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Identification of a mitotic Rac-GEF, Trio, that counteracts MgcRacGAP function during cytokinesis.

Cannet A, Schmidt S, Delaval B, Debant A - Mol. Biol. Cell (2014)

Bottom Line: Moreover, Trio depletion specifically rescues the cytokinesis failure induced by MgcRacGAP depletion.Of importance, we demonstrate that this rescue is mediated by the Trio-Rac1 pathway, using GEF-dead mutants of Trio and a specific inhibitor of Rac1 activation by Trio.Overall this work identifies for the first time a GEF controlling Rac1 activation in dividing cells that counteracts MgcRacGAP function in cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Signaling and Cytoskeleton Dynamics Group, University of Montpellier, 34293 Montpellier, France.

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Inhibition of Rac1 activation by Trio rescues the cytokinesis failure induced by MgcRacGAP depletion. (A) HeLa cells were treated for 24 h with DMSO or ITX3 alone or in combination with MgcRacGAP siRNA. Western blot showing the amount of Trio and MgcRacGAP. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (B) Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green) to monitor the presence of multinucleated cells after MgcRacGAP depletion upon DMSO or ITX3 treatments. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows the quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM. (C) Immunofluorescence of HeLa cells (left) cotransfected with MgcRacGAP siRNA and Ctrl or Trio siRNAs. GFP, GFP-Trio siRNA resistant, GFP-D1d siRNA resistant (GEFD1 dead mutant of Trio unable to activate Rac1/RhoG), or GFP-D2d siRNA resistant (GEFD2 dead mutant of Trio unable to activate RhoA) was transfected together with the indicated siRNA to monitor which mutant form of Trio can suppress the rescue. HeLa cells were stained with DAPI (nuclei, blue) and α-tubulin (red) to monitor multinucleated cells. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows the quantification of the percentage of multinucleated cells after codepletion of Trio and MgcRacGAP and expression of mutant forms of Trio. Five hundred cells were counted per experiment; three independent experiments. Mean ± SEM. *p < 0.05.
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Figure 5: Inhibition of Rac1 activation by Trio rescues the cytokinesis failure induced by MgcRacGAP depletion. (A) HeLa cells were treated for 24 h with DMSO or ITX3 alone or in combination with MgcRacGAP siRNA. Western blot showing the amount of Trio and MgcRacGAP. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (B) Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green) to monitor the presence of multinucleated cells after MgcRacGAP depletion upon DMSO or ITX3 treatments. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows the quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM. (C) Immunofluorescence of HeLa cells (left) cotransfected with MgcRacGAP siRNA and Ctrl or Trio siRNAs. GFP, GFP-Trio siRNA resistant, GFP-D1d siRNA resistant (GEFD1 dead mutant of Trio unable to activate Rac1/RhoG), or GFP-D2d siRNA resistant (GEFD2 dead mutant of Trio unable to activate RhoA) was transfected together with the indicated siRNA to monitor which mutant form of Trio can suppress the rescue. HeLa cells were stained with DAPI (nuclei, blue) and α-tubulin (red) to monitor multinucleated cells. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows the quantification of the percentage of multinucleated cells after codepletion of Trio and MgcRacGAP and expression of mutant forms of Trio. Five hundred cells were counted per experiment; three independent experiments. Mean ± SEM. *p < 0.05.

Mentions: To investigate directly whether Trio depletion rescues the cytokinesis failure induced by MgcRacGAP depletion by controlling Rac1 activation, we examined whether the increase in multinucleated cells induced by MgcRacGAP depletion could be suppressed using ITX3, the specific inhibitor of Rac1 activation by Trio (Bouquier et al., 2009). siRNA depletion of MgcRacGAP upon ITX3 treatment was controlled by Western blot analysis and immunofluorescence staining (Figure 5A and Supplemental Figure S4B). Strikingly, ITX3 treatment rescued the number of multinucleated cells induced by MgcRacGAP depletion (Figure 5B). This demonstrates that inhibition of Rac1 activation by Trio rescues the cytokinesis failure induced by MgcRacGAP depletion by decreasing Rac1 activity.


