Components And Kinetics In Exocytosis

胞吐作用的组成和动力学

• 批准号: 8149275
• 负责人: JOSHUA ZIMMERBERG
• 金额: $ 170.28万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149275
• 关键词: Exocytosis; Kinetics

Insulin regulates glucose transport through recruitment of GLUT4 to the plasma membrane (PM) where these transporters facilitate glucose uptake. The current model of GLUT4 recycling proposes a complex regulated system of GLUT4 cycling among specialized GLUT4 storage vesicles (GSV), intracellular compartments, and PM. Insulin has been variably reported to regulate GLUT4 recycling in several ways. However, despite this large number of processes apparently affected by insulin, recent experimental work by us and others strongly suggests that the main site of regulation of GLUT4 recycling and glucose uptake occurs at PM. To probe both GLUT4 organization in PM and its relationship to insulin-regulated recycling, we investigated GLUT4 dynamics in isolated rat adipose cells. We find: 1) clusters are generated by fusion with retention of GLUT4 in nascent domains; 2) GLUT4 is internalized at these domains after subsequent recruitment of clathrin, and 3) insulin induces a burst of GLUT4 exocytosis that mostly bypasses these domains and disperses GLUT4 directly into PM. The relationship between the spatial and temporal organization of plasma membrane (PM) glucose transporters is key to the regulation of cell metabolism. In addition to the complex recycling of GLUT4 among endosomal compartments, GLUT4-storage vesicles (GSV), and PM, we now know that their spatial organization in PM, where they facilitate glucose transport, depends upon insulin in a time-dependent fashion. The predominance in the basal state of PM GLUT4 cluster formation upon exocytosis of GSV gives way with insulin stimulation to a shift in GSV fusion to GLUT4 dispersal in PM. Further, GLUT4 clusters are found to nucleate clathrin assembly where most of the GLUT4 internalization from the cell surface then takes place. Thus, GLUT4 clusters represent a molecular organization mediating the transition between GLUT4 delivery and withdrawal from PM, depending upon insulin. While no molecular mechanism for GLUT4 clustering has been established, it was often attributed to accumulation of GLUT4 either in clathrin-coated pits or caveolae. Caveolar structures have been proposed to play some intermediate role in GLUT4 internalization. In our experiments we did not detect any significant co-localization of GLUT4 and caveolin in either basal or insulin-stimulated cells, which is consistent with another recent study arguing against a direct role for caveolae in GLUT4 recycling. While the role of clathrin in the recycling of GLUT4 is well supported, no evidence exists suggesting direct involvement of clathrin in GLUT4 clustering. We were able to separately measure the overall co-localization of GLUT4 and clathrin, as well as co-localization of surface-exposed GLUT4 with clathrin. Surprisingly, we found that surface-exposed GLUT4 have much less co-localization with clathrin than total GLUT4. Thus, a specific accumulation of GLUT4 in