Function of an Unconventional Myosin in Tetrahymena

四膜虫中非常规肌球蛋白的功能

• 批准号: 0130624
• 负责人: Ray Gavin
• 金额: $ 35.99万
• 依托单位: CUNY Brooklyn College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0130624&HistoricalAwards=false
• 关键词: Function; Unconventional; Myosin; Tetrahymena

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.

Myo 1 p是Gavin博士及其同事在模型原生动物嗜热四膜虫（Tetrahymena thermophila）中发现的一种新型非常规肌球蛋白，它在两个基本细胞过程中发挥作用：内吞/吞噬作用和核运动。在最近完成的一个项目中，Gavin实验室使用靶向基因破坏来创建缺乏功能性MYO 1基因的四膜虫菌株。在转化的MYO 1细胞中，食物泡（内体）的形成速度比野生型细胞慢，大核在无丝分裂细胞分裂过程中经常不能正确伸长，导致DNA与后代的分离严重不平等。在这个更新项目中，将进行实验以直接和特异性地将Myo 1 p与内吞作用和大核伸长的机制联系起来。Myo 1 p功能在四膜虫的两个一般假设将进行测试。(1)假设：MYO 1是内体成熟和内体再循环所必需的。内体形成的速率可以部分地通过胞质溶胶中的内体成熟速率和随后的内体在被称为细胞直肠的膜结构处的再循环来调节。如果Myo 1 p参与内体成熟，MYO 1敲除菌株可能在新生内体的扩增和/或酸化中表现出缺陷。如果Myo 1 p参与细胞直肠的内体回收，则MYO 1敲除菌株的内体降解速率可能会降低。(2)假设：大核的伸长是由微管、肌动蛋白微丝和Myo 1 p的协同作用介导的。人们可以设想至少有两种非相互排斥的假定分子机制的安排，可以为大核延伸提供动力。伸长机制可能集中在大核内部或膜的中间区附近。或者，延长的机器可以集中在整个周边的大核膜上或周围的细胞质中。 为了验证这些假设，必须获得全长MYO 1序列并开发新的试剂。因此，项目I将包括MYO 1测序的完成，针对四膜虫肌动蛋白和Myo 1 p的多克隆抗体的产生，以及针对三种四膜虫蛋白质：肌动蛋白、微管蛋白和Myo 1 p的GFP表达构建体的产生。 项目II重点关注Myo 1 p在内吞作用中的功能。研究的三个领域是：（1）肌动蛋白和Myo 1 p定位于内吞途径的组成部分，（2）内体的成熟，和（3）内体在细胞直肠的再循环。将使用延时共聚焦显微镜研究表达GFP-肌动蛋白或GFP-Myo 1 p的细胞和表达GFP-肌动蛋白的MYO 1敲除细胞。荧光聚苯乙烯珠和膜标记染料将用于内体成熟和内体回收的测定中。在项目III中，表达GFP-肌动蛋白、GFP-微管蛋白或GFP-Myo 1 p的细胞和表达GFP-肌动蛋白或GFP-微管蛋白的MYO 1敲除细胞的共聚焦显微镜将确定肌动蛋白微丝、微管和Myo 1 p是否是能够为大核伸长提供动力的分子机制的组成部分。体外运动试验将获得大核Myo 1 p动力运动的直接证据。Myo 1 p的功能研究有可能扩大我们对内吞/吞噬和核运动的理解，并可能揭示非常规肌球蛋白在这些过程中的作用的新机制。内吞作用/吞噬作用的研究远远超出了四膜虫模型的影响。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进食是重要的，并且在后生动物中，它是免疫系统的重要组成部分。大核运动的潜在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，并且已知涉及细胞骨架元件。