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Figure 8


Fig. 8. Schematic summary of morphological changes on polarized processes of neuronal precursors observed in situ. (A) Control EGFP-expressing cells elongated a long leading process. The tip of the process formed protrusions in a spatially restricted manner. (B) Expression of N17-Cdc42 induced retraction of the major existing process and abrogated the monopolar morphology. (C) Perturbation of polarity effectors of Cdc42 (Par6-NT, S9A-GSK, APC{Delta}CT) generated branched protrusions on the terminus of the leading process. Expression of KD-PAK also induced branching of the terminal protrusion. The duration of these branches were short; but the resulting processes often displayed a curved shape at the points the branches formed. (D) Depolymerization of microtubules by nocodazole caused fragmentation of the leading process. Altered orientation of TLP was often observed during the fragmentation. (E) Overexpression of microtubule plus-end binding protein EB3-GFP induced non-directional extension of the leading process. (F) Dominant-negative mutant of class IA PI3K ({Delta}p85{alpha} PI3K) promoted random migration of rhombic-lip-derived cells. This mutant disturbed formation of long leading process as well as perturbed directional migration. (G) Kinase-dead mutant of class IB PI3K (KD-p110{gamma} PI3K) affected directional extension but somewhat reduced the size of TLP. Overexpression of PTEN also reduced the size of TLP. The resulting TLPs extended normally compared with control EGFP cells. (H) Inhibition of actomyosin contraction by blebbistatin reduced the degree of spreading of TLP. Perturbation of the Rho-Rho-kinase pathway by expression of C3 toxin or DN-Rho-kinase also suppressed spreading of TLP. (I) C124A-PTEN increased the terminal spreading of leading process. Overexpression of PAK also enhanced the terminal protrusion and resulted in a well-developed growth cone-like structure. (J) Overexpression of Rac produced an aberrant multiply protrusive structure on the TLP. This structure displayed rapid extension and retraction of microspikes rather than lamella. (K) Perturbing the function of Rac (N17-Rac) as well as RTK (AG1478, ErbB4{Delta}IC) suppressed generation of protrusions at the TLP.