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-配体复合体和营养物质的一个基本机制是在分子筛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、细胞分化和细胞内环境的稳定。CME蛋白的突变会导致癌症、高胆固醇血症和神经退行性疾病。质膜张力(PMT)的增加同样会影响CME的效率。肌动蛋白皮质和质膜之间的相互作用造成了75%的张力。例如，在上皮细胞中，CME在顶膜上的寿命较慢，其特点是PMT高于基底膜，具有较低的PMT。药物抑制肌动蛋白细胞骨架聚合对顶端细胞膜的影响更为严重，提示肌动蛋白是CME中对抗PMT的主要作用力。然而，哺乳动物CME中肌动蛋白功能对PMT增加的反应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。我将在人类诱导的多能干细胞来源的成纤维细胞中测试这一假设，以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片上不同大小的粘合剂表面，以限制其扩散，导致细胞骨架排列的受控改变，从而导致PMT。我将使用活细胞荧光显微镜来测量CME组分和肌动蛋白的荧光寿命，以确定对PMT量化变化的反应。荧光标记的蛋白质是从宿主实验室通过基因组编辑建立的内源染色体位置表达的。其次，我的目标是鉴定CME机制中能够感知PMT变化的分子成分。通过新的CRISPR干涉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，将使用双色三维超分辨率显微镜对肌动蛋白和网状蛋白结构进行观察，以揭示与CME位点相关的肌动蛋白在该过程的特定时间步长中的组织。拟议的项目将揭示在哺乳动物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力。我的发现将促进对机械信号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人员，我想研究参与细胞分化和增殖的信号通路是如何在三维微环境中受到机械线索的影响的。

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.