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Investigation of Clathrin-Mediated Endocytosis Using Genome Editing in Somatic and Pluripotent Human Cells

  • Author(s): Cheng, Aaron T
  • Advisor(s): Drubin, David G
  • et al.
Abstract

Clathrin-mediated endocytosis (CME) is the best-studied and predominant pathway by which portions of the plasma membrane and extracellular material are internalized. Traditional methods using ectopically overexpressed endocytic fluorescent protein fusions indicated that mammalian CME is a dynamic but inefficient and heterogeneous process. Here, I present three studies that utilize genome editing to probe the dynamics of endocytic proteins expressed from their endogenous loci.

First, I describe a proof-of-principle study establishing the feasibility and utility of zinc finger nucleases (ZFNs) for integration of fluorescent markers at user-specified genomic sites for the study of protein dynamics. Together with use of live-cell fluorescence microscopy, we monitored two key components of the endocytic machinery, clathrin and dynamin. Genome-edited somatic cells exhibited enhanced endocytic function, dynamics, and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to protein levels. This work also establishes genome editing as a robust tool to faithfully report protein localization and dynamics.

Genome-edited cells display stable fluorescent marker integration and uniform protein fusion expression across a cell population. This allowed us to perform global analyses on live cells to probe the requirement for actin in CME. We monitored dynamin dynamics and found that, like yeast, endocytosis is highly regular at late stages despite heterogeneity in clathrin lifetime. Impairment of actin dynamics by small molecule inhibitors revealed that F-actin turnover is critical for the robust recruitment of dynamin. Additionally, dynamin recruitment is dependent on its own concentration. Together, these data more clearly define the mechanisms that underlie CME.

Previous work using somatic cell lines suggested that endocytic dynamics vary among different cell types. The ability to monitor protein fusions at endogenous levels afforded me the opportunity to establish a cell-based developmental system using human pluripotent stem cells (hPSCs) to directly probe if and how endocytic dynamics change upon cellular differentiation. I engineered hPSCs to express CLTA-RFP at endogenous levels and found that these cells displayed long-lived CLTA-RFP structures at the plasma membrane that co-localized with adhesions, implying that components of adhesion complexes and/or the actin cytoskeleton that it engages may influence clathrin dynamics. Strikingly, upon differentiation into fibroblast-like cells, CLTA-RFP behavior became highly dynamic. Collectively, these data show that endocytic dynamics can and do change upon cellular differentiation and suggest a role for endocytosis in defining cell function and fate.

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