Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells

Abstract

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.

Dual Role of BAR Domain-containing Proteins in Regulating Vesicle Release Catalyzed by the GTPase, Dynamin-2

“…However, given the commonality of protein interaction motifs involved and because of the necessity of overexpressing fluorescently tagged proteins, it is likely that higher concentrations of one partner might displace others, thereby disrupting and/or blurring the normal sequence of events. Recent developments in genome-editing and tagging endocytic proteins at their endogenous loci in mammalian cells (37), when coupled to more sensitive methods for detection and tracking (4), should provide clearer insights into these hierarchical relationships.…”

Cell-cycle regulation of formin-mediated actin cable assembly

“…The interactive 3D surface plot plugin of Image J was used for actin cable pattern demonstration by measuring the surface fluorescent signal intensity. The lifetimes of actin patches were measured by Imaris software (Bitplane Scientific) as previously described (116).…”

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…Unlike yeast cells, until recently it was not possible to replace endogenous genes in mammalian cells with their FP tagged cognates (but see Doyon et al 2011). Instead low overexpression levels of endocytic accessory proteins were used, although this naturally raised concerns over potential artifacts caused by protein overexpression (Doyon et al 2011; although see Aguet et al 2013).…”

“…Instead low overexpression levels of endocytic accessory proteins were used, although this naturally raised concerns over potential artifacts caused by protein overexpression (Doyon et al 2011; although see Aguet et al 2013). Nonetheless, the recruitment of FP tagged endocytic accessory proteins was measured relative to single productive scission events in one cell type (mouse fibroblasts) with a temporal resolution of 2 sec (Merrifield et al 2005;Taylor et al 2011Taylor et al , 2012.…”