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: 8729508
• 负责人: PAUL FORSCHER
• 金额: $ 36.03万
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8729508
• 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; Image; 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; public health relevance; 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起关键作用，这对所有形式的真核细胞运动和正常的大脑发育至关重要。尽管有大量关于控制cofilin活性的信号通路的文献，但由于缺乏对cofilin的实时分析，评估活细胞中cofilin的功能一直受到阻碍