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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 regulates focal adhesion dynamics. (a) Serum-starved PC3 cells stimulated with 10% FCS for 1 h to promote spreading were stained for F-actin and with antibodies to phospho-paxillin to show focal adhesions and focal complexes. Boxed regions (middle panels) are shown at higher magnification in the right panels. (b,c) Graphs show the number of phospho-paxillin-positive focal adhesions/focal complexes (FA) per cell or area occupied by FA relative to the total spread area. (d) PC3 cells under normal migratory conditions (1% FCS) were stained for F-actin and with antibodies to phospho-paxillin to show FA. Boxed regions (middle panels) are shown at higher magnification in the right panels. (e,f) Number of phospho-paxillin-positive FA per cell or area occupied by FA relative to the total spread area in normal migratory conditions. Results in graphs are from at least 10 cells from two different experiments. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001, **p < 0.01, *p < 0.05. (g) (i) Representative interference reflection microscopy images of PC3 cells after siRNA transfection. (ii) Dynamics images show a thresholded maximal projection of the same cells over 70 frames (15 s frame−1). (iii) Detail: enlargement of boxed regions in (ii). (iv) Graph shows percentage of cells with stable focal contacts in interference reflection microscopy movies; n > 20 cells per condition. (a,d,g) Scale bars, 20 µm.
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RSOB120076F7: RhoB regulates focal adhesion dynamics. (a) Serum-starved PC3 cells stimulated with 10% FCS for 1 h to promote spreading were stained for F-actin and with antibodies to phospho-paxillin to show focal adhesions and focal complexes. Boxed regions (middle panels) are shown at higher magnification in the right panels. (b,c) Graphs show the number of phospho-paxillin-positive focal adhesions/focal complexes (FA) per cell or area occupied by FA relative to the total spread area. (d) PC3 cells under normal migratory conditions (1% FCS) were stained for F-actin and with antibodies to phospho-paxillin to show FA. Boxed regions (middle panels) are shown at higher magnification in the right panels. (e,f) Number of phospho-paxillin-positive FA per cell or area occupied by FA relative to the total spread area in normal migratory conditions. Results in graphs are from at least 10 cells from two different experiments. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001, **p < 0.01, *p < 0.05. (g) (i) Representative interference reflection microscopy images of PC3 cells after siRNA transfection. (ii) Dynamics images show a thresholded maximal projection of the same cells over 70 frames (15 s frame−1). (iii) Detail: enlargement of boxed regions in (ii). (iv) Graph shows percentage of cells with stable focal contacts in interference reflection microscopy movies; n > 20 cells per condition. (a,d,g) Scale bars, 20 µm.

Mentions: Because the level of total surface β1 integrin was reduced by RhoB depletion (figure 2c), integrin-mediated adhesions could be affected by RhoB. Cells were stained with antibodies to phospho-paxillin, which localizes to focal adhesions and smaller focal complexes [20]. Cells were analysed during both acute serum-induced spreading that stimulates focal complex and adhesion formation (collectively called FA here), and under the normal migratory conditions (1% foetal calf serum, FCS) used for other experiments (compare figure 7a and d). RhoB-depleted cells had a reduced spread area following acute serum-induced spreading, as observed for cells under normal conditions (see electronic supplementary material, figure S2b; figure 1a). Serum-stimulated RhoB-depleted cells had less FA/cell than control cells (figure 7b), suggesting a defect in the formation or increased turnover of new FA. In contrast, under the steady-state normal migratory conditions, the number of FA/cell was similar between the two populations (figure 7e). The area occupied by FA compared with the total spread area measured was increased in RhoB knockdown cells in both conditions, because the spread area of these cells is lower than control cells (figure 7c,f).Figure 7.


RhoB regulates cell migration through altered focal adhesion dynamics.

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

RhoB regulates focal adhesion dynamics. (a) Serum-starved PC3 cells stimulated with 10% FCS for 1 h to promote spreading were stained for F-actin and with antibodies to phospho-paxillin to show focal adhesions and focal complexes. Boxed regions (middle panels) are shown at higher magnification in the right panels. (b,c) Graphs show the number of phospho-paxillin-positive focal adhesions/focal complexes (FA) per cell or area occupied by FA relative to the total spread area. (d) PC3 cells under normal migratory conditions (1% FCS) were stained for F-actin and with antibodies to phospho-paxillin to show FA. Boxed regions (middle panels) are shown at higher magnification in the right panels. (e,f) Number of phospho-paxillin-positive FA per cell or area occupied by FA relative to the total spread area in normal migratory conditions. Results in graphs are from at least 10 cells from two different experiments. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001, **p < 0.01, *p < 0.05. (g) (i) Representative interference reflection microscopy images of PC3 cells after siRNA transfection. (ii) Dynamics images show a thresholded maximal projection of the same cells over 70 frames (15 s frame−1). (iii) Detail: enlargement of boxed regions in (ii). (iv) Graph shows percentage of cells with stable focal contacts in interference reflection microscopy movies; n > 20 cells per condition. (a,d,g) Scale bars, 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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RSOB120076F7: RhoB regulates focal adhesion dynamics. (a) Serum-starved PC3 cells stimulated with 10% FCS for 1 h to promote spreading were stained for F-actin and with antibodies to phospho-paxillin to show focal adhesions and focal complexes. Boxed regions (middle panels) are shown at higher magnification in the right panels. (b,c) Graphs show the number of phospho-paxillin-positive focal adhesions/focal complexes (FA) per cell or area occupied by FA relative to the total spread area. (d) PC3 cells under normal migratory conditions (1% FCS) were stained for F-actin and with antibodies to phospho-paxillin to show FA. Boxed regions (middle panels) are shown at higher magnification in the right panels. (e,f) Number of phospho-paxillin-positive FA per cell or area occupied by FA relative to the total spread area in normal migratory conditions. Results in graphs are from at least 10 cells from two different experiments. Boxes of box and whisker plots show median, 25th and 75th percentile; whiskers show 95th percentile; ***p < 0.001, **p < 0.01, *p < 0.05. (g) (i) Representative interference reflection microscopy images of PC3 cells after siRNA transfection. (ii) Dynamics images show a thresholded maximal projection of the same cells over 70 frames (15 s frame−1). (iii) Detail: enlargement of boxed regions in (ii). (iv) Graph shows percentage of cells with stable focal contacts in interference reflection microscopy movies; n > 20 cells per condition. (a,d,g) Scale bars, 20 µm.
Mentions: Because the level of total surface β1 integrin was reduced by RhoB depletion (figure 2c), integrin-mediated adhesions could be affected by RhoB. Cells were stained with antibodies to phospho-paxillin, which localizes to focal adhesions and smaller focal complexes [20]. Cells were analysed during both acute serum-induced spreading that stimulates focal complex and adhesion formation (collectively called FA here), and under the normal migratory conditions (1% foetal calf serum, FCS) used for other experiments (compare figure 7a and d). RhoB-depleted cells had a reduced spread area following acute serum-induced spreading, as observed for cells under normal conditions (see electronic supplementary material, figure S2b; figure 1a). Serum-stimulated RhoB-depleted cells had less FA/cell than control cells (figure 7b), suggesting a defect in the formation or increased turnover of new FA. In contrast, under the steady-state normal migratory conditions, the number of FA/cell was similar between the two populations (figure 7e). The area occupied by FA compared with the total spread area measured was increased in RhoB knockdown cells in both conditions, because the spread area of these cells is lower than control cells (figure 7c,f).Figure 7.

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