Ration as expected [35]. Coating also affected cell morphology with cells on fibronectin appearing flatter and cells on Matrigel rounder (Fig. 4B). Collagen I coating stimulated roundish cell bodies but also stimulated long extensions. Knockdown of p114RhoGEF had a variable effect on the morphology of cells on coated surfaces and led to more spread cells on Matrigel, but the effect was not as pronounced as on non-coated dishes (Fig. 4B). Strikingly, cells on fibronectin were not only flat but also formed leading edges with pronounced lamellipodia; on Matrigel, cells moved preferentially as roundish cells. We next quantified the effect of depletion of p114RhoGEF and GEF-H1 on migration of cells plated on different matrixes. Cells were transfected in large cultures on uncoated surfaces and then plated on the different matrices to ensure equal depletion efficiencies. Figure 4C shows that depletion of p114RhoGEF did not affect migration on fibronectin, whereas GEF-H1 depletionstill attenuated it. On collagen and Matrigel, however, p114RhoGEF depletion inhibited migration effectively and was strongest when cells were plated on the latter substrate on which cells had the roundest morphology. GEF-H1 depletion inhibited migration on all matrices. p114RhoGEF thus regulates cell locomotion in a matrix-dependent manner and seems to affect amoeboid and not mesenchymal-like migration.Regulation of Tumor Cell Locomotion in 3D MatricesInvasion of tumor cells into 3D matrices occurs either by mesenchymal- or amoeboid-like locomotion, with the latter being independent of MMPs [3,6]. Given the phenotypes observed upon p114RhoGEF depletion in 2D migration, one would thus expect that invasion is p114RhoGEF dependent and that inhibition of MMPs does not affect p114RhoGEF-dependent invasion. To test this we performed first standard invasion assays across matrix coated filter inserts [1,26]. Figure 5A shows that MDA-MB-231 cells efficiently invaded Matrigel coated filters. If p114RhoGEF had been depleted, however, invasion was drastically inhibited. Depletion of GEFH1 also reduced invasion as expected [36,37]. Quantification revealed that depletion of p114RhoGEF indeed inhibited invasion by about 60 percent (Fig. 5B). p114RhoGEF is thus required for efficient invasion. We next embedded siRNA transfected cells into 3D matrices and recorded time-lapse movies to follow the movement and to quantify locomotion within the matrices. Figure 5C shows that the effect on cell morphology was not as striking as in 2D cultures.Cortical Myosin Regulation and Cell MigrationFigure 2. Regulation of single cell migration by p114RhoGEF. MDA-MB-231 cells were transfected with GSK429286A web siRNAs as GSK962040 site indicated (p114RG siRNA-1 and siRNA-2 refer to distinct individual siRNAs; siRNA-p refers to a pool of the two siRNAs). Expression of indicated proteins was analyzed by immunoblotting (A), effect on total levels of active RhoA by G-LISA assay (B; shown are means 6 1SD, n = 4), and migration by time-lapse microscopy over 5 hours (C,D). Migration distances were quantified by single cell tracking. Panel D shows means 6 1SD of four different fields (20 cells were analyzed for each field). doi:10.1371/journal.pone.0050188.gHowever, control siRNA transfected cells had again more roundish and cylindrical cell bodies and formed comparatively short extensions, typical for various forms of amoeboid-like, actinomyosin-driven movement [5]. Upon p114RhoGEF deple-tion, however, cells again appeared flatter and p.Ration as expected [35]. Coating also affected cell morphology with cells on fibronectin appearing flatter and cells on Matrigel rounder (Fig. 4B). Collagen I coating stimulated roundish cell bodies but also stimulated long extensions. Knockdown of p114RhoGEF had a variable effect on the morphology of cells on coated surfaces and led to more spread cells on Matrigel, but the effect was not as pronounced as on non-coated dishes (Fig. 4B). Strikingly, cells on fibronectin were not only flat but also formed leading edges with pronounced lamellipodia; on Matrigel, cells moved preferentially as roundish cells. We next quantified the effect of depletion of p114RhoGEF and GEF-H1 on migration of cells plated on different matrixes. Cells were transfected in large cultures on uncoated surfaces and then plated on the different matrices to ensure equal depletion efficiencies. Figure 4C shows that depletion of p114RhoGEF did not affect migration on fibronectin, whereas GEF-H1 depletionstill attenuated it. On collagen and Matrigel, however, p114RhoGEF depletion inhibited migration effectively and was strongest when cells were plated on the latter substrate on which cells had the roundest morphology. GEF-H1 depletion inhibited migration on all matrices. p114RhoGEF thus regulates cell locomotion in a matrix-dependent manner and seems to affect amoeboid and not mesenchymal-like migration.Regulation of Tumor Cell Locomotion in 3D MatricesInvasion of tumor cells into 3D matrices occurs either by mesenchymal- or amoeboid-like locomotion, with the latter being independent of MMPs [3,6]. Given the phenotypes observed upon p114RhoGEF depletion in 2D migration, one would thus expect that invasion is p114RhoGEF dependent and that inhibition of MMPs does not affect p114RhoGEF-dependent invasion. To test this we performed first standard invasion assays across matrix coated filter inserts [1,26]. Figure 5A shows that MDA-MB-231 cells efficiently invaded Matrigel coated filters. If p114RhoGEF had been depleted, however, invasion was drastically inhibited. Depletion of GEFH1 also reduced invasion as expected [36,37]. Quantification revealed that depletion of p114RhoGEF indeed inhibited invasion by about 60 percent (Fig. 5B). p114RhoGEF is thus required for efficient invasion. We next embedded siRNA transfected cells into 3D matrices and recorded time-lapse movies to follow the movement and to quantify locomotion within the matrices. Figure 5C shows that the effect on cell morphology was not as striking as in 2D cultures.Cortical Myosin Regulation and Cell MigrationFigure 2. Regulation of single cell migration by p114RhoGEF. MDA-MB-231 cells were transfected with siRNAs as indicated (p114RG siRNA-1 and siRNA-2 refer to distinct individual siRNAs; siRNA-p refers to a pool of the two siRNAs). Expression of indicated proteins was analyzed by immunoblotting (A), effect on total levels of active RhoA by G-LISA assay (B; shown are means 6 1SD, n = 4), and migration by time-lapse microscopy over 5 hours (C,D). Migration distances were quantified by single cell tracking. Panel D shows means 6 1SD of four different fields (20 cells were analyzed for each field). doi:10.1371/journal.pone.0050188.gHowever, control siRNA transfected cells had again more roundish and cylindrical cell bodies and formed comparatively short extensions, typical for various forms of amoeboid-like, actinomyosin-driven movement [5]. Upon p114RhoGEF deple-tion, however, cells again appeared flatter and p.
