11 research outputs found
UNDERSTANDING THE ROLE OF MITOTIC KINESIN MKLP2 AND AURORA B KINASE IN CYTOKINETIC ABSCISSION
Ph.DDOCTOR OF PHILOSOPHY (DUKE
Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis
10.1371/journal.pone.0064826PLoS ONE86
Cdk1 Coordinates Timely Activation of MKlp2 Kinesin with Relocation of the Chromosome Passenger Complex for Cytokinesis
The chromosome passenger complex (CPC) must relocate from anaphase chromosomes to the cell equator for successful cytokinesis. Although this landmark event requires the mitotic kinesin MKlp2, the spatiotemporal mechanistic basis remains elusive. Here, we show that phosphoregulation of MKlp2 by the mitotic kinase Cdk1/cyclin B1 coordinates proper mitotic transition with CPC relocation. We identified multiple Cdk1/cyclin B1 phosphorylation sites within the stalk and C-terminal tail that inhibit microtubule binding and bundling, oligomerization/clustering, and chromosome targeting of MKlp2. Specifically, inhibition of these abilities by Cdk1/cyclin B1 phosphorylation is essential for proper early mitotic progression. Upon anaphase onset, however, reversal of Cdk1/cyclin B1 phosphorylation promotes MKlp2-CPC complex formation and relocates the CPC from anaphase chromosomes for successful cytokinesis. Thus, we propose that phosphoregulation of MKlp2 by Cdk1/cyclin B1 ensures that activation of MKlp2 kinesin and relocation of the CPC occur at the appropriate time and space for proper mitotic progression and genomic stability
MKlp2 localizes to the growing furrow in a myosin-II-dependent manner, and it is required for cell polarization during monopolar cytokinesis.
<p>(<b>A</b>) Immunofluorescence analysis of HeLa cells before (panel a) or during monopolar cytokinesis (panels b-d). Asynchronously growing HeLa cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 15 min before fixation in ice-cold methanol. (<b>B, C</b>) HeLa cells were transfected with the indicated siRNAs for 20 h before the addition of monastrol, and monopolar cytokinesis was induced as described in panel <b>A</b>. For panel <b>B</b>, HeLa cells stably expressing GFP-α-tubulin were used. For panels <b>A–C</b>, images were acquired using 3D-SIM. (<b>D</b>) The average percentages calculated based on three independent experiments using non-polarized, polarized (determined by cortically localized Aurora B from panel <b>C</b>) or furrowed cells (determined by myosin-II) are shown with error bars (<i>n</i>>100 cells per condition). (<b>E</b>) Time-lapse live-cell imaging. HeLa cells were transfected with the indicated siRNAs together with the vectors encoding GFP-UtrCH to monitor cortical changes and were subjected to monopolar cytokinesis. Arrows indicate the site where a typical polarized furrow was formed and progressed. (<b>F</b>) Monopolar cells (from panel <b>E</b>) were scored as cells that completed monopolar cytokinesis (polarized MPC) or failed without polarizing the cortex (non-polarized with no MPC). The average percentages based on three independent experiments (total <i>n</i>>100 per condition, +/− standard deviation) are shown. Images were acquired using 2D-SIM. White bars represent 5 µm.</p
Recruiting Aurora B to the cell cortex and the growing furrow by MKlp2 is required for stable polarization and furrow formation during monopolar cytokinesis.
<p>(<b>A–C</b>) Immunofluorescence analysis of Dox-inducible HeLa cells undergoing monopolar cytokinesis. Flag-MKlp2 expression was induced with Dox for 20 h after the transfection of MKlp2 siRNA. After Dox-treatment, cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 20 min before fixation in ice-cold methanol. (<b>A, B</b>) Flag-MKlp2(1-890) localized to the cell cortex and at the growing furrow together with Aurora B (A, panels b-d) and INCENP (<b>B</b>, panels a, b), but Flag-MKlp2(1-842) only localized to the ends of monopolar spindles together with Aurora B (<b>A</b>, panels f-h) and INCENP (<b>B</b>, panels c, d). (<b>C</b>) In contrast to Aurora B and INCENP in panels <b>A</b> and <b>B</b>, centralspindlin and PRC1 were efficiently localized to the ends of monopolar spindles in cells expressing Flag-MKlp2(1-890) and MKlp2(1-842). This finding indicates that Flag-MKlp2(1-842) is selectively defective in targeting Aurora B and INCENP (thus, most likely the CPC) from the monopolar spindles to the cell cortex. (<b>D</b>) The average percentages based on three independent experiments of non-polarized, polarized (determined by the direction of Flag-MKlp2 and the monopolar spindles towards the cell cortex) or furrowed cells (determined by the presence of Flag-MKlp2 and myosin-II in the furrow) expressing Flag-MKlp2 (<i>n</i>>100 per condition, +/− standard deviation) are shown.</p
MKlp2 is a novel binding partner of myosin-II <i>in vivo</i> and <i>in vitro</i>.
