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

肌球蛋白 II 之间的功能串扰

• 批准号: 8915750
• 负责人: PAUL FORSCHER
• 金额: $ 36.42万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8915750
• 关键词: ADP-G-actin; ATP phosphohydrolase; Acceleration; Actins; Acute; Address; Advanced Malignant Neoplasm; Affect; Affinity; Aplysia; Biochemical; Biological; Biological Assay; Biology; Brain; Calcineurin; Cells; Clinical; Computer Simulation; Coupling; Cytoplasmic Tail; Data; Development; Electron Microscopy; Engineering; Eukaryotic Cell; F-Actin; G Actin; Growth; Growth Cones; Growth Factor; Growth Factor Receptors; Health; In Vitro; Label; Life; Light; Literature; Maleimides; Measures; Mechanics; Microfilaments; Microscopy; Myosin ATPase; Myosin Type II; Neoplasm Metastasis; Nerve Regeneration; Neurites; Neurodegenerative Disorders; Neurons; Normal Cell; Pattern; Peripheral; Phospholipase C; Phosphorylation; Physics; Play; Process; Protein Family; Proteins; Publishing; Regulation; Reporting; Research; Rho-associated kinase; Role; Running; Serotonin; Signal Pathway; Signal Transduction; Site; Spinal cord injury; Structure; System; Testing; Time; Treatment Protocols; Trefoil Motif; Update; axon growth; axon regeneration; base; cancer cell; cell motility; cell type; cofilin; kinematics; light microscopy; mathematical model; neuronal growth; neurotropic; non-muscle myosin; novel; quantitative imaging; research study; response; simulation; tool; trend

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.

描述（由申请人提供）：ADF/Cofilin 蛋白家族在肌动蛋白丝周转中发挥着关键作用，而肌动蛋白丝周转对所有形式的真核细胞运动和正常大脑发育至关重要。尽管有大量关于控制丝切蛋白活性的信号通路的文献，但由于缺乏对丝切蛋白的实时检测，评估活细胞中的丝切蛋白功能受到阻碍 活动。在目标中，我将利用肌动蛋白丝的内在 ATP 酶活性和高 活性肌动蛋白丝切蛋白与 ADP-肌动蛋白亚基的亲和力，使用定量荧光散斑显微镜 (qFSM) 测定活体神经元中的丝动蛋白丝切蛋白活性和动态。海兔丝切蛋白将在带有 AlexaFluor 标签的特定位点进行衍生化，并在体外验证其生化活性和功能。然后将 AlexaFluor-apCofilins 和 AlexaFluor-G-actin 注射到海兔神经元中，并在不同 apCofilin 活性的条件下记录低水平的 qFSM 和斑点动态。 apCofilin 与肌动蛋白散斑运动学、散斑寿命、 并对营业额动态进行分析。对丝切蛋白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 依赖性机械力会影响丝切蛋白切断活性；因此，我们将研究肌球蛋白 II 活性是否直接影响神经突生长过程中的丝切蛋白活性和肌动蛋白动态。这种强大的丝切蛋白活性测定可移植到其他细胞类型，并将提供一种有价值的新工具 用于解决细胞运动过程的调节，包括轴突生长和再生。 神经突生长的特点是中央（C）和外周（P）细胞质生长锥域的协调前进。我们最近报道，血清素 (5-HT) 通过涉及生长锥 P 结构域中磷脂酶 C (PLC) 依赖性 Ca 释放和钙调神经磷酸酶 (CN) 依赖性丝切蛋白激活的机制，使神经突生长速度加快约 300%。 5-HT 刺激的生长伴随着 P 结构域中逆行肌动蛋白丝流的 CN 依赖性增加。当背景非肌肉肌球蛋白 II 活性受到抑制时，5-HT 继续触发丝切蛋白激活并增加逆行肌动蛋白流，但不再发生 C 结构域前进。因此，肌球蛋白 II 活性对于将 P 结构域中肌动蛋白跑步的增加与 C 结构域的前进进行功能耦合是必要的。在目标 II-III 中，我们解释了为什么会这样。我们之前已经将 Rho 激酶 (ROCK) 与肌球蛋白 II 依赖性 C 结构域收缩性的调节联系起来，并将研究 ROCK 在协调 C 和 P 结构域功能中的作用。实验旨在将正在研究的细胞骨架机制推广到许多其他利用 PLC 信号传导的生长因子受体。预计这些研究对于了解与脑和/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。