Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway

BACKGROUND: Cytokinesis is the final step of cell division taking place at the end of mitosis during which the cytoplasmic content and replicated chromosomes of a cell are equally partitioned between the two daughter cells. This process is achieved by the formation and the ingression of an actomyosin contractile ring under the control of equatorial microtubules. The mechanisms of contractile ring formation are not fully understood but involve recruitment of preexisting actin filaments and de novo actin polymerisation. RESULTS: In this study, we evaluated the role of the actin nucleation factor, Arp2/3 complex, during cytokinesis. We found that the Arp2/3 complex is recruited late to the cleavage furrow suggesting a potential involvement of Arp2/3 complex during this process. Furthermore, wiskostatin a potent inhibitor of N-WASP activity towards the Arp2/3 complex blocked cytokinesis without affecting mitosis. Nonetheless, this inhibition could not be reproduced using alternative approaches targeting the N-WASP/Arp2/3 complex pathway. CONCLUSION: We conclude that the wiskostatin induced defective cytokinesis does not occur through the inhibition of the N-WASP/Arp2/3 pathway. Wiskostatin is likely to either directly target other proteins required for cytokinesis progression or alternately wiskostatin bound to N-WASP could affect the activity of other factors involved in cytokinesis

Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis

Phosphorylation of the Ran GTPase on Serine-135 by PAK4 changes Ran’s association with its regulatory proteins and its ability to induce microtubule asters

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway

Abstract Background Cytokinesis is the final step of cell division taking place at the end of mitosis during which the cytoplasmic content and replicated chromosomes of a cell are equally partitioned between the two daughter cells. This process is achieved by the formation and the ingression of an actomyosin contractile ring under the control of equatorial microtubules. The mechanisms of contractile ring formation are not fully understood but involve recruitment of preexisting actin filaments and de novo actin polymerisation. Results In this study, we evaluated the role of the actin nucleation factor, Arp2/3 complex, during cytokinesis. We found that the Arp2/3 complex is recruited late to the cleavage furrow suggesting a potential involvement of Arp2/3 complex during this process. Furthermore, wiskostatin a potent inhibitor of N-WASP activity towards the Arp2/3 complex blocked cytokinesis without affecting mitosis. Nonetheless, this inhibition could not be reproduced using alternative approaches targeting the N-WASP/Arp2/3 complex pathway. Conclusion We conclude that the wiskostatin induced defective cytokinesis does not occur through the inhibition of the N-WASP/Arp2/3 pathway. Wiskostatin is likely to either directly target other proteins required for cytokinesis progression or alternately wiskostatin bound to N-WASP could affect the activity of other factors involved in cytokinesis.</p

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway-1

or indicated concentrations of wiskostatin. Cells were harvested at indicated time points and DNA content determined by FACS analysis. A similar scale was used for each histogram. This result is representative of at least 3 independent experiments. B. HeLa cells synchronised as previously described were treated with vehicle (-) or 10 μM wiskostatin (+) and harvested at indicated time points. Cyclin B1, phosphorylation of Ser 10 of histone H3 and cofilin levels were determined by immunoblot analysis with specific antibodies. This result is representative of at least 5 independent experiments. C. HeLa cells stably expressing GFP-tagged histone H2B were synchronised as previously described, treated with vehicle (Control) or 10 μM wiskostatin (Wiskostatin 10 μM) and fixed after 300 minutes. F-actin was stained and nuclei per cell determined. Percentage of binucleated cells was then calculated. 536 cells treated with vehicle and 569 cells treated with wiskostatin were count from three independent experiments. Error bars represent standard error of the mean (SEM). D. Assynchronous HeLa cells stably expressing GFP-tagged histone H2B were treated with vehicle (Control) or 5 μM wiskostastin (Wiskostatin 5 μM) for 24 hours and were then fixed. Percentages of binucleated cells were determined as previously indicated. 629 cells treated with vehicle and 640 cells treated with wiskostatin were count from three independent experiments. Error bars represent SEM. Representative images from time lapse movies of HeLa cells stably expressing GFP-tagged histone H2B synchronised as previously described, treated with vehicle (E) or 10 μM wiskostatin (F). Images represent merged between DIC and fluorescent (Histone H2B) images. Time indicated as hours: minutes.<p><b>Copyright information:</b></p><p>Taken from "Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway"</p><p>http://www.biomedcentral.com/1471-2121/9/42</p><p>BMC Cell Biology 2008;9():42-42.</p><p>Published online 30 Jul 2008</p><p>PMCID:PMC2527559.</p><p></p