clathrin-coated pits is unlikely; it is more likely that these two molecules co-localize in intracellular compartments. Together with the fact that the majority of GLUT4 clusters exists at PM without showing any co-localization with clathrin, our data indicate that clathrin itself cannot account for the existence and formation of GLUT4 clusters at PM. The delivery of GLUT4 to PM through insulin-regulated exocytosis has been well documented by both biochemistry and live-cell imaging. Using TIRF microscopy, a number of groups detected single GSV fusion events using as a criterion for fusion the post-fusion dispersal of fluorescently labeled GLUT4-GFP. However, the number of fusion events measured microscopically was fewer than the number expected from biochemical and physiological approaches. Surprisingly, we found that many fusion events were not associated with dispersal of GLUT4 from the site of fusion, explaining why the number of fusion events detected only by GLUT4 dispersal was underestimated. Fusion-with-retention was predominant in the basal state where we observed that almost all fusion events detected by IRAP-pHluorin flash were not accompanied by dispersal of GLUT4 into PM. The result of this fusion-with-retention is the creation of de novo GLUT4 clusters at PM. Consistent with previous results, insulin increased the overall number of fusion events. Interestingly, insulin not only affected the number of fusion events, but also dramatically shifted the mode of fusion towards fusion with dispersal of GLUT4 into PM. This finding is also consistent with the pronounced increase of diffuse HA-antibody staining of PM and corresponding shift in the relative amount of GLUT4 from clusters to monomers. This observation further implies that while clustered and monomeric GLUT4 co-exist in a steady state, the relative amounts of GLUT4 in these pools can be differentially regulated by insulin. Interestingly, the insulin-stimulated increase of GLUT4 fusion was transient, and after a pronounced peak at 2-3 min, the fusion frequency declined to a level only slightly above the basal. While old models of GLUT4 recycling predict an over-all increase of GLUT4 recycling in response to insulin, our results fit the prediction of a quantum release model. However, while the older model considers insulin to regulate the size of the active pool available for GLUT4 recycling, our model includes all GLUT4 in the recycling process and addresses the existence of GLUT4 clusters as a distinct pool. One important feature of our model, based on the experimental observations, is the restriction of GLUT4 internalization to the clusters. Our data suggest that the major part of GLUT4 internalization occurs from clusters via recruitment of clathrin, while other clathrin-coated pits outside the clusters do not efficiently endocytose GLUT4. This organization of GLUT4 recycling provides flexibility to upregulate PM GLUT4 (in monomeric states) separately from GLUT4 available for endocytosis (clustered). Taken together, the data presented in this study suggest that GLUT4 clusters may function as intermediate