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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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Trio depletion decreases Rac1 activity in dividing cells and leads to F-actin remodeling defects. (A) Quantification of GTP-bound Rac1 in control and Trio#2 siRNA–treated mitotic cells. Western blot showing the amount of Rac1 bound to GST-CRIB (Active Rac1) and the total level of Rac1 expression (Total Rac1). Immunoblot, anti-Rac1 antibody. Molecular weight is indicated in kilodaltons. Histogram (right), relative Active Rac1/Total Rac1 ratio. Mitotic index, 60%. Three independent experiments. Mean ± SEM. *p < 0.01. (B) Western blot showing the amount of Trio, Rac1, or p34-Arc. p34-Arc is used to control for Arp2/3 complex disruption. siRNA conditions include control, Trio#1, Trio#2, Rac1, or Arp3. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (C) HeLa cells were transfected with control siRNA, Trio#1 siRNA, Trio#2 siRNA, Rac1 siRNA, or Arp3 siRNA. HeLa cells were 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. (D) Graph 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. For all siRNA conditions compared with control siRNA, *p < 00001. (E) Graph showing the percentage of cells with F-actin remodeling defects. n > 30 anaphase cells; three independent experiments. Mean ± SEM.
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Figure 3: Trio depletion decreases Rac1 activity in dividing cells and leads to F-actin remodeling defects. (A) Quantification of GTP-bound Rac1 in control and Trio#2 siRNA–treated mitotic cells. Western blot showing the amount of Rac1 bound to GST-CRIB (Active Rac1) and the total level of Rac1 expression (Total Rac1). Immunoblot, anti-Rac1 antibody. Molecular weight is indicated in kilodaltons. Histogram (right), relative Active Rac1/Total Rac1 ratio. Mitotic index, 60%. Three independent experiments. Mean ± SEM. *p < 0.01. (B) Western blot showing the amount of Trio, Rac1, or p34-Arc. p34-Arc is used to control for Arp2/3 complex disruption. siRNA conditions include control, Trio#1, Trio#2, Rac1, or Arp3. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (C) HeLa cells were transfected with control siRNA, Trio#1 siRNA, Trio#2 siRNA, Rac1 siRNA, or Arp3 siRNA. HeLa cells were 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. (D) Graph 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. For all siRNA conditions compared with control siRNA, *p < 00001. (E) Graph showing the percentage of cells with F-actin remodeling defects. n > 30 anaphase cells; three independent experiments. Mean ± SEM.

Mentions: To directly test whether Trio could function as a GEF of Rac1 in dividing cells, the amount of activated Rac1 was monitored by pull-down assay in synchronized mitotic cells. Compared to control siRNA–treated cells, Trio depletion reduced by half the amount of activated Rac1 in mitotic cells (Figure 3A), showing that Trio activates Rac1 in mitosis. Strikingly, Trio depletion (Figure 3B) led to defects in F-actin cytoskeleton remodeling in both interphase (unpublished data) and dividing cells (Figure 3C). More specifically, in anaphase cells, the F-actin staining at the cortex was significantly reduced in Trio-depleted cells compared with control cells (Figure 3C, MP). This decrease in F-actin staining was particularly obvious at the bottom plane (Figure 3, C–E, BP and inset), where cells are in contact with the substrate. Of interest, Trio depletion phenocopied the depletion of Rac1, consistent with a role for the Trio-Rac1 pathway in controlling F-actin remodeling in dividing cells (Figure 3, B–E). Trio depletion also phenocopied the depletion of actin-related protein 3 (Arp3; Figure 3, B–E), a component of the Arp2/3 complex whose depletion disrupts the Arp2/3 complex (Steffen et al., 2006). Arp2/3 is known to nucleate branched actin filaments downstream of Rac1 (Pollard, 2007). Taken together, these results suggest that Trio could control F-actin remodeling during cell division by controlling the Rac1-Arp2/3 pathway.


