Molecular mechanisms of force production and force sensing during clathrin-mediated endocytosis

网格蛋白介导的内吞作用过程中力产生和力传感的分子机制

• 批准号: 9913559
• 负责人: Julien Berro
• 金额: $ 31.85万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9913559
• 关键词: 3-Dimensional; Actins; Address; Affect; Behavior; Biochemical; Biological Assay; Biophysical Process; Caliber; Cell Wall; Cell membrane; Cells; Clathrin; Communication; Crosslinker; Cytoskeletal Proteins; Cytoskeleton; Data; Data Analyses; Defect; Disease; Electron Microscopy; Endocytic Vesicle; Endocytosis; Eukaryotic Cell; Filament; Fimbrin; Fission Yeast; Fluorescence Microscopy; Generations; Goals; In Vitro; Individual; Light; Malignant Neoplasms; Mammalian Cell; Measures; Mechanics; Mediating; Membrane; Metabolic syndrome; Methods; Microfilaments; Microfluidic Microchips; Microscopy; Modeling; Molecular; Monitor; Motor; Motor Activity; Mutation; Myosin ATPase; Myosin Type I; Neuropathy; Nutrient; Output; Pathway interactions; Physiological Processes; Physiology; Process; Production; Proteins; Quantitative Microscopy; Role; SM 22 muscle protein; Shapes; Signal Transduction; Surface; Techniques; Testing; Time; Update; Vesicle; Virus; Work; Yeasts; biophysical properties; coated pit; crosslink; design; experience; experimental study; improved; in silico; in vivo; innovation; mathematical model; mechanotransduction; motor impairment; nanoscale; novel therapeutics; pathogen; polymerization; prevent; public health relevance; receptor; remote location; simulation

 DESCRIPTION (provided by applicant): Eukaryotic cells ubiquitously use clathrin-mediated endocytosis (CME) to internalize nutrients, receptors and recycle plasma membrane. Defects in endocytosis are implicated in multiple diseases such as cancer, neuropathies, and metabolic syndromes and the CME machinery can be hijacked by some pathogens to infect cells. During CME, the well-conserved endocytic machinery has to overcome membrane tension to shape a ~50-nm diameter vesicle from the flat plasma membrane. When membrane tension is high, a dynamic actin cytoskeleton is necessary for CME to proceed. Despite intensive studies on most of the endocytic proteins, it remains unknown 1) how the actin network produces the forces necessary to deform the plasma membrane and 2) how its dynamic behavior adapts to different membrane tension. In this project, we will address these questions in fission yeast (S. pombe) using an innovative strategy combining experiments and mathematical modeling. We will focus our work on understanding the roles of actin assembly, the motor protein myosin-I (Myo1p), and actin filament crosslinkers fimbrin (Fim1p) and transgelin (Stg1p) during CME. In Aim 1, we will develop a 3D mathematical model taking into account individual filaments to quantitatively evaluate different molecular mechanisms for force generation and force sensing during CME, and to generate experimentally testable predictions. In Aim 2, we will use quantitative microscopy, a method we and others have developed to count locally and globally the absolute copy number of protein molecules in live cells, in order to elucidate the role of myosin-I and filament crosslinkers in force generation. In Aim 3, we will use quantitative microscopy to evaluate the molecular mechanisms by which the endocytic machinery adapts to varying membrane tension. The experimental results we will collect in aims 2 and 3 will be interpreted in light of our mathematical model developed in aim 1, which will, in turn, be continuously updated to take into account the new information and quantitative constraints we will acquire in our experiments. During this project, we will determine the roles of the actin meshwork and myosin-Is during CME and uncover new force production and mechano-sensing mechanisms at the nanometer scale that have never been characterized before.

 描述（由申请人提供）：真核细胞普遍使用网格蛋白介导的内吞作用（CME）来内化营养物质、受体并回收质膜。内吞作用的缺陷与癌症、神经病和代谢综合征等多种疾病有关，并且 CME 机制可能被某些病原体劫持来感染细胞。在 CME 过程中，保守的内吞机制必须克服膜张力，从平坦的质膜上形成直径约 50 nm 的囊泡。当膜张力较高时，动态肌动蛋白细胞骨架对于 CME 的进行是必需的。尽管对大多数内吞蛋白进行了深入研究，但仍然未知：1）肌动蛋白网络如何产生使质膜变形所需的力；2）其动态行为如何适应不同的膜张力。在这个项目中，我们将使用实验和数学建模相结合的创新策略来解决裂殖酵母（S. pombe）中的这些问题。我们的工作重点是了解肌动蛋白组装、运动蛋白肌球蛋白-I (Myo1p) 以及肌动蛋白丝交联剂纤维蛋白 (Fim1p) 和转凝胶蛋白 (Stg1p) 在 CME 过程中的作用。在目标 1 中，我们将开发一个考虑单个细丝的 3D 数学模型，以定量评估 CME 期间力产生和力传感的不同分子机制，并生成可通过实验测试的预测。在目标 2 中，我们将使用定量显微镜，这是我们和其他人开发的一种方法，用于计算活细胞中蛋白质分子的局部和全局绝对拷贝数，以阐明肌球蛋白-I 和细丝交联剂在力产生中的作用。在目标 3 中，我们将使用定量显微镜来评估内吞机制适应不同膜张力的分子机制。我们在目标 2 和 3 中收集的实验结果将根据我们在目标 1 中开发的数学模型进行解释，而该模型又将不断更新，以考虑我们将在实验中获得的新信息和定量约束。在这个项目中，我们将确定肌动蛋白网络和肌球蛋白-Is 在 CME 过程中的作用，并揭示纳米尺度上以前从未表征过的新的力产生和机械传感机制。