hubs from the time of GLUT4 exocytosis until their internalization. In the basal state, these domains appear to play the major role in regulating the recycling of GLUT4 between PM and the intracellular pool of GSV. In the insulin-stimulated state, a rapid increase of PM GLUT4 is achieved by an increase in GSV fusion, particularly events with full and immediate release of GLUT4 molecules diffusely into PM. However, the rate of internalization and recycling of GLUT4 into GSV must now include a new parameter, the time it takes for GLUT4 to reach an uptake site; the rate-limiting step in this trafficking process remains to be determined. This kinetics, through GLUT4 hubs, must ultimately determine the new equilibrium GLUT4 activity level set by insulin stimulation. Thus, they are of particular interest in pathological states in which the relationship between insulin blood levels and GLUT4 activity is disrupted.

胰岛素通过募集GLUT4募集到质膜（PM）来调节葡萄糖转运，这些转运蛋白可促进葡萄糖摄取。 GLUT4回收的当前模型提出了一个复杂的调节GLUT4循环系统，在专用GLUT4储存囊泡（GSV），细胞内隔室和PM之间。据报道，胰岛素可以通过多种方式调节GLUT4回收。 然而，尽管显然受胰岛素影响了大量的过程，但我们和其他人最近的实验性工作表明，GLUT4回收和葡萄糖摄取的主要部位发生在PM处。 为了探测PM中的GLUT4组织及其与胰岛素调节的回收的关系，我们研究了分离的大鼠脂肪细胞中的GLUT4动力学。 我们发现：1）簇是通过融合而产生的，将GLUT4保留在新生的结构域中； 2）在随后募集网格蛋白后，在这些结构域中将GLUT4内部化，3）胰岛素诱导了glut4胞外增生爆发，主要绕过这些结构域并将Glut4直接分散到PM中。 质膜（PM）葡萄糖转运蛋白的空间和时间组织之间的关系是调节细胞代谢的关键。 除了内体隔室，GLUT4存储囊泡（GSV）和PM之间的GLUT4复杂的回收外，我们现在知道它们在PM中的空间组织（它们促进葡萄糖转运，都取决于胰岛素，都以时间依赖性方式取决于胰岛素。 GSV胞吐作用后PM Glut4簇的基础状态的主要占主导地位，并通过胰岛素刺激使GSV融合到PM中的GLUT4分散体的转移。此外，发现GLUT4簇是对网格蛋白组装的核定，其中大多数GLUT4从细胞表面进行。因此，GLUT4簇代表了一个分子组织，该分子组织介导GLUT4递送和从PM提取的过渡，具体取决于胰岛素。 虽然尚未建立用于GLUT4聚类的分子机制，但通常归因于粘液蛋白涂层的凹坑或小窝中的Glut4的积累。已经提出了洞穴结构在GLUT4内部化中起中间作用。在我们的实验中，我们没有在基底或胰岛素刺激的细胞中检测到Glut4和Caveolin的任何显着共定位，这与另一项最近的研究一致，该研究反对Caveolae在GLUT4回收中的直接作用。 虽然clathrin在GLUT4回收中的作用得到了很好的支持，但尚无证据表明网格蛋白直接参与GLUT4聚类。我们能够分别测量GLUT4和网格蛋白的总体共定位，以及与网格蛋白的表面暴露GLUT4的共定位。出乎意料的是，我们发现表面暴露的GLUT4与网格蛋白的共定位少于总GLUT4。因此，不太可能在网格蛋白涂层的坑中特异性地积累。这两个分子更有可能在细胞内室中共定位。随着大多数GLUT4簇在PM存在而没有与网格蛋白共定位的事实，我们的数据表明，网格蛋白本身无法解释PM处GLUT4簇的存在和形成。 生物化学和活细胞成像都很好地证明了通过胰岛素调节的胞吐作用将GLUT4传递到PM。使用TIRF显微镜，许多组检测到单个GSV融合事件，使用作为融合的标准，用于融合后融合后分布的荧光标记为GLUT4-GFP。但是，微观测量的融合事件数量少于生化和生理方法预期的数量。令人惊讶的是，我们发现许多融合事件与融合部位的glut4分散无关，这解释了为什么仅低估了仅通过GLUT4分散检测到的融合事件数量。在基础状态下，退休融合是主要的，我们观察到几乎所有由IRAP-Phluorin闪光检测到的融合事件都不伴随着将GLUT4散布到PM中的融合事件。 保留这种融合的结果是在PM上创建了Novo Glut4簇。 与以前的结果一致，胰岛素增加融合事件的总数。有趣的是，胰岛素不仅影响了融合事件的数量，而且还会显着将融合方式转移到与GLUT4分散的融合中。这一发现也与PM的弥漫性HA抗体染色的明显增加以及GLUT4从簇为单体相对的相对量的相应变化。该观察结果进一步意味着，虽然聚类和单体GLUT4在稳定状态下共存，但这些池中的GLUT4的相对量可以通过胰岛素差异地调节。 有趣的是，Glut4融合的胰岛素刺激的增加是短暂的，在2-3分钟时明显的峰值后，融合频率下降到仅略高于基础的水平。虽然GLUT4回收的旧模型预测GLUT4响应胰岛素的回收量增加了，但我们的结果符合量子释放模型的预测。但是，尽管较旧的模型考虑胰岛素来调节可用于GLUT4回收的活动池的大小，但我们的模型在回收过程中包括所有GLUT4，并将GLUT4簇的存在作为一个独特的池。 基于实验观察结果，我们模型的一个重要特征是将GLUT4内在化限制到群集中。我们的数据表明，GLUT4内部化的主要部分是通过募集网状蛋白从簇中发生的，而簇外的其他网状蛋白涂层的坑则不能有效地内吞Glut4。该GLUT4回收组织提供了灵活性，可与可用于内吞作用的GLUT4分别上调PM Glut4（在单体状态下）（聚类）。 综上所述，这项研究中提供的数据表明，GLUT4簇从GLUT4胞吞作用到其内在化可能起到中间引线的作用。在基础状态下，这些域似乎在调节PM和GSV细胞内池之间的GLUT4回收方面起着主要作用。在胰岛素刺激的状态下，PM GLUT4的迅速增加是通过GSV融合的增加来实现的，尤其是将GLUT4分子完全释放到PM中的事件。但是，现在必须将GLUT4的内在化和回收率包括一个新参数，GLUT4到达吸收位点所需的时间；在此贩运过程中的限制步骤仍有待确定。这种动力学通过GLUT4集线器必须最终确定胰岛素刺激设定的新平衡GLUT4活性水平。因此，它们在胰岛素血液水平与GLUT4活性之间的关系中断的病理状态中特别感兴趣。