Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis

  1. Michael M Lacy
  2. David Baddeley
  3. Julien Berro  Is a corresponding author
  1. Yale University, United States

Abstract

Actin dynamics generate forces to deform the membrane and overcome the cell's high turgor pressure during clathrin-mediated endocytosis (CME) in yeast, but precise molecular details are still unresolved. Our previous models predicted that actin filaments of the endocytic meshwork continually polymerize and disassemble, turning over multiple times during an endocytic event, similar to other actin systems. We applied single-molecule speckle tracking in live fission yeast to directly measure molecular turnover within CME sites for the first time. In contrast with the overall ~20-sec lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 sec, and similarly high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins' motions. These results indicate that endocytic proteins turn over up to five times during the formation of an endocytic vesicle, and suggest revising quantitative models of force production.

Data availability

All data generated or analysed during this study are included in the manuscript and the supporting file "SuppFile4sets.mat" (Matlab data file).

Article and author information

Author details

  1. Michael M Lacy

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0498-2817
  2. David Baddeley

    Nanobiology Institute, Yale University, West Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Julien Berro

    Department of Molecular Biophysics and Biochemistry, Department of Cell Biology, Yale University, New Haven, United States
    For correspondence
    julien.berro@yale.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9560-8646

Funding

National Institute of General Medical Sciences (R01GM115636)

  • Michael M Lacy
  • Julien Berro

National Institute of General Medical Sciences (T32GM008283)

  • Michael M Lacy

Yale Program in Physics Engineering and Biology

  • Michael M Lacy
  • David Baddeley
  • Julien Berro

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Pekka Lappalainen, University of Helsinki, Finland

Publication history

  1. Received: October 3, 2019
  2. Accepted: December 18, 2019
  3. Accepted Manuscript published: December 19, 2019 (version 1)
  4. Version of Record published: January 23, 2020 (version 2)

Copyright

© 2019, Lacy 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

  • 2,617
    Page views
  • 420
    Downloads
  • 8
    Citations

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

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. Michael M Lacy
  2. David Baddeley
  3. Julien Berro
(2019)
Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis
eLife 8:e52355.
https://doi.org/10.7554/eLife.52355

Further reading

    1. Cell Biology
    Robert J Tower et al.
    Research Article

    De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema. Using this technique, we generated a gene signature with high specificity for the blastema in both our spatial data, as well as other previously published single-cell RNA-sequencing transcriptomic studies. To elucidate potential mechanisms distinguishing regenerative from non-regenerative healing, we applied spatial transcriptomics to an aging model. Consistent with other forms of repair, our digit amputation mouse model showed a significant impairment in regeneration in aged mice. Contrasting young and aged mice, spatial analysis revealed a metabolic shift in aged blastema associated with an increased bioenergetic requirement. This enhanced metabolic turnover was associated with increased hypoxia and angiogenic signaling, leading to excessive vascularization and altered regenerated bone architecture in aged mice. Administration of the metabolite oxaloacetate decreased the oxygen consumption rate of the aged blastema and increased WNT signaling, leading to enhanced in vivo bone regeneration. Thus, targeting cell metabolism may be a promising strategy to mitigate aging-induced declines in tissue regeneration.

    1. Cell Biology
    Wenyang Li et al.
    Research Article

    Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.