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RhoB regulates cell migration through altered focal adhesion dynamics.

Vega FM, Colomba A, Reymond N, Thomas M, Ridley AJ - Open Biol (2012)

Bottom Line: RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration.They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions.We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK.

ABSTRACT
The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

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RhoB and GEF-H1 affect cell morphology, but not microtubule dynamics. (a) Confocal images showing F-actin and α-tubulin distribution in PC3 cells transfected with siRNAs targeting GEF-H1, RhoA or control siRNA. Scale bars, 20 µm. (b) Graph shows quantification of cell area (mean ± s.e.m.) relative to siControl from two different experiments with >40 cells per condition; **p < 0.01. (c) Quantification of microtubule tips per unit area from movies of EB3-GFP-expressing RhoB-depleted or control cells. Graph shows mean ± s.e.m. from 10 cells in two different experiments.
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RSOB120076F5: RhoB and GEF-H1 affect cell morphology, but not microtubule dynamics. (a) Confocal images showing F-actin and α-tubulin distribution in PC3 cells transfected with siRNAs targeting GEF-H1, RhoA or control siRNA. Scale bars, 20 µm. (b) Graph shows quantification of cell area (mean ± s.e.m.) relative to siControl from two different experiments with >40 cells per condition; **p < 0.01. (c) Quantification of microtubule tips per unit area from movies of EB3-GFP-expressing RhoB-depleted or control cells. Graph shows mean ± s.e.m. from 10 cells in two different experiments.

Mentions: Integrin-mediated adhesion and lamellipodial dynamics are regulated by microtubule dynamics in addition to actin polymerization [13,14]. We therefore investigated whether the effects of RhoB were due to changes in microtubules. RhoB-depleted cells did not have a different microtubule organization, although microtubules appeared condensed around the periphery in some cases owing to the reduced spreading (figure 5a). Results from a siRNA screen indicated that knockdown of the RhoGEF GEF-H1 (officially known as ARHGEF2) could affect cell morphology (FM Vega 2011, unpublished data). GEF-H1 is best known as an activator of RhoA [15,16], but can also act as a GEF for RhoB [17]. GEF-H1 depletion in PC3 cells reduced cell spread area, similar to RhoB knockdown and clearly different to the elongated phenotype observed in RhoA-depleted cells (figure 5a [8]). GEF-H1 binds directly to microtubules, and GEF-H1 knockdown in HeLa cells was reported to increase the number of microtubules reaching the cell periphery [16]. We therefore analysed microtubule dynamics in PC3 cells transfected with EB3-GFP, which localizes to microtubule tips. From the analysis of movies, we did not observe any change in the number of EB3-GFP3-positive microtubule tips at the cell edge (see figure 5c; electronic supplementary material, figure S2a). In addition, neither RhoB nor GEF-H1 depletion affected the levels of stabilized acetylated tubulin (see electronic supplementary material, figure S1d,e). RhoB can interact with mDia formins, which are known to regulate both actin polymerization and microtubule dynamics [18]. However, mDia1-depleted PC3 cells had an elongated morphology (figure 6), consistent with our previous results [19]. This did not resemble the reduced spreading phenotype of RhoB depletion. The RhoB knockdown phenotype was also very different to that of PC3 cells depleted of the Rho-kinases ROCK1 or ROCK2 [8]. It is therefore unlikely that either mDia1 or ROCKs mediate RhoB-dependent morphological changes in these cells.Figure 5.


RhoB regulates cell migration through altered focal adhesion dynamics.

Vega FM, Colomba A, Reymond N, Thomas M, Ridley AJ - Open Biol (2012)

RhoB and GEF-H1 affect cell morphology, but not microtubule dynamics. (a) Confocal images showing F-actin and α-tubulin distribution in PC3 cells transfected with siRNAs targeting GEF-H1, RhoA or control siRNA. Scale bars, 20 µm. (b) Graph shows quantification of cell area (mean ± s.e.m.) relative to siControl from two different experiments with >40 cells per condition; **p < 0.01. (c) Quantification of microtubule tips per unit area from movies of EB3-GFP-expressing RhoB-depleted or control cells. Graph shows mean ± s.e.m. from 10 cells in two different experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376739&req=5

RSOB120076F5: RhoB and GEF-H1 affect cell morphology, but not microtubule dynamics. (a) Confocal images showing F-actin and α-tubulin distribution in PC3 cells transfected with siRNAs targeting GEF-H1, RhoA or control siRNA. Scale bars, 20 µm. (b) Graph shows quantification of cell area (mean ± s.e.m.) relative to siControl from two different experiments with >40 cells per condition; **p < 0.01. (c) Quantification of microtubule tips per unit area from movies of EB3-GFP-expressing RhoB-depleted or control cells. Graph shows mean ± s.e.m. from 10 cells in two different experiments.
Mentions: Integrin-mediated adhesion and lamellipodial dynamics are regulated by microtubule dynamics in addition to actin polymerization [13,14]. We therefore investigated whether the effects of RhoB were due to changes in microtubules. RhoB-depleted cells did not have a different microtubule organization, although microtubules appeared condensed around the periphery in some cases owing to the reduced spreading (figure 5a). Results from a siRNA screen indicated that knockdown of the RhoGEF GEF-H1 (officially known as ARHGEF2) could affect cell morphology (FM Vega 2011, unpublished data). GEF-H1 is best known as an activator of RhoA [15,16], but can also act as a GEF for RhoB [17]. GEF-H1 depletion in PC3 cells reduced cell spread area, similar to RhoB knockdown and clearly different to the elongated phenotype observed in RhoA-depleted cells (figure 5a [8]). GEF-H1 binds directly to microtubules, and GEF-H1 knockdown in HeLa cells was reported to increase the number of microtubules reaching the cell periphery [16]. We therefore analysed microtubule dynamics in PC3 cells transfected with EB3-GFP, which localizes to microtubule tips. From the analysis of movies, we did not observe any change in the number of EB3-GFP3-positive microtubule tips at the cell edge (see figure 5c; electronic supplementary material, figure S2a). In addition, neither RhoB nor GEF-H1 depletion affected the levels of stabilized acetylated tubulin (see electronic supplementary material, figure S1d,e). RhoB can interact with mDia formins, which are known to regulate both actin polymerization and microtubule dynamics [18]. However, mDia1-depleted PC3 cells had an elongated morphology (figure 6), consistent with our previous results [19]. This did not resemble the reduced spreading phenotype of RhoB depletion. The RhoB knockdown phenotype was also very different to that of PC3 cells depleted of the Rho-kinases ROCK1 or ROCK2 [8]. It is therefore unlikely that either mDia1 or ROCKs mediate RhoB-dependent morphological changes in these cells.Figure 5.

Bottom Line: RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration.They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions.We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK.

ABSTRACT
The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

Show MeSH
Related in: MedlinePlus