP puncta were tracked and imaged in wild-type and efa-6(tm3124) embryos. Each image shows the position of EBP-2–GFP puncta in an area of the cortex at the indicated times. Scale bar, 4 μm. () Residency time of EBP-2–GFP puncta at the cortex (number of plus ends; 63, wild-type, 117 efa-6(tm3124)). EBP-2–GFP puncta were tracked over time as shown in . () Microtubu! le dynamics in wild-type and efa-6(tm3124) embryos. Dynamics were quantified by imaging embryos expressing the EBP-2–GFP reporter. Data are means ± s.e.m. with a confidence interval of 0.95. Microtubule plus-end residence at the cortex, astral microtubule nucleation rate, and the astral microtubule polymerization rate were determined at pronuclear meeting, the numbers of microtubule plus ends at the cortex was determined 15 s after pronuclear meeting. * Figure 4: Determination of the EFA-6 microtubule-regulating residues. () Effect of EFA-6 domain deletion on spindle oscillations. Left: schematic representations of the indicated EFA-6 constructs. EFA-6.a; wild-type EFA-6 fused to GFP, and the construct used to make the mutants and other constructs. EFA-6.a(E447K); Glu 447 of EFA-6 substituted with a lysine residue. sec7Δ, CCΔ, C-termΔ, PHΔ and N-termΔ; deletion of the indicated domains, residues 684–816 (C-termΔ) or residues 2–352 (N-termΔ) from EFA-6. N-term–CAAX; amino-acid residues 1–353 of EFA-6 fused to the CAAX peptide. N-term–SAAX; amino-acid residues 1–353 of EFA-6 fused to SAAX, a mutant CAAX domain. Conserved motif–CAAX; a start codon and amino-acid residues 26–60 of EFA-6 fused to CAAX. Plasmids encoding GFP or the constructs were transformed into efa-6(tm3124) mutant worms. Middle: spindle pole transverse positions were determined every 1 s for 152 s during anaphase. Right: fluorescence microscopy images of a representative embryo (mid-embryonic z section) d! emonstrating localization of the GFP-tagged proteins at telophase/cytokinesis (for the wild-type embryo, a DIC image is presented). () Top: alignment of the conserved EFA-6 N-terminal motif from several animal phyla and species. Bottom: schematic representation of the position of the domain in the context of the full-length proteins of the nematode C. elegans, the insect Drosophila melanogaster and the mollusk Lottia gigantea. () Images of mCherry–TBA-1-labelled cortical microtubules in one-cell embryos were obtained by merging three z sections (each section 0.5 μm apart). Dashed red line indicates the embryo boundary in the bottom left image. Scale bars, 10 μm. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions GenBank * NP_001097874.1 * XP_001943684.1 * XP_001603899.1 * XP_969387.1 * XP_002425811.1 * XP_001865870.1 * ABIR01001549 * XP_001899191.1 * NP_001122818.1 * ABLG01000531.1_contig530 * NCBI_GNO_6700040 * scaffold_117:275944-280349 * fgenesh2_pg.C_sca_5000157 * CJ976727 * PPA21096 Author information * Accession codes * Author information * Supplementary information Affiliations * Institute of Molecular Biology, 1229 University of Oregon, Eugene, Oregon 97403, USA. * Sean M. O'Rourke, * Sara N. Christensen & * Bruce Bowerman Contributions S.M.O. and B.B. designed the experiments and wrote the paper. S.M.O. conceived the project, performed the experiments and analysed the data. Microscopy and strain generation were also contributed by S.N.C. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Sean M. O'Rourke (seanor@molbio.uoregon.edu) or * Bruce Bowerman (bbowerman@molbio.uoregon.edu) Supplementary information * Accession codes * Author information * Supplementary information Movies * Supplementary Movie 1 (3M) Supplementary Information * Supplementary Movie 2 (2M) Supplementary Information * Supplementary Movie 3 (3M) Supplementary Information * Supplementary Movie 4 (2M) Supplementary Information * Supplementary Movie 5 (8M) Supplementary Information * Supplementary Movie 6 (6M) Supplementary Information * Supplementary Movie 7 (1M) Supplementary Information PDF files * Supplementary Information (2M) Supplementary Information Additional data - AKAP-Lbc enhances cyclic AMP control of the ERK1/2 cascade
- ncb 12(12):1242-1249 (2010)
