An extracellular-matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration - PubMed (original) (raw)

Figure 3

βPix acts through Cdc42 to suppress and localize RhoA activity during migration in 3D collagen. (a, b) RhoA activity determined using GST-RBD binding from NS and βPix shRNA-expressing HFFs migrating in fibronectin or fibrillar collagen environments; collagen-specific increases (40-60%) in RhoA activity with loss of βPix (mean ± s.e.m, n = 3 independent western blots, _t_-tests). (c, d) Similarly, knockdown of Cdc42, but not Rac1, during migration on fibrillar collagen leads to increased intracellular RhoA activity (mean ± s.e.m, n = 3 independent western blots, one-way ANOVA with Bonferroni correction). (e) Maximum projections of confocal stacks of live fibroblast migration expressing a RhoA biosensor on fibronectin (FN) or fibrillar collagen (FIB COL). Knockdown of βPix on collagen results in overall elevation of RhoA activity accompanied by a loss of front-back segregation of RhoA activity. Pseudocolor intensity scales were identical for each matrix condition; scale bars, 25 μm. White arrows designate direction of leading edge protrusions. (f) Average integrated whole cell RhoA FRET intensity on FN versus FIB COL. n = 10 cells for NS FN, βPix sh#2 FN, NS FIB COL, and βPix sh#2 FIB COL were assessed across three independent experiments (mean ± s.e.m., _t_-test). (g) Quantification RhoA FRET polarization index on FN versus FIB COL. n = 10 cells for NS FN, βPix sh#2 FN, NS FIB COL, and βPix sh#2 FIB COL were assessed across three independent experiments (mean ± s.e.m.,_t_-test). (h) Phase contrast timelapse images (Supplementary Movie 4) of an HFF expressing low levels of GFP-RhoAQ63L in 3D collagen reveal rounded morphology, spatially and temporally deregulated protrusions (white arrowheads) and loss of persistent migration. Scale bars, 25 μm. (i) Quantification of cell elliptical factor (maximal length/width) in cells low-expressing GFP-RhoAQ63L in 3D collagen. n = 30, 35, and 29 cells for NS, βPix sh#2, and RhoQ63L were assessed across three independent experiments. (j) Quantification of cell protrusions in cells with low-level GFP-RhoAQ63L expression in 3D collagen. n = 36, 36, and 29 cells for NS, βPix sh#2, and RhoQ63L were assessed across three independent experiments. (k) Quantification of cell velocity in cells with low GFP-RhoAQ63L expression in 3D collagen. n = 25, 24, and 21 cells for NS, βPix sh#2, and RhoQ63L were assessed across three independent experiments. (l) βPix shRNA fibroblasts were cultured overnight in 3D collagen gels in the presence of cell-permeable C3 transferase (2 μg/mL) or blebbistatin (25 μM). n = 25, 24, 20, and 20 cells for NS, βPix sh#2, βPix+C3, and βPix+Blebb were assessed across three independent experiments. For (i-l), data given as mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction. Statistical source data can be found in Supplementary Table 2, *** P < 0.001, * P < 0.05.