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: 9325076
• 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 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9325076
• 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; In Vitro; Injectable; Label; 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; 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; Update; axon growth; axon regeneration; base; cancer cell; cell motility; cell type; cofilin; experimental study; kinematics; light microscopy; mathematical model; mechanical force; neuronal growth; neurotropic; non-muscle myosin; novel; portability; public health relevance; quantitative imaging; response; simulation; tool; treadmill; trend; 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; In Vitro; Injectable; Label; 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; 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; Update; axon growth; axon regeneration; base; cancer cell; cell motility; cell type; cofilin; experimental study; kinematics; light microscopy; mathematical model; mechanical force; neuronal growth; neurotropic; non-muscle myosin; novel; portability; public health relevance; quantitative imaging; response; simulation; tool; treadmill; 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）：蛋白质的ADF/Cofilin家族在肌动蛋白细丝周转中起着至关重要的作用，这对于所有形式的真核细胞运动和正常脑发育至关重要。尽管关于控制Cofilin活性的信号传导途径的大量文献，但由于缺乏Cofilin的实时测定，评估活细胞中的Cofilin功能受到了阻碍 活动。为了目标，我将利用肌动蛋白丝和高的固有ATPase活性 活性Cofilin对ADP-肌动蛋白亚基的亲和力，使用定量荧光斑点显微镜（QFSM）实施活性神经元中的Cofilin活性和动力学测定法。 Aplysia cofilins将在具有Alexafluor标签的特定位点衍生化，并在体外验证其生化活性和功能。然后将在不同的apcofilin活性条件下记录的QFSM和Speckle动力学的Alexafluor-apcofilins和Alexafluor-G-肌动蛋白将被注入Aplysia神经元和低水平。 apcofilin vs肌动蛋白斑点运动学，斑点寿命， 并将分析营业额动态。对Cofilin活性的影响将与通过光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依赖性的机械力会影响Cofilin的切断活性。因此，我们将研究肌球蛋白II活性在神经突生长过程中是否直接影响Cofilin活性和肌动蛋白动力学。强大的Cofilin活性测定方法可用于其他细胞类型，将提供有价值的新工具 用于解决细胞运动过程的调节，包括轴突生长和再生。 神经突生长的特征是中央（C）和周围（P）细胞质生长锥域的协调前进。我们最近报道说，血清素（5-HT）通过涉及磷脂酶C（PLC）依赖性CA释放和钙调蛋白（CN）依赖cofilin在生长锥P域中的磷脂酶C（PLC）C（CN）依赖性激活的机制加速了神经突生长的速率。 5-HT刺激的产物伴随着P域中逆行肌动蛋白丝流的依赖性依赖性增加。当背景非肌肉肌球蛋白II活性被抑制时，5-HT继续触发Cofilin的激活并增加逆行肌动蛋白流量，但C结构域不再发生。因此，肌球蛋白II活性对于随着C域的前进而在P域中肌动蛋白踏板的功能耦合增加是必需的。在AIM II-III中，我们解决了为什么这样。我们以前已经牵涉到Rho激酶（ROCK）参与肌球蛋白II依赖性C结构域的收缩力的调节，并将研究岩石在C和P结构域功能协调中的作用。提出了实验，以将研究的细胞骨架机制推广到使用PLC信号传导的许多其他生长因子受体。预计这些研究对了解与脑和/或脊髓损伤有关的神经退行性疾病和神经再生具有重要的临床意义。