Imaging and Modeling the Dynamics of Clathrin-Mediated Endocytosis

Abstract

Clathrin-mediated endocytosis (CME) plays a central role in cellular homeostasis and is mediated by clathrin-coated pits (CCPs). Live-cell imaging has revealed a remarkable heterogeneity in CCPassembly kinetics, which can be used as an intrinsic source of mechanistic information on CCP regulation but also poses several major problems for unbiased analysis of CME dynamics. The backbone of unveiling the molecular control of CME is an imagingbased inventory of the full diversity of individual CCP behaviors, which requires detection and tracking of structural fiduciaries and regulatory proteins with an accuracy of .99.9%, despite very low signals. This level of confidence can only be achieved by combining appropriate imaging modalities with self-diagnostic computational algorithms for image analysis and data mining.

Selective regulation of clathrin-mediated epidermal growth factor receptor signaling and endocytosis by phospholipase C and calcium

“…The set of CLSs includes CCPs as well as other clathrin structures such as subthreshold CLSs (sCLSs), of which the latter do not give rise to bona fide CCPs (Aguet et al. , 2013; Mettlen and Danuser, 2014; Kadlecova et al. , 2017).…”

“…Furthermore, this method allows further resolution of each CLS as either a bona fide CCP or an sCLS, as previously described (Aguet et al. , 2013; Mettlen and Danuser, 2014; Kadlecova et al. , 2017).…”

Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis

“…Overexpression of fluorescent fusion proteins changes the stoichiometry of CME proteins, and can perturb normal CME dynamics and function (Doyon et al ., 2011; Mettlen and Danuser, 2014). Additionally, changes in CME adaptor protein levels affect CME productivity (Loerke et al ., 2009; Mettlen et al ., 2009).…”

“…Our use of genome-edited cells provides a possible explanation for this discrepancy. Overexpression of a fluorescent fusion protein on top of expression of the endogenous protein can render endocytosis less efficient (Doyon et al ., 2011; Mettlen and Danuser, 2014). This discrepancy could also be explained if our approach combining two-color imaging and machine learning is more effective at identification of authentic CME sites than approaches taken previously (Aguet et al ., 2013).…”

Endocytosis: Past, Present, and Future

“…We begin with several articles that describe our current understanding of the molecular machinery and mechanisms driving entry of cargo (e.g., receptors and bound ligands) into the cell, by clathrin-dependent and -independent endocytosis (Traub and Bonifacino 2013;Kirchhausen et al 2014;Mayor et al 2014;Merrifield and Kaksonen 2014;Mettlen and Danuser 2014). Following internalization, the endocytosed material is shuttled along an ultrastructurally complex and dynamic pathway of tubular and vesicular compartments collectively known as endosomes, and described in the article by Klumperman and Raposo (2014), from which they can be sorted for recycling back to the cell surface or delivered to lysosomes for degradation.…”