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

The Trio-Rac1 pathway is important to control F-actin remodeling in dividing cells. (A) Rescue experiment performed by transfecting HeLa cells with GFP or GFP-DH1PH1 (domain of Trio known to strongly activate Rac1) and Ctrl or Trio siRNAs as indicated. Cells were stained with rhodamine-phalloidin (actin, red) and DAPI (DNA, blue) to monitor F-actin remodeling defects. Middle planes (MPs) and bottom planes are shown. Z-stack maximum projection, 0881 μm. Scale bar, 5 μm; inset, 2 μm. (B) Western blot showing the amount of Trio and GFP or GFP-DH1-PH1. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (C) Graph showing the percentage of cells with F-actin remodeling defects at the bottom plane. n > 30 anaphase cells/condition; three independent experiments. Mean ± SEM. (D) HeLa cells were treated with DMSO or ITX3 and stained with rhodamine-phalloidin to monitor the F-actin cytoskeleton (red) and DAPI (DNA, blue) to identify anaphase cells. Middle planes (MPs) and bottom planes (BPs) are shown to visualize actin remodeling defects. Z-stack maximum projection, 0881 μm. Scale bar, 5 μm; inset, 2 μm. Graph (middle) showing the average F-actin intensity (bottom single plane) in each condition. Data from three independent experiments were pooled, and the actin intensity for each condition was normalized to the average intensity in control cells. Mean ± SEM. *p < 00001. Graph (far right) shows the percentage of cells with F-actin remodeling defects. n > 30 anaphase cells; three independent experiments. Mean ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263449&req=5

Figure 4: The Trio-Rac1 pathway is important to control F-actin remodeling in dividing cells. (A) Rescue experiment performed by transfecting HeLa cells with GFP or GFP-DH1PH1 (domain of Trio known to strongly activate Rac1) and Ctrl or Trio siRNAs as indicated. Cells were stained with rhodamine-phalloidin (actin, red) and DAPI (DNA, blue) to monitor F-actin remodeling defects. Middle planes (MPs) and bottom planes are shown. Z-stack maximum projection, 0881 μm. Scale bar, 5 μm; inset, 2 μm. (B) Western blot showing the amount of Trio and GFP or GFP-DH1-PH1. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (C) Graph showing the percentage of cells with F-actin remodeling defects at the bottom plane. n > 30 anaphase cells/condition; three independent experiments. Mean ± SEM. (D) HeLa cells were treated with DMSO or ITX3 and stained with rhodamine-phalloidin to monitor the F-actin cytoskeleton (red) and DAPI (DNA, blue) to identify anaphase cells. Middle planes (MPs) and bottom planes (BPs) are shown to visualize actin remodeling defects. Z-stack maximum projection, 0881 μm. Scale bar, 5 μm; inset, 2 μm. Graph (middle) showing the average F-actin intensity (bottom single plane) in each condition. Data from three independent experiments were pooled, and the actin intensity for each condition was normalized to the average intensity in control cells. Mean ± SEM. *p < 00001. Graph (far right) shows the percentage of cells with F-actin remodeling defects. n > 30 anaphase cells; three independent experiments. Mean ± SEM.

Mentions: To confirm that Trio activates Rac1 to control F-actin remodeling in dividing cells, we performed rescue experiments using the GEFD1 domain of Trio (green fluorescent protein [GFP]–DH1PH1; Figure 4, A–C, and Supplemental Figure S3). This domain was previously shown to strongly activate Rac1 (Bellanger et al., 1998). Expression of GFP-DH1PH1, insensitive to Trio siRNAs (Supplemental Figure S3), efficiently rescued the F-actin cytoskeleton remodeling defects observed in Trio-depleted anaphase cells (Figure 4, A–C). To demonstrate further the involvement of the Trio-Rac1 pathway in controlling F-actin remodeling in dividing cells, we used ITX3, a specific inhibitor of Rac1 activation by Trio (Bouquier et al., 2009). ITX3 treatment phenocopied Trio depletion (Figure 4D). Taken together, these results demonstrate that Trio functions as a GEF of Rac1 during cell division and that the Trio-Rac1-Arp2/3 pathway is important to control F-actin cytoskeleton remodeling 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)

