Fig. 6. PAK, Rho and actomyosin system regulate spreading of protrusions. (A) A kinase-dead mutant of PAK (KD-PAK) was co-expressed with EGFP in cerebellar rhombic-lip-derived neuronal precursors. EGFP fluorescent images were captured every 2 minutes for 2 hours (see supplementary material Movie 12), and images at 0, 40, 80, 120 minutes are shown. KD-PAK created unstable branches (arrowheads) and caused frequent changes in the growing direction of the leading process. (B) Overexpression of PAK induced a large, spread growth-cone-like protrusion at the TLPs and inhibited their extension. These large protrusions were relatively stable (see supplementary material Movie 13). (C) Cerebellar explants electroporated with EGFP were pre-cultured for 24 hours, before treatment with 100 µM blebbistatin, a myosin II ATPase inhibitor, for 15 minutes. Inhibition of myosin II reduced formation of spread lamella on the TLPs and decreased the rate of their extension (see supplementary material Movie 14). (D) C3-toxin-expressing neuronal precursors showed reduced spreading of leading protrusions. Termini of their leading process often displayed an angular shape and irregularly extended protrusion in non-directional manner (arrows; see supplementary material Movie 15). (E) Expression of DN-Rho-kinase reduced spreading of leading protrusions and perturbed their coordinated extension. Bar, 10 µm.