Understanding the role of myosin in endocytosis

了解肌球蛋白在内吞作用中的作用

基本信息

批准号：
8509523
负责人：
Eric Brecher Lewellyn
金额：
$ 5.22万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8509523
关键词：
Actins Affect Area Automobile Driving Binding Biological Assay Cell Surface Receptors Cell membrane Cell physiology Cells Cellular biology Clathrin Complex Dictyostelium Disease Endocytic Vesicle Endocytosis Eukaryotic Cell F-Actin Fluorescence Microscopy Generations Health Human In Vitro Infection Length Life Lipid Binding Malignant Neoplasms Measures Mediating Membrane Membrane Lipids Microfilaments Modeling Molecular Motor Movement Mutation Myosin ATPase Myosin Type I Pathway interactions Phenotype Phosphatidylinositol 4,5-Diphosphate Phospholipids Positioning Attribute Process Proteins Recruitment Activity Recycling Research Research Design Role Saccharomyces cerevisiae Saccharomycetales Series Site Stroke Tail Techniques Testing Time Variant Viral Virus Yeasts arm cancer type experience fluorescence imaging fungus improved insight macromolecule member mutant particle pathogen polymerization prevent protein protein interaction

项目摘要

Clathrin-mediated endocytosis is a conserved pathway in eukaryotic cells for internalizing external macromolecules, membrane lipids, and cell surface receptors. Perturbation of this process is associated with multiple types of cancer and endocytic internalization is used by multiple viruses during infection. Therefore, understanding the underlying molecular mechanisms will improve our understanding of multiple disease processes besides providing fundamental insights into an essential part of cell biology. The yeast Saccharomyces cerevisiae is an ideal model for endocytosis because it is easily manipulated experimentally. Actin polymerization is critically required for endocytic internalization. Interestingly, the class I myosins Myo3p and Myo5p (Myo3/5) are essential for this inward movement in yeast and metazoan cells. Deletion of these proteins or mutation of the motor domain prevents internalization of either actin or endocytic vesicles. This is surprising because actin polymerization in the absence of myosin is sufficient to propel intracellular movement of pathogens like lysteria and propel membrane projections. Therefore, we propose to determine how Myo3/5 contributes to endocytosis. Specific Aims: The motor domain and the tail homology domain 1 (TH1) of Myo3/5 contribute to endocytosis by unknown mechanisms. Therefore, we propose the following specific aims: Aim I. To understand how the Myo3/5 motor domain contributes to endocytosis; Aim II. To determine the roles of the TH1 domain of Myo3/5. Study Design: Aim 1. We hypothesize that the motor domain both enhances the force generated by actin polymerization by transiently binding actin filaments and directly pushes on the actin network to drive internalization. To test this hypothesis, we propose to generate a series of motor domain mutations that will alter the translocation of actin using strategies that have been validated in multiple other myosins. These mutations will cause the translocation distance to increase, decrease, or become reversed while preserving the ability of the motor to transiently bind actin and hydrolyze ATP. This strategy will allow us to independently evaluate whether Myo3/5 contribute to internalization by transiently binding actin filaments, directly pushing on actin filaments, or both mechanisms. Discriminating between these two models has not been possible previously because previous motor mutations disrupted both the ability to translocate and the ability to transiently bind actin. Aim II. We hypothesize that the TH1 domain contributes to localization of Myo3/5 by binding to acidic phospholipids in the inner leaflet of the plasma membrane. To test this hypothesis, we will also evaluate whether Myo3/5 bind to acidic phospholipids in vitro through the TH1 domain. We will also determine how TH1 domain mutations affect the endocytosis phenotype in live cells. Whether the TH1 domain functions by binding acidic phospholipids or by an unanticipated mechanism, our assays will allow us to fully characterize the phenotype and generate new models if appropriate.

网格蛋白介导的内吞作用是真核细胞中的一种保守途径，用于内化外部大分子，膜脂质和细胞表面受体。该过程的扰动与多种类型的癌症有关，并且在感染过程中使用多种病毒使用内吞内在化。因此，了解潜在的分子机制将提高我们对多种疾病过程的理解，除了为细胞生物学的重要组成部分提供基本见解。酿酒酵母的酵母糖含量是内吞作用的理想模型，因为它很容易通过实验进行操作。肌动蛋白聚合至关重要的内吞内在化是必需的。有趣的是，I类Myosins myo3p和Myo5p（Myo3/5）对于在酵母和后生细胞中的内向运动至关重要。这些蛋白质的缺失或运动结构域的突变阻止肌动蛋白或内吞囊泡的内在化。这是令人惊讶的，因为在没有肌球蛋白的情况下，肌动蛋白聚合足以推动病原体（如溶裂和推动膜突显）的细胞内运动。因此，我们建议确定肌3/5如何促进内吞作用。具体目的：MyO3/5的运动域和尾部同源域1（Th1）通过未知机制导致内吞作用。因此，我们提出以下特定目的：目标I.了解MyO3/5运动域如何对内吞作用有效；目标II。确定Myo3/5的Th1域的作用。研究设计：目标1。我们假设运动域既可以通过瞬时结合肌动蛋白丝来增强肌动蛋白聚合产生的力，又可以直接推向肌动蛋白网络以驱动内在化。为了检验这一假设，我们建议生成一系列运动域突变，这些突变将使用已在多种其他肌动物中验证的策略来改变肌动蛋白的易位。这些突变将导致易位距离增加，减小或逆转，同时保持电动机瞬时结合肌动蛋白和水解ATP的能力。该策略将使我们能够独立评估MyO3/5是否通过瞬时结合的肌动蛋白丝，直接推动肌动蛋白丝或两种机制来促进内在化。以前不可能区分这两个模型，因为先前的运动突变破坏了转运能力和瞬时结合肌动蛋白的能力。目标II。我们假设TH1结构域通过与质膜内部小叶中的酸性磷脂结合来促进Myo3/5的定位。为了检验这一假设，我们还将评估MyO3/5是否通过TH1结构域与酸性磷脂结合。我们还将确定TH1结构域突变如何影响活细胞中的内吞作用。无论是通过结合酸性磷脂的结合还是意外机制来起作用，我们的测定方法都可以使我们能够充分表征表型并在适当的情况下生成新模型。