The Endosome System and Membrane Trafficking

细胞间的通讯经常通过穿梭小泡进行。草履虫内体系统的独特特征将通过使用单抗、共聚焦荧光显微镜、快速冷冻干燥细胞片段的复制品、冰冻切片和Lowicryl切片的免疫金标记法以及分离细胞器的高分辨率扫描电子显微镜来探索。探针的进程将从包被的凹坑到早期的内小体，然后到达下一个隔室，它可能是酸性小体，也可能不是。初步研究表明，后一种转运是通过早期内体产生的90 nm网状蛋白包裹的穿梭小泡进行的。结合单抗的选择性标记、抗原阻断和胰酶消化技术将用于跟踪质膜驻留抗原C6在细胞表面的再现，C6从包被的凹坑通过190 nm的前内体小泡进入早期内吞体室，但不进入90 nm的早期内体衍生的穿梭小泡。另一种针对酸性小体的单抗将用于研究酸性小体膜的生物发生及其与内体或收缩空泡系统的关系。最后，将使用快速冷冻、冷冻干燥技术和高分辨率扫描电子显微镜来可视化和研究与该细胞的内吞液体、吞噬小体-溶酶体和收缩空泡系统相关的膜的真实胞液表面。几乎所有真核细胞都有一个内膜系统，它通过膜的边界从细胞质中分离出一系列功能和生物化学上截然不同的细胞内隔间。虽然这些隔室在功能和生化上是不同的，但众所周知，它们彼此之间以及与细胞外空间(通过细胞的质膜与细胞质隔开)直接或间接地通过膜融合事件进行通信和交换内容。这种内部膜系统是细胞从环境中吸收物质并进行酶处理的途径，也是向环境分泌产物的途径。了解这个系统是如何运作的，以及它是如何被调控的，是细胞生物学的一个主要目标。在这个项目中，单细胞自由生物草履虫被用作一个易于操作的模型来研究细胞如何在选定的隔室之间通信，它们如何包装驻留蛋白并将其返回到它们的居家隔室或膜，以及它们真实的细胞膜表面的地形如何与隔室的功能相关。

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.