Limits...
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.

Show MeSH

Related in: MedlinePlus

Trio depletion rescues the cytokinesis failure induced by the depletion of MgcRacGAP. (A) Western blot showing the amount of Trio and MgcRacGAP 30 h after siRNA transfection. siRNA conditions include control siRNA (siCtrl) and two independent siRNAs against Trio (siTrio#1, siTrio#2) alone or in combination with MgcRacGAP siRNA (siMgcRacGAP). 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 visualize multinucleated cells upon MgcRacGAP depletion. siRNA conditions, as indicated. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM. (C) Frames from movies showing cells undergoing cytokinesis. Cytokinesis failure is observed in MgcRacGAP siRNA–treated cells but not in control, Trio2, or Trio2/MgcRacGAP siRNA–treated cells. Asterisks, multinucleated cells. Time (minutes) is indicated in the upper left corner. Scale bar, 20 μm. Graph shows quantification of the percentage of dividing cells undergoing cytokinesis failure at the end of the first division after siRNA transfection. n > 30 cells/experiment; three independent experiments. Mean ± SEM. (D) Western blot showing the amount of Trio and Ect2 30 h after siRNA transfection. siRNA conditions include control siRNA (siCtrl) and Trio siRNA alone or in combination with Ect2 siRNA (siEct2). α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green) to visualize multinucleated cells upon Ect2 depletion. siRNA conditions, as indicated. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263449&req=5

Figure 2: Trio depletion rescues the cytokinesis failure induced by the depletion of MgcRacGAP. (A) Western blot showing the amount of Trio and MgcRacGAP 30 h after siRNA transfection. siRNA conditions include control siRNA (siCtrl) and two independent siRNAs against Trio (siTrio#1, siTrio#2) alone or in combination with MgcRacGAP siRNA (siMgcRacGAP). 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 visualize multinucleated cells upon MgcRacGAP depletion. siRNA conditions, as indicated. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM. (C) Frames from movies showing cells undergoing cytokinesis. Cytokinesis failure is observed in MgcRacGAP siRNA–treated cells but not in control, Trio2, or Trio2/MgcRacGAP siRNA–treated cells. Asterisks, multinucleated cells. Time (minutes) is indicated in the upper left corner. Scale bar, 20 μm. Graph shows quantification of the percentage of dividing cells undergoing cytokinesis failure at the end of the first division after siRNA transfection. n > 30 cells/experiment; three independent experiments. Mean ± SEM. (D) Western blot showing the amount of Trio and Ect2 30 h after siRNA transfection. siRNA conditions include control siRNA (siCtrl) and Trio siRNA alone or in combination with Ect2 siRNA (siEct2). α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green) to visualize multinucleated cells upon Ect2 depletion. siRNA conditions, as indicated. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM.

Mentions: To confirm that Trio depletion could rescue the cytokinesis failure induced by MgcRacGAP depletion, we used two independent siRNAs to deplete Trio in combination with MgcRacGAP siRNA in HeLa cells. Protein depletion was controlled by Western blot analysis and immunofluorescence staining (Figure 2A and Supplemental Figure S2C). Of importance, both Trio siRNAs efficiently rescued the number of multinucleated cells induced by MgcRacGAP depletion, demonstrating that the observed phenotype was indeed specific to Trio depletion (Figure 2B).


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)

Trio depletion rescues the cytokinesis failure induced by the depletion of MgcRacGAP. (A) Western blot showing the amount of Trio and MgcRacGAP 30 h after siRNA transfection. siRNA conditions include control siRNA (siCtrl) and two independent siRNAs against Trio (siTrio#1, siTrio#2) alone or in combination with MgcRacGAP siRNA (siMgcRacGAP). 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 visualize multinucleated cells upon MgcRacGAP depletion. siRNA conditions, as indicated. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM. (C) Frames from movies showing cells undergoing cytokinesis. Cytokinesis failure is observed in MgcRacGAP siRNA–treated cells but not in control, Trio2, or Trio2/MgcRacGAP siRNA–treated cells. Asterisks, multinucleated cells. Time (minutes) is indicated in the upper left corner. Scale bar, 20 μm. Graph shows quantification of the percentage of dividing cells undergoing cytokinesis failure at the end of the first division after siRNA transfection. n > 30 cells/experiment; three independent experiments. Mean ± SEM. (D) Western blot showing the amount of Trio and Ect2 30 h after siRNA transfection. siRNA conditions include control siRNA (siCtrl) and Trio siRNA alone or in combination with Ect2 siRNA (siEct2). α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green) to visualize multinucleated cells upon Ect2 depletion. siRNA conditions, as indicated. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4263449&req=5

Figure 2: Trio depletion rescues the cytokinesis failure induced by the depletion of MgcRacGAP. (A) Western blot showing the amount of Trio and MgcRacGAP 30 h after siRNA transfection. siRNA conditions include control siRNA (siCtrl) and two independent siRNAs against Trio (siTrio#1, siTrio#2) alone or in combination with MgcRacGAP siRNA (siMgcRacGAP). 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 visualize multinucleated cells upon MgcRacGAP depletion. siRNA conditions, as indicated. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM. (C) Frames from movies showing cells undergoing cytokinesis. Cytokinesis failure is observed in MgcRacGAP siRNA–treated cells but not in control, Trio2, or Trio2/MgcRacGAP siRNA–treated cells. Asterisks, multinucleated cells. Time (minutes) is indicated in the upper left corner. Scale bar, 20 μm. Graph shows quantification of the percentage of dividing cells undergoing cytokinesis failure at the end of the first division after siRNA transfection. n > 30 cells/experiment; three independent experiments. Mean ± SEM. (D) Western blot showing the amount of Trio and Ect2 30 h after siRNA transfection. siRNA conditions include control siRNA (siCtrl) and Trio siRNA alone or in combination with Ect2 siRNA (siEct2). α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green) to visualize multinucleated cells upon Ect2 depletion. siRNA conditions, as indicated. Asterisks, multinucleated cells. Scale bar, 20 μm. Graph shows quantification of the percentage of multinucleated cells. n = 500 cells; three independent experiments. Mean ± SEM.
Mentions: To confirm that Trio depletion could rescue the cytokinesis failure induced by MgcRacGAP depletion, we used two independent siRNAs to deplete Trio in combination with MgcRacGAP siRNA in HeLa cells. Protein depletion was controlled by Western blot analysis and immunofluorescence staining (Figure 2A and Supplemental Figure S2C). Of importance, both Trio siRNAs efficiently rescued the number of multinucleated cells induced by MgcRacGAP depletion, demonstrating that the observed phenotype was indeed specific to Trio depletion (Figure 2B).

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