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Uncovering the roles of the three tropomyosin isoforms in Drosophila S2 Cells

揭示三种原肌球蛋白亚型在果蝇 S2 细胞中的作用

基本信息

批准号：
9326018
负责人：
Johnny Rodriguez
金额：
$ 3.44万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9326018
关键词：
ATP phosphohydrolase Actin-Binding Protein Actins Address Adopted Affect Affinity Alpha Cell Animal Model Binding Biological Assay Biological Models California Cell Line Cell Maintenance Cell Shape Cell division Cell membrane Cell physiology Cells Cellular Morphology Collaborations Complex Data Dimensions Drosophila genus Drosophila melanogaster Electron Microscopy Elongation Factor Eukaryotic Cell Exhibits Filament Fission Yeast Fluorescence Microscopy Genes Immunodeficiency and Cancer Immunologic Deficiency Syndromes Individual Literature Mammalian Cell Measures Microfilaments Molecular Conformation Motor Muscle Contraction Mutate Mutation Myopathy Myosin ATPase Myosin Type II Neoplasm Metastasis Process Property Protein Isoforms Proteins RNA Interference Recruitment Activity Regulation Research Resolution Role San Francisco Site Skeleton Structure Tropomyosin Universities base cancer therapy cell assembly cell motility cell type daughter cell experimental study in vitro Assay insight lead-binding proteins new therapeutic target non-muscle myosin polymerization public health relevance therapeutic development three dimensional structure tumor

项目摘要

DESCRIPTION (provided by applicant): Actin subunits assemble into many different three-dimensional structures that are central to processes such as cellular division and motility, yet how a cell assembles different cytoskeletal structures using actin as the basic building block remains unclear. I hypothesize that tropomyosin isoforms determine which actin structures are assembled by regulating which accessory factors can gain access to actin filaments. This argument is supported by data that eukaryotic cells can express multiple different tropomyosin isoforms, where each isoform is sorted to a unique actin structure. Although mammalian tropomyosins are being characterized, the fact that mammalian non-muscle cells express over forty different tropomyosin isoforms has made tropomyosin research very complex. Our lab has chosen Drosophila melanogaster S2 cells as the model organism for understanding why eukaryotic non-muscle cells express multiple tropomyosin isoforms. The S2 cell line is the quintessential model system for the study of tropomyosins because S2 cells are easy to grow, are highly susceptible to gene inhibition using RNAi, have well-characterized actin networks, and most importantly, express only three tropomyosin isoforms in comparison to mammalian cells. Recent data from the Mullins lab discovered that, like mammalian cells, the Drosophila tropomyosin isoforms each localize to distinct actin structures in S2 cells. When these individual tropomyosins are mutated, the cell exhibits cell morphology and cell division abnormalities, suggesting that all three tropomyosin isoforms are required. The main objective of this project is to identify the functions of the three tropomyosin isoforms. In the first part of my proposal, I wil uncover the assembly mechanism of tropomyosin on actin filaments using a single filament Total Internal Reflection Fluorescence (TIRF) microscopy. Since the species the actin was purified from significantly influences tropomyosin activity, I will use only Drosophila-based components. In the second part of my proposal, I will identify the relationship between tropomyosins Tm1A and Tm1J and their relationship with myosin II and formin, respectively. I will use gliding assays to study myosin binding and activity and single filament TIRF microscopy to visualize the activity of formins and tropomyosins. This proposal will be the first to characterize every single tropomyosin informs for a given cell type, revealing the roles of tropomyosin isoforms in eukaryotic cells. In addition, understanding tropomyosins will also reveal how they assemble different actin structures.

描述（由申请人提供）：肌动蛋白亚基组装成许多不同的三维结构，这些结构是细胞分裂和运动等过程的核心，但细胞如何使用肌动蛋白作为基本构建块组装不同的细胞骨架结构仍不清楚。我推测，原肌球蛋白亚型决定哪些肌动蛋白结构组装调节哪些辅助因子可以获得肌动蛋白丝。真核细胞可以表达多种不同的原肌球蛋白同种型，其中每种同种型被分选为独特的肌动蛋白结构，这一论点得到了数据的支持。虽然哺乳动物原肌球蛋白的特点，哺乳动物的非肌肉细胞表达超过40种不同的原肌球蛋白异构体的事实，使原肌球蛋白的研究非常复杂。我们的实验室选择果蝇S2细胞作为模式生物，以了解为什么真核非肌肉细胞表达多种原肌球蛋白亚型。S2细胞系是研究原肌球蛋白的典型模型系统，因为S2细胞易于生长，对使用RNAi的基因抑制高度敏感，具有良好表征的肌动蛋白网络，最重要的是，与哺乳动物细胞相比，仅表达三种原肌球蛋白同种型。Mullins实验室的最新数据发现，与哺乳动物细胞一样，果蝇原肌球蛋白亚型各自定位于S2细胞中不同的肌动蛋白结构。当这些单独的原肌球蛋白发生突变时，细胞表现出细胞形态和细胞分裂异常，这表明需要所有三种原肌球蛋白亚型。本项目的主要目的是确定三种原肌球蛋白异构体的功能。在第一部分中，我将利用单丝全内反射荧光显微镜（TIRF）来揭示原肌球蛋白在肌动蛋白丝上的组装机制。由于纯化肌动蛋白的物种显著影响原肌球蛋白活性，因此我将仅使用基于果蝇的组分。在我的建议的第二部分中，我将确定原肌球蛋白Tm1A和Tm1J之间的关系，以及它们与肌球蛋白II和肌球蛋白II的关系，分别。我将使用滑行试验来研究肌球蛋白的结合和活性，并使用单丝TIRF显微镜来观察formins和tropomyosins的活性。这一建议将是第一个表征每一个单一的原肌球蛋白通知一个给定的细胞类型，揭示原肌球蛋白亚型在真核细胞中的作用。此外，了解原肌球蛋白也将揭示它们如何组装不同的肌动蛋白结构。