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Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Carter, Stephen D. and Mamede, João I. and Hope, Thomas J. and Jensen, Grant J. (2020) Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo. Proceedings of the National Academy of Sciences of the United States of America, 117 (47). pp. 29702-29711. ISSN 0027-8424. https://resolver.caltech.edu/CaltechAUTHORS:20191108-075710149

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Abstract

Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.1920323117DOIArticle
https://www.pnas.org/content/suppl/2020/11/05/1920323117.DCSupplementalPublisherSupporting Information
https://doi.org/10.1101/835322DOIDiscussion Paper
ORCID:
AuthorORCID
Carter, Stephen D.0000-0002-4237-4276
Mamede, João I.0000-0002-6048-1876
Hope, Thomas J.0000-0001-7183-8319
Jensen, Grant J.0000-0003-1556-4864
Alternate Title:Correlated cryogenic fluorescence microscopy and electron cryotomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo
Additional Information:© 2020 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). Edited by John M. Coffin, Tufts University, Boston, MA, and approved September 23, 2020 (received for review November 19, 2019). PNAS first published November 5, 2020. We thank Sarah Speed (Division of Biology and Biological Engineering, California Institute of Technology) for valuable help in segmenting cryo-tomograms and Catherine Oikonomou (Division of Biology and Biological Engineering, California Institute of Technology) for insights and comments on the manuscript. This work was supported in part by NIH Grant AI150464 (to G.J.J. and T.J.H.). Data Availability: All study data are included in the article and SI Appendix. Author contributions: S.D.C., J.I.M., T.J.H., and G.J.J. designed research; S.D.C. and J.I.M. performed research; S.D.C., J.I.M., T.J.H., and G.J.J. contributed new reagents/analytic tools; S.D.C., J.I.M., T.J.H., and G.J.J. analyzed data; and S.D.C. and G.J.J. wrote the paper. The authors declare no competing interest. This article is a PNAS Direct Submission. This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1920323117/-/DCSupplemental.
Funders:
Funding AgencyGrant Number
NIHAI150464
Subject Keywords:cryo-CLEM/ECT; TRIM5α-body; hexagonal nets; autophagy; endoplasmic reticulum
Issue or Number:47
Record Number:CaltechAUTHORS:20191108-075710149
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20191108-075710149
Official Citation:Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo. Stephen D. Carter, João I. Mamede, Thomas J. Hope, Grant J. Jensen. Proceedings of the National Academy of Sciences Nov 2020, 117 (47) 29702-29711; DOI: 10.1073/pnas.1920323117
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99748
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:08 Nov 2019 22:11
Last Modified:25 Nov 2020 19:11

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