Developmental Cell
Volume 56, Issue 22, 22 November 2021, Pages 3146-3159.e5
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Article
De novo endocytic clathrin coats develop curvature at early stages of their formation

https://doi.org/10.1016/j.devcel.2021.10.019Get rights and content
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Highlights

  • Endocytic clathrin coats develop curvature starting from early stages of their formation

  • Formation of clathrin pits does not depend on a late-stage flat-to-curved transition

  • Membrane tension does not alter the mechanism of curvature generation by clathrin pits

  • CALM adaptors form distinct clusters beneath the clathrin plaques

Summary

Sculpting a flat patch of membrane into an endocytic vesicle requires curvature generation on the cell surface, which is the primary function of the endocytosis machinery. Using super-resolved live cell fluorescence imaging, we demonstrate that curvature generation by individual clathrin-coated pits can be detected in real time within cultured cells and tissues of developing organisms. Our analyses demonstrate that the footprint of clathrin coats increases monotonically during the formation of pits at different levels of plasma membrane tension. These findings are only compatible with models that predict curvature generation at the early stages of endocytic clathrin pit formation. We also found that CALM adaptors associated with clathrin plaques form clusters, whereas AP2 distribution is more homogenous. Considering the curvature sensing and driving roles of CALM, we propose that CALM clusters may increase the strain on clathrin lattices locally, eventually giving rise to rupture and subsequent pit completion at the edges of plaques.

Keywords

clathrin-mediated endocytosis
fluorescence live cell microscopy
super-resolution imaging
membrane tension

Data and code availability

  • Microscopy data reported in this paper will be shared by the lead contact upon request.

  • Custom MATLAB codes developed for manual and automated analysis of clathrin coat traces will be available from the lead contact upon request.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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