Characterization of a Dynamin-Related Protein Involved in Endocytosis in Tetrahymena Thermophila

所有真核细胞都通过从质膜上吸收分子与周围环境进行交流。这种摄取称为内吞作用，可以满足多种需求，包括营养和质膜本身的再循环。一个细胞可以使用几种不同的内吞作用模式。虽然已经发现了许多参与内吞作用的蛋白质，但对内吞作用的确切机制以及对多种途径的调节和协调的了解仍然是零碎的。本项目旨在剖析纤毛原生动物嗜热四膜虫的两条摄取途径。第一种途径是专门用于营养的，并允许这些细胞吸收大颗粒，如细菌，其过程可能类似于人类免疫细胞的颗粒摄取。第二种途径没有被很好地描述，但它可能与膜回收有关。初步数据表明，这两种途径似乎共享一些蛋白质，尤其是一种叫做动力蛋白的蛋白质。这是一个令人惊讶的结果，因为动力蛋白在内吞作用中的功能以前被认为仅限于高等真核细胞。因此，本项目的第一个目的是利用电子显微镜确认动力蛋白在四膜虫中的定位。为了确定动力蛋白是否影响细胞形态，将对动力蛋白基因被破坏的细胞进行电子显微镜观察。第二个目的是分析动力蛋白的作用，并区分动力蛋白功能丧失后的直接和间接影响。这一目标将通过对温度触发的快速失活敏感的诱变变体取代动力蛋白来实现。第三个目标是了解动力蛋白是如何精确定位到其作用部位的。这将通过创建由动力蛋白的小部分组成的微型蛋白质来研究，并询问其中哪些包含足够的信息以进行适当的定位。该项目将为研究生和本科生提供研究训练。

All eukaryotic cells communicate with their surroundings by taking up molecules from the plasma membrane. This uptake, known as endocytosis, can fulfill a variety of requirements including nutrition and recycling of the plasma membrane itself. A cell can use several different modes of endocytosis. Although many proteins involved in endocytosis have been discovered, knowledge of the precise mechanisms underlying endocytosis, and understanding the regulation and coordination of multiple pathways are still fragmentary. This project is aimed at dissecting two pathways of uptake in the ciliated protozoan Tetrahymena thermophila. The first pathway is specialized for nutrition, and allows these cells to take up large particles, such as bacteria, by a process that may resemble particle uptake in human immune cells. The second pathway has been less well described, but it may be involved in membrane recycling. Preliminary data suggest that both pathways appear to share some proteins, in particular a protein called dynamin. This was a surprising result because the function of dynamin in endocytosis was previously believed to be restricted to higher eukaryotic cells. A first aim of this project is therefore to confirm the localization of dynamin in Tetrahymena by using electron microscopy. To determine whether dynamin affects cellular morphology, electron microscopy will be performed on cells in which the dynamin gene has been disrupted. A second aim is to analyze the roles of dynamin and distinguish between direct and indirect effects following loss of dynamin function. This aim will be achieved by replacing dynamin with mutagenized variants that are sensitive to rapid temperature-triggered inactivation. A third aim is to understand how dynamin is accurately targeted to its site of action. This will be investigated by creating mini-proteins consisting of small parts of the dynamin protein, and asking which of these contains sufficient information for proper localization. This project will provide research training for graduate and undergraduate students.