摘要
细胞分泌活动是神经信号传导和内分泌激素释放等细胞生理功能的基础，是在时空上严格协调的胞吐与胞吞的循环过程，需要包括细胞信号整合、膜动力学改变、细胞骨架重组等一系列复杂的分子机制参与调控。过去的25年，参与囊泡循环调控的许多蛋白质被发现和确认了，但是在何时何地怎样调控还不清楚。本文在胰腺细胞和胰岛素分泌的INS-1细胞中研究了囊泡循环胞吞途径。在3丁3-L1脂肪细胞中区分了葡萄糖转运蛋白4 CGLU丁4>囊泡在转运融合过程中经过的不同步骤及其调节机制。最后发展了一种能跟踪GLU丁4转运过程的新的探针。
结合膜电容测量，光学成像以及分子生物学技术，我们研究了胰腺p细胞中网格蛋白非依赖的快速胞吞和网格蛋白依赖的慢速胞吞。以前的报道中提出不同的dynamin业型分别介导网格蛋白依赖和网格蛋白非依赖的胞吞途径。然而，我们在胰腺p细胞中利用电生理和成像技术都发现，神经兀中特异的dynamin1和普遍表达的dynamin2同时参与网格蛋白非依赖和网格蛋白依赖的胞吞。这两种胞吞的膜回收幅值都与此前的胞吐成线性相关，i fu b‘它们都需要dynamin蛋白。我们进一步证明了网格蛋白非依赖的胞吞直接由高钙浓度促发，而并不是传统的“kiss and run" o
GLU丁4在胰岛素作用下的转运上膜是血糖调控的一个关键途径。然而始终缺少很好的功能检测方法可以将GLU丁4储存囊泡转运及融合过程中所经过的一系列过程区分开来，因此不能清楚解释整个过程中的分子调控机制。在本研究中，开发了一种可以实时跟踪单个GLU丁4囊泡，自动区分并系统的在体研究质膜上囊泡锚定、启动、融合过程的方法，并发现了尽管P13K和它的下游效应物Rab GAP AS160调抓‘了囊泡的锚定过程，胰岛素调控的关键步骤却是在囊泡锚定在细胞膜下之后，使囊泡具有融合能力的启动过程。进一步，我们还发现Akt磷酸化AS160并不GLU丁4转运的主要调控步骤，在GLU丁4储存囊泡锚定在质膜之后，有可能受到其它的Akt底物或者另外的信号途径的调控。
然而利用GLU丁4-EGFP跟踪融合事件非常耗时，需要专门的训练并目‘可能低估融合速率。因此堕需一种直接而方便有效的在单细胞水平上面检测融合事件的方法。这里我们发展了一个新的跟踪GLU丁4转运过程的探针，它是基十两种荧光的比率检测。这个探针在检测单个GLU丁4囊泡的融合事件方面比现在的技术灵敏了一个数量级。在3丁3-L1月旨肪细胞中，第一次检测到胰岛素的作用使GLU丁4囊泡的融合速率增加了大约40倍。另外这个探针还能用来跟踪GLU丁4囊泡在融合前的状态。利用这个探针进一步发展的筛选策略将会有利十发现GLU丁4转运途径中的关键分子或者新的药物靶定。
关键词:分泌胞吞，胰岛素，囊泡，葡萄糖转运蛋白4，锚定，启动，融合
Abstract
Secretion forms the basis for signal transmission between neurons andhormone release from endocrine cells. It must undergo repeated rounds of exocytosis, endocytosis, refilling and mobilization, a phenomenon called recycling. The temporal and spatial coordination of exo-endocytic recycling requires a complex molecular machinery that integrates cell signaling, dynamic changes
within membranes, and cytoskeletal rearrangements. Over the past 25 years, many proteins of this machinery have been identified and characterized. However, it is still not clear when and where do these proteins function? Now we studied vesicle recycling pathways in mouse pancreatic p cells and the insulin-secreting INS一1 cells. In addition, we dissected the multiple steps in vesicle translocation and
elucidated the mechanisms of insulin induced membrane protein GLUT4 trafficking in 3T3一L1 adipocytes. Finally we developed a novel probe to monitor the translocation process of GLUT4 based on dual-emission ratiometric fluorescence measurement.
By combining patch-clamp capacitance recording, optical method and specific molecular interventions, we characterized a clathrin一independent fast endocytosis and a clathrin-dependent slow endocytosis in pancreatic p cells. The amplitudes of both fast and slow membrane retrievals were linearly correlated with preceding exocytosis, and dynamin was required for both processes. We further
demonstrated that the clathrin-independent endocytic pathway was directly riggered by enhanced elevation in [Ca2+]i，and was not a results of "kiss and run". Different dynamin isoforms have previously proposed to mediate clathrin-dependent and independent endocytosis, respectively. However, we showed that neuron-specific dynamin 1 and ubiquitous dynamin 2 simultaneously participated in the clathrin一independent and dependent membrane retrievals in pancreatic p cells, as confirmed electrophysiologically and optically. Therefore, dentification of novel factors other than different dynamin isoforms is required to dissect the fast and slow endocytosis and evaluation their contributions to normal vesicle recycling in the future.
