Fig. 2. Lsc/p115 RhoGEF and LARG increase stress fibers and localizes to focal adhesions on FN. (A) Domain structure of full-length p115 RhoGEF [p115(FL)] and the different mutants used in this study. The DH-dead p115(4A) mutant contains alanine point substitutions (*) of four residues (E423, K567, L570, N603) in the DH domain that are important for the catalytic exchange reaction. The p115(N) mutant lacks the N-terminus of the protein containing the RGS domain. All constructs were cloned into N-terminal GFP- or V5-tagged vectors. (B,C) REF52 fibroblasts were transfected with vector encoding either GFP-p115(FL) or GFP-LARG(FL). 24 hours post transfection, cells were serum-starved, held in suspension for 2 hours and plated onto FN-coated coverslips for the times indicated. (B) The cells were then fixed and stained with phalloidin to visualize F-actin. Arrows in the top panels point to the tight cortical actin bundles known as arcs. Arrows in the bottom panels point to the discrete patches of p115 RhoGEF or LARG localization. Bar, 40 µm. (C) The cells were fixed and stained with antibody against paxillin to visualize focal adhesions. The images represent 0.3 µm confocal sections at the ventral surface of the cells. Arrows point to areas of colocalization between paxillin-containing focal adhesions and the discrete patches of p115 RhoGEF or LARG localization.