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 decreases Rac1 activity in dividing cells and leads to F-actin remodeling defects. (A) Quantification of GTP-bound Rac1 in control and Trio#2 siRNA–treated mitotic cells. Western blot showing the amount of Rac1 bound to GST-CRIB (Active Rac1) and the total level of Rac1 expression (Total Rac1). Immunoblot, anti-Rac1 antibody. Molecular weight is indicated in kilodaltons. Histogram (right), relative Active Rac1/Total Rac1 ratio. Mitotic index, 60%. Three independent experiments. Mean ± SEM. *p < 0.01. (B) Western blot showing the amount of Trio, Rac1, or p34-Arc. p34-Arc is used to control for Arp2/3 complex disruption. siRNA conditions include control, Trio#1, Trio#2, Rac1, or Arp3. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (C) HeLa cells were transfected with control siRNA, Trio#1 siRNA, Trio#2 siRNA, Rac1 siRNA, or Arp3 siRNA. HeLa cells were 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. (D) Graph 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. For all siRNA conditions compared with control siRNA, *p < 00001. (E) Graph showing the percentage of cells with F-actin remodeling defects. n > 30 anaphase cells; three independent experiments. Mean ± SEM.
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Figure 3: Trio depletion decreases Rac1 activity in dividing cells and leads to F-actin remodeling defects. (A) Quantification of GTP-bound Rac1 in control and Trio#2 siRNA–treated mitotic cells. Western blot showing the amount of Rac1 bound to GST-CRIB (Active Rac1) and the total level of Rac1 expression (Total Rac1). Immunoblot, anti-Rac1 antibody. Molecular weight is indicated in kilodaltons. Histogram (right), relative Active Rac1/Total Rac1 ratio. Mitotic index, 60%. Three independent experiments. Mean ± SEM. *p < 0.01. (B) Western blot showing the amount of Trio, Rac1, or p34-Arc. p34-Arc is used to control for Arp2/3 complex disruption. siRNA conditions include control, Trio#1, Trio#2, Rac1, or Arp3. α-Tubulin, loading control. Molecular weight is indicated in kilodaltons. (C) HeLa cells were transfected with control siRNA, Trio#1 siRNA, Trio#2 siRNA, Rac1 siRNA, or Arp3 siRNA. HeLa cells were 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. (D) Graph 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. For all siRNA conditions compared with control siRNA, *p < 00001. (E) Graph showing the percentage of cells with F-actin remodeling defects. n > 30 anaphase cells; three independent experiments. Mean ± SEM.
Mentions: To directly test whether Trio could function as a GEF of Rac1 in dividing cells, the amount of activated Rac1 was monitored by pull-down assay in synchronized mitotic cells. Compared to control siRNA–treated cells, Trio depletion reduced by half the amount of activated Rac1 in mitotic cells (Figure 3A), showing that Trio activates Rac1 in mitosis. Strikingly, Trio depletion (Figure 3B) led to defects in F-actin cytoskeleton remodeling in both interphase (unpublished data) and dividing cells (Figure 3C). More specifically, in anaphase cells, the F-actin staining at the cortex was significantly reduced in Trio-depleted cells compared with control cells (Figure 3C, MP). This decrease in F-actin staining was particularly obvious at the bottom plane (Figure 3, C–E, BP and inset), where cells are in contact with the substrate. Of interest, Trio depletion phenocopied the depletion of Rac1, consistent with a role for the Trio-Rac1 pathway in controlling F-actin remodeling in dividing cells (Figure 3, B–E). Trio depletion also phenocopied the depletion of actin-related protein 3 (Arp3; Figure 3, B–E), a component of the Arp2/3 complex whose depletion disrupts the Arp2/3 complex (Steffen et al., 2006). Arp2/3 is known to nucleate branched actin filaments downstream of Rac1 (Pollard, 2007). Taken together, these results suggest that Trio could control F-actin remodeling during cell division by controlling the Rac1-Arp2/3 pathway.

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