The Trio-Rac1 pathway is important to control F-actin remodeling in dividing cells. (A) Rescue experiment performed by transfecting HeLa cells with GFP or GFP-DH1PH1 (domain of Trio known to strongly activate Rac1) and Ctrl or Trio siRNAs as indicated. Cells were stained with rhodamine-phalloidin (actin, red) and DAPI (DNA, blue) to monitor F-actin remodeling defects. Middle planes (MPs) and bottom planes are shown. Z-stack maximum projection, 0881 μm. Scale bar, 5 μm; inset, 2 μm. (B) Western blot showing the amount of Trio and GFP or GFP-DH1-PH1. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (C) Graph showing the percentage of cells with F-actin remodeling defects at the bottom plane. n > 30 anaphase cells/condition; three independent experiments. Mean ± SEM. (D) HeLa cells were treated with DMSO or ITX3 and stained with rhodamine-phalloidin to monitor the F-actin cytoskeleton (red) and DAPI (DNA, blue) to identify anaphase cells. Middle planes (MPs) and bottom planes (BPs) are shown to visualize actin remodeling defects. Z-stack maximum projection, 0881 μm. Scale bar, 5 μm; inset, 2 μm. Graph (middle) showing the average F-actin intensity (bottom single plane) in each condition. Data from three independent experiments were pooled, and the actin intensity for each condition was normalized to the average intensity in control cells. Mean ± SEM. *p < 00001. Graph (far right) shows the percentage of cells with F-actin remodeling defects. n > 30 anaphase cells; three independent experiments. Mean ± SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: The Trio-Rac1 pathway is important to control F-actin remodeling in dividing cells. (A) Rescue experiment performed by transfecting HeLa cells with GFP or GFP-DH1PH1 (domain of Trio known to strongly activate Rac1) and Ctrl or Trio siRNAs as indicated. Cells were stained with rhodamine-phalloidin (actin, red) and DAPI (DNA, blue) to monitor F-actin remodeling defects. Middle planes (MPs) and bottom planes are shown. Z-stack maximum projection, 0881 μm. Scale bar, 5 μm; inset, 2 μm. (B) Western blot showing the amount of Trio and GFP or GFP-DH1-PH1. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (C) Graph showing the percentage of cells with F-actin remodeling defects at the bottom plane. n > 30 anaphase cells/condition; three independent experiments. Mean ± SEM. (D) HeLa cells were treated with DMSO or ITX3 and stained with rhodamine-phalloidin to monitor the F-actin cytoskeleton (red) and DAPI (DNA, blue) to identify anaphase cells. Middle planes (MPs) and bottom planes (BPs) are shown to visualize actin remodeling defects. Z-stack maximum projection, 0881 μm. Scale bar, 5 μm; inset, 2 μm. Graph (middle) showing the average F-actin intensity (bottom single plane) in each condition. Data from three independent experiments were pooled, and the actin intensity for each condition was normalized to the average intensity in control cells. Mean ± SEM. *p < 00001. Graph (far right) shows the percentage of cells with F-actin remodeling defects. n > 30 anaphase cells; three independent experiments. Mean ± SEM.
Mentions: To confirm that Trio activates Rac1 to control F-actin remodeling in dividing cells, we performed rescue experiments using the GEFD1 domain of Trio (green fluorescent protein [GFP]–DH1PH1; Figure 4, A–C, and Supplemental Figure S3). This domain was previously shown to strongly activate Rac1 (Bellanger et al., 1998). Expression of GFP-DH1PH1, insensitive to Trio siRNAs (Supplemental Figure S3), efficiently rescued the F-actin cytoskeleton remodeling defects observed in Trio-depleted anaphase cells (Figure 4, A–C). To demonstrate further the involvement of the Trio-Rac1 pathway in controlling F-actin remodeling in dividing cells, we used ITX3, a specific inhibitor of Rac1 activation by Trio (Bouquier et al., 2009). ITX3 treatment phenocopied Trio depletion (Figure 4D). Taken together, these results demonstrate that Trio functions as a GEF of Rac1 during cell division and that the Trio-Rac1-Arp2/3 pathway is important to control F-actin cytoskeleton remodeling 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