Functional crosstalk between myosin II & cofilin in regulation of neuronal growth

描述（由申请人提供）：ADF/Cofilin蛋白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真核细胞运动和正常脑发育至关重要。尽管有大量关于控制cofilin活性的信号传导途径的文献，但由于缺乏cofilin的真实的时间测定，评估cofilin在活细胞中的功能受到阻碍 活动目的是利用肌动蛋白丝固有的ATP酶活性， 本发明的目的是通过测定活性cofilin对ADP-肌动蛋白亚基的亲和力来使用定量荧光斑点显微术（qFSM）实施活神经元中cofilin活性和动力学的测定。将在特定位点用AlexaFluor标签衍生化Aaplasia cofilins，并在体外验证其生化活性和功能。然后将AlexaFluor-apCofilins和AlexaFluor-G-肌动蛋白注射到失智症神经元中，并在不同apCofilin活性的条件下记录低水平的qFSM和斑点动力学。apCofilin与肌动蛋白斑点运动学，斑点寿命， 并对流动动态进行分析。对cofilin活性的影响将与通过光学和电子显微镜评估的肌动蛋白丝结构相关。据报道，肌球蛋白II依赖的机械力会影响cofilin切割活性，因此，我们将研究肌球蛋白II活性是否直接影响cofilin活性和肌动蛋白动力学在神经突生长。这种稳定的cofilin活性检测方法可移植到其他类型的细胞中，并将提供一种有价值的新工具 用于解决包括轴突生长和再生的细胞运动过程的调节。 神经突生长的特征在于中央（C）和外周（P）细胞质生长锥结构域的协调推进。我们最近报道，5-羟色胺（5-HT）通过磷脂酶C（PLC）依赖性Ca释放和钙调神经磷酸酶（CN）依赖性激活生长锥P结构域中的cofilin的机制，使轴突生长速度加快约300%。5-HT刺激的生长伴随着CN依赖性的增加，在P域的逆行肌动蛋白丝流。当背景非肌肉肌球蛋白II活性被抑制时，5-HT继续触发cofilin激活和增加逆行肌动蛋白流，但C结构域前进不再发生。因此，肌球蛋白II的活性是必要的功能耦合增加肌动蛋白在P结构域与先进的C结构域的研磨。在目标二至三中，我们讨论了为什么会这样。我们以前曾暗示Rho激酶（ROCK）在调节肌球蛋白II依赖的C结构域收缩性，并将研究ROCK在协调C和P结构域功能的作用。实验提出了推广的细胞骨架机制正在研究的许多其他生长因子受体，利用PLC信号。预计这些研究对于理解与脑和/或脊髓损伤相关的神经退行性疾病和神经再生具有重要的临床意义。

DESCRIPTION (provided by applicant): The ADF/Cofilin family of proteins plays a critical role in actin filament turnover essential to all forms of eukaryotic cell motility and normal brain development. Despite a vast literature on signaling pathways controlling cofilin activity, assessing cofilin function in living cells has been hampered by lack of real time assays of cofilin activity. In Aim I will take advantage of the intrinsic ATPase activity of actin filaments and high affinity of active cofilin for ADP-actin subunits to implement an assay for cofilin activity and dynamics in living neurons using quantitative fluorescent speckle microscopy (qFSM). Aplysia cofilins will be derivatized at specific sites with AlexaFluor tags and their biochemical activity and functionality verified in vitro. AlexaFluor-apCofilins and AlexaFluor-G-actin will then be injected into Aplysia neurons and low levels for qFSM and speckle dynamics recorded under conditions of varying apCofilin activity. apCofilin vs actin speckle kinematics, speckle lifetimes, and turnover dynamics will be analyzed. Effects on cofilin activity will be correlated with actin filament structure assessed by light and electron microscopy. Myosin II dependent mechanical forces have been reported to affect cofilin severing activity; thus, we will investigate whether Myosin II activity directly affect cofilin activity and actin dynamics during neurite outgrowth. Ths robust cofilin activity assay is portable to other cells types and will provide a valuable new tool for addressing regulation of cell motility processes including axon growth and regeneration. Neurite outgrowth is characterized by coordinated advance of the central (C) and peripheral (P) cytoplasmic growth cone domains. We recently reported that serotonin (5-HT) accelerates rates of neurite outgrowth by ~300% via a mechanism involving phospholipase C (PLC) dependent Ca release and calcineurin (CN) dependent activation of cofilin in the growth cone P domain. 5-HT stimulated outgrowth was accompanied by CN dependent increases in retrograde actin filament flow in the P domain. When background non-muscle Myosin II activity was inhibited, 5-HT continued to trigger cofilin activation and increases in retrograde actin flow but C domain advance no longer occurred. Thus, myosin II activity is necessary for functionally coupling increases in actin treadmilling in the P domain with advance of the C domain. In Aim II-III we address why this is so. We have previously implicated Rho kinase (ROCK) in regulation of Myosin II dependent C domain contractility and will investigate a role for ROCK in coordination of C and P domain function. Experiments are proposed to generalize the cytoskeletal mechanisms being studied to the many other growth factor receptors that utilize PLC signaling. These studies are predicted to have significant clinical implications for understanding neurodegenerative disease and nerve regeneration related to brain and/or spinal cord injury.