Clathrin-independent pathways do not contribute significantly to endocytic flux.

Vassilis Bitsikas,Benjamin J Nichols,Ivan R Corrêa

doi:10.7554/elife.03970

Abstract

Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and function of alternative pathways have been controversial. Here we apply universal chemical labelling of plasma membrane proteins to define all primary endocytic vesicles, and labelling of specific proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and stringent discrimination between surface-connected and intracellular membranes. We find that at least 95% of the earliest detectable endocytic vesicles arise from clathrin-coated pits. GPI-anchored proteins, candidate cargoes for alternate pathways, are also found to enter the cell predominantly via coated pits. Experiments employing a mutated clathrin adaptor reveal distinct mechanisms for sorting into coated pits, and thereby explain differential effects on the uptake of transferrin and GPI-anchored proteins. These data call for a revision of models for the activity and diversity of endocytic pathways in mammalian cells.

Highlights

Endocytosis has central roles in many cell biological processes (Le Roy and Wrana, 2005; Doherty and McMahon, 2009)
Dissection of clathrin-mediated endocytosis benefited greatly from signature cargoes such as the transferrin receptor, which are efficiently concentrated in the forming endocytic vesicle, and from the fact that the forming vesicle is marked in space and time by transient assemblies of clathrin, adaptors and associated proteins (Pearse, 1982; Dautry-Varsat et al, 1983; Doxsey et al, 1987; Ehrlich et al, 2004)
We examined co-localisation with flotillin 1 (Glebov et al, 2006; Stuermer, 2011), caveolin 1 (Rothberg et al, 1992; Schneider et al, 2008; Parton and Howes, 2010), GRAF1-GFP (Lundmark et al, 2008), and ARF6-GFP (Naslavsky et al, 2003, 2004) after 90 s and 15 min of endocytosis at 37°C (Figure 7, Figure 7—figure supplement 1)

Summary

Introduction

Endocytosis has central roles in many cell biological processes (Le Roy and Wrana, 2005; Doherty and McMahon, 2009). One central difficulty in defining clathrin-independent endocytic pathways has been the paucity of rigorously validated endocytic markers and pathway-specific cargoes. Dissection of clathrin-mediated endocytosis benefited greatly from signature cargoes such as the transferrin receptor, which are efficiently concentrated in the forming endocytic vesicle, and from the fact that the forming vesicle is marked in space and time by transient assemblies of clathrin, adaptors and associated proteins (Pearse, 1982; Dautry-Varsat et al, 1983; Doxsey et al, 1987; Ehrlich et al, 2004). Clathrin independent endocytosis has largely been defined by morphological criteria, and by the persistent uptake of cargoes that may utilise multiple pathways following perturbation of the clathrin machinery (Saslowsky et al, 2010; Engel et al, 2011; Cho et al, 2012)

Methods

Results

Conclusion