Nature Cell Biology | Letter AKAP-Lbc enhances cyclic AMP control of the ERK1/2 cascade * F. Donelson Smith1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lorene K. Langeberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Cristina Cellurale2 Search for this author in: * NPG journals * PubMed * Google Scholar * Tony Pawson3 Search for this author in: * NPG journals * PubMed * Google Scholar * Deborah K. Morrison4 Search for this author in: * NPG journals * PubMed * Google Scholar * Roger J. Davis2 Search for this author in: * NPG journals * PubMed * Google Scholar * John D. Scott1scottjdw@u.washington.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Cell BiologyVolume: 12,Pages:1242–1249Year published:(2010)DOI:doi:10.1038/ncb2130Received15 April 2010Accepted13 October 2010Published online21 November 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mitogen-activated protein kinase (MAPK) cascades propagate a variety of cellular activities1. Processive relay of signals through RAF–MEK–ERK modulates cell growth and proliferation2, 3. Signalling through this ERK cascade is frequently amplified in cancers, and drugs such as sorafenib (which is prescribed to treat renal and hepatic carcinomas) and PLX4720 (which targets melanomas) inhibit RAF kinases4, 5. Natural factors that influence ERK1/2 signalling include the second messenger cyclic AMP6, 7. However, the mechanisms underlying this cascade have been difficult to elucidate. We demonstrate that the A-kinase-anchoring protein AKAP-Lbc and the scaffolding protein kinase suppressor of Ras (KSR-1) form the core of a signalling network that efficiently relay signals from RAF, through MEK, and on to ERK1/2. AKAP-Lbc functions as an enhancer of ERK signalling by securing RAF in the vicinity of MEK1 and synchronizing protein kinase A (PKA)-mediated phosphorylation of Ser 838! on KSR-1. This offers mechanistic insight into cAMP-responsive control of ERK signalling events. View full text Figures at a glance * Figure 1: Characterization of AKAP-Lbc–KSR-1 interactions. () Lysates from HEK293 cells transfected with empty vector or a plasmid encoding Flag-AKAP-Lbc were subject to immunoprecipitation (IP) with anti-Flag antibodies. Proteins were resolved by SDS–PAGE and Coomassie staining, and identified by MS/MS spectrometry. () Lysates from HEK293 cells expressing HA–KSR-1, and transfected with control vector or vector encoding Flag–AKAP-Lbc, were immunoprecipitated using anti-Flag and proteins were identified by immunoblotting. () NIH3T3 cells were transfected with control vectors or vectors encoding HA–KSR-1. Cell lysates were immunoprecipitated with anti-HA, and indicated proteins were identified by immunoblotting. () NIH3T3 lysate and pre-immune or anti-AKAP-Lbc immunoprecipitates were immunoblotted with antibodies against KSR-1 (top) and AKAP-Lbc (bottom). () NIH3T3 lysate and control immunoglobulin G (IgG) or anti-AKAP-Lbc immunoprecipitates were immunoblotted with antibodies against the indicated proteins. () Schematic repres! entation of AKAP-Lbc fragments used to construct GST fusion proteins for the pulldown experiments. Shaded fragment indicates KSR-1-binding fragment, as determined in and . () GST–AKAP-Lbc fragments were used as bait in pulldown experiments using HEK293 lysates. KSR-1 binding was detected by immunoblot (top). GST fusion proteins were resolved by SDS–PAGE and Ponceau S staining (bottom). () GST–AKAP-Lbc fragments were used as bait in pulldown experiments with in vitro-translated recombinant KSR-1. KSR-1 binding was detected by immunoblot (top). GST-fusion proteins were resolved by SDS–PAGE and Ponceau S staining (bottom). () Schematic representation of KSR-1 fragments used in . Shaded fragment indicates AKAP-Lbc-binding fragment as determined in . () Pyo-tagged KSR-1 fragments were co-expressed with Flag–AKAP-Lbc in HEK293 cells. Complexes were immunoprecipitated with anti-Pyo. Proteins were detected by immunoblotting with antibodies against the indicated proteins. ! () Schematic representation of EKAR, a FRET-based reporter for! ERK activity16. () Time-lapse microscopy images of FRET signals in HEK293 cells expressing EKAR alone (bottom), or with AKAP-Lbc–mCherry (top), at indicated times after addition of EGF (min). Scale bars, 10 μm. () Quantification of normalized YFP/CFP ratio from a FRET experiment as performed in . Black and white bars indicate addition of EGF and UO126, respectively. