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（由申请人提供）：2型糖尿病的主要缺陷是周围组织的胰岛素抵抗，主要是肌肉和脂肪组织。胰岛素刺激的葡萄糖摄取是由胰岛素反应性葡萄糖转运蛋白（GLUT4），GLUT4囊泡转运和质膜融合的介导的，对于葡萄糖稳态至关重要。为了进行囊泡融合，必须以时间和空间方式破坏质膜的皮质细胞骨架网络。尽管最近的研究表明，GLUT4囊泡易位和膜重塑需要肌动蛋白的重组，但对这些过程所需的摩托力知之甚少。常规的非肌肉肌球蛋白II是一种基于肌动蛋白的运动蛋白，已显示可促进各种细胞类型的囊泡运输。该提案的目的是表征肌球蛋白II在胰岛素介导的GLUT4囊泡运输脂肪细胞中的作用，通过识别负责调节GLUT4运输的肌球蛋白II同工型并确定其作用和调节方式。我们将使用3T3-L1脂肪细胞细胞培养模型来研究肌球蛋白II在GLUT4运输中的作用，因为使用该细胞系阐明了脂肪细胞中胰岛素刺激的葡萄糖摄取的许多已知性。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，但不损害GLUT4转移到质膜。我们还表明，脂肪细胞同时表达肌球蛋白IIA和IIB同工型，并且在胰岛素刺激后将肌球蛋白IIA募集到质膜上。根据我们的发现，我们假设在胰岛素刺激时激活肌球蛋白II并易位到细胞皮层，以促进与质膜融合的GLUT4。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受了激素作用，囊泡运输和葡萄糖代谢的基本概念。该项目将允许学生使用标准分子，细胞和生化技术来获得这些概念的实践实验室经验。该项目还允许学生整合教室中教授的关键概念，以解决实验室中与生理相关的问题。因此，该提案的总体目的是深入了解调节胰岛素刺激的GLUT4囊泡运输过程中细胞骨架动态重组的因素，以便为旨在恢复胰岛素敏感性和葡萄糖稳态的药物疗法提供潜在的药物治疗。