CELL SECRETION AND MEMBRANE FORMATION IN THE PANCREAS

胰腺中的细胞分泌和膜形成

• 批准号: 2749420
• 负责人: JAMES Douglas JAMIESON
• 金额: $ 35.24万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749420
• 关键词: acinar cell; binding proteins; cell membrane; chimeric proteins; complementary DNA; electron microscopy; exocytosis; immunocytochemistry; immunoprecipitation; intracellular transport; laboratory mouse; laboratory rabbit; laboratory rat; membrane biogenesis; membrane fusion; molecular cloning; northern blottings; phosphorylation; protein isoforms; protein structure function; secretory protein; synaptotagmin; syntaxin; tissue /cell culture; western blottings

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.

胰腺腺泡细胞在我们实验室已经研究了很多年， 一个模型，用于检查细胞内的运输途径，在非- 神经内分泌调节的分泌）细胞。我们实验室的最新数据 表明胰腺腺泡细胞含有几种免疫反应性 分泌囊泡和靶血浆的SNARE复合物的成员 在酵母和神经细胞中发现的膜蛋白。几 这些可能是新的哺乳动物同源物，包括v-SNARE的同种型 （cellubrevin），tSNARE（syntaxin）和控制膜的蛋白质 融合（Rab 3D，突触结合蛋白）。这些信息提供了基础， 未来的研究中，我们将检查这些蛋白质的作用， 这种非神经内分泌调节的分泌细胞。具体目标是： 首先，我们将描述参与调控的膜蛋白， 胞吐作用沿着以下路线进行：1.全面完成 Rab 3D，腺泡细胞低Mr GTP结合蛋白的表征 这是颗粒特异性的，可能作为一个分子开关， 调节胞吐作用; 2.我们将继续进行分子表征 腺泡中v-和t-SNARE及相关蛋白（突触结合蛋白）的表达 细胞和它们在隔室中的共同联合沿着分泌 通路这将通过cDNA的分子克隆进行， 免疫金免疫细胞化学3.我们将确定SNARE蛋白 在强烈的促分泌素刺激后重新定位在细胞中， 与高尔基体的扩增相关;以及4.我们计划评估 使用透化细胞的腺泡细胞中SNARE蛋白的功能 其允许探针（针对SNARE等和肉毒杆菌的抗体）进入 毒素）。将分析它们对分泌途径的影响 包括小泡纤维素在受调节的 组成性分泌囊泡和胞吐作用。二是 研究膜融合对分泌的分子机制， 酶原颗粒/质膜融合的无细胞测定途径 和组成性分泌囊泡的形成。这将使我们能够 在受控条件下直接测试抗体的效果 针对SNARE、突触结合蛋白、Rab 3D和其他与 膜靶向和融合。第三，腺泡细胞的胞吐作用是 与大量的分泌颗粒膜迁移到 通过膜回收补偿的顶面，可能 高尔基复合体我们将研究的路线和动力学的检索 用免疫金电子显微镜从刺激的胰腺小叶 以及抗胞吐和胞吞细胞膜成分的抗体 隔间腺泡细胞循环小泡的免疫分离 通过体外促分泌素的刺激， 补偿性膜修复过程中膜蛋白的结合。 将特别注意包被囊泡的形成和微囊泡的形成。 突触结合蛋白与SNARE复合物蛋白质的关系 膜回收