Elucidating the molecular mechanism of actin function in mammalian clathrin-mediated endocytosis: plasma membrane tension as a potential regulator

细胞摄取受体-配体复合物和营养物质的一个重要机制涉及在网格蛋白介导的内吞作用（CME）过程中内陷质膜。CME调节重要的信号通路，以确保适当的细胞间通讯、细胞分化和细胞稳态。CME蛋白的突变可导致癌症、高胆固醇血症和神经退行性疾病。增加质膜张力（PMT）同样会干扰CME效率。下层肌动蛋白皮层和质膜之间的相互作用占张力的75%。例如，在上皮细胞中，顶膜的CME寿命较慢，其特征是PMT较高，而基底侧膜的PMT较低。药物治疗对肌动蛋白细胞骨架聚合的抑制作用对顶细胞膜的影响更为严重，这表明肌动蛋白是CME中抗PMT的主要动力来源。然而，肌动蛋白在哺乳动物CME中响应PMT增加的功能的分子基础尚不清楚。我假设某些蛋白质可以感知质膜张力，并以多步骤的方式参与肌动蛋白机制。我将在人类诱导多能干细胞衍生的成纤维细胞中测试这一假设，保证干净的遗传背景，以最高的灵敏度研究内吞过程。首先，我将把成纤维细胞暴露在玻璃盖上不同大小的粘附表面上，以限制其扩散，导致细胞骨架排列的可控改变，从而导致PMT。我将通过使用活细胞荧光显微镜测量CME成分和肌动蛋白的荧光寿命来确定对定量PMT变化的响应。荧光标记的蛋白质在宿主实验室通过基因组编辑建立的内源性染色体位点上表达。其次，我的目标是确定CME机制中可以感知PMT变化的分子成分。通过新的CRISPR干扰方法敲除识别特定膜曲率的蛋白质将测试它们可能的膜张力传感功能。第三，利用双色三维超分辨显微镜对肌动蛋白和网格蛋白的结构进行观察，揭示与CME位点相关的肌动蛋白在特定时间步长的组织结构。该项目将揭示哺乳动物CME过程中感知PMT变化的分子机制，并将阐明肌动蛋白如何施加力。我的发现将促进对机械线索如何转化为生化途径并影响细胞过程的基本理解。在未来，作为一名独立的研究者，我想研究三维微环境中机械信号如何影响细胞分化和增殖的信号通路。

An essential mechanism for the cell to take up receptor-ligand complexes and nutrients involves invaginating the plasma membrane in the process of clathrin-mediated endocytosis (CME). CME regulates important signaling pathways to ensure proper cell-cell communication, cell differentiation and cell homeostasis. Mutations in CME proteins can cause cancer, hypercholesterolemia and neurodegenerative diseases. Increasing plasma membrane tension (PMT) can likewise perturb CME efficiency. The interaction between the underlying actin cortex and the plasma membrane contributes to 75% to the tension. In epithelial cells for instance, CME lifetimes are slower on the apical membrane, which is characterized by higher PMT than the basolateral membrane, possessing lower PMT. Inhibition of actin cytoskeleton polymerization by drug treatment shows a more severe effect on the apical cell membrane, implicating actin as the major force generator against PMT in CME. However, the molecular basis for actin function in mammalian CME in response to increasing PMT is not clear. I hypothesize that certain proteins can sense the plasma membrane tension and engage the actin machinery in a multi-step manner. I will test this hypothesis in human induced pluripotent stem cell derived fibroblasts guaranteeing a clean genetic background to investigate the endocytic process with highest sensitivity.First I will expose the fibroblasts to adhesive surfaces with different sizes on glass coverslips to constrain its spreading, causing controlled alteration in cytoskeletal arrangement and thus PMT. I will determine the response to quantified PMT changes by using live cell fluorescence microscopy to measure fluorescence lifetimes of CME components and actin. The fluorescently tagged proteins are expressed from their endogenous chromosomal loci established by genome editing in the host lab.Second, I aim to identify the molecular components in the CME machinery that can sense PMT changes. Knocking down proteins that recognize specific membrane curvature by the novel CRISPR interference approach will test their possible membrane tension sensing function.Third, dual-color three-dimensional superresolution microscopy on the actin and clathrin structures will be used to reveal the organization of actin associated with CME sites during specific time steps of the process.The proposed project will reveal the molecular machinery that senses PMT changes during mammalian CME and will elucidate how actin exerts force. My findings will advance fundamental understanding of how mechanical cues are translated into biochemical pathways and impact cellular processes. In the future as an independent researcher I want to investigate how signaling pathways involved in cell differentiation and proliferation are influenced by mechanical cues in three-dimensional microenvironments.