VESICLE TRAFFICKING AND PANCREATIC ACINAR CELL SECRETION

本研究的目的是明确正常和异常胰酶分泌的分子机制。我们的重点是胰腺腺泡细胞，以及支持激动剂诱导的酶原颗粒的形成和从跨高尔基网络(TGN)运输到顶膜以释放后续酶的机制。使用原代腺泡细胞制剂和我们开发的一种新的培养的胰腺腺泡细胞模型，我们进行了以下新的观察。(I)CCK刺激诱导高尔基体的囊泡形成，同时增加酶原颗粒的运输；(Ii)激动剂刺激后，特定的囊泡外壳和细胞骨架蛋白被招募到腺泡细胞TGN；(Iii)微管(MT)相关的运动酶Kinesin与酶原颗粒相关，并与MTs一起，是颗粒运动所必需的。这项研究将利用最先进的显微成像技术对表达GFP蛋白结构的活体和固定腺泡细胞进行成像，结合生物化学和分子学方法来验证以下中心假设：腺泡细胞中特定的外壳和马达蛋白在分泌刺激期间被招募和激活，以介导酶原颗粒的适当形成和从TGN到顶腔的运输。我们预测，这种酶原运输机制的破坏将导致腺泡细胞中新生蛋白酶的错误分选，并导致随后的胰腺炎。因此，我们提出了三个具体目标。首先，我们将定义腺泡细胞TGN的结构，并观察其在刺激分泌过程中的变化。将进行机制研究，以抑制运动和外套功能，并随后评估对TGN结构和酶原颗粒形成的影响。其次，我们将确定哪些马达蛋白支持酶原颗粒向根尖管腔的运输，并测试这些马达在分泌过程中是否上调。第三，我们将直接测试外壳和马达蛋白的破坏或超刺激是否会导致异常错误的分选和随后的酵素释放到细胞质中。据我们所知，这将是第一批操纵和观察活体腺泡细胞分泌过程中囊泡形成和运输的研究之一。我们相信，这项研究将对了解胰腺腺泡细胞的功能和疾病做出重要贡献。

The objective of this proposal is to define the molecular mechanisms involved in both normal and abnormal pancreatic enzyme secretion. Our focus is on the pancreatic acinar cell and the mechanisms that support the agonist-induced formation and transport of zymogen granules from the trans-Golgi network (TGN) to the apical membrane for subsequent enzyme release. Using both primary acini preparations and a novel cultured pancreatic acinar cell model that we developed, we have made the following novel observations. (i), CCK-stimulation induces vesiculation of the Golgi apparatus while increasing zymogen granule transport, (ii), specific vesicle-coat and cytoskeletal proteins are recruited to the acinar cell TGN following agonist stimulation, and (iii), the microtubule (Mt) associated motor enzyme kinesin is associated with zymogen granules and, along with Mts, is required for granule movement. This study will utilize state-of-the-art microscopic imaging techniques of living and fixed acinar cells expressing GFP-protein constructs, combined with biochemical and molecular methods to test the following CENTRAL HYPOTHESIS: specific coat and motor proteins in the acinar cell are recruited and activated during a secretory stimulus to mediate the appropriate formation and transport of zymogen granules from the TGN to the apical lumen. We predict that disruption of this zymogen transport machinery will lead to missorting of nascent proteases in the acinar cell and subsequent pancreatitis. Thus, we propose three SPECIFIC AIMS. First, we will define the structure of the acinar cell TGN and observe how it changes during stimulated secretion. Mechanistic studies will be conducted to inhibit motor and coat function and subsequently assess the effects on TGN structure and zymogen granule formation. Second, we will define which motor proteins support the transport of zymogen granules to the apical lumen and test whether these motors are upregulated during secretion. Third, we will directly test if a disruption of coat and motor proteins, or suprastimulation, leads to an aberrant missorting and subsequent release of zymogens into the cytoplasm. To our knowledge this will be one of the first studies to manipulate and view vesicle formation and transport in living acinar cells as they secrete. We are confident that this study will make important contributions essential to understanding pancreatic acinar cell function and disease.