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家族蛋白在肌动蛋白细丝周转中起关键作用，肌动蛋白细丝对所有形式的肌动蛋白都是必不可少的。 真核细胞的运动性。尽管有大量关于控制粘连蛋白活性的信号通路的文献，但评估 这种重要的蛋白质在活细胞中的功能因缺乏实时的胶着蛋白检测而受到阻碍 功能。使用一种新的实时检测方法同时评估粘附素活性和肌动蛋白动力学 定量荧光散斑显微镜(QFSM)，我们发现机械应力施加在 跑步式肌动蛋白网络增加了肌动蛋白的活性，显著影响了肌动蛋白的周转率，这种影响依赖于 压力的程度。低压力水平与肌动蛋白周转率和跑步率的增加有关， 与趋化生长有关；相反，超过临界阈值的压力水平会导致灾难性的 肌动蛋白网络密度降低，导致轴突回缩。我们一直在研究机械效应 5-羟色胺(5-羟色胺)介导的神经突起生长反应中胶粘蛋白活性的研究 G(Q)亚型GPCRs，激活磷脂酶C产生IP3和DAG信号。5-羟色胺引发IP3 依赖于细胞内钙释放和钙→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中PKC依赖的增加 活动。这进而产生局部网络应力和cofilin的机械催化活化，从而导致局部 F-肌动蛋白结构和网络周转率的变化。对肌动蛋白结构的影响也取决于 PKC激活。PKC还有其他已知的作用，包括调节生长锥体中的微管(MT)动态 由于MT是内质网/钙库的运输底物，MT动态调节功能地形图 依赖IP3的钙释放参与5-羟色胺依赖的生长。PKC还可以增强基于整合素的细胞 粘附性，从而影响牵引力参与神经元生长。我们建议将PKC作为一种 协调的信号节点：1)肌球蛋白II的收缩，2)肌动蛋白通过cofilin机械催化的转化，3)钙 通过调节微管/内质网动力学释放地形，以及4)最终，牵引力产生 在轴突生长过程中。这些研究将提供一个机制框架，以了解cofilin如何使 趋化生长反应中肌动蛋白动力学和肌球蛋白II收缩之间的功能串扰。 这一结果将对PKC在神经元生长和修复中的关键作用产生有趣的影响。 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.