The Role of Myosin in Vesicle Trafficking

描述(申请人提供)：2型糖尿病的主要缺陷是周围组织，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应答的葡萄糖转运体(GLUT4)介导的，GLUT4囊泡的移位和与质膜的融合是葡萄糖动态平衡的关键。为了发生囊泡融合，质膜上的皮质细胞骨架网络必须以时间和空间的方式被破坏。虽然最近的研究证实肌动蛋白重组是GLUT4囊泡移位和膜重塑所必需的，但对这些过程所需的运动力知之甚少。传统的非肌肉肌球蛋白，肌球蛋白II是一种以肌动蛋白为基础的马达蛋白，已被证明促进各种细胞类型的囊泡运输。本研究的目的是通过鉴定负责调节GLUT4转运的肌球蛋白II异构体，并确定其作用模式和调控方式，来表征肌球蛋白II在胰岛素介导的GLUT4囊泡转运中的作用。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转运中的作用，因为许多已知的胰岛素刺激的脂肪细胞葡萄糖摄取都是使用该细胞系来阐明的。我们的初步研究表明，肌球蛋白II的抑制会损害GLUT4介导的葡萄糖摄取，但不会损害GLUT4到质膜的转位。我们还表明，脂肪细胞表达肌球蛋白IIA和IIB亚型，并且在胰岛素刺激下，肌球蛋白IIA被招募到质膜上。根据我们的发现，我们假设肌球蛋白II在胰岛素刺激下被激活，并移位到细胞皮质，以促进GLUT4与质膜的融合。这个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荷尔蒙作用、囊泡运输和葡萄糖代谢的基本概念。这个项目将允许学生使用标准的分子、细胞和生物化学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，以解决实验室中与生理相关的问题。因此，这项建议的总体目的是深入了解在胰岛素刺激的GLUT4囊泡运输过程中调控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治疗提供潜在的药物靶点。

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.