Mechanical catalysis of calcineurin dependent cofilin activity during chemotropic axon growth: a new role for PKC in coordinating actin dynamics and myosin II contractility

ADF/cofilin蛋白质家族在肌动蛋白丝周转中起关键作用，肌动蛋白丝周转对所有形式的免疫缺陷都是必不可少的。 真核细胞运动尽管有大量关于控制cofilin活性的信号传导途径的文献，但评估cofilin活性的信号传导途径仍然是困难的。 由于缺乏对cofilin的真实的时间测定，这一重要蛋白在活细胞中的功能受到阻碍 功能使用一种新的真实的时间测定法同时评估cofilin活性和肌动蛋白动力学， 定量荧光散斑显微镜（qFSM），我们已经发现，机械应力施加在 研磨肌动蛋白网络增加了cofilin活性，对肌动蛋白周转率有显著影响， 压力的程度。低应力水平与肌动蛋白周转率和细胞研磨率的增加有关， 与趋化性生长有关;相反，超过临界阈值的应力水平会导致灾难性的 肌动蛋白网络密度降低导致神经突收缩。我们一直在研究机械效应 在5-羟色胺（5-HT）诱发的神经突生长反应的背景下， G（q）亚型GPCR，其激活磷脂酶C以产生IP 3和DAG信号。5-HT引起IP 3 依赖于从细胞内储存的Ca释放和通过Ca→钙调神经磷酸酶信号级联激活cofilin。 我们现在有证据表明，DAG的产生导致非肌肉肌球蛋白II的PKC依赖性增加， 活动这反过来又产生局部网络应力和cofilin的机械催化活化，导致局部神经元损伤。 F-肌动蛋白结构和网络周转率的改变。对肌动蛋白结构的影响也取决于 PKC激活。PKC还具有其他已知的作用，包括调节生长中的微管（MT）动力学 圆锥体。MT是ER/Ca库的转运底物;因此，MT动力学调节ER/Ca库的功能。 IP 3依赖性Ca释放的拓扑结构是5-HT依赖性生长所必需的。最后，PKC可以 增强基于整联蛋白的细胞粘附，从而影响生长锥所必需的牵引力 提前我们建议研究PKC作为一个信号节点，协调：1）肌球蛋白II收缩性，2） 通过cofilin机械催化的肌动蛋白周转，3）通过调节微管/ER的Ca释放拓扑 动力学，以及4）最终，在刺激轴突生长期间的牵引力。这些研究将提供一个 理解cofilin如何使肌动蛋白动力学之间的功能串扰和 肌球蛋白II在趋化性生长反应中的收缩性。研究结果将产生有趣的影响 关于PKC在神经元生长和神经变性中的关键作用。

The ADF/cofilin family of proteins play a critical role in actin filament turnover essential to all forms of eukaryotic cell motility. Despite a vast literature on signaling pathways controlling cofilin activity, assessing the function of this important protein in living cells has been hampered by lack of real time assays of cofilin function. Using a novel real time assay for assessing cofilin activity and actin dynamics simultaneously by quantitative fluorescent speckle microscopy (qFSM), we have discovered that mechanical stress imposed on treadmilling actin networks increases cofilin activity with dramatic effects on actin turnover rates that depend on the level of stress. Low stress levels are associated with increases in actin turnover and treadmilling rates that are associated with chemotropic growth; in contrast, stress levels above a critical threshold lead to catastrophic decreases in actin network density resulting in neurite retraction. We have been studying mechanical effects on cofilin activity in the context of serotonin (5-HT) evoked neurite growth responses mediated by classical G(q) subtype GPCRs, which activate phospholipase C to generate IP3 and DAG signals. 5-HT evokes IP3 dependent Ca release from intracellular stores and cofilin activation by a Ca→calcineurin signaling cascade. We now have evidence that DAG production results in PKC dependent increases in non-muscle myosin II activity. This in turn generates local network stress and mechano-catalytic activation of cofilin resulting in local alteration of F-actin structure and network turnover rates. Effects on actin structure also depend on the level of PKC activation. PKC has other known roles including regulation of microtubule (MT) dynamics in growth cones. MTs are the transport substrate for ER/Ca stores; thus, MT dynamics regulate the functional topography of IP3 dependent Ca release that is essential for 5-HT dependent growth. Finally, PKC can potentiate integrin based cell adhesion and thereby affect traction forces that are necessary for growth cone advance. We propose to investigate PKC as a signaling node that coordinates: 1) myosin II contractility, 2) actin turnover via cofilin mechano-catalysis, 3) Ca release topography via regulation of microtubule/ER dynamics, and 4) ultimately, traction forces during stimulated axon growth. These studies will provide a mechanistic framework for understanding how cofilin enables functional crosstalk between actin dynamics and myosin II contractility during chemotropic growth responses. The results will have interesting implications regarding the key role PKC plays in neuronal growth and neurodegeneration.