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

 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、受体和再循环质膜。内吞作用的缺陷与多种疾病如癌症、神经病和代谢综合征有关，并且CME机制可以被一些病原体劫持以感染细胞。在CME过程中，高度保守的内吞机制必须克服膜张力，从平坦的质膜形成直径约50 nm的囊泡。当膜张力高时，动态肌动蛋白细胞骨架是CME进行所必需的。尽管对大多数内吞蛋白进行了深入的研究，但仍不清楚1）肌动蛋白网络如何产生使质膜变形所需的力以及2）其动态行为如何适应不同的膜张力。在这个项目中，我们将解决这些问题在裂变酵母（S。pombe）使用结合实验和数学建模的创新策略。我们将把我们的工作重点放在理解肌动蛋白组装，马达蛋白肌球蛋白-I（Myo 1 p），肌动蛋白丝交联剂fim 1 p（Fim 1 p）和transgelin（Stg 1 p）在CME期间的作用。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期间力产生和力感测的不同分子机制，并生成实验可检验的预测。在目标2中，我们将使用定量显微镜，我们和其他人已经开发的方法来计算局部和全局的活细胞中的蛋白质分子的绝对拷贝数，以阐明肌球蛋白-I和丝交联剂在力的产生中的作用。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。我们将在目标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，反过来，该模型将不断更新，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在CME过程中的作用，并在纳米尺度上发现以前从未被表征过的新的力产生和机械传感机制。

 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.