CAVEOLAE--STRUCTURE/FUNCTION IN INTESTINAL ENDOCYTOSIS

腹泻仍然是1998年儿童死亡的主要原因， 5岁的世界各地，和致炎性大肠杆菌（如那些引起 霍乱毒素，CT）占近20%的可识别 - 是的确定CT作用的细胞机制可能有助于 改进治疗和预防措施的发展 对CT和与CT密切相关的E.大肠杆菌不耐热毒素 本提案的总体目标是阐明结构， 洗涤剂不溶性糖脂膜微区的功能 （DIGs）在霍乱毒素转运到模型人中的作用 肠上皮细胞（T84细胞）。我们的首要目标是 确定DIG在将CT靶向极化细胞中的作用 信号转导所必需的区室（如通过 C1-分泌反应，可以电测量）。炭疽 水肿毒素（AT）诱导T84细胞分泌C1反应 类似于CT，尽管具有不同的动力学。然而，在这方面， AT不通过DIG与细胞表面结合。嵌合毒素 CT和AT的匹配细胞结合和酶单位将 用于确定通过DIG进入细胞是否对CT至关重要 引发T84细胞分泌反应的能力。的能力 引起分泌反应以及分泌动力学 将产生关于通过DIG进入细胞的重要性的信息 CT对极化上皮细胞的作用。第二个具体目标是 以鉴定与CT特异性DIG相关的膜蛋白。我们 建议首先确定以下方面的作用， 肌动蛋白在维持DIG连接到我们的模型系统，Triton X- 100个浸提T84单层细胞。其次，我们计划调查CT- 通过化学交联和共交联， 与CT密切相关蛋白的免疫沉淀及其临床意义 受体神经节苷脂GM 1。

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.