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 2: AKAP-Lbc anchors RAF. () Lysates from HEK293 cells expressing B-Raf and either GFP (left) or AKAP-Lbc–GFP (right) were immunoprecipitated with anti-GFP. The indicated proteins were identified by immunoblotting with antibodies against the indicated proteins. Bottom two panels indicate immunoblot of input lysate. () NIH3T3 lysate and pre-immune or anti-AKAP-Lbc immunoprecipitates were immunoblotted with antibodies against B-Raf (top) and AKAP-Lbc (bottom). () Schematic representation of the AKAP-Lbc fragments used to construct GST fusion proteins for the pulldown experiments. Shaded area indicates B-RAF binding fragments, as determined in . () GST–AKAP-Lbc fragments were used as bait in pulldown experiments using in vitro-translated B-Raf. B-Raf binding was detected by immunoblot (top). GST-fusion proteins were resolved by SDS–Page and Ponceau S staining (bottom). () Cells were transfected, and lysates were immunoprecipitated, as in . The immunoprecipitated complexes were then used in a kinas! e assay, with kinase-inactive GST–MEK1 as a substrate. Top: immunoblot of kinase assay, using antibodies against the indicated proteins. MEK1 phosphorylation was assessed with antibodies specific to MEK phosphorylated at Ser 218 and Ser 222. Bottom: the phosphorylated MEK-1 band was quantified by densitometry and normalized to the control cells. Data are means ± s.e.m. A.U.; arbitrary units. () HEK293 cells were transfected with vectors encoding HA–MEK1 and then co-transfected with vectors encoding AKAP-Lbc–GFP and HA–KSR-1, or empty vector controls, as indicated. Cell lysates were immunoprecipitated with anti-GFP, resolved by SDS−PAGE and immunoblotted with antibodies against MEK1, AKAP-Lbc or KSR-1, as indicated. MEK1 levels in lysates were confirmed by immunoblotting (bottom). Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 3: AKAP-Lbc enhances signal relay through the ERK kinase cascade. () HEK293 cells were transfected with vectors encoding HA–MEK1 and Flag–B-Raf along with increasing amounts of plasmid encoding AKAP-Lbc–GFP. Top: immunoblot of cell lysates used to detect the indicated proteins. Bottom: quantification of the phosphorylated MEK1 band (relative to total MEK) by densitometry (data are means ± s.e.m.). (–) NIH3T3 cells were transfected with a vector encoding AKAP-Lbc–GFP (right) or control vector (left). Cells were fixed and immunostained with antibodies against phosphorylated ERK1/2 (, ), or GFP fluorescence was observed (, ). is a merge of and , and is a merge of and ; in both DAPI was used as a nuclear marker. Scale bars, 20 μm. () Time-lapse microscopy images of FRET signals in HEK293 cells expressing EKAR and transfected with control siRNA oligonucleotides (top) or siRNA oligonucleotides against AKAP-Lbc, at indicated times after addition of EGF (min). Scale bars, 20 μm. () Quantification of normalized YFP/CFP ratio from a FR! ET experiment as performed in . Black bar indicates addition of EGF. Data are means ± s.e.m. Inset: immunoblot of lysates from cells transfected with control or AKAP-Lbc siRNA. () HEK293 cells were transfected with vectors expressing EKAR and AKAP-Lbc–mCherry. Cells were pre-treated with Ht31 peptide (red) or vehicle as a control (black) before treatment with EGF and FRET recording. Normalized YFP/CFP ratios are indicated. Data are means ± s.e.m. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 4: PKA phosphorylation of KSR-1. () A plasmid encoding KSR-1 was transfected into HEK293 cells, and cells were treated with forskolin and IBMX, and H-89, as indicated. Top: cell lysates were immunoblotted with antibodies against the indicated proteins. Bottom: ERK1/2 activation was measured by quantification of the phosphorylated ERK1/2 bands by densitometry. Values were normalized to total ERK1/2 levels. Data are means ± s.e.m. () Lysates from HEK293 cells expressing HA–KSR-1 were immunopreciptated with anti-HA. Top: autoradiograph of immunoprecipitated complexes incubated with [γ-32P]ATP. Immunoprecipitates were also incubated with cAMP and PKI (to block PKA activity) as indicated. Loading controls are also shown. Bottom: bands from the autoradiograph were quantified by densitometry. Data are means ± s.e.m. () HA–KSR-1 was co-expressed with AKAP-Lbc or AKAP-LbcΔPKA in HEK293 cells. Cell lysates were immunoprecipitated with anti-HA. Top: autoradiograph of immunocomplexes incubated with [γ-32P]ATP,! and with cAMP and PKI as indicated. Loading controls are shown. Bottom: bands from the autoradiograph were quantified by densitometry. Data are means ± s.e.m. () Sequence alignment of a conserved consensus PKA phosphorylation site in KSR-1. The target serine is indicated. () Lysates of cells expressing wild-type (WT) KSR-1 or a S838A mutant were immunoprecipitated with anti-HA. Top: autoradiograph of immunocomplexes incubated with [γ-32P]ATP, and with PKA and PKI as indicated. Loading controls are shown. Bottom: bands from the autoradiograph were quantified by