Genetic Dissection of Phagocytosis in Tetrahymena Thermophila

拟议的研究旨在进一步了解吞噬作用，细胞吞噬和处理颗粒的过程。 过去的研究表明，吞噬作用是一个涉及许多蛋白质的多步骤过程。尽管如此，许多这些蛋白质的身份和/或功能仍然未知或知之甚少。拟议的研究将利用纤毛原生动物嗜热四膜虫作为解剖吞噬作用的模型。T.长期以来，嗜热菌一直被用作研究许多真核生物过程的模式生物，并且存在用于该生物的一系列强有力的遗传和分子遗传工具。在自然界中，四膜虫有效地吞噬其他微生物以满足其营养需求，但该过程是有条件的非必需的，因为在营养培养基上可以无吞噬作用地生长。本项目的具体目标是：（1）分离和鉴定一组吞噬作用突变体。一种有效的反义核糖体诱变程序将与两种筛选/选择程序结合使用，以鉴定影响吞噬过程中各种步骤的基因。(2)描述吞噬作用中的突变体。这将包括鉴定突变体中受影响的基因，并产生基因敲除菌株以确定无效突变的表型。此外，将使用许多细胞学程序分析突变体，以深入了解吞噬作用的哪个阶段受到影响，定量吞噬作用中缺陷的水平，并确定鉴定的基因产物的亚细胞位置。(3)开展旨在确定和表征四膜虫吞噬体蛋白质组主要蛋白质组分的研究。计划进行两类研究。首先，已经确定的吞噬体蛋白在小鼠中的作用将通过分离四膜虫中的基因同源物，然后构建和分析基因敲除来研究。第二，吞噬体纯化的方法已经适应于四膜虫，并通过质谱法进行初步分析的四膜虫吞噬体蛋白质组。这将最终导致的吞噬体蛋白的保守集的鉴定，并设置许多吞噬体组件的功能分析的阶段。该项目将作为研究生科学培训的工具，并为处于职业生涯早期阶段的学生提供夏季研究经验。

The proposed studies seek to further understanding of phagocytosis, the process by which cells engulf and process particulates. Past studies indicate that phagocytosis is a multi-step process involving scores of proteins. Nonetheless, the identity and/or function of many of these proteins remain unknown or poorly understood. The proposed studies will utilize the ciliated protozoan Tetrahymena thermophila as a model for dissecting phagocytosis. T. thermophila has long been used as a model organism for the study of many eukaryotic processes, and a strong array of genetic and molecular genetic tools exist for the organism. In nature, Tetrahymena efficiently engulfs other microorganisms to meet its nutritional needs, but the process is conditionally non-essential, as growth without phagocytosis is possible on nutrient medium. The specific aims for the project are to: (1) Isolate and characterize a panel of phagocytosis mutants. An efficient antisense ribosome mutagenesis procedure will be used in combination with two screening/selection procedures to identify genes that affect a variety of steps in the process of phagocytosis. (2) Characterize mutants in phagocytosis. This will include identifying the affected gene in mutants, and generating gene knockout strains to determine the phenotype of null mutations. In addition, mutants will be analyzed using a number of cytological procedures to gain insight into which stage of phagocytosis is affected, to quantitate the level of the defect in phagocytosis, and to determine the subcellular location of identified gene products. (3) Initiate studies aimed at identifying and characterizing the major protein components of the Tetrahymena phagosome proteome. Two types of studies are planned. First, the role of phagosome proteins already identified in the mouse will be investigated by isolating gene homologs in Tetrahymena and then constructing and analyzing gene knockouts. Second, methods of phagosome purification have been adapted to Tetrahymena, and a preliminary analysis of the Tetrahymena phagosome proteome by mass spectrometry will be carried out. This will ultimately lead to the identification of the conserved set of phagosome proteins, and set the stage for functional analysis of many phagosome components. The project will serve as a vehicle for the scientific training of graduate students, as well as providing summer research experiences for students at earlier stages of their careers.