A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane
Abstract
Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the 'endosomal sorting complex required for transport' machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.
Additional Information
© 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). Submitted 5 May 2020; Accepted 9 February 2021; Published 26 March 2021. We thank A. A. Hyman and I. Poser for providing the HeLa cell line stably expressing CHMP4B-EGFP. We thank A. Collazo and S. Wilbert for technical assistance with confocal microscopy. We also thank S. Chen and A. Malyutin for technical assistance with cryo-EM. The bulk of the confocal imaging was performed at the Biological Imaging Facility, and EM was performed at the Beckman Institute Resource Center for Transmission Electron Microscopy, both at Caltech. This work was supported by funding from the NIH (P50 AI150464 awarded to G.J.J.). Author contributions: W.Y.Y. performed light microscopy experiments and prepared sample for cryo-ET with help from S.K.M. S.K.M. performed CLEM, cryo-ET, and data analyses. S.K.M. prepared the manuscript with help from W.Y.Y., C.M.O., and G.J.J. Y.C. performed the Western blot assays for Myo1a and Vps4B knockdowns. The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the first and corresponding authors.Attached Files
Published - eabc6345.full.pdf
Submitted - 2019.12.23.887638v1.full.pdf
Supplemental Material - abc6345_Movie_S1.mp4
Supplemental Material - abc6345_Movie_S2.mp4
Supplemental Material - abc6345_Movie_S3.mp4
Supplemental Material - abc6345_SM.pdf
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Additional details
- Alternative title
- Plasma membrane damage removal by F-actin-mediated shedding from repurposed filopodia
- PMCID
- PMC7997517
- Eprint ID
- 100447
- Resolver ID
- CaltechAUTHORS:20200102-095248499
- NIH
- P50 AI150464
- Created
-
2020-01-02Created from EPrint's datestamp field
- Updated
-
2023-07-20Created from EPrint's last_modified field
- Caltech groups
- Division of Biology and Biological Engineering (BBE)