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

描述(申请人提供)：ADF/cofilin蛋白家族在肌动蛋白细丝周转中发挥关键作用，肌动蛋白细丝对所有形式的真核细胞运动和正常大脑发育至关重要。尽管有大量文献介绍了控制粘附素活性的信号通路，但由于缺乏对粘附素的实时检测，评估活细胞中的粘附素功能一直受到阻碍 活动。在AIM中，我将利用肌动蛋白细丝的固有ATPase活性和高 活性粘附素与ADP-肌动蛋白亚基的亲和力利用定量荧光散斑显微镜(QFSM)实现活神经元中粘附素活性和动力学的分析。Aplysia cofilin将通过AlexaFluor标签在特定位置进行衍生，其生化活性和功能将在体外得到验证。然后将AlexaFluor-apCofilins和AlexaFluor-G-actin注射到海兔神经元中，并在不同apCoflin活性的条件下记录低水平的QFSM和斑点动力学。ApCoflin与肌动蛋白斑点运动学，斑点寿命， 并对员工的离职动态进行了分析。通过光镜和电子显微镜评估肌动蛋白细丝结构，将会对肌丝蛋白活性产生影响。肌球蛋白II依赖的机械力已被报道影响粘连蛋白的切断活性，因此，我们将研究肌球蛋白II活性是否直接影响神经突起生长过程中的粘连蛋白活性和肌动蛋白动力学。这种可靠的粘附素活性测定方法可移植到其他类型的细胞，并将提供一种有价值的新工具 用于研究细胞运动过程的调节，包括轴突生长和再生。轴突突起的特征是中央(C)和外周(P)胞质生长锥域的协调推进。我们最近报道，5-羟色胺(5-HT)通过磷脂酶C(PLC)依赖的钙释放和钙调神经磷酸酶(CN)依赖的钙调神经磷酸酶(CN)激活生长锥P区的cofilin，使轴突生长速度加快约300%。5-羟色胺刺激的突起伴随着CN依赖的P区肌动蛋白细丝逆行流动的增加。当背景非肌肉肌球蛋白II活性被抑制时，5-羟色胺继续触发粘连蛋白激活并增加肌动蛋白逆行流动，但不再发生C结构域推进。因此，肌球蛋白II的活性是随着C结构域的推进而在P区肌动蛋白踏板上的功能偶联增加所必需的。在AIM II-III中，我们阐述了为什么会这样。我们先前已经发现Rho Kinase(ROCK)参与调节肌球蛋白II依赖的C结构域的收缩能力，并将研究ROCK在协调C和P结构域功能中的作用。实验建议将正在研究的细胞骨架机制推广到许多其他利用PLC信号的生长因子受体。这些研究预计将对了解与脑和/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.