Abstract
Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, "disassembly asymmetry score classification (DASC)", that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.
Highlights
Clathrin-mediated endocytosis (CME) is the major pathway for cellular uptake of macro-molecular cargo [1]
Overexpression of eGFP-CLCa ensures near stoichiometric incorporation of fluorescently-labeled CLC into clathrin triskelia by displacing both endogenous CLCa and CLCb
For all conditions, ≥19 independent movies were collected and the eGFP intensities of >200,000 clathrin structures per condition were tracked over time using total internal reflection fluorescence microscopy (TIRFM) and established automated image analysis pipelines [10, 23]
Summary
Clathrin-mediated endocytosis (CME) is the major pathway for cellular uptake of macro-molecular cargo [1]. It is accomplished by concentrating cell surface receptors into specialized 100-200 nm wide patches at the plasma membrane created by a scaffold of assembled clathrin triskelia [2]. Numerous endocytic accessory proteins (EAPs), which modulate various aspects of CCP assembly and maturation, contribute to the formation of clathrin-coated vesicles (CCVs) that transport cargo to the cell interior. Perturbed EAP functions can be physiologically consequential, e.g. CALM is identified as associated to Alzheimer’s disease [11] and SNX9 is correlated to cancer and other human diseases [12]. We question whether measuring internalization by biochemical assays is sufficient for determining the actual phenotypes of missing EAP functions, and thereby further supporting clinical studies of the EAPs in more complex models
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