Genetic Dissection of Phagocytosis in Tetrahymena Thermophila

提出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。提出的研究将利用纤毛原生动物嗜热四膜虫作为解剖吞噬的模型。t . thermophila一直被用作生物模型研究许多真核生物的过程,和一系列强烈的遗传和分子遗传工具存在的有机体。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，但这一过程是有条件的非必需的，因为在营养培养基上生长而不吞噬是可能的。该项目的具体目标是：(1)分离和表征一组吞噬突变体。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，以确定影响吞噬过程中各种步骤的基因。(2)描述吞噬突变体的特征。这将包括在突变体中识别受影响的基因，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来分析突变体，以深入了解吞噬作用的哪个阶段受到影响，量化吞噬缺陷的水平，并确定已鉴定基因产物的亚细胞位置。(3)开展旨在鉴定和表征四膜虫吞噬体蛋白质组主要蛋白质成分的研究。计划进行两类研究。首先，我们将通过在四膜虫中分离基因同源物，然后构建和分析基因敲除来研究在小鼠中已经鉴定出的吞噬体蛋白的作用。其次，吞噬体纯化方法已适应于四膜虫，并将采用质谱法对四膜虫吞噬体蛋白质组进行初步分析。这将最终导致鉴定一组保守的吞噬体蛋白，并为许多吞噬体成分的功能分析奠定基础。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暑期研究经验。

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.