Function of an Unconventional Myosin in Tetrahymena

Myo1p是Gavin博士及其同事在模式原生动物嗜热四膜虫中发现的一种新颖的、非常规的肌球蛋白，它在两个基本的细胞过程中起作用：内吞/吞噬和核运动。在最近完成的一个项目中，加文实验室使用靶向基因破坏技术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，食物液泡（核内体）的形成速度比野生型细胞慢，并且在无丝分裂细胞分裂过程中，巨核经常不能正常延长，导致DNA向后代的分离严重不平等。在这个更新项目中，将进行实验来直接和具体地将Myo1p与内吞作用和大核延伸机制联系起来。对于Myo1p在四膜虫中的功能，我们将测试两种一般假设。(1)假设：核内体成熟和核内体循环都需要MYO1。核内体的形成速度可以部分地通过细胞质中核内体的成熟速度和随后在称为细胞原质的膜结构中核内体的再循环来调节。如果Myo1p参与了核内体的成熟，myo1敲除菌株可能在新生核内体的扩张和/或酸化方面表现出缺陷。如果Myo1p参与胞内体的再循环，那么myo1敲除菌株的内体降解率可能会降低。(2)假设：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分子机制安排，可以为大核延伸提供动力。延伸机制可能集中在大核内部或膜的中间区域附近。或者，延伸机制可能集中在整个大核的外围，直接在大核膜上或在周围的细胞质中。为了验证这些假设，必须获得全长MYO1序列并开发新的试剂。因此，项目1将包括完成MYO1的测序，生成抗四膜虫肌动蛋白和Myo1p的多克隆抗体，并创建三种四膜虫蛋白：肌动蛋白、微管蛋白和Myo1p的GFP表达构建体。项目二主要研究Myo1p在胞吞作用中的功能。将进行三个领域的研究：(1)肌动蛋白和Myo1p定位于内吞途径的组成部分，(2)核内体的成熟，(3)核内体在细胞原蛋白处的再循环。用延时共聚焦显微镜研究表达GFP-actin或GFP-Myo1p的细胞和表达GFP-actin的myo1敲除细胞。荧光聚苯乙烯珠和膜标记染料将用于核内体成熟和核内体回收的测定。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敲除细胞进行共聚焦显微镜观察，将确定肌动蛋白微丝、微管和Myo1p是否为促进大核延伸的分子机制的组成部分。体外运动试验将获得myo1p驱动的大核运动的直接证据。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，并可能揭示非常规肌球蛋白在这些过程中作用的新机制。内吞作用/吞噬作用的研究的意义远远超出了四膜虫模型。细胞对表面受体的调节是通过内吞作用介导的，吞噬过程对许多原生生物的摄食很重要，在后生动物中，它是免疫系统的重要组成部分。巨核运动的潜在基础可能与核迁移和定位有关，核迁移和定位发生在许多细胞类型的发育过程中，并且已知涉及细胞骨架元件。

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.