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

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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.