Identification of a mitotic Rac-GEF, Trio, that counteracts MgcRacGAP function during cytokinesis.

Cannet A, Schmidt S, Delaval B, Debant A - Mol. Biol. Cell (2014)

Inhibition of Rac1 activation by Trio rescues the cytokinesis failure induced by MgcRacGAP depletion. (A) HeLa cells were treated for 24 h with DMSO or ITX3 alone or in combination with MgcRacGAP siRNA. Western blot showing the amount of Trio and MgcRacGAP. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (B) Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green) to monitor the presence of multinucleated cells after MgcRacGAP depletion upon DMSO or ITX3 treatments. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows the quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM. (C) Immunofluorescence of HeLa cells (left) cotransfected with MgcRacGAP siRNA and Ctrl or Trio siRNAs. GFP, GFP-Trio siRNA resistant, GFP-D1d siRNA resistant (GEFD1 dead mutant of Trio unable to activate Rac1/RhoG), or GFP-D2d siRNA resistant (GEFD2 dead mutant of Trio unable to activate RhoA) was transfected together with the indicated siRNA to monitor which mutant form of Trio can suppress the rescue. HeLa cells were stained with DAPI (nuclei, blue) and α-tubulin (red) to monitor multinucleated cells. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows the quantification of the percentage of multinucleated cells after codepletion of Trio and MgcRacGAP and expression of mutant forms of Trio. Five hundred cells were counted per experiment; three independent experiments. Mean ± SEM. *p < 0.05.
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Figure 5: Inhibition of Rac1 activation by Trio rescues the cytokinesis failure induced by MgcRacGAP depletion. (A) HeLa cells were treated for 24 h with DMSO or ITX3 alone or in combination with MgcRacGAP siRNA. Western blot showing the amount of Trio and MgcRacGAP. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (B) Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green) to monitor the presence of multinucleated cells after MgcRacGAP depletion upon DMSO or ITX3 treatments. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows the quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM. (C) Immunofluorescence of HeLa cells (left) cotransfected with MgcRacGAP siRNA and Ctrl or Trio siRNAs. GFP, GFP-Trio siRNA resistant, GFP-D1d siRNA resistant (GEFD1 dead mutant of Trio unable to activate Rac1/RhoG), or GFP-D2d siRNA resistant (GEFD2 dead mutant of Trio unable to activate RhoA) was transfected together with the indicated siRNA to monitor which mutant form of Trio can suppress the rescue. HeLa cells were stained with DAPI (nuclei, blue) and α-tubulin (red) to monitor multinucleated cells. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows the quantification of the percentage of multinucleated cells after codepletion of Trio and MgcRacGAP and expression of mutant forms of Trio. Five hundred cells were counted per experiment; three independent experiments. Mean ± SEM. *p < 0.05.
Mentions: To investigate directly whether Trio depletion rescues the cytokinesis failure induced by MgcRacGAP depletion by controlling Rac1 activation, we examined whether the increase in multinucleated cells induced by MgcRacGAP depletion could be suppressed using ITX3, the specific inhibitor of Rac1 activation by Trio (Bouquier et al., 2009). siRNA depletion of MgcRacGAP upon ITX3 treatment was controlled by Western blot analysis and immunofluorescence staining (Figure 5A and Supplemental Figure S4B). Strikingly, ITX3 treatment rescued the number of multinucleated cells induced by MgcRacGAP depletion (Figure 5B). This demonstrates that inhibition of Rac1 activation by Trio rescues the cytokinesis failure induced by MgcRacGAP depletion by decreasing Rac1 activity.

Bottom Line: Moreover, Trio depletion specifically rescues the cytokinesis failure induced by MgcRacGAP depletion.Of importance, we demonstrate that this rescue is mediated by the Trio-Rac1 pathway, using GEF-dead mutants of Trio and a specific inhibitor of Rac1 activation by Trio.Overall this work identifies for the first time a GEF controlling Rac1 activation in dividing cells that counteracts MgcRacGAP function in cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Signaling and Cytoskeleton Dynamics Group, University of Montpellier, 34293 Montpellier, France.

Show MeSH
Related in: MedlinePlus