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IP3和DAG信号。5-HT引起IP3 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。蛋白激酶C还具有其他已知的作用，包括调节生长锥中的微管（MT）动力学 由于MT是ER/Ca库的转运底物，MT动力学调节功能地形 IP3依赖性Ca释放参与5-HT依赖性生长。PKC还可以增强整合素基础细胞 粘附，从而影响参与神经元生长的牵引力。我们建议研究PKC作为一种 协调以下的信号传导节点：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）Ca 通过调控微管/ER动力学释放拓扑结构，以及4）最终，产生牵引力 在轴突生长的过程中。这些研究将为理解cofilin如何使 趋化性生长反应中肌动蛋白动力学和肌球蛋白II收缩性之间的功能性串扰。 这些结果将对PKC在神经元生长中的关键作用产生重要影响， 神经退行性疾病

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 and since MTs are the transport substrate for ER/Ca stores, MT dynamics regulate the functional topography of IP3 dependent Ca release involved in 5-HT dependent growth. PKC can also potentiate integrin based cell adhesion and thereby affect traction forces involved in neuronal growth. 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 force production during 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 neurodegenerative disease.