The Role of Myosin in Vesicle Trafficking

肌球蛋白在囊泡运输中的作用

• 批准号: 7012988
• 负责人: YASHOMATI M PATEL
• 金额: $ 20.93万
• 依托单位: UNIVERSITY OF NORTH CAROLINA GREENSBORO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012988
• 关键词: 3T3 cells; adipocytes; cytoskeleton; enzyme induction /repression; glucose transport; glucose transporter; hormone regulation /control mechanism; insulin; intracellular transport; isozymes; membrane fusion; myosins; phosphorylation; protein localization; vesicle /vacuole

DESCRIPTION (provided by applicant): The major defect in type 2 diabetes is insulin resistance of peripheral tissues, primarily muscle and adipose tissue. Insulin-stimulated glucose uptake is mediated by the insulin-responsive glucose transporter, (GLUT4), GLUT4 vesicle translocation and fusion with the plasma membrane are critical for glucose homeostasis. In order for vesicle fusion to occur, the cortical cytoskeletal networks at the plasma membrane have to be disrupted in a temporal and spatial manner. While recent studies have established that actin reorganization is required for GLUT4 vesicle translocation and membrane remodeling, little is known about the motor forces required for these processes. The conventional nonmuscle myosin, myosin II is an actin-based motor protein that has been shown to facilitate vesicle trafficking in various cell types. The goal of this proposal is to characterize the role of myosin II in insulin-mediated GLUT4 vesicle trafficking in adipocytes by identifying the myosin II isoform responsible for regulating GLUT4 trafficking and determining its mode of action and regulation. We will use the 3T3-L1 adipocyte cell culture model to investigate the role of myosin II in GLUT4 trafficking since much of what is known about insulin-stimulated glucose uptake in adipocytes has been elucidated using this cell line. Our preliminary studies show that inhibition of myosin II impairs GLUT4- mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and translocates to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. This project lends itself extremely well to student involvement. Undergraduate students are exposed to the fundamental concepts of hormone action, vesicle transport and glucose metabolism early in the Biology curriculum. This project will allow students to gain practical laboratory experience of these concepts using standard molecular, cellular and biochemical techniques. This project also allows students to integrate key concepts taught in the classroom to address a physiologically relevant question in the laboratory. Thus the overall aim of this proposal is to gain insight on the factors regulating the dynamic reorganization of the cytoskeleton during insulin-stimulated GLUT4 vesicle trafficking in order to provide potential pharmaceutical targets for drug therapies aimed at restoring insulin sensitivity and glucose homeostasis.

描述（申请人提供）：2型糖尿病的主要缺陷是外周组织（主要是肌肉和脂肪组织）的胰岛素抵抗。胰岛素刺激的葡萄糖摄取是由胰岛素响应性葡萄糖转运蛋白 (GLUT4) 介导的，GLUT4 囊泡易位以及与质膜的融合对于葡萄糖稳态至关重要。为了发生囊泡融合，质膜处的皮质细胞骨架网络必须以时间和空间的方式被破坏。虽然最近的研究已经确定 GLUT4 囊泡易位和膜重塑需要肌动蛋白重组，但人们对这些过程所需的动力知之甚少。传统的非肌肉肌球蛋白，肌球蛋白 II 是一种基于肌动蛋白的运动蛋白，已被证明可以促进各种细胞类型中的囊泡运输。该提案的目标是通过鉴定负责调节 GLUT4 运输的肌球蛋白 II 亚型并确定其作用和调节模式，来表征肌球蛋白 II 在脂肪细胞中胰岛素介导的 GLUT4 囊泡运输中的作用。我们将使用 3T3-L1 脂肪细胞培养模型来研究肌球蛋白 II 在 GLUT4 运输中的作用，因为有关脂肪细胞中胰岛素刺激的葡萄糖摄取的大部分知识已使用该细胞系阐明。我们的初步研究表明，抑制肌球蛋白 II 会损害 GLUT4 介导的葡萄糖摄取，但不会损害 GLUT4 向质膜的易位。我们还表明，脂肪细胞表达肌球蛋白 IIA 和 IIB 亚型，并且肌球蛋白 IIA 在胰岛素刺激下被募集至质膜。根据我们的研究结果，我们假设肌球蛋白 II 在胰岛素刺激下被激活，并转移到细胞皮质以促进 GLUT4 与质膜的融合。该项目非常适合学生参与。本科生在生物学课程的早期就接触到激素作用、囊泡运输和葡萄糖代谢的基本概念。该项目将使学生能够使用标准分子、细胞和生化技术获得这些概念的实际实验室经验。该项目还允许学生整合课堂上教授的关键概念，以解决实验室中的生理相关问题。因此，该提案的总体目标是深入了解胰岛素刺激的 GLUT4 囊泡运输过程中细胞骨架动态重组的调节因素，以便为旨在恢复胰岛素敏感性和葡萄糖稳态的药物治疗提供潜在的药物靶标。