The Role of Myosin in Vesicle Trafficking

描述（由申请人提供）：2型糖尿病是美国最常见的糖尿病。导致2型糖尿病的主要因素是外周组织的胰岛素抵抗，主要是骨骼肌和脂肪组织。胰岛素介导含有胰岛素反应性葡萄糖转运蛋白（GLUT4）的囊泡向质膜的易位和融合；这个过程对于正常的葡萄糖稳态至关重要。因此，鉴定和表征调节GLUT4功能的细胞因子和过程对于理解胰岛素敏感性受损的分子机制至关重要。虽然胰岛素信号通过调节肌动蛋白重组和细胞骨架重塑来协调GLUT4囊泡的易位和融合，但对这些过程所需的细胞收缩力知之甚少。该建议的主要目的是深入了解在胰岛素刺激的GLUT4囊泡运输过程中调节细胞骨架动态重组的机制。肌凝蛋白家族，特别是肌凝蛋白II已被证明可调节肌动蛋白丝重组以促进囊泡运输。我们的初步研究表明，肌球蛋白IIA异构体在胰岛素刺激下被激活并招募到质膜上，以促进glut4介导的葡萄糖摄取。我们还发现胰岛素通过肌球蛋白轻链激酶（MLCK）调节肌球蛋白IIA，这是一种Ca2+调节的激酶，可促进肌球蛋白IIA的激活和向质膜募集。此外，我们发现肌球蛋白II活性的抑制会损害GLUT4在质膜上的适当插入。先前的研究表明，胰岛素刺激的GLUT4易位和与质膜融合需要细胞内Ca2+。基于我们的研究结果，我们假设Ca2+介导肌球蛋白IIA的激活和募集到细胞皮层，以促进GLUT4与质膜的融合。因此，本提案的具体目的是定义和表征Ca2+在调节肌球蛋白IIA在脂肪细胞中GLUT4囊泡运输中的作用，并确定肌球蛋白IIA调节肌动蛋白重组的机制。

DESCRIPTION (provided by applicant): Type 2 diabetes is the most prevalent form of diabetes in the United States. The major contributing factor to the development of type 2 diabetes is insulin resistance of peripheral tissues, primarily skeletal muscle and adipose tissue. Insulin mediates the translocation and fusion of insulin-responsive glucose transporter (GLUT4)-containing vesicles to the plasma membrane; this process is critical for proper glucose homeostasis. Thus, the identification and characterization of cellular factors and processes regulating GLUT4 function are critical to understanding the molecular mechanisms underlying impaired insulin sensitivity. While insulin signaling coordinates the translocation and fusion of GLUT4 vesicles by regulating actin reorganization and cytoskeletal remodeling little is known about the cellular contractile forces required for these processes. The broad aim of this proposal is to gain insight on the mechanisms regulating the dynamic reorganization of the cytoskeleton during insulin-stimulated GLUT4 vesicle trafficking. The myosin family of protein, specifically myosin II has been shown to regulate actin filament reorganization to facilitate vesicle trafficking. Our preliminary studies show that the myosin IIA isoform is activated and recruited to the plasma membrane upon insulin stimulation to facilitate GLUT4-mediated glucose uptake. We also have found that insulin regulates myosin IIA via myosin light chain kinase (MLCK), a Ca2+ regulated kinase to promote myosin IIA activation and recruitment to the plasma membrane. Furthermore, we show that inhibition of myosin II activity impairs the proper insertion of GLUT4 at the plasma membrane. Previous studies have shown a requirement for intracellular Ca2+ for insulin stimulated GLUT4 translocation and fusion with the plasma membrane. Based on our findings, we hypothesize that Ca2+ mediates the activation and recruitment of myosin IIA to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. Thus, the specific aims of this proposal are to define and characterize the role of Ca2+ on the regulation of myosin IIA in GLUT4 vesicle trafficking in adipocytes, and to determine the mechanisms by which myosin IIA regulates actin reorganization. PUBLIC HEALTH RELEVANCE: Type 2 diabetes is a major public health issue in the United States affecting approximately 6% of the population. The prevalence of type 2 diabetes is even higher among African Americans, Hispanics, Native Americans, Asian Americans, and the elderly. A particularly alarming trend is the recent dramatic rise in the incidence of type 2 diabetes in children. With the increasing incidence of type 2 diabetes it is critical to identify factors contributing to the development of this disease in order to design effective treatments. The proposed research will provide critical information for understanding the molecular mechanism(s) involved in the development of in type 2 diabetes.