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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 affects cell morphology. (a) Quantification of cell area and cell perimeter of siRNA-transfected PC3 cells (mean ± s.d. from four different experiments and >200 cells). Black bars denote siControl; light grey bars denote siRhoB-1; dark grey bars denote siRhoB-2. Values are shown relative to siControl (**p < 0.01). (b) Phase-contrast images of RhoB-depleted or control cells from the indicated cell lines. Scale bars, 40 µm. (c) Orthogonal view of a representative siControl or siRhoB-treated cell. Scale bars, z = 9.5 µm. (d) F-actin distribution in siRNA-transfected PC3 cells plated on uncoated plastic or glass, or plastic coated with Matrigel or fibronectin. Scale bars, 20 µm.
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RSOB120076F1: RhoB affects cell morphology. (a) Quantification of cell area and cell perimeter of siRNA-transfected PC3 cells (mean ± s.d. from four different experiments and >200 cells). Black bars denote siControl; light grey bars denote siRhoB-1; dark grey bars denote siRhoB-2. Values are shown relative to siControl (**p < 0.01). (b) Phase-contrast images of RhoB-depleted or control cells from the indicated cell lines. Scale bars, 40 µm. (c) Orthogonal view of a representative siControl or siRhoB-treated cell. Scale bars, z = 9.5 µm. (d) F-actin distribution in siRNA-transfected PC3 cells plated on uncoated plastic or glass, or plastic coated with Matrigel or fibronectin. Scale bars, 20 µm.

Mentions: Using specific siRNAs, we found that RhoB-depleted PC3 cells had a reduced spread area and perimeter (figure 1a,b). They were also generally less flattened on the substrate (figure 1c). Similarly, RhoB depletion reduced spreading in LnCAP and DU145 prostate cancer cells (figure 1b), MDA-MB-231 breast cancer cells and primary endothelial cells (not shown); thus, the RhoB phenotype is not specific to a single cell type. RhoB depletion did not alter cell diameter in suspension, indicating that the lower spread area was not due to reduced size (see electronic supplementary material, figure S1a). The effect of RhoB on spreading did not depend on the substrate: RhoB-depleted cells were less spread on uncoated plastic or glass, or plastic coated with the extracellular matrix protein fibronectin or Matrigel (figure 1d). These results point to a central role of RhoB in the regulation of cell shape.Figure 1.


RhoB regulates cell migration through altered focal adhesion dynamics.

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

RhoB affects cell morphology. (a) Quantification of cell area and cell perimeter of siRNA-transfected PC3 cells (mean ± s.d. from four different experiments and >200 cells). Black bars denote siControl; light grey bars denote siRhoB-1; dark grey bars denote siRhoB-2. Values are shown relative to siControl (**p < 0.01). (b) Phase-contrast images of RhoB-depleted or control cells from the indicated cell lines. Scale bars, 40 µm. (c) Orthogonal view of a representative siControl or siRhoB-treated cell. Scale bars, z = 9.5 µm. (d) F-actin distribution in siRNA-transfected PC3 cells plated on uncoated plastic or glass, or plastic coated with Matrigel or fibronectin. Scale bars, 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120076F1: RhoB affects cell morphology. (a) Quantification of cell area and cell perimeter of siRNA-transfected PC3 cells (mean ± s.d. from four different experiments and >200 cells). Black bars denote siControl; light grey bars denote siRhoB-1; dark grey bars denote siRhoB-2. Values are shown relative to siControl (**p < 0.01). (b) Phase-contrast images of RhoB-depleted or control cells from the indicated cell lines. Scale bars, 40 µm. (c) Orthogonal view of a representative siControl or siRhoB-treated cell. Scale bars, z = 9.5 µm. (d) F-actin distribution in siRNA-transfected PC3 cells plated on uncoated plastic or glass, or plastic coated with Matrigel or fibronectin. Scale bars, 20 µm.
Mentions: Using specific siRNAs, we found that RhoB-depleted PC3 cells had a reduced spread area and perimeter (figure 1a,b). They were also generally less flattened on the substrate (figure 1c). Similarly, RhoB depletion reduced spreading in LnCAP and DU145 prostate cancer cells (figure 1b), MDA-MB-231 breast cancer cells and primary endothelial cells (not shown); thus, the RhoB phenotype is not specific to a single cell type. RhoB depletion did not alter cell diameter in suspension, indicating that the lower spread area was not due to reduced size (see electronic supplementary material, figure S1a). The effect of RhoB on spreading did not depend on the substrate: RhoB-depleted cells were less spread on uncoated plastic or glass, or plastic coated with the extracellular matrix protein fibronectin or Matrigel (figure 1d). These results point to a central role of RhoB in the regulation of cell shape.Figure 1.

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