Function of an Unconventional Myosin in Tetrahymena

Myo1p是Gavin博士和他的同事在模型原生动物嗜热四膜虫中发现的一种新的、非传统的肌球蛋白，它被认为在两个基本的细胞过程中发挥作用：吞噬/吞噬和核运动。在最近完成的一个项目中，加文实验室使用定向基因破坏创造了一种缺乏功能性MYO1基因的四膜虫菌株。在转化的MYO1细胞中，食物空泡(内体)的形成速度慢于野生型细胞，而且在无丝分裂细胞分裂过程中，大核经常不能正常伸长，导致DNA严重不均匀地分离到后代。在这个更新项目中，将进行实验，将Myo1p直接和特定地连接到内吞和大核延长的机制。我们将测试四膜虫中Myo1p功能的两个一般假设。(1)假说：MYO1是内体成熟和内体循环所必需的。内小体形成的速度可以部分地由胞质中内小体成熟的速度和随后内小体在被称为细胞直肠的膜结构上的循环来调节。如果Myo1p参与内小体成熟，MYO1基因敲除菌株可能在新生内小体的扩张和/或酸化方面表现出缺陷。如果Myo1p参与细胞直肠的内体循环，MYO1基因敲除菌株的内体降解率可能会降低。(2)假说：大核伸长是由微管、肌动蛋白微丝和Myo1p协同作用介导的。人们可以设想至少有两种非互斥的安排，用于可能推动大核延长的分子机制。伸长机可以集中在大核内部或膜的中区附近。或者，延长的机制可以集中在整个大核的外围，或者直接集中在大核膜上，或者集中在周围的细胞质中。为了验证这些假说，必须获得全长的MYO1序列，并开发新的试剂。因此，项目I将包括完成MYO1测序，产生针对四膜虫肌动蛋白和Myo1p的多克隆抗体，并为三种四膜虫蛋白：肌动蛋白、微管蛋白和Myo1p创建GFP表达载体。项目II的重点是Myo1p在内吞作用中的作用。将进行三个方面的研究：(1)肌动蛋白和Myo1p在内吞途径中的定位，(2)内小体的成熟，(3)细胞直肠的内小体循环。表达GFP-肌动蛋白或GFP-Myo1p的细胞和表达GFP-肌动蛋白的MYO1基因敲除细胞将用时间推移共聚焦显微镜观察。荧光聚苯乙烯微珠和膜标记染料将用于内体成熟和内体回收的检测。在项目III中，对表达GFP-肌动蛋白、GFP-微管蛋白或GFP-Myo1p的细胞和表达GFP-肌动蛋白或GFP-微管蛋白的MYO1基因敲除细胞的共聚焦显微镜将确定肌动蛋白微丝、微管和Myo1p是否是推动大核延长的分子机制的组件。体外运动性分析将获得Myo1p驱动的大核运动的直接证据。对Myo1p的功能研究有可能扩大我们对内吞/吞噬和核运动的理解，并可能揭示非传统肌球蛋白在这些过程中所起作用的新机制。对内吞/吞噬作用的研究具有远远超出四膜虫模型的意义。细胞对表面受体的调节是由内吞作用介导的，吞噬过程对于许多原生动物的摄食是重要的，在后生动物中是免疫系统的重要组成部分。大核运动的基本基础可能与许多细胞类型在发育过程中发生的核迁移和定位有关，并已知涉及细胞骨架元素。

Myo1p is a novel, unconventional myosin discovered by Dr. Gavin and colleagues in the model protozoan Tetrahymena thermophila that has been implicated as playing a role in two fundamental cellular processes: endocytosis/phagocytosis and nuclear motility. In a recently completed project, the Gavin laboratory used targeted gene disruption to create a Tetrahymena strain that lacked a functional MYO1 gene. In transformed MYO1 cells, food vacuoles (endosomes) formed at a slower rate than in wild-type cells, and the macronucleus frequently failed to elongate properly during amitotic cell division, resulting in grossly unequal segregation of DNA to progeny. In this renewal project, experiments will be performed to directly and specifically link Myo1p to the machinery for both endocytosis and macronuclear elongation. Two general hypotheses for Myo1p function in Tetrahymena will be tested. (1) Hypothesis: MYO1 is required for both endosome maturation and endosome recycling. The rate of endosome formation could be regulated, in part, by the rate of endosome maturation in the cytosol and subsequent endosome recycling at the membrane structure known as the cytoproct. If Myo1p is involved in endosome maturation, a MYO1-knockout strain might exhibit defects in expansion and/or acidification of nascent endosomes. If Myo1p is involved in endosome recycling at the cytoproct, a MYO1-knockout strain might have a reduced rate of endosome degradation. (2) Hypothesis: Macronuclear elongation is mediated by cooperative action of microtubules, actin microfilaments, and Myo1p. One can envision at least two, non-mutually exclusive, arrangements for the putative molecular machinery that could power macronuclear elongation. The elongation machinery could be concentrated near the midzone of the macronuclear interior or membrane. Alternatively, the machinery for elongation could be concentrated throughout the periphery of the macronucleus either directly on the macronuclear membrane or in the surrounding cytosol. In order to test these hypotheses, a full-length MYO1 sequence will have to be acquired and new reagents developed. Therefore, Project I will involve completion of MYO1 sequencing, generation of polyclonal antibodies against Tetrahymena actin and Myo1p, and creation of GFP- expression constructs for three Tetrahymena proteins: actin, tubulin, and Myo1p. Project II focuses on Myo1p function in endocytosis. Three areas of investigation will be pursued: (1) localization of actin and Myo1p to components of the endocytic pathway, (2) maturation of endosomes, and (3) endosome recycling at the cytoproct. Cells expressing either GFP-actin or GFP-Myo1p and MYO1-knockout cells expressing GFP-actin will be investigated with time-lapse confocal microscopy. Fluorescent polystyrene beads and membrane marker dyes will be used in assays for endosome maturation and endosome recycling. In project III, confocal microscopy of cells expressing either GFP-actin, GFP-tubulin, or GFP-Myo1p and MYO1-knockout cells expressing either GFP-actin or GFP-tubulin will determine whether actin microfilaments, microtubules, and Myo1p are components of a molecular machinery that could power macronuclear elongation. An in vitro motility assay will obtain direct evidence for Myo1p-powered movement of macronuclei. Functional studies of Myo1p have the potential for expanding our understanding of endocytosis /phagocytosis and nuclear motility and may reveal novel mechanisms for the role of unconventional myosins in these processes. Studies of endocytosis/phagocytosis have implications far beyond the Tetrahymena model. Cell regulation of surface receptors is mediated by endocytosis, and the phagocytic process is important for the feeding of many protists, and in metazoa it is an essential part of the immune system. The underlying basis for macronuclear motility may be related to nuclear migration and positioning that take place during development in many cell types and are known to involve cytoskeletal elements.