Function of an Unconventional Myosin in Tetrahymena

四膜虫中非常规肌球蛋白的功能

• 批准号: 0517083
• 负责人: Ray Gavin
• 金额: $ 41.99万
• 依托单位: CUNY Brooklyn College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517083&HistoricalAwards=false
• 关键词: Function; Unconventional; Myosin; Tetrahymena

Intellectual Merit:This project concerns in depth functional analysis of a novel unconventional myosin from the free living protozoan model organism, Tetrahymena thermophila. Myosins are a group of protein biomolecular motors that are generally associated with subcellular microfilaments made of a protein called actin; linkage of myosin molecules to various other subcellular particles or other structures results in movement of those particles or structures along the actin microfilament. Perhaps the most well-known cellular function of "conventional" myosin is that of muscle contraction, in which a linear array of myosin molecules move along actin filaments to cause shortening of entire cells. However, myosins are also involved in many other subcellular motility functions, ranging from the movement of vesicles or granules along the length of cellular extensions such as axons to the belt-like constriction of cell membrane during cell division that results in the formation of two daughter cells from the original dividing cell. In recent years, quite a few different molecular kinds (classes) of myosins have been discovered that are "unconventional" (i.e., different from the two myosin classes found in high abundance in animal tissues). In this project, the myosin under study is Myo1p, a divergent unconventional myosin from Tetrahymena that is not assigned to any of the known myosin classes. In prior NSF-funded work, Dr. Gavin disovered MYO1 and characterized the phenotype from a MYO1 knockout. Results from that work demonstrated that Myo1p is involved in two fundamental cellular processes of this protozoan: phagocytosis and nuclear motility. The predicted full-length Myo1p primary structure has a molecular mass of 210,889 Daltons and contains a region of predicted coiled coil, a 136-aa myosin tail homology 4 (MyTH4) motif, a 308-aa Band 4.1, ezrin, radixin, moesin homology (FERM) motif, a putative calmodulin-binding (IQ) motif that is located in the tail domain rather than the usual neck-domain location, and a 124-aa C-terminus. In the MYO1 knockout strain, the rate of phagosome formation was reduced, and macronuclear elongation often failed to be completed. Further studies of the knockout strain revealed that phagosomes moved randomly in the cytosol in contrast to directed movement toward the posterior end in wild-type cells. A conclusion of these studies of phagocytosis is that directed motility of phagosomes requires actin filaments and Myo1p. How phagosome-associated actin and Myo1p interact to effect directed (as opposed to random) motility of phagosomes is unknown and is the focus of this new project. The general hypothesis is that conserved and/or non-conserved regions in the tail domain of Myo1p target this myosin to its sites of action where MyTH4 and/or FERM mediates organization of actin filaments. If Myo1p is localized and linked to the site of action solely by the tail domain, over-expression of tail domain fragments that contain sufficient targeting information would replace endogenous Myo1p and thereby inhibit Myo1p function. Strains with inducible expression of tagged, tail-domain fragments that contain conserved motifs and/or nonconserved regions will be constructed. Germline gene replacements would construct different strains that express a tagged truncated Myo1p in which regions of the tail domain have been deleted. Anti-epitope tag antibodies and antibodies directed against regions of the tail domain will localize tail domain fragments. Immunostaining with antibodies directed against the Myo1p motor domain will demonstrate whether or not over-expressed tail fragments have replaced endogenous Myo1p. Double labeling with anti-Myo1p and anti-actin antibodies will localize Myo1p in relation to actin in wild-type and transformant cells. It is anticipated that Myo1p would localize either transiently or permanently to phagosomes or phagosome-associated actin and to regions near the macronucleus. Functional analyses will employ assays for phagosome motility and a morphometric analysis for identification of macronuclear aberrations in mass cultures of Tetrahymena. Immunoprecipitation and co-sedimentation assays will determine whether or not specific tail domain motifs associate with actin and induce bundling or cross-linking of actin in vitro. These studies have implications far beyond the Tetrahymena model. Internalization of particulates and fluids through phagocytosis and endocytosis is of fundamental importance to diverse cell types including protozoa, apoptotic cells, and professional phagocytes of the immune system. The underlying basis for macronuclear elongation may be related to nuclear migration and positioning that take place during development in many cell types and are known to involve cytoskeletal elements.Broader Impacts: This project will provide research training for several Brooklyn College undergraduates and would integrate a cell biology laboratory course. Outreach to a new science high school geared to educating minority urban students will involve high school students engaged in research in the PI's laboratory and workshops for high school teachers.

