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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 knockdown impairs spreading. (a) PC3 cell adhesion after 20 min and (b) spread area after 30 min on uncoated plastic, or plastic coated with Matrigel or fibronectin. Black bars denote siControl, grey bars denote siRhoB. Graphs show mean ± s.e.m. (n = 5 with >100 cells per condition; **p < 0.01). (c) Analysis of β1 and β3 integrin cell surface expression in PC3 cells. Surface proteins were biotinylated, pulled down with streptavidin beads and then analysed by immunoblotting with β1 (black bars) and β3 (grey bars) integrin antibodies and GADPH antibodies as a loading control. (i) Representative Western blot; (ii) mean levels ± s.e.m. of surface integrin levels relative to total and expressed as percentage of siControl-1 levels. n = 6. *p < 0.05, **p < 0.01.
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RSOB120076F2: RhoB knockdown impairs spreading. (a) PC3 cell adhesion after 20 min and (b) spread area after 30 min on uncoated plastic, or plastic coated with Matrigel or fibronectin. Black bars denote siControl, grey bars denote siRhoB. Graphs show mean ± s.e.m. (n = 5 with >100 cells per condition; **p < 0.01). (c) Analysis of β1 and β3 integrin cell surface expression in PC3 cells. Surface proteins were biotinylated, pulled down with streptavidin beads and then analysed by immunoblotting with β1 (black bars) and β3 (grey bars) integrin antibodies and GADPH antibodies as a loading control. (i) Representative Western blot; (ii) mean levels ± s.e.m. of surface integrin levels relative to total and expressed as percentage of siControl-1 levels. n = 6. *p < 0.05, **p < 0.01.

Mentions: The reduced spread area of RhoB-depleted cells could reflect decreased adhesion. RhoB depletion did not alter cell adhesion to uncoated plastic, Matrigel or fibronectin (figure 2a), whereas there was already a significant decrease in the spread area 30 min after seeding on plastic or fibronectin (figure 2b). Differences on Matrigel were not significant at 30 min, although reduced spreading was clearly observed at later points (see electronic supplementary material, figure S1b).Figure 2.


RhoB regulates cell migration through altered focal adhesion dynamics.

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

RhoB knockdown impairs spreading. (a) PC3 cell adhesion after 20 min and (b) spread area after 30 min on uncoated plastic, or plastic coated with Matrigel or fibronectin. Black bars denote siControl, grey bars denote siRhoB. Graphs show mean ± s.e.m. (n = 5 with >100 cells per condition; **p < 0.01). (c) Analysis of β1 and β3 integrin cell surface expression in PC3 cells. Surface proteins were biotinylated, pulled down with streptavidin beads and then analysed by immunoblotting with β1 (black bars) and β3 (grey bars) integrin antibodies and GADPH antibodies as a loading control. (i) Representative Western blot; (ii) mean levels ± s.e.m. of surface integrin levels relative to total and expressed as percentage of siControl-1 levels. n = 6. *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120076F2: RhoB knockdown impairs spreading. (a) PC3 cell adhesion after 20 min and (b) spread area after 30 min on uncoated plastic, or plastic coated with Matrigel or fibronectin. Black bars denote siControl, grey bars denote siRhoB. Graphs show mean ± s.e.m. (n = 5 with >100 cells per condition; **p < 0.01). (c) Analysis of β1 and β3 integrin cell surface expression in PC3 cells. Surface proteins were biotinylated, pulled down with streptavidin beads and then analysed by immunoblotting with β1 (black bars) and β3 (grey bars) integrin antibodies and GADPH antibodies as a loading control. (i) Representative Western blot; (ii) mean levels ± s.e.m. of surface integrin levels relative to total and expressed as percentage of siControl-1 levels. n = 6. *p < 0.05, **p < 0.01.
Mentions: The reduced spread area of RhoB-depleted cells could reflect decreased adhesion. RhoB depletion did not alter cell adhesion to uncoated plastic, Matrigel or fibronectin (figure 2a), whereas there was already a significant decrease in the spread area 30 min after seeding on plastic or fibronectin (figure 2b). Differences on Matrigel were not significant at 30 min, although reduced spreading was clearly observed at later points (see electronic supplementary material, figure S1b).Figure 2.

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