<p>(<b>A</b>) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. (<b>B</b>–<b>D</b>) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. (<b>E</b>) Autoradiography of <i>in vitro</i>-translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total <i>in vitro</i>-translated product is shown. (<b>F</b>) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with <i>in vitro</i>-polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.</p
MKlp2 promotes and maintains efficient ingression of the cleavage furrow.
<p>(<b>A</b>) Synchronized HeLa cells transfected with the indicated siRNAs were subjected to time-lapse live-cell imaging. Arrows indicate the ingressed cleavage furrow. (<b>B</b>) The cytokinesis progression of cells (<i>n</i>>40) that were selected at random and the time they spent in ingression before regression is plotted (top graph). All HeLa cells depleted of MKlp1 or MKlp2 formed a cleavage furrow and ingressed followed by furrow regression and cytokinesis failure, while none of the control cells showed furrow regression after ingression. The average duration of the furrow in ingression is based on three independent experiments (total <i>n</i>>100 per condition, +/− standard deviation) (bottom graph). To determine the statistical significance of the duration of the furrow in ingression Student’s <i>t</i>-test was performed. <i>P</i> values are indicated. (<b>C</b>) Immunoblot analysis of total cell lysates from panel <b>A</b>. C: control, M1: MKlp1, M2: MKlp2. Relative band intensities to control siRNA are shown in the bottom of each panel. (<b>D</b>) Immunofluorescence analysis using asynchronously grown HeLa cells was performed at 30 h after transfection with the indicated siRNAs. Cells in anaphase are shown. Images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm.</p
MKlp2 recruits and provides continuous Aurora B kinase activity to the growing furrow for furrow propagation and completion.
<p>(<b>A–D</b>) Time-lapse live-cell images. HeLa cells were transfected with expression vectors encoding mCherry-MKlp2 and GFP-UtrCH (cortical marker), and 24 h after transfection, the cells were subjected to monopolar cytokinesis. In panel <b>A</b>, arrows indicate where a typical polarized furrow was completed. Asterisks indicate the site where mCherry-MKlp2 accumulated at the cell cortex and the furrow. Arrow heads in panels <b>A</b>, <b>C</b> and <b>D</b> denote the sites of the growing furrow before adding ZM447439 or hesperadin. For panel <b>B</b>, Purv A and ZM447439 were added concurrently. For panels <b>C</b> and <b>D</b>, ZM447439 or hesperadin was added sequentially after purvalanol A treatment for 10 min. The fate of the same cell was continuously monitored. White bars represent 5 µm. (<b>E</b>) Monopolar cells (from <b>A–D</b>) were scored as cells that completed monopolar cytokinesis (polarized MPC), formed ectopic furrows without a polarized cortex (non-polarized with ectopic furrowing), no significant furrowing activity without a polarized cortex (non-polarized without furrowing), or formed polarized furrows but subsequently regressed (regressed after polarization). The average percentages based on three independent experiments (total <i>n</i>>100 per condition, +/− standard deviation) are shown. (<b>F</b>) Proposed model. (a) In monopolar cytokinesis, inhibition of Cdk1 triggers a symmetry-breaking reaction that initiates polarization of the monopolar spindles and the cell cortex. Although monopolar spindles are symmetrically positioned, they become asymmetric upon Cdk1 inhibition. Thus, the original event triggering the asymmetry of monopolar spindles remains unclear. (b) During this reaction, MKlp2 mediates Aurora B translocation from the monopolar spindles (or the spindle midzone) towards the cell cortex (or the equatorial cortex) in an interdependent manner. At the ends of monopolar spindles that contact the cell cortex, MKlp2 may act as the bridge between the asymmetrically polarizing monopolar spindles and the actomyosin filaments at the cell cortex. This event may stabilize the polarizing monopolar spindles towards the cell cortex. In turn, it facilitates a rapid accumulation of MKlp2-Aurora B to the cell cortex in a polarized manner. This cortical accumulation of MKlp2-Aurora B promotes furrow formation (or might constrict contractile ring in bipolar cytokinesis) via the kinase activity of Aurora B towards currently unidentified target(s). (c) Once the furrow is formed, MKlp2 targets Aurora B to actomyosin filaments in the gap region between the stably polarized monopolar spindles and the furrowing cortical cap. This event may continuously provide Aurora B kinase activity to the growing furrow for propagation and completion. Interestingly, the kinase activity of Aurora B at the growing furrow may also be essential for maintaining the monopolar spindles polarized towards the furrow (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064826#pone.0064826.s010" target="_blank">Movie S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064826#pone.0064826.s011" target="_blank">S2</a>), indicating that positive feedback loops may exist that maintain polarization between the monopolar spindles and the growing furrow.</p
Flexibility of NS5 methyltransferase-polymerase linker region is essential for dengue virus replication
We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5’s conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication