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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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A siRNA screening approach identifies Trio as a potential GEF of Rac1 in dividing cells. HeLa cells were transfected with MgcRacGAP siRNA (siMgcRacGAP) in combination with control siRNA, Rac1 siRNA, or siRNAs targeting the indicated GEFs (SMART pools) and scored for the presence of multinucleated cells 48 h posttransfection. (A) Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green). Asterisks, multinucleated cells. Scale bar, 20 μm. (B) Quantification of the percentage of multinucleated cells. Five hundred cells were counted per experiment. Three independent experiments. Mean ± SEM. *p < 0.02.
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Figure 1: A siRNA screening approach identifies Trio as a potential GEF of Rac1 in dividing cells. HeLa cells were transfected with MgcRacGAP siRNA (siMgcRacGAP) in combination with control siRNA, Rac1 siRNA, or siRNAs targeting the indicated GEFs (SMART pools) and scored for the presence of multinucleated cells 48 h posttransfection. (A) Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green). Asterisks, multinucleated cells. Scale bar, 20 μm. (B) Quantification of the percentage of multinucleated cells. Five hundred cells were counted per experiment. Three independent experiments. Mean ± SEM. *p < 0.02.

Mentions: To identify a RhoGEF regulating Rac1 activity in dividing cells, we performed a small interfering RNA (siRNA) screening approach in HeLa cells using SMARTpool siRNA oligonucleotides. Seventeen Rac GEFs belonging to the diffuse B-cell lymphoma (Dbl) family (Rossman et al., 2005) were depleted by siRNA alone or in combination with MgcRacGAP siRNAs in order to identify the ones able to rescue the cytokinesis failure induced by MgcRacGAP depletion (Figure 1 and Supplemental Figure S1). Of note, depletion of Rac-GEFs alone did not lead to a significant increase in the number of multinucleated cells (unpublished data). As previously described, 48 h after siRNA treatment, MgcRacGAP depletion resulted in the appearance of multinucleated cells, consistent with cytokinesis failure (Figure 1 and Supplemental Figure S1; Bastos et al., 2012). Codepletion of MgcRacGAP and Rac1 efficiently rescued the number of multinucleated cells (Figure 1), confirming that MgcRacGAP is acting as a GAP for Rac1 at the cleavage furrow of HeLa cells (Bastos et al., 2012). Of importance, codepletion of MgcRacGAP and Trio, a GEF characterized primarily for its role in axon outgrowth and guidance via RhoG/Rac1 activation (Steven et al., 1998; Bateman et al., 2000; Newsome et al., 2000; Estrach et al., 2002; Briançon-Marjollet et al., 2008; DeGeer et al., 2013), resulted in a strong decrease in the number of multinucleated cells (Figure 1). This result indicated that Trio depletion could rescue the cytokinesis failure induced by MgcRacGAP depletion. Of note, codepletion of MgcRacGAP and αPix also resulted in the decrease of multinucleated cells, but to a lower extent than Trio depletion. Of interest, the extent of the rescue induced by Trio depletion was similar to the one obtained with the codepletion of Rac1 (Figure 1). Even if the involvement of other GEFs activating Rac1 during cell division cannot be excluded, our screening approach identifies Trio as a major GEF of Rac1 in dividing cells.


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)

A siRNA screening approach identifies Trio as a potential GEF of Rac1 in dividing cells. HeLa cells were transfected with MgcRacGAP siRNA (siMgcRacGAP) in combination with control siRNA, Rac1 siRNA, or siRNAs targeting the indicated GEFs (SMART pools) and scored for the presence of multinucleated cells 48 h posttransfection. (A) Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green). Asterisks, multinucleated cells. Scale bar, 20 μm. (B) Quantification of the percentage of multinucleated cells. Five hundred cells were counted per experiment. Three independent experiments. Mean ± SEM. *p < 0.02.
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Related In: Results  -  Collection

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Figure 1: A siRNA screening approach identifies Trio as a potential GEF of Rac1 in dividing cells. HeLa cells were transfected with MgcRacGAP siRNA (siMgcRacGAP) in combination with control siRNA, Rac1 siRNA, or siRNAs targeting the indicated GEFs (SMART pools) and scored for the presence of multinucleated cells 48 h posttransfection. (A) Immunofluorescence of HeLa cells stained with DAPI (nuclei, red) and α-tubulin (green). Asterisks, multinucleated cells. Scale bar, 20 μm. (B) Quantification of the percentage of multinucleated cells. Five hundred cells were counted per experiment. Three independent experiments. Mean ± SEM. *p < 0.02.
Mentions: To identify a RhoGEF regulating Rac1 activity in dividing cells, we performed a small interfering RNA (siRNA) screening approach in HeLa cells using SMARTpool siRNA oligonucleotides. Seventeen Rac GEFs belonging to the diffuse B-cell lymphoma (Dbl) family (Rossman et al., 2005) were depleted by siRNA alone or in combination with MgcRacGAP siRNAs in order to identify the ones able to rescue the cytokinesis failure induced by MgcRacGAP depletion (Figure 1 and Supplemental Figure S1). Of note, depletion of Rac-GEFs alone did not lead to a significant increase in the number of multinucleated cells (unpublished data). As previously described, 48 h after siRNA treatment, MgcRacGAP depletion resulted in the appearance of multinucleated cells, consistent with cytokinesis failure (Figure 1 and Supplemental Figure S1; Bastos et al., 2012). Codepletion of MgcRacGAP and Rac1 efficiently rescued the number of multinucleated cells (Figure 1), confirming that MgcRacGAP is acting as a GAP for Rac1 at the cleavage furrow of HeLa cells (Bastos et al., 2012). Of importance, codepletion of MgcRacGAP and Trio, a GEF characterized primarily for its role in axon outgrowth and guidance via RhoG/Rac1 activation (Steven et al., 1998; Bateman et al., 2000; Newsome et al., 2000; Estrach et al., 2002; Briançon-Marjollet et al., 2008; DeGeer et al., 2013), resulted in a strong decrease in the number of multinucleated cells (Figure 1). This result indicated that Trio depletion could rescue the cytokinesis failure induced by MgcRacGAP depletion. Of note, codepletion of MgcRacGAP and αPix also resulted in the decrease of multinucleated cells, but to a lower extent than Trio depletion. Of interest, the extent of the rescue induced by Trio depletion was similar to the one obtained with the codepletion of Rac1 (Figure 1). Even if the involvement of other GEFs activating Rac1 during cell division cannot be excluded, our screening approach identifies Trio as a major GEF of Rac1 in dividing cells.

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