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

摘要 真核细胞使用网格蛋白介导的内吞作用（CME）来内化营养素、受体和细胞因子。 回收它们的质膜。内吞作用的缺陷与多种疾病有关， 因为癌症、神经病变和代谢综合征，CME机制可以被劫持， 病原体感染细胞。将膜变形为约50 nm的内吞囊泡需要 60多种蛋白质的编排组装和拆卸。因为芝加哥商品交易所的机器 连续交换和CME是衍射限制，精确的分子机制， 部队的生产仍然难以实现。基于肌动蛋白聚合的模型已经被 但他们实际上可以产生的力量也是1-2个数量级 低在这个项目中，我们将根据我们在上一个资助期内的发现， 在CME现场产生力的新机制，并确定 调节膜张力，这是内吞作用的关键参数。在目标1中，我们 假设交联的高动态肌动蛋白丝可以产生大量的力 以持续的方式。我们将在体外表征fimalloy的生物物理特性， 数学建模揭示了通过交联 动态肌动蛋白丝，并测试模型的预测通过单分子跟踪在 CME网站。Aim 2将专注于发现快速的分子机制， 我们在上一个资助期间发现的内吞蛋白的交换。我们将测试不同的 假设使用数学建模，测量fimmunoglobulin的分离的依赖性 速率作为力的函数，并确定内吞蛋白的交换是否取决于 处于内吞作用阶段。在目标3中，我们将描述 质膜张力及其对CME的影响。我们将使用光学镊子来确定 膜张力是否是局部和暂时调节的，并使用数学建模 为了了解膜张力的局部降低如何调节 内吞作用总而言之，我们的研究结果将揭示动态力生产的新范式 以及影响多种细胞过程的膜张力控制。

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.