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway-0

Etails). Two hours after release from RO3306 block cells were permeabilised and fixed. Cells were treated for indirect Alexa 555 localisation of Arp3 (a, e, and i) and Alexa 350 localisation of β-tubulin (c, g and k) with specific antibodies. F-actin was visualised with FITC-coupled phalloidin (b, f, and j). Merged images are presented (d, h and l). Images are representative of the different stages of mitosis: anaphase (a-d), late telophase (e-h) and cytokinesis (i-l). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway"</p><p>http://www.biomedcentral.com/1471-2121/9/42</p><p>BMC Cell Biology 2008;9():42-42.</p><p>Published online 30 Jul 2008</p><p>PMCID:PMC2527559.</p><p></p

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway-2

P-tagged β-tubulin were synchronised in prometaphase with nocodazole and released after a shake-off in presence of vehicle (Cont) or 10 μM wiskostatin (Wisko). 120 minutes after release cells were fixed and F-actin (a and e) and DNA (c and g) stained respectively with Alexa 555-coupled phalloidin and DAPI. β-tubulin (b and f) was directly visualised. Merged images (d and h) are presented. B. Wiskostatin alters Arp3 localisation. HeLa cells were synchronised in G2/M by thymidine and RO3306 double block. 120 minutes after release cells were fixed and stained as described in Figure 1. Images a and e represent staining of Arp3, b and f staining of F-actin and c and g staining of β-tubulin. Merged images (d and h) are presented. Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway"</p><p>http://www.biomedcentral.com/1471-2121/9/42</p><p>BMC Cell Biology 2008;9():42-42.</p><p>Published online 30 Jul 2008</p><p>PMCID:PMC2527559.</p><p></p

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway-3

or Arp3 or N-WASP + Arp3 or ECT2. 72 hours after transfection cells were harvested and lysed. N-WASP, Arp3, ECT2, p34 and β-tubulin protein levels were determined by immunoblot analysis using specific antibodies. B. The DNA content of the same HeLa cells was determined by flow cytometry analysis. Arrows indicate abnormal DNA content in ECT2 knockdown cells. A similar scale was used for each histogram. These results are representative of at least three independent experiments. C. Representative DIC images from time-lapse movies of HeLa cells transfected with water (Ø), siRNA directed against N-WASP (N-WASP), against Arp3 (Arp3), against Arp3 and N-WASP (Arp3 + N-WASP) or against ECT2. Images started to be acquired 24 hours post-transfection for 40 hours. Coloured stars indicate the nuclei of dividing cells and of their daughter cells. Time indicated as hours: minutes.<p><b>Copyright information:</b></p><p>Taken from "Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway"</p><p>http://www.biomedcentral.com/1471-2121/9/42</p><p>BMC Cell Biology 2008;9():42-42.</p><p>Published online 30 Jul 2008</p><p>PMCID:PMC2527559.</p><p></p

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway-4

Metaphase with nocodazole and released after shake-off in presence of vehicle (control), 10 mM NaN+ 6 mM deoxyglucose (ΔATP) or 10 μM wiskostatin (wiskostatin). After indicated time points cells were harvested and lysed in boiling water for 10 minutes. Cellular ATP contents were determined on clarified lysates using ATP determination kit (see Methods). Cellular ATP level at time point 0 from each treatment was defined as 100%. Graph represents mean of three independent experiments. Error bars represent SEM. ATP depletion delays mitosis. B. HeLa cells synchronised and treated as previously described were harvested at indicated time points and DNA content analysed by flow cytometry. C. In similarly treated cells cyclin B1 and γ-tubulin levels were determined by immunoblot analysis using specific antibodies.<p><b>Copyright information:</b></p><p>Taken from "Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway"</p><p>http://www.biomedcentral.com/1471-2121/9/42</p><p>BMC Cell Biology 2008;9():42-42.</p><p>Published online 30 Jul 2008</p><p>PMCID:PMC2527559.</p><p></p

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway-5

Etails). Two hours after release from RO3306 block cells were permeabilised and fixed. Cells were treated for indirect Alexa 555 localisation of Arp3 (a, e, and i) and Alexa 350 localisation of β-tubulin (c, g and k) with specific antibodies. F-actin was visualised with FITC-coupled phalloidin (b, f, and j). Merged images are presented (d, h and l). Images are representative of the different stages of mitosis: anaphase (a-d), late telophase (e-h) and cytokinesis (i-l). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway"</p><p>http://www.biomedcentral.com/1471-2121/9/42</p><p>BMC Cell Biology 2008;9():42-42.</p><p>Published online 30 Jul 2008</p><p>PMCID:PMC2527559.</p><p></p