Components And Kinetics In Exocytosis

1.由于葡萄糖代谢的标志是胰岛素刺激的葡萄糖转运蛋白-4（GLUT 4）向质膜（PM）的递送，而膜蛋白组织的标志是其结构域结构，因此我们继续研究胰岛素对脂肪细胞PM中GLUT 4组织的影响。 果蝇（Drosophilamelanogaster）是研究发育和疾病基因的一个极好的模式系统。然而，由于昆虫和哺乳动物之间的代谢差异，其对生理系统的适用性不太清楚。由于胰岛素信号与糖尿病和其他疾病的相关性，已经在哺乳动物中进行了研究，但哺乳动物和昆虫之间的途径有许多相似之处。例如，果蝇胰岛素样肽的缺失导致“糖尿病”果蝇循环糖水平升高。这种情况是否反映了在哺乳动物中观察到的糖摄取到外周组织中的失败尚不清楚，这取决于苍蝇是否具有安装类胰岛素依赖性糖摄取反应的机制。在这里，我们问果蝇脂肪细胞是否有能力响应胰岛素与糖转运蛋白的调节贩运。产生了表达人葡萄糖转运蛋白4（GLUT 4）的转基因果蝇，该糖转运蛋白主要在胰岛素应答组织中表达。在脂肪体中表达后，通过共聚焦和全内反射荧光显微镜（TIRFM）监测GLUT 4细胞内运输和定位。我们发现，脂肪体细胞对胰岛素的反应是增加GLUT 4的运输和转运到质膜。虽然这些反应的幅度在标准饮食饲养的动物中相对较弱，但在限糖饮食饲养的动物中大大增强，这表明喂食标准饮食的苍蝇具有胰岛素抵抗性。我们的研究结果表明，苍蝇是有能力动员易位糖转运蛋白的细胞表面响应胰岛素。他们认为，果蝇脂肪细胞对胰岛素有反应，当动物暴露于恒定的高水平糖时，这些途径被下调。最后，这些研究是第一次使用TIRFM监测果蝇中的胰岛素信号通路，证明了TIRFM的标签糖转运蛋白监测昆虫中的信号通路的实用性。 2.各种膜功能单元，如受体，转运蛋白和通道，其作用必然涉及捕获扩散分子，通常被组织成多聚体复合物，在细胞和细胞器膜上形成簇。这些功能单位本身通常是几个完整蛋白质的寡聚体，它们具有自己的对称性。根据对称性，它们在不同的填充晶格上形成簇。此外，局部膜不均匀性，例如，所谓的膜域、筏、茎等，导致甚至在相同堆积晶格上的结构内的不同图案。团簇中的单元竞争扩散分子并相互屏蔽。在这里，我们提出了一个通用的方法，允许一个量化的筛选效果。该方法是用来推导简单的近似公式给出的捕获率的扩散分子的吸收剂的集群上的晶格的不同的包装对称性。所得到的结果描述的捕集率从形成集群的单个吸收剂的有效集体率的总和的平滑变化。后者显示了单个吸收体的捕获效率如何随着簇中吸收体的数量增加和/或吸收体间距离减小而降低。数值试验表明，由理论预测的速率和从布朗动力学模拟得到的不同形状和大小的集群之间的协议。 3.一个简单的近似公式推导出的速率常数，描述了稳态通量的扩散粒子通过一个集群的完全吸收盘上，否则反射平面壁，假设磁盘中心占据相邻网站的一个正方形晶格。磁盘簇捕获的一个显著特征是位于簇外围的磁盘屏蔽了簇中心的磁盘。圆盘对扩散粒子的这种竞争使得在一般情况下不可能找到速率常数的精确解析解。为了导出近似公式，我们使用最近建议的方法A。M.别列日科夫斯基湖弗吉尼亚州达杜格Lizzelf，J. Zimmerberg，and S. M. Bezrukov，J.Chem.Phys.136，211102（2012），其基于通过有效均匀的部分吸收斑点替换盘簇。该公式显示了速率常数如何取决于簇的大小和形状。为了检查公式的准确性，我们将其预测与从布朗动力学模拟获得的速率常数的值进行比较。对18个不同形状和大小的团簇进行了比较，结果表明理论预测与数值计算结果吻合较好。

1. Since the hallmark of glucose metabolism is insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) and the hallmark of membrane protein organization is its domain structure, we continue to examine insulin's effect on GLUT4 organization in PM of adipose cells. The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin-signaling pathways in Drosophila, demonstrating the utility of TIRFM of tagged sugar transporters to monitor signaling pathways in insects. 2. Various membrane functional units such as receptors, transporters, and channels, whose action necessarily involves capturing diffusing molecules, are often organized into multimeric complexes forming clusters on the cell and organelle membranes. These functional units themselves are usually oligomers of several integral proteins, which have their own symmetry. Depending on the symmetry, they form clusters on different packing lattices. Moreover, local membrane inhomogeneities, e.g., the so-called membrane domains, rafts, stalks, etc., lead to different patterns even within the structures on the same packing lattice. Units in the cluster compete for diffusing molecules and screen each other. Here we propose a general approach that allows one to quantify the screening effects. The approach is used to derive simple approximate formulas giving the trapping rates of diffusing molecules by clusters of absorbers on lattices of different packing symmetries. The obtained results describe smooth variation of the trapping rate from the sum of the rates of individual absorbers forming the cluster to the effective collective rate. The latter shows how the trapping efficiency of an individual absorber decreases as the number of absorbers in the cluster increases and/or the inter-absorber distance decreases. Numerical tests demonstrate good agreement between the rates predicted by the theory and obtained from Brownian dynamics simulations for clusters of different shapes and sizes. 3. A simple approximate formula is derived for the rate constant that describes steady-state flux of diffusing particles through a cluster of perfectly absorbing disks on the otherwise reflecting flat wall, assuming that the disk centers occupy neighboring sites of a square lattice. A distinctive feature of trapping by a disk cluster is that disks located at the cluster periphery shield the disks in the center of the cluster. This competition of the disks for diffusing particles makes it impossible to find an exact analytical solution for the rate constant in the general case. To derive the approximate formula, we use a recently suggested approach A. M. Berezhkovskii, L. Dagdug, V. A. Lizunov, J. Zimmerberg, and S. M. Bezrukov, J. Chem. Phys. 136, 211102 (2012), which is based on the replacement of the disk cluster by an effective uniform partially absorbing spot. The formula shows how the rate constant depends on the size and shape of the cluster. To check the accuracy of the formula, we compare its predictions with the values of the rate constant obtained from Brownian dynamics simulations. The comparison made for 18 clusters of various shapes and sizes shows good agreement between the theoretical predictions and numerical results.