The Role of Myosin in Vesicle Trafficking

描述(申请人提供)：2型糖尿病是美国最常见的糖尿病类型。导致2型糖尿病发展的主要因素是周围组织，主要是骨骼肌和脂肪组织的胰岛素抵抗。胰岛素介导含有胰岛素反应性葡萄糖转运体(GLUT4)的囊泡移位和融合到质膜；这一过程对适当的葡萄糖动态平衡至关重要。因此，识别和表征调节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囊泡的移位和融合，但对这些过程所需的细胞收缩力量知之甚少。这项建议的主要目的是深入了解在胰岛素刺激的GLUT4囊泡运输过程中细胞骨架动态重组的调节机制。肌球蛋白家族的蛋白质，特别是肌球蛋白II，已被证明调节肌动蛋白细丝重组以促进囊泡运输。我们的初步研究表明，在胰岛素刺激下，肌球蛋白IIA亚型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)来调节肌球蛋白IIA，MLCK是一种钙离子调节的激酶，可以促进肌球蛋白IIA的激活和募集到质膜上。此外，我们还发现，肌球蛋白II活性的抑制会损害GLUT4在质膜上的正确插入。以前的研究表明，胰岛素刺激的GLUT4转位和与质膜的融合需要细胞内的钙离子。根据我们的发现，我们假设钙离子介导肌球蛋白IIA的激活和募集到细胞皮质，以促进GLUT4与质膜的融合。因此，本研究的具体目的是确定和表征钙离子对肌球蛋白IIA在脂肪细胞GLUT4囊泡运输中的调节作用，并确定肌球蛋白IIA调节肌动蛋白重组的机制。 公共卫生相关性：在美国，2型糖尿病是一个主要的公共卫生问题，影响着大约6%的人口。在非洲裔美国人、西班牙裔美国人、土著美国人、亚裔美国人和老年人中，2型糖尿病的患病率甚至更高。一个特别令人担忧的趋势是，最近儿童2型糖尿病的发病率急剧上升。随着2型糖尿病发病率的增加，为了设计有效的治疗方法，确定导致这种疾病发展的因素是至关重要的。这项拟议的研究将为理解2型糖尿病发生的分子机制(S)提供关键信息。

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.