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.

胰岛素通过将 GLUT4 招募到质膜 (PM) 来调节葡萄糖转运，这些转运蛋白促进葡萄糖摄取。目前的 GLUT4 循环模型提出了一个在专门的 GLUT4 储存囊泡 (GSV)、细胞内区室和 PM 之间进行 GLUT4 循环的复杂调节系统。据报道，胰岛素可以通过多种方式调节 GLUT4 的再循环。 然而，尽管大量过程显然受到胰岛素的影响，但我们和其他人最近的实验工作强烈表明，GLUT4 循环和葡萄糖摄取的主要调节位点发生在下午。 为了探究 PM 中 GLUT4 的组织及其与胰岛素调节循环的关系，我们研究了离体大鼠脂肪细胞中的 GLUT4 动态。 我们发现：1）簇是通过在新生域中保留 GLUT4 的融合而生成的； 2) 在随后招募网格蛋白后，GLUT4 在这些结构域内内化，并且 3) 胰岛素诱导 GLUT4 胞吐作用的爆发，该胞吐作用大部分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细胞代谢调节的关键。 除了在内体区室、GLUT4 储存囊泡 (GSV) 和 PM 之间复杂的 GLUT4 循环外，我们现在知道它们在 PM 中的空间组织（促进葡萄糖转运）以时间依赖性方式依赖于胰岛素。 GSV 胞吐作用时 PM GLUT4 簇形成的基础状态的优势随着胰岛素刺激而让位于 GSV 融合向 PM 中 GLUT4 分散的转变。此外，发现 GLUT4 簇使网格蛋白组装成核，然后大部分 GLUT4 从细胞表面内化发生。因此，GLUT4簇代表了介导GLUT4递送和PM撤出之间转变的分子组织，这取决于胰岛素。 虽然尚未建立 GLUT4 聚集的分子机制，但通常归因于 GLUT4 在网格蛋白包被的小凹或小窝中的积累。已经提出小凹结构在 GLUT4 内化中发挥一些中间作用。在我们的实验中，我们没有在基底细胞或胰岛素刺激细胞中检测到 GLUT4 和小窝蛋白的任何显着共定位，这与最近的另一项研究一致，该研究反对小窝蛋白在 GLUT4 回收中的直接作用。 虽然网格蛋白在 GLUT4 回收中的作用得到充分支持，但没有证据表明网格蛋白直接参与 GLUT4 聚类。我们能够分别测量 GLUT4 和网格蛋白的整体共定位，以及表面暴露的 GLUT4 与网格蛋白的共定位。令人惊讶的是，我们发现表面暴露的 GLUT4 与网格蛋白的共定位比总 GLUT4 少得多。因此，GLUT4 在网格蛋白包被的小凹中的特异性积累是不可能的。这两种分子更有可能共定位于细胞内区室中。加上大多数 GLUT4 簇存在于 PM 且未显示与网格蛋白任何共定位的事实，我们的数据表明网格蛋白本身不能解释 GLUT4 簇在 PM 的存在和形成。 生物化学和活细胞成像均已充分记录了通过胰岛素调节的胞吐作用将 GLUT4 传递至 PM 的过程。使用 TIRF 显微镜，许多小组检测到单个 GSV 融合事件，使用荧光标记的 GLUT4-GFP 的融合后分散作为融合标准。然而，用显微镜测量的融合事件的数量少于生化和生理学方法预期的数量。令人惊讶的是，我们发现许多融合事件与 GLUT4 从融合位点的扩散无关，这解释了为什么仅通过 GLUT4 扩散检测到的融合事件的数量被低估了。保留融合在基础状态中占主导地位，我们观察到几乎所有由 IRAP-pHluorin flash 检测到的融合事件都不伴随 GLUT4 分散到 PM 中。 这种融合与保留的结果是在 PM 处从头创建 GLUT4 簇。 与之前的结果一致，胰岛素增加了融合事件的总数。有趣的是，胰岛素不仅影响融合事件的数量，而且还通过将 GLUT4 分散到 PM 中，显着地将融合模式转变为融合。这一发现也与 PM 弥散性 HA 抗体染色的显着增加以及 GLUT4 相对量从簇到单体的相应转变相一致。这一观察结果进一步表明，虽然簇状 GLUT4 和单体 GLUT4 以稳定状态共存，但这些池中 GLUT4 的相对量可以通过胰岛素进行差异调节。 有趣的是，胰岛素刺激的 GLUT4 融合增加是短暂的，在 2-3 分钟达到明显峰值后，融合频率下降至仅略高于基础水平的水平。虽然旧的 GLUT4 回收模型预测 GLUT4 回收响应胰岛素的总体增加，但我们的结果符合量子释放模型的预测。然而，虽然旧模型认为胰岛素可以调节可用于 GLUT4 回收的活动池的大小，但我们的模型包括回收过程中的所有 GLUT4，并将 GLUT4 簇的存在作为一个独特的池来解决。 根据实验观察，我们模型的一个重要特征是 GLUT4 内化对簇的限制。我们的数据表明，GLUT4 内化的主要部分是通过招募网格蛋白从簇发生的，而簇外的其他网格蛋白包被的凹坑不能有效地内吞 GLUT4。这种 GLUT4 回收组织提供了上调 PM GLUT4（单体状态）与可用于内吞作用（簇状）的 GLUT4 分开的灵活性。 总而言之，本研究中提供的数据表明，GLUT4 簇可能作为从 GLUT4 胞吐作用直至其内化的中间枢纽。在基础状态下，这些结构域似乎在调节 PM 和 GSV 细胞内库之间的 GLUT4 循环中发挥着主要作用。在胰岛素刺激状态下，PM GLUT4 的快速增加是通过 GSV 融合的增加实现的，特别是完全且立即释放 GLUT4 分子扩散到 PM 中的事件。然而，GLUT4 内化和再循环到 GSV 中的速率现在必须包含一个新参数，即 GLUT4 到达摄取位点所需的时间；这一贩运过程中的限速步骤仍有待确定。通过 GLUT4 中枢的这种动力学最终必须确定由胰岛素刺激设定的新的平衡 GLUT4 活性水平。因此，他们对胰岛素血液水平和 GLUT4 活性之间的关系被破坏的病理状态特别感兴趣。