LYOSOME--MEDIATED CELL INVASION BY TRYPANOSOMA CRUZI

我们实验室最近的研究揭示了一种新的细胞凋亡机制， 原生动物克氏锥虫的入侵 在进入非- 吞噬哺乳动物细胞，宿主溶酶体被招募到寄生虫 附着位点，并逐渐与质膜融合， 含有寄生物的液泡。在我们寻找信号传导的过程中 在这个过程中，我们发现感染性锥鞭毛体， 从宿主细胞胞内储存中动员Ca 2+，诱导 细胞质游离Ca 2+浓度的短暂增加， 各种哺乳动物细胞类型。 宿主细胞中的快速重排 皮质肌动蛋白细胞骨架，一种已知导致T. cruzi入侵，观察到锥鞭毛体诱导的结果， Ca 2+瞬变。这些瞬变现象的预防 抑制寄生虫侵入宿主细胞。 我们现在计划直接 研究T. cruzi诱导的细胞内游离钙 加上瞬时和动员和融合的溶酶体与 哺乳动物细胞的质膜。 具体来说，我们将：1）使用 视频显微镜分析，检查宿主细胞胞浆游离Ca 2+的作用 加上对募集和/或质膜融合的瞬变， T. cruzi; 2）研究主要PKC的作用 底物、Ca 2 +-钙调素和肌动蛋白结合蛋白MARCKS在 宿主细胞事件导致溶酶体融合和T.克鲁齐入侵; 3） 使用透化的哺乳动物细胞系统来研究 参与Ca 2+调节的溶酶体胞吐作用的机制， 特别是NSF/SNAP/SNARE融合复合物的作用， 突触结合蛋白、膜联蛋白和单体和三聚体GTP结合蛋白; 4)将溶酶体胞吐测定中获得的数据与T. cruzi侵袭，使用特异性抑制剂和细胞显微注射。 这些研究将推进我们对不寻常的细胞入侵的认识 重要的人类病原体T.克鲁兹，以及 哺乳动物中涉及溶酶体的基本膜运输事件 细胞

Recent studies from our laboratory uncovered a novel mechanism for cell invasion by the protozoan Trypanosoma cruzi. Prior to entry into non- phagocytic mammalian cells, host lysosomes are recruited to the parasite attachment site and gradually fuse with the plasma membrane, originating the parasite-containing vacuole. In our search for signal transduction events involved in this process, we found that infective trypomastigotes mobilize Ca2 plus from host cell intracellular stores, inducing transient increases in the cytosolic free Ca2 plus concentration of a variety of mammalian cell types. Rapid rearrangements in the host cell cortical actin cytoskeleton, a condition known to result in enhanced T. cruzi invasion, are observed as a consequence of trypomastigote-induced Ca2 plus-transients. Prevention of these transients significantly inhibits host cell invasion by the parasites. We now plan to directly investigate the link between the T. cruzi-induced intracellular free Ca2 plus transients and the mobilization and fusion of lysosomes with the plasma membrane in mammalian cells. Specifically, we will: 1) using a video-microscopy assay, examine the role of host cell cytosolic free Ca2 plus transients on the recruitment and/or plasma membrane fusion of lysosomes induced by T. cruzi; 2) investigate the role of the major PKC substrate, Ca2 plus-calmodulin and actin-binding protein MARCKS in the host cell events leading to lysosome fusion and T. cruzi invasion; 3) use a permeabilized mammalian cell system to investigate the molecular mechanisms involved in Ca2 plus-regulated lysosome exocytosis, specifically the role of the NSF/SNAP/SNARE fusion complex, synaptotagmin, annexins and monomeric and trimeric GTP-binding proteins; 4) correlate the data obtained in the lysosome exocytosis assay with T. cruzi invasion, using specific inhibitors and cellular microinjection. These studies will advance our knowledge of the unusual cell invasion mechanism utilized by the important human pathogen T. cruzi, as well as of fundamental membrane traffic events involving lysosomes in mammalian cells.