The Endosome System and Membrane Trafficking

细胞区室之间的通信经常通过以下方式发生： 穿梭囊泡。 核内体系统的独特特征 草履虫将通过使用单克隆抗体进行探索， 共聚焦荧光显微镜，快速冷冻的复制品- 干燥的细胞碎片，冷冻切片的免疫金标记， Lowicryl切片和高分辨率扫描电子显微镜 分离的细胞器。 探测器的过程将从 包被的小凹到早期的内体并继续到下一个隔室， 其可以是或许多不是酸体。 初步研究表明 后一种转运是通过90 nm网格蛋白包被的穿梭物进行的 早期内体产生的小泡。 选择性标记，抗原 封闭和胰蛋白酶消化技术， 单克隆抗体将用于跟踪在 细胞表面的质膜驻留抗原C6， 通过190 nm从包被的小凹进入早期内体室 前内体囊泡，但不进入90 nm早期内体， 衍生的穿梭囊泡。 另一种特异于酸体的mAb， 将用于研究酸体膜的生物发生 以及它们与内体或收缩泡的关系 系统. 最后，速冻、冻干技术和高 分辨率扫描电子显微镜将用于可视化 并研究真正的细胞膜表面与 内体、吞噬体-溶酶体和收缩泡系统 这个细胞。 %%% 几乎所有的真核细胞都含有一个内部膜系统 它创造了一系列功能和生化上不同的 细胞内隔室通过其 膜边界 尽管这些隔间在功能上 而且在生物化学上是不同的，所以我们知道它们是“交流”的。 与对方交换内容， 细胞外空间（通过细胞的细胞壁与细胞质分离） 质膜）直接或间接通过膜融合 事件 这种内部膜系统是细胞 从周围环境中获取材料并进行加工 酶促地，以及向环境分泌产物。 了解这个系统是如何运作的，以及它是如何被监管的， 是细胞生物学的一个主要目标 在这个项目中，单- 草履虫是一种自由生活的生物， 一个可操作的模型来研究细胞如何在选定的细胞之间进行通信， 它们如何包装并将驻留蛋白质返回到 他们的家庭隔间或膜，以及他们的地形如何， 真正的胞质膜表面与隔室的 功能

Communication between cellular compartments frequently occurs by shuttle vesicles. The unique features of the endosomal system of Paramecium will be explored by the use of monoclonal antibodies, confocal fluorescence microscopy, replicas of quick-freeze freeze- dried cell fragments, immunogold labeling of cryosections and Lowicryl sections, and high resolution scanning electron microscopy of isolated organelles. The course of probes will be followed from coated pits to early endosomes and on to the next compartment, which may or many not be the acidosomes. Initial studies indicate that this latter transport is via 90 nm clathrin-coated shuttle vesicles arising from early endosomes. Selective labeling, antigen blocking and trypsinization techniques in conjunction with a monoclonal antibody will be used to follow the reappearance on the cell surface of a plasma membrane resident antigen, C6, which enters the early endosome compartment from coated pits via 190 nm preendosomal vesicles but does not enter the 90 nm early endosome- derived shuttle vesicles. Another mAb, specific for acidosomes, will be used to study the biogenesis of the acidosomal membranes and their relationship to the endosomal or contractile vacuole systems. Finally, quick-freeze, freeze-dry techniques and high resolution scanning electron microscopy will be used to visualize and study the true cytosolic surfaces of membranes associated with the endosomal, phagosome-lysosome, and contractile vacuole systems of this cell. %%% Virtually all eukaryotic cells contain an internal membrane system which creates a series of functionally and biochemically distinct intracellular compartments isolated from the "cytoplasm" by their membrane boundaries. Although these compartments are functionally and biochemically distinct, it is known that they "communicate" with and exchange contents with each other and with the extracellular space (isolated from the cytoplasm by the cell's plasma membrane) either directly or indirectly via membrane fusion events. This internal membrane system is the route by which cells take up materials from their environment and process them enzymatically, as well as secrete products to the environment. Understanding how this system functions, and how it is regulated, is a major goal in cell biology. In this project, the single- celled freeliving organism, Paramecium, is used as a readily manipulable model to study how cells communicate between selected compartments, how they package and return resident proteins to their home compartment or membrane, and how the topography of their true cytosolic membrane surface is related to the compartment's function.