Abstract

Clathrin-mediated endocytosis is an essential process that forms vesicles from the plasma membrane. Although most of the protein components of the endocytic protein machinery have been thoroughly characterized, their organization at the endocytic site is poorly understood. We developed a fluorescence microscopy method to track the average positions of yeast endocytic proteins in relation to each other with a time precision below 1 s and with a spatial precision of ~10 nm. With these data, integrated with shapes of endocytic membrane intermediates and with superresolution imaging, we could visualize the dynamic architecture of the endocytic machinery. We showed how different coat proteins are distributed within the coat structure and how the assembly dynamics of N-BAR proteins relate to membrane shape changes. Moreover, we found that the region of actin polymerization is located at the base of the endocytic invagination, with the growing ends of filaments pointing toward the plasma membrane.

Article and author information

Author details

  1. Andrea Picco

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Markus Mund

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Jonas Ries

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. François Nédélec

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Marko Kaksonen

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    For correspondence
    kaksonen@embl.de
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University, United States

Version history

  1. Received: August 28, 2014
  2. Accepted: February 10, 2015
  3. Accepted Manuscript published: February 12, 2015 (version 1)
  4. Version of Record published: March 12, 2015 (version 2)

Copyright

© 2015, Picco 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

  • 6,535
    Page views
  • 1,207
    Downloads
  • 86
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

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. Andrea Picco
  2. Markus Mund
  3. Jonas Ries
  4. François Nédélec
  5. Marko Kaksonen
(2015)
Visualizing the functional architecture of the endocytic machinery
eLife 4:e04535.
https://doi.org/10.7554/eLife.04535

Share this article

https://doi.org/10.7554/eLife.04535

Further reading

    1. Structural Biology and Molecular Biophysics
    Nikaela W Bryan, Aamir Ali ... Ben E Black
    Research Article Updated

    The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as ‘fuzzy’. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation – a heterotrimer of INCENP, Survivin, and Borealin – we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Karolina Honzejkova, Dalibor Kosek ... Tomas Obsil
    Research Article

    Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.