Role of Rho- and Ca^<2+>-dependent signaling mechanisms in the regulation of neuronal actin cytoskeleton

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We here investigated the molecular signaling mechanisms underlying Rho- and Ca^<2+>-dependent modifications of neuronal actin cytoskeleton, as well as their physiological downstream consequences.In order to identify a CaMK subtype involved, in dendrite formation and outgrowth, a systematic RNAi screening was carried out in a cortical neuronal culture system. No effect was seen on dendrites when CaMKIα, CaMKII, or CaMKIV were knocked down by shRNA. In contrast, shRNA of CLICK-III/CaMKIγ produced a robust decrease in dendritic growth, and this was accompanied with a drop in Rac activity. Further experiments suggested an essential role for a CLICK-III/CaMKIγ-STEF-Rac pathway during cortical dendritogenesis (Takemoto-Kimura et al., under revision)..We next examined the potential impact of two CaMK-like kinases, CLICK-I/DCaMKL1 and CLICK-II/DCaMKL2, on dendritic morphology. Overexpression of splice variants containing an intact doublecortin domain revealed a strong bundling of microtubules, but little effect on actin cytoskeleton (Ohmae et al. J.Biol.Chem. 2006).During the course of these studies, gene delivery protocols involving lipofection, adenoviral infection or gene gun were optimized. This led to striking discovery of spine morphological aberrance in PSD-95-mutant-expressing neurons (Nonaka et al., J.Neurosci. 2006). We also found that actin cytoskeleton and calcium dynamics may regulate cortactin or Shank localization in either hippocampal or Purkinje cell dendrites (Iki et al. Eur.J.Neurosci., 2005; Fuse et al. in preparation).Finally, in collaboration with the Narumiya laboratory, we determined that Rho-mDia1-dependent regulation of actin cytoskeleton plays a critical role in the localization of Apc and Src complexes at distinct polarized positions in a migrating cell.