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

我们在这里研究了Rho-和Ca^<2+>-依赖性神经元肌动蛋白细胞骨架修饰的分子信号机制，以及它们的生理下游后果。为了确定参与树突形成和生长的CaMK亚型，在皮质神经元培养系统中进行了系统的RNAi筛选。当CaMKIα、CaMKII或CaMKIV被shRNA敲除时，树突未见影响。相比之下，CLICK-III/CaMKIγ的shRNA产生了树突生长的强烈减少，这伴随着Rac活性的下降。进一步的实验表明，CLICK-III/CaMKIγ-STEF-Rac通路在皮层树突发生过程中发挥了重要作用（Takemoto-Kimura等人，正在修订中）。接下来，我们研究了两种camk样激酶CLICK-I/DCaMKL1和CLICK-II/DCaMKL2对树突形态的潜在影响。含有完整双皮质素结构域的剪接变异体的过表达揭示了微管的强捆绑，但对肌动蛋白细胞骨架的影响很小（Ohmae等）。J.Biol.Chem。2006)。在这些研究过程中，优化了涉及脂肪感染、腺病毒感染或基因枪的基因传递方案。这导致在表达psd -95突变的神经元中惊人地发现脊柱形态异常（Nonaka et al., j.n neurosci .）。2006)。我们还发现肌动蛋白细胞骨架和钙动力学可能调节海马或浦肯野细胞树突的接触或Shank定位（Iki等）。Eur.J.Neurosci。, 2005;Fuse等人在准备中)。最后，在Narumiya实验室的合作下，我们确定了rho - mdia1依赖性的肌动蛋白细胞骨架调控在Apc和Src复合物在迁移细胞中不同极化位置的定位中起着关键作用。

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.