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Fig. 7. Rac3-expressing cells show defective cell-matrix adhesions, which can be restored by direct activation of integrins by Rap^G12V. (A) Fluorescent images showing paxillin distribution in N1E-115 cells expressing Rac1 or Rac3. Cells were transiently transfected, cultured in serum-containing medium for 24 hours and subsequently serum-starved for 12 hours to trigger increase in cell-matrix adhesions, which precedes the outgrowth of protrusions and differentiation. Panels on the right show enlargements of the boxed regions in the middle panels. Note the absence of paxillin-rich structures in Rac3-expressing cells (middle and right panel). (B) Depletion of Rac3 is accompanied with increased cell-matrix adhesion and paxillin staining. N1E-115 cells were transfected with shRac3 (together with 5:1 eGFP), cultured for 24 hours and stained for paxillin. (C) Parental N1E-115 cells and N1E-115 cells stably expressing Rac3 were transiently transfected with control vector or pcDNA3.1/Rap^G12V. Cells were subsequently starved for 1 hour, and stained for paxillin. Note the restoration of the paxillin-rich structures in N1E-115/Rac3 cells that express Rap^G12V (middle and right panels). (D) Bar graph representing the quantification of the experiment shown in C. At least 100 cells were counted per sample, derived from two independent experiments.

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