智力优点：该项目涉及对来自自由生活的原生动物模型生物——嗜热四膜虫的新型非常规肌球蛋白进行深入的功能分析。 肌球蛋白是一组蛋白质生物分子马达，通常与由肌动蛋白构成的亚细胞微丝相关。肌球蛋白分子与各种其他亚细胞颗粒或其他结构的连接导致这些颗粒或结构沿着肌动蛋白微丝运动。 也许“传统”肌球蛋白最著名的细胞功能是肌肉收缩，其中肌球蛋白分子的线性阵列沿着肌动蛋白丝移动，导致整个细胞缩短。 然而，肌球蛋白还参与许多其他亚细胞运动功能，从囊泡或颗粒沿着细胞延伸长度（如轴突）的运动到细胞分裂过程中细胞膜的带状收缩，导致原始分裂细胞形成两个子细胞。 近年来，已经发现了相当多不同的“非常规”肌球蛋白分子种类（类别）（即与动物组织中大量发现的两种肌球蛋白类别不同）。 在这个项目中，所研究的肌球蛋白是 Myo1p，一种来自四膜虫的非常规肌球蛋白，不属于任何已知的肌球蛋白类别。 在之前 NSF 资助的工作中，Gavin 博士发现了 MYO1 并表征了 MYO1 敲除的表型。 这项工作的结果表明，Myo1p 参与了这种原生动物的两个基本细胞过程：吞噬作用和核运动。 预测的全长 Myo1p 一级结构的分子量为 210,889 道尔顿，包含预测的卷曲线圈区域、136-aa 肌球蛋白尾同源 4 (MyTH4) 基序、308-aa 带 4.1、ezrin、radixin、moesin 同源 (FERM) 基序、位于尾部结构域的假定钙调蛋白结合 (IQ) 基序 而不是通常的颈部结构域位置，以及 124 个氨基酸的 C 末端。在MYO1敲除菌株中，吞噬体形成速率降低，大核伸长常常无法完成。 对敲除菌株的进一步研究表明，吞噬体在胞质溶胶中随机移动，而野生型细胞中的吞噬体则定向向后端移动。这些吞噬作用研究的结论是，吞噬体的定向运动需要肌动蛋白丝和 Myo1p。吞噬体相关的肌动蛋白和 Myo1p 如何相互作用以影响吞噬体的定向（而不是随机）运动尚不清楚，这是这个新项目的重点。 一般假设是，Myo1p 尾部结构域中的保守和/或非保守区域将该肌球蛋白靶向至 MyTH4 和/或 FERM 介导肌动蛋白丝组织的作用位点。如果Myo1p仅通过尾部结构域定位并连接到作用位点，则包含足够靶向信息的尾部结构域片段的过度表达将取代内源性Myo1p，从而抑制Myo1p功能。将构建具有包含保守基序和/或非保守区域的标记尾部结构域片段的诱导表达的菌株。种系基因替换将构建表达带标记的截短 Myo1p 的不同菌株，其中尾部结构域区域已被删除。抗表位标签抗体和针对尾部结构域区域的抗体将定位尾部结构域片段。使用针对 Myo1p 运动结构域的抗体进行免疫染色将证明过度表达的尾部片段是否已取代内源性 Myo1p。抗 Myo1p 和抗肌动蛋白抗体的双重标记将定位野生型和转化细胞中与肌动蛋白相关的 Myo1p。预计 Myo1p 将暂时或永久定位于吞噬体或吞噬体相关肌动蛋白以及大核附近的区域。功能分析将采用吞噬体运动测定和形态测定分析来鉴定四膜虫大规模培养物中的大核畸变。免疫沉淀和共沉淀测定将确定特定尾部结构域基序是否与肌动蛋白相关并在体外诱导肌动蛋白的成束或交联。这些研究的意义远远超出了四膜虫模型。 通过吞噬作用和内吞作用实现颗粒和液体的内化对于多种细胞类型（包括原生动物、凋亡细胞和免疫系统的专业吞噬细胞）至关重要。大核伸长的根本基础可能与许多细胞类型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素。 更广泛的影响：该项目将为布鲁克林学院的几名本科生提供研究培训，并将整合细胞生物学实验室课程。 面向一所旨在教育少数民族城市学生的新科学高中的推广活动将让高中生参与 PI 实验室的研究工作，并为高中教师举办讲习班。