CAVEOLAE--STRUCTURE/FUNCTION IN INTESTINAL ENDOCYTOSIS

腹泻仍然是儿童死亡的主要原因。 世界范围内5岁以下儿童和致毒性腹泻(如由 霍乱毒素，CT)占可识别的近20% 腹泻。确定CT作用的细胞机制可能有助于 改进治疗方法和预防措施的发展 抗CT和密切相关的大肠杆菌不耐热毒素LT。 这项提案的总体目标是澄清结构和 洗涤剂不溶性糖脂膜微结构域的功能 (挖掘)霍乱毒素向模型人类的运输 肠上皮细胞(T84细胞)。我们的第一个具体目标是 确定DIGS在将CT靶向极化细胞中所起的作用 信号转导所需的隔室(如 C1--可用电学测量的分泌反应)。炭疽病 水肿型毒素(AT)诱导T84细胞的C1型分泌反应 与CT相似，但动力学不同。然而， AT不通过挖孔结合到细胞表面。嵌合毒素 匹配CT和AT的细胞结合和酶单位将是 用于确定通过地址进入单元格是否对CT至关重要 诱导T84细胞分泌反应的能力。有能力 引起分泌反应以及分泌的动力学 将提供有关通过数字输入单元格的重要性的信息 CT对极化上皮的作用。第二个具体目标是 鉴定与CT特异性DIGs相关的膜蛋白。我们 建议通过首先确定…的角色来实现这一点 肌动蛋白在维持与我们模型系统的DIG连接中，Triton X- 100个提取的T84单层。其次，我们计划调查CT- 通过化学交联法和共沉淀法获得特定的DIG组分 CT及其相关蛋白的免疫沉淀 受体神经节苷脂GM1。

Diarrhea remains the leading cause of death of children under the age of 5 worldwide, and toxigenic diarrheas (such as those caused by cholera toxin, CT) account for nearly 20% of identifiable diarrheas. Determining cellular mechanisms of CT action may aid in the development of improved therapies and preventative measures against CT and the closely related E. coli heat-labile toxin, LT. The overall goal of this proposal is to elucidate the structure and function of detergent-insoluble glycolipid membrane microdomains (DIGs) in the transport of cholera toxin into model human intestinal epithelia (T84 cells). Our first specific aim is to determine the role DIGs play in targeting CT into polarized cell compartments necessary for signal transduction (as evidenced by a C1- secretory response that can be measured electrically). Anthrax edema toxin (AT) elicits a C1- secretory response from T84 cells similar to that of CT, although with different kinetics. However, AT does not bind via DIGs to the cell surface. Chimeric toxins matching cell-binding and enzymatic units from CT and AT will be used to determine if entry to the cell via DIGs is critical to CT's ability to elicit T84 cell secretory responses. The ability to elicit a secretory response as well as the kinetics of secretion will yield information about the importance of cell entry via DIGs to CT action upon polarized epithelia. The second specific aim is to identify membrane proteins associated with CT-specific DIGs. We propose to accomplish this by first characterizing the role of actin in maintaining DIG attachment to our model system, Triton X- 100 extracted T84 monolayers. Secondly, we plan to investigate CT- specific DIG components by chemical cross-linking and co- immunoprecipitation of proteins closely associated with CT and its receptor ganglioside GM1.