Function of an Unconventional Myosin in Tetrahymena

智力价值：这个项目涉及对自由生活的原生动物模式生物四膜虫嗜热四膜虫的一种新的非传统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，通常与由一种名为肌动蛋白的蛋白质组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肌动蛋白微丝运动。也许“传统的”肌球蛋白最广为人知的细胞功能是肌肉收缩，在这一过程中，肌球蛋白分子的线性阵列沿着肌动蛋白细丝移动，导致整个细胞缩短。然而，肌球蛋白也参与了许多其他的亚细胞运动功能，从沿着细胞延伸的长度移动的囊泡或颗粒，如轴突，到细胞分裂过程中细胞膜的带状收缩，导致从最初的分裂细胞形成两个子细胞。近年来，许多不同的分子类型(类)的肌球蛋白被发现是“非常规的”(即，不同于在动物组织中发现的高丰度的两类肌球蛋白)。在这个项目中，正在研究的肌球蛋白是Myo1p，一种与四膜虫不同的非传统肌球蛋白，没有被归类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，并从MYO1基因敲除中鉴定了其表型。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：吞噬和核运动。预测的全长Myo1p一级结构具有210,889道尔顿的分子质量，包含一个预测的卷曲区域，一个136-AA肌球蛋白尾部同源4(MyTH4)基序，一个308-AA带4.1，Ezrin，Radix，Moesin同源(FERM)基序，一个可能位于尾部区域而不是通常的颈区位置的钙调蛋白结合(IQ)基序，以及一个124-AA的C末端。在MYO1基因敲除株中，吞噬小体形成率降低，大核伸长常常无法完成。对敲除菌株的进一步研究表明，吞噬体在胞浆中随机移动，而不是在野生型细胞中定向向后端移动。这些吞噬作用研究的结论是，吞噬小体的定向运动需要肌动蛋白细丝和Myo1p。吞噬小体相关的肌动蛋白和Myo1p如何相互作用以影响吞噬小体的定向(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尾部区域的保守和/或非保守区域将这种肌球蛋白靶向其作用部位，在那里MyTH4和/或FERM介导肌动蛋白细丝的组织。如果Myo1p仅通过尾部结构域定位并与作用部位相连，则含有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的功能。将构建含有保守基序和/或非保守区的带有标签的尾部结构域片段可诱导表达的菌株。生殖系基因替换将构建不同的菌株，表达尾部区域已被删除的标签截断的Myo1p。抗表位标签抗体和针对尾域区域的抗体将定位尾域片段。用针对Myo1p运动区的抗体进行免疫染色将证明过度表达的尾部片段是否取代了内源性Myo1p。用抗Myo1p和抗肌动蛋白抗体进行双重标记，将在野生型和转化细胞中定位Myo1p与肌动蛋白的关系。预计Myo1p将暂时或永久地定位于吞噬小体或与吞噬小体相关的肌动蛋白以及大核附近的区域。功能分析将使用吞噬小体运动性分析和形态计量学分析来鉴定四膜虫大规模培养中的大核异常。免疫沉淀和共沉淀分析将确定特定的尾部结构域基序是否与肌动蛋白结合并在体外诱导肌动蛋白的捆绑或交联。这些研究的意义远远超出了四膜虫模型。通过吞噬和内吞作用使颗粒和液体内化对于包括原生动物、凋亡细胞和免疫系统的专业吞噬细胞在内的各种细胞类型都是至关重要的。大核延长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，并已知涉及细胞骨架元素。广泛影响：该项目将为布鲁克林学院的几名本科生提供研究培训，并将整合一门细胞生物学实验室课程。对一所面向城市少数民族学生的新理科高中的宣传将包括高中生在PI的实验室进行研究，并为高中教师举办讲习班。

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.