DASC, a sensitive classifier for measuring discrete early stages in clathrin-mediated endocytosis

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.

Integrating protein copy numbers with interaction networks to quantify stoichiometry in mammalian endocytosis

“…This reduction of the alternate cargo on the membrane would indicate increased internalization with DAB2 removed 75 . We note that this compensation in which cargo is taken up is not found with all experimental knockdowns of adaptor proteins 88 , because the adaptors also can contribute to efficient clathrin coat assembly. Removal of PICALM, for example, reduced transferrin receptor uptake 88 .…”

“…We note that this compensation in which cargo is taken up is not found with all experimental knockdowns of adaptor proteins 88 , because the adaptors also can contribute to efficient clathrin coat assembly. Removal of PICALM, for example, reduced transferrin receptor uptake 88 . DAB2 perhaps operates more independently of AP-2 to coordinate uptake of its own cargo, as it does not need AP-2 binding to internalize LDL receptors 75 .…”

“…The exact extent to which complexes will change upon knockdown depend on binding strengths and dynamics, but our results predict that an experimental knockdown of DAB2, for example, would be significantly more impactful in affecting CME dynamics in HeLa cells than in fibroblast, as it is much more abundant and out-competes other adaptors in binding to AP-2. A recent experimental study demonstrated how single KD of multiple endocytic adaptor proteins in epithelial cells impacted clathrin coated structure formation, finding a surprising lack of correlation between the impact on early CME stages and the amount of bulk transferrin uptake 88 . Our analysis further suggests where specific changes might be expected to occur on a protein-by-protein basis, made possible by our analysis of every protein, lipid, and cargo response to perturbations to the network.…”

Evolving models for assembling and shaping clathrin-coated pits

“…However, early live-cell studies revealed that a significant portion (30–50%) of nascent CCPs fail to mature and spontaneously abort ( Ehrlich et al, 2004 ; Loerke et al, 2009 ). Several factors appear to be required to stabilize nascent CCPs, including conformational changes in AP2 ( Kadlecova et al, 2017 ) required to enhance PI(4,5)P 2 and cargo binding ( Edeling et al, 2006 ; Jackson et al, 2010 ; Kelly et al, 2014 ), cargo loading ( Ehrlich et al, 2004 ; Kadlecova et al, 2017 ; Loerke et al, 2009 ), and several “pioneer” EAPs, so named because they act during early stages of CME ( Cocucci et al, 2012 ; Ma et al, 2016 ; Ritter et al, 2013 ; Wang et al, 2020 ).…”

“…Among the pioneer EAPs required for early stabilization of nascent CCPs are the scaffold proteins Eps15 and Fcho1 ( Cocucci et al, 2012 ; Ma et al, 2016 ; Wang et al, 2020 ), both of which bind AP2 and each other through multiple low-affinity binding motifs and protein interaction domains. Interestingly, a recent study has revealed that Eps15 coassembles with substoichiometric amounts of Fcho1 to form phase-separated liquid-like protein droplets ( Day et al, 2019 Preprint ), a property shared by its yeast homologue, Ede1 ( Kozak and Kaksonen, 2019 Preprint ).…”