densitometry. Data are means ± s.e.m. Uncropped images of blots are shown in Supplementary Information, Fig. S7. * Figure 5: Phosphorylation of KSR-1 on Ser 838 controls ERK1/2 signalling. () KSR-1 immunoprecipitates from KSR-1−/− mouse embryonic fibroblasts (MEFs) and MEFs expressing wild-type KSR-1 were screened for AKAP-Lbc, PKA RII and KSR-1 by immunoblotting. () Time-course of EGF-stimulated ERK1/2 activity in KSR-1−/− MEFs and MEFs expressing wild-type KSR-1 or KSR-1S838A. Starved cells were treated with EGF for the indicated times. ERK activation was assessed by immunoblotting for phosphorylated ERK1/2, ERK1/2, KSR-1 and AKAP-Lbc. () Quantification of phosphorylated ERK1/2 bands by densitometry, from experiment carried out as in (data are means ± s.e.m.). () Time-lapse microscopy images of FRET signals in HEK293 cells expressing EKAR, and AKAP-Lbc and wild-type KSR-1 (top), or AKAP-Lbc and KSR-1S838A (bottom), at indicated times after addition of EGF (min). Scale bars, 20 μm. () Quantification of YFP/CFP ratios for experiment performed as in . Black bar indicates addition of EGF. Data are means ± s.e.m. () HEK293 cells expressing wild-type KS! R-1 or KSR-1S838A were starved and stimulated with forskolin/IBMX, as indicated. ERK activation was measured by immunoblotting of the cell lysates. () Quantification of phosphorylated ERK1/2 bands by densitometry from experiments performed in . Data are normalized to the density of the total ERK1/2 bands. Data are means ± s.e.m. (asterisk indicates P < 0.05, paired t-test). () KSR-1−/− MEFs and MEFs expressing wild-type or KSR-1S838A were treated with forskolin and IBMX, and H-89, as indicated. ERK activation was assessed by immunoblotting for phosphorylated ERK1/2, ERK1/2, KSR-1 and AKAP-Lbc. () Quantification of phosphorylated ERK1/2 bands by densitometry from experiments performed in . Data are normalized to the density of the total ERK1/2 bands. Data are means ± s.e.m. (asterisk indicates P < 0.001, ANOVA). ( Immunofluorescence microscopy analysis of ERK activation. KSR-1−/− MEFs (–), and MEFs expressing KSR-1 (–) or KSR-1S838A (–) were starved, treated ! with forskolin/IBMX for 10 min, fixed, and immunostained for p! hosphorylated ERK1/2 and total ERK1/2, as indicated. Scale bars, 20 μm. () Schematic representation of the AKAP-Lbc–KSR-1 core unit directing growth factor and cAMP signals through the ERK signalling network. Uncropped images of blots are shown in Supplementary Information, Fig. S7. Author information * Author information * Supplementary information Affiliations * Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, 1959 Pacific Avenue NE, Seattle, WA 98195, USA. * F. Donelson Smith, * Lorene K. Langeberg & * John D. Scott * Howard Hughes Medical Institute, University of Massachusetts, Program in Molecular Medicine, 373 Plantation Street Worcester, MA 01605, USA. * Cristina Cellurale & * Roger J. Davis * Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 1084-600 University Avenue, Toronto, Ontario, M5G 1X5, Canada. * Tony Pawson * Laboratory of Cell and Developmental Signalling, NCI-Frederick, Building 560, Room 22-90B, Frederick, MD 21702, USA. * Deborah K. Morrison Contributions F.D.S and L.K.L performed all experiments. F.D.S., L.K.L and J.D.S. designed and analysed all experiments and wrote the manuscript. T.P. performed mass spectrometry. D.K.M. generated KSR-1 rescue MEFs. R.J.D. and C.C. developed and characterized the FRET reporters. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * John D. Scott (scottjdw@u.washington.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Information (805K) Supplementary Information Additional data - The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing
- ncb 12(12):1249 (2010)
Nature Cell Biology | Erratum The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing * Joon-Il Jun Search for this author in: * NPG journals * PubMed * Google Scholar * Lester F. Lau Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature Cell BiologyVolume: 12,Page:1249Year published:(2010)DOI:doi:10.1038/ncb1210-1249 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Cell Biol.12 DOI:10.1038/ncb2070; published online 6 June 2010; corrected online, 12 November 2010 In the version of this article initially published online and in print, the labels BSA and CCN1 in Fig. 2a were swapped. The correct labelling is shown below. This error has been corrected in both the HTML and PDF versions of the article. Additional data
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