Function of an Unconventional Myosin in Tetrahymena

学术成就：本计画关注一种新的非传统肌球蛋白的深入功能分析，这种肌球蛋白来自于自由生活的原生动物模式生物，嗜热四膜虫。 肌球蛋白是一组蛋白质生物分子马达，其通常与由称为肌动蛋白的蛋白质制成的亚细胞微丝相关联;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"常规"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沿着肌动蛋白丝移动，导致整个细胞缩短。 然而，肌球蛋白也参与许多其他亚细胞运动功能，范围从囊泡或颗粒沿着细胞延伸部（如轴突）的长度的运动到细胞分裂期间细胞膜的带状收缩（导致从原始分裂细胞形成两个子细胞）。 近年来，已经发现了相当多的不同分子种类（类别）的肌球蛋白是"非常规的"（即，不同于在动物组织中大量发现的两类肌球蛋白）。 在这个项目中，正在研究的肌球蛋白是Myo1p，一种来自四膜虫的分歧的非常规肌球蛋白，没有被分配到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MYO 1，并从MYO 1敲除中表征了表型。 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一级结构具有210，889道尔顿的分子量，并且包含预测的卷曲螺旋区域、136-aa肌球蛋白尾同源性4（MyTH 4）基序、308-aa带4.1、埃兹蛋白、根蛋白、膜突蛋白同源性（FERM）基序、位于尾结构域而不是通常的颈结构域位置的推定的钙调蛋白结合（IQ）基序，和124-aa的C-末端。在MYO 1基因敲除菌株中，吞噬体形成的速率降低，并且大核延伸常常不能完成。 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直接向后端移动。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。 一般的假设是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其作用位点，其中MyTH4和/或FERM介导肌动蛋白丝的组织。如果Myo1p仅通过尾部结构域定位并连接到作用位点，则含有足够靶向信息的尾部结构域片段的过表达将取代内源性Myo1p，从而抑制Myo1p功能。将构建含有保守基序和/或非保守区的标记的尾结构域片段的诱导型表达的菌株。生殖系基因替换将构建表达标记的截短Myo1p的不同菌株，其中尾部结构域的区域已被删除。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。用针对Myo1p运动结构域的抗体进行免疫染色将证明过表达的尾部片段是否已取代内源性Myo1p。用抗Myo1p和抗肌动蛋白抗体的双标记将定位Myo1p相对于野生型和哺乳动物细胞中的肌动蛋白。预计Myo1p将暂时或永久地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试验和形态测定分析，以鉴定大规模培养的四膜虫中的大核畸变。免疫沉淀和共沉淀分析将确定是否特定的尾部结构域基序与肌动蛋白相关，并诱导捆绑或交联的肌动蛋白在体外。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的颗粒和液体的内化对于包括原生动物、凋亡细胞和免疫系统的专职吞噬细胞在内的多种细胞类型具有根本重要性。大核伸长的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素。更广泛的影响：本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。 对一所面向少数民族城市学生的新科学高中的外联活动将涉及高中学生在PI的实验室和高中教师讲习班中从事研究。

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.