Dimensionality, matrix elastic behavior, and RhoA–ROCK–myosin II govern the mode of normal cell migration. (A) During cell migration on flat or fibrillar 2D surfaces, lamellipodia form the leading edge of adherent fibroblasts. The presence of lamellipodia was confirmed by a prominent rim of F-actin and cortactin, along with active Rac1 and Cdc42 with PIP3 at the leading edge. In a 3D ECM, HFFs can use either lobopodia- or lamellipodia-based 3D migration. (B) The choice to migrate using lobopodia- or lamellipodia-based migration can be represented by a decision tree consisting of three questions: what is the dimensionality of the matrix, what is the level of RhoA activity, and is the 3D matrix linearly elastic? Lobopodia-based 3D migration predominates in linear elastic ECM and may use high actomyosin contraction downstream of RhoA–ROCK–myosin II to increase intracellular pressure and push the leading edge forward in combination with integrin-mediated adhesion. When RhoA–ROCK–myosin II activity is diminished, either in nonlinear ECM or through treatment of cells with RhoA siRNA or specific inhibitors, cells form lamellipodia with actin polymerization to advance the leading edge. Both modes of migration involve elongated cells that form 3D matrix adhesions, but the distribution of active Rac1, Cdc42, and PIP3 distinguishes the two modes.