权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：
9126867
负责人：
Johnny Rodriguez
金额：
$ 3.39万
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/9126867
关键词：
ATP phosphohydrolase Actin-Binding Protein Actins Address Adopted Affect Animal Model Binding Biological Assay Biological Models California Cell Line Cell Shape Cell division Cell membrane Cell physiology Cells Cellular Morphology Collaborations Complex Data Drosophila genus Drosophila melanogaster Electron Microscopy Elongation Factor Eukaryotic Cell Exhibits Filament Fluorescence Microscopy Genes Individual Lead Literature Maintenance 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 Sorting - Cell Movement Structure Syndrome Tropomyosin Universities Yeasts base cancer therapy cell assembly cell motility cell type daughter cell in vitro Assay insight new therapeutic target non-muscle myosin polymerization public health relevance research study 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(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件组装不同的细胞骨架结构仍不清楚。我假设原肌球蛋白的异构体通过调节哪些辅助因子可以获得肌动蛋白细丝来决定哪些肌动蛋白结构被组装。数据支持这一论点，即真核细胞可以表达多种不同的原肌球蛋白亚型，其中每种亚型都被归类为独特的肌动蛋白结构。虽然哺乳动物的原肌球蛋白是特征性的，但哺乳动物非肌肉细胞表达40多种不同的原肌球蛋白亚型的事实使原肌球蛋白的研究变得非常复杂。本实验室选择果蝇黑腹果蝇S2细胞作为研究真核非肌肉细胞表达多种原肌球蛋白亚型的模式生物。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RNAi的基因抑制高度敏感，具有良好的肌动蛋白网络，最重要的是，与哺乳动物细胞相比，S2细胞只表达三种原肌球蛋白亚型。来自穆林斯实验室的最新数据发现，像哺乳动物细胞一样，果蝇原肌球蛋白的每个亚型都定位于S2细胞中不同的肌动蛋白结构。当这些单独的原肌球蛋白突变时，细胞表现出细胞形态和细胞分裂异常，这表明所有三种原肌球蛋白亚型都是必需的。这个项目的主要目标是确定三种原肌球蛋白亚型的功能。在我的建议的第一部分，我将用单丝全内反射荧光显微镜揭示原肌球蛋白在肌动蛋白细丝上的组装机制。由于肌动蛋白是从对原肌球蛋白活性有显著影响的物种中提纯出来的，我将只使用以果蝇为基础的成分。在我的提案的第二部分，我将确定原肌球蛋白Tm1a和Tm1J之间的关系，以及它们分别与肌球蛋白II和福尔明的关系。我将使用滑动试验研究肌球蛋白的结合和活性，并使用单丝TIRF显微镜观察福尔马林和原肌球蛋白的活性。这项提案将首次对特定细胞类型的每一种原肌球蛋白进行表征，揭示原肌球蛋白异构体在真核细胞中的作用。此外，了解原肌球蛋白还将揭示它们如何组装不同的肌动蛋白结构。