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

摘要 真核细胞使用网状蛋白介导的内吞作用(CME)来内化营养物质、受体和 回收他们的质膜。内吞作用缺陷与各种疾病有关，如 作为癌症、神经病和代谢综合征，以及CME机制可以被劫持 病原体感染细胞。将膜变形为~50 nm的内吞囊泡需要 60种蛋白质的编排组装和拆卸。因为芝加哥商品交易所的机制 连续交换和CME是衍射受限的，精确的分子机制 部队生产仍然难以捉摸。基于肌动蛋白聚合的模型已经被 但他们实际能产生的力量也是1-2个数量级 很低。在这个项目中，我们将在上一次资助期间的发现的基础上进行研究 CME现场的新的力量生产机制和确定的机制 膜张力的调节，这是内吞作用的关键参数。在目标1中，我们 假设交联高度动态的肌动蛋白细丝可以产生很大的力 以可持续的方式。我们将在体外表征Fimbrin的生物物理性质，使用 用数学模型揭示交联剂持力采油机理 动态肌动蛋白细丝，并通过单分子跟踪测试模型的预测 芝加哥商品交易所网站。目标2将专注于发现FAST背后的分子机制 我们在上一次资助期发现的内吞蛋白交换。我们将测试不同的 假设采用数学建模，测量纤毛膜脱离的依赖性 作为力的函数的速率，并确定内吞蛋白的交换是否取决于 在内吞阶段。在目标3中，我们将描述监管的机制 质膜张力及其对继续医学教育的影响。我们将使用光学镊子来确定 膜张力是否局部和时间上调节，并使用数学模型 要了解膜张力的局部减少如何调节所需的力 内吞作用。总而言之，我们的结果将揭示动力生产的新范式 以及膜张力控制，这将影响各种细胞过程。

ABSTRACT Eukaryotic cells use clathrin-mediated endocytosis (CME) to internalize nutrients, receptors and recycle their plasma membrane. Defects in endocytosis are implicated in various diseases such as cancer, neuropathies, and metabolic syndromes, and the CME machinery can be hijacked by pathogens to infect cells. Deforming the membrane into ~50-nm endocytic vesicles requires the choregraphed assembly and disassembly of 60+ proteins. Because the CME machinery continuously exchanges and CME is diffraction-limited, the precise molecular mechanisms for force production have remained elusive. Models based on actin polymerization have been proposed but the amount of force they can realistically produce is 1-2 orders of magnitude too low. In this project, we will build on the discoveries we made during last funding period to study new mechanisms of force production at the CME site and determine mechanisms for the regulation of membrane tension, which is a key parameter for endocytosis. In aim 1, we hypothesize that crosslinking highly dynamic actin filaments can produce large amounts of force in a sustained way. We will characterize the biophysical properties of fimbrin in vitro, use mathematical modeling to uncover mechanisms of sustained force production by crosslinking of dynamic actin filaments, and test the model’s predictions by single-molecule tracking at the CME site. Aim 2 will focus on discovering the molecular mechanisms underlying the fast exchange of endocytic proteins we discovered during last funding period. We will test different hypotheses using mathematical modeling, measure the dependence of fimbrin’s detachment rate as a function of force, and determine whether the exchange of endocytic protein depends on the stage of endocytosis. In aim 3, we will characterize mechanisms for the regulation of plasma membrane tension and its influence on CME. We will use optical tweezers to determine whether membrane tension is locally and temporally regulated and use mathematical modeling to understand how a local reduction in membrane tension modulates the forces required for endocytosis. Altogether, our results will uncover new paradigms for dynamic force production and membrane tension control that will impact a variety of cellular processes.