Fig. 5. PIP₃ signaling controls direction and magnitude of protrusion formation. (A) Class IA PI3K mutant, p85, 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 9), and images at 0, 40, 80, 120 minutes are shown. p85-expressing neuronal precursors do not form long leading process and show random migration. Since these cells no longer extend their leading process directionally toward the ventral midline, images of cell soma-rich region near the rhombic lip are shown. (B) Dynamics of Class IB PI3K mutant, KD-p110-expressing neuronal precursors visualized with co-expressed EGFP. These cells do not show any apparent defect in directed extension of leading processes (see supplementary material Movie 10). (C) Overexpression of PTEN reduced the protrusive activity on the tip of the leading processes. However, it had no apparent effect on rate and direction of process extension. (D) Phosphatase-dead PTEN mutant, C124A-PTEN, upregulated the protrusive activity at the process tips. This significantly perturbed the extension of leading process (see supplementary material Movie 11). Bar, 10 µm.