CELL SECRETION AND MEMBRANE FORMATION IN THE PANCREAS

胰腺腺泡细胞在我们实验室已经研究了很多年 一种用于研究非肿瘤细胞内转运途径的模型 神经内分泌调节分泌)细胞。我们实验室的最新数据 提示胰腺腺泡细胞含有几种免疫反应物 分泌囊泡和靶血浆的SNARE复合体成员 在酵母和神经细胞中发现的膜蛋白。其中几个 这些可能是新的哺乳动物同源物，包括v-SNARS的异构体 (细胞短缩蛋白)、tSNARE(合成素)和控制膜的蛋白质 融合(Rab3D，synaptopagmin)。此信息提供了以下依据 在未来的研究中，我们将研究这些蛋白质在 这种非神经内分泌调节的分泌细胞。具体目标是： 首先，我们将对参与调控的膜蛋白进行鉴定 胞吐作用：1.我们将完成 腺泡细胞低MR GTP结合蛋白Rab3D的特性研究 这是颗粒特有的，很可能是一种分子开关 调控的胞吐作用；2.我们将继续进行分子表征 腺泡内v-和t-SNARs及其相关蛋白(Synaptopagmin)的表达 细胞及其在沿着分泌物的隔间中的共同结合 路径。这将通过cDNA的分子克隆和 免疫金免疫细胞化学。3.我们将确定SNARE蛋白是否 在强烈促分泌剂刺激后在细胞内重新定位，即 与高尔基体扩增有关：和4.我们计划评估 利用通透性细胞研究SNARE蛋白在腺泡细胞中的作用 允许探针(抗圈套抗体等)和肉毒杆菌进入 毒素)。我们将分析它们对分泌途径的影响。 包括细胞短链蛋白在调节和调节细胞成熟中的作用 构成分泌囊泡和胞吐作用。第二，我们将 探讨膜融合对分泌物影响的分子机制 酶原颗粒/质膜融合的无细胞检测途径 并形成结构性分泌小泡。这将使我们能够 在受控条件下直接检测抗体的效果 抗SNARS、SYNAPTOAGMIN、Rab3D和其他与 膜靶向和融合。第三，腺泡细胞的胞吐作用是 与分泌颗粒膜的大量移位有关 根尖表面被膜修复所补偿，很可能 高尔基情结。我们将研究取回的路径和动力学 用免疫金电子显微镜观察刺激的胰腺小叶 以及针对胞外和胞内的膜成分的抗体 车厢。从腺泡细胞中分离回收囊泡的免疫分离 在体外被促分泌剂刺激应该能让我们识别 代偿膜恢复过程中膜蛋白之间的联系。 将特别注意包被囊泡的形成和 大鼠SNARE复合体蛋白与突触素的关系 膜回收。

The pancreatic acinar cell has been studies in our lab for many years as a model for examining the intracellular transport pathway in a non- neuroendocrine regulated secretor) cell. Recent data from our laboratory indicate that the pancreatic acinar cell contains several immunoreactive members of the SNARE complex of secretory vesicle and target plasma membrane proteins discovered in yeast and neuronal cells. Several of these may be novel mammalian homologues and include isoforms of v-SNAREs (cellubrevin), tSNAREs (syntaxin), and proteins that control membrane fusion (Rab3D, synaptotagmin). This information provides the basis for future studies in which we will examine the role of these proteins in this nonneuroendocrine regulated secretory cell. The Specific Aims are: First, we will characterize membrane proteins involved in regulated exocytosis along the following lines: 1. We will complete the characterization of Rab3D, the acinar cell low Mr GTP binding protein that is granule-specific and likely serves as a molecular switch for regulated exocytosis; 2. We will pursue the molecular characterization of v- and t-SNAREs and associated proteins (synaptotagmin) in the acinar cell and their co-association in compartments along the secretory pathway. This will be carried out by molecular cloning of cDNAs and immunogold immunocytochemistry. 3. We will determine if SNARE proteins relocated in the cell following intense secretagogue stimulation that is associated with amplification of the Golgi: and 4. We plan to assess the function of SNARE proteins in the acinar cell using permeabilized cells which allow entry of probes (antibodies against SNAREs etc. and botulinal toxins). They will be analyzed for effects on the secretory pathway including the function of cellubrevin in maturation of regulated and constitutive secretory vesicles and on exocytosis. Second, we will examine the molecular mechanisms of membrane fusion on the secretory pathway using cell free assays for zymogen granule/plasma membrane fusion and formation of constitutive secretory vesicles. This will allow us to test directly under controlled conditions the effects of antibodies against SNAREs, synaptotagmin, Rab3D and other proteins implicated in membrane targeting and fusion. Third, exocytosis from the acinar cell is associated with massive relocation of secretory granule membranes to the apical surface which is compensated for by membrane retrieval, likely to the Golgi complex. We will examine the route and kinetics of retrieval from stimulated pancreatic lobules using immunogold electron microscopy and antibodies against membrane components of the exocytic and endocytic compartments. Immunoisolation of recycling vesicles from acinar cells stimulated by secretagogues in vitro should allow us to identify associations of membrane proteins during compensatory membrane retrieval. Particular attention will be paid to formation of coated vesicles and the relationship of synaptotagmin and proteins of the SNARE complex in membrane recycling.