Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo
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.
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.Attached Files
Published - 29702.full.pdf
Submitted - 835322.full.pdf
Supplemental Material - pnas.1920323117.sapp.pdf
Supplemental Material - pnas.1920323117.sm01.mp4
Supplemental Material - pnas.1920323117.sm02.mp4
Supplemental Material - pnas.1920323117.sm03.mp4
Supplemental Material - pnas.1920323117.sm04.mp4
Supplemental Material - pnas.1920323117.sm05.mp4
Supplemental Material - pnas.1920323117.sm06.mp4
Supplemental Material - pnas.1920323117.sm07.mp4
Supplemental Material - pnas.1920323117.sm08.mp4
Supplemental Material - pnas.1920323117.sm09.mp4
Supplemental Material - pnas.1920323117.sm10.mp4
Supplemental Material - pnas.1920323117.sm11.mp4
Files
Name | Size | Download all |
---|---|---|
md5:f6ce73451667d9e1c78cd4864195ee77
|
16.3 MB | Preview Download |
md5:767ab32505595502ec32e2531bdbabf0
|
11.4 MB | Download |
md5:0d25d74c4459017b50f88ea4958ff327
|
32.9 MB | Download |
md5:0a801637eee4c88ee886f695c94d897e
|
44.4 MB | Download |
md5:bcbfa349eb9d904f25cfac5c31a310ea
|
127.0 MB | Download |
md5:79e1ba08f93252810d4451a920f83ea3
|
20.6 MB | Download |
md5:c71d36e6d5e974f2ced4c15a1443ef00
|
52.5 MB | Download |
md5:ac198c46a0eb603f425bcc961b647620
|
71.3 MB | Download |
md5:5ae4e233ed7b8eb20a2c9eab5b03c204
|
7.1 MB | Download |
md5:ffcec0a48be59cea8ecb173608b9e1f6
|
8.5 MB | Download |
md5:6c80d9af639e06c1e98116127ce960a5
|
3.0 MB | Preview Download |
md5:3db0cb9cc3a95d5da21c6b1e05648271
|
2.0 MB | Preview Download |
md5:40206fe3a54434021313fc6c6ffd2fbe
|
30.2 MB | Download |
md5:883109e83cfa45ae05afc9012e6044c6
|
70.5 MB | Download |
Additional details
- Alternative title
- Correlated cryogenic fluorescence microscopy and electron cryotomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo
- PMCID
- PMC7703684
- Eprint ID
- 99748
- Resolver ID
- CaltechAUTHORS:20191108-075710149
- AI150464
- NIH
- Created
-
2019-11-08Created from EPrint's datestamp field
- Updated
-
2023-06-01Created from EPrint's last_modified field
- Caltech groups
- Division of Biology and Biological Engineering