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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 is required for Rac1-driven lamellipodial protrusion. (a) (i) Kymographs of representative lamellipodial regions of migrating siRNA-transfected PC3 cells from the electronic supplementary material, movie S1. Black bars denote kymograph region. Scale bars, 20 µm. (ii) Lamellipodial protrusion rate measured from five to seven cells per condition, mean ± s.d. (b) (i) Representative images and (ii) quantification of GFP-positive PC3 cells after transfection with GFP-Rac1-L61 or control GFP and the indicated siRNAs. Scale bars, 20 µm. n > 300 cells per condition from three independent experiments; boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; **p < 0.01, ***p < 0.001.
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RSOB120076F3: RhoB is required for Rac1-driven lamellipodial protrusion. (a) (i) Kymographs of representative lamellipodial regions of migrating siRNA-transfected PC3 cells from the electronic supplementary material, movie S1. Black bars denote kymograph region. Scale bars, 20 µm. (ii) Lamellipodial protrusion rate measured from five to seven cells per condition, mean ± s.d. (b) (i) Representative images and (ii) quantification of GFP-positive PC3 cells after transfection with GFP-Rac1-L61 or control GFP and the indicated siRNAs. Scale bars, 20 µm. n > 300 cells per condition from three independent experiments; boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; **p < 0.01, ***p < 0.001.

Mentions: Analysis of time-lapse movies indicated that RhoB-depleted PC3 cells had a defect in spreading and lamellipodium stability, although dynamic membrane ruffles were frequently observed (see figure 3a and electronic supplementary material, movie S1). Multiple short protrusions were extended with intensive ruffling, but did not develop into a proper extended lamellipodium or a persistent leading edge. The speed of protrusion extension was similar between RhoB-depleted and control cells, but these protrusions were less persistent (figure 3a). The behaviour of the rear of migrating cells also appeared different: when a cell changed direction, the previous protrusion was now behind the cell and was then suddenly retracted, such that the rear and nucleus jumped forward. Altered adhesion, at the rear, can contribute to lamellipodium stabilization and polarization [10].Figure 3.


RhoB regulates cell migration through altered focal adhesion dynamics.

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

RhoB is required for Rac1-driven lamellipodial protrusion. (a) (i) Kymographs of representative lamellipodial regions of migrating siRNA-transfected PC3 cells from the electronic supplementary material, movie S1. Black bars denote kymograph region. Scale bars, 20 µm. (ii) Lamellipodial protrusion rate measured from five to seven cells per condition, mean ± s.d. (b) (i) Representative images and (ii) quantification of GFP-positive PC3 cells after transfection with GFP-Rac1-L61 or control GFP and the indicated siRNAs. Scale bars, 20 µm. n > 300 cells per condition from three independent experiments; boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; **p < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120076F3: RhoB is required for Rac1-driven lamellipodial protrusion. (a) (i) Kymographs of representative lamellipodial regions of migrating siRNA-transfected PC3 cells from the electronic supplementary material, movie S1. Black bars denote kymograph region. Scale bars, 20 µm. (ii) Lamellipodial protrusion rate measured from five to seven cells per condition, mean ± s.d. (b) (i) Representative images and (ii) quantification of GFP-positive PC3 cells after transfection with GFP-Rac1-L61 or control GFP and the indicated siRNAs. Scale bars, 20 µm. n > 300 cells per condition from three independent experiments; boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; **p < 0.01, ***p < 0.001.
Mentions: Analysis of time-lapse movies indicated that RhoB-depleted PC3 cells had a defect in spreading and lamellipodium stability, although dynamic membrane ruffles were frequently observed (see figure 3a and electronic supplementary material, movie S1). Multiple short protrusions were extended with intensive ruffling, but did not develop into a proper extended lamellipodium or a persistent leading edge. The speed of protrusion extension was similar between RhoB-depleted and control cells, but these protrusions were less persistent (figure 3a). The behaviour of the rear of migrating cells also appeared different: when a cell changed direction, the previous protrusion was now behind the cell and was then suddenly retracted, such that the rear and nucleus jumped forward. Altered adhesion, at the rear, can contribute to lamellipodium stabilization and polarization [10].Figure 3.

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