Secretion-associated dynamic of the plasma membrane

描述(由申请人提供)：这项建议将填补我们对分泌物中关键事件的理解的空白，即胞吐和内吞耦合过程中质膜形态和生化变化的性质。当被钙离子触发时，神经内分泌细胞通过致密的核心颗粒与质膜融合将生物胺和神经肽释放到细胞外空间。然后通过内吞作用回收颗粒膜以供重复使用。一系列精心策划的活动是正确执行这些任务的基础。在胞吐之前，颗粒与质膜蛋白和脂类之间的生化相互作用启动颗粒进行融合；肌醇磷脂Pl(4，5)P2在这些事件中起关键作用。然后，将各自的膜破裂并合并，形成融合孔。胞吐后，发生其他相互作用，使质膜变形，恢复颗粒膜成分。近年来，一组被认为在外吞/内吞作用中起重要作用的蛋白质已被发现。其中一些被预测会改变膜的形态，要么在胞吐之前，如突触凝集素，要么在胞吐之后，如亲内素和两栖菌素。这些变化的时间和位置可能受到它们与PI(4，5)P2相互作用的影响。颗粒膜在进入质膜或被内吞作用恢复之前保持其曲率的时间长短也是不确定的。由于质膜事件的快速、高分辨率检测一直很困难，因此对这些过程的阐明仍然缺乏。这一提议背后的统一假设是，对分泌至关重要的质膜潜在的生化和生理变化导致膜构象的变化。其具体目的是：1)确定与胞吐和内吞作用相关的质膜的形态动力学；2)确定PI(4，5)P2在调节对分泌至关重要的质膜特性中的作用。这些实验将利用肾上腺嗜铬细胞，以及基于共聚焦和TIR的光学方法。预计我们的研究结果将为分泌性反应的调节和动态提供基本的见解。基于嗜铬细胞模型与其他神经内分泌细胞以及神经元中突触前通路的相似性，它们也将具有广泛的相关性。更全面地了解外/内吞作用的机制将有助于开发合理的心血管、内分泌和神经疾病的治疗策略。

DESCRIPTION (provided by applicant): This proposal will fill a gap in our understanding of key events in secretion, namely, the nature of changes in plasma membrane morphology and biochemistry during the coupled processes of exocytosis and endocytosis. When triggered by Ca2+, neuroendocrine cells release biogenic amines and neuropeptides into the extracellular space through fusion of dense core granules with the plasma membrane. Granule membranes are then retrieved for reuse via endocytosis. A precisely orchestrated series of events underlie the proper execution of these tasks. Prior to exocytosis, biochemical interactions between granule and plasma membrane proteins and lipids occur priming the granule for fusion; the inositol phospholipid Pl(4,5) P2 is critical for these events. The respective membranes are then ruptured and merged to form a fusion pore. After exocytosis, other interactions occur to deform the plasma membrane and recover granule membrane constituents. In recent years, a cohort of proteins thought to be important in exo-/endocytosis has been identified. Some of these are predicted to alter membrane morphology, either prior to exocytosis, such as synaptotagmin, or after exocytosis, such as endophilin and amphiphysin. The timing and the location of these changes may be influenced by their interaction with PI(4,5)P2. There is also uncertainty about the length of time the granule membrane retains its curvature before it flattens into the plasma membrane or is retrieved by endocytosis. Because rapid, high resolution detection of plasma membrane events has been difficult, the elucidation of these processes is still lacking. The unifying hypothesis behind this proposal is that the underlying biochemical and physiological changes in the plasma membrane important for secretion result in changes of membrane conformation. The Specific Aims are designed to: 1) Determine the morphological dynamics of the plasma membrane associated with exocytosis and endocytosis; 2) Determine the role of PI(4,5)P2 in regulating properties of the plasma membrane important for secretion. The experiments will utilize adrenal chromaffin cells, and confocal and TIR-based optical methods. It is anticipated that the results of our studies will provide fundamental insights into the regulation and dynamics of the secretory response. They will also have broad relevance based on the similarity of the chromaffin cell model to other neuroendocrine cells and to presynaptic pathways in neurons. A more complete understanding of the mechanisms underlying exo-/endocytosis will help drive the development of rational strategies for the treatment of cardiovascular, endocrine and neurological diseases.