Clathrin-independent pathways do not contribute significantly to endocytic flux

  1. Vassilis Bitsikas
  2. Ivan R Corrêa
  3. Benjamin J Nichols  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom
  2. New England Biolabs, Inc., United States

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.

Article and author information

Author details

  1. Vassilis Bitsikas

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  2. Ivan R Corrêa

    New England Biolabs, Inc., Ipswich, United States
    Competing interests
    Ivan R Corrêa, An employee of New England Biolabs Inc. New England Biolabs Inc. has a commercial interest in successful application of reagents used in this study.
  3. Benjamin J Nichols

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    For correspondence
    ben@mrc-lmb.cam.ac.uk
    Competing interests
    No competing interests declared.

Copyright

© 2014, Bitsikas et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 6,754
    views
  • 884
    downloads
  • 152
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Vassilis Bitsikas
  2. Ivan R Corrêa
  3. Benjamin J Nichols
(2014)
Clathrin-independent pathways do not contribute significantly to endocytic flux
eLife 3:e03970.
https://doi.org/10.7554/eLife.03970

Share this article

https://doi.org/10.7554/eLife.03970

Further reading

    1. Cell Biology
    2. Developmental Biology
    Sofía Suárez Freire, Sebastián Perez-Pandolfo ... Mariana Melani
    Research Article

    Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.