Format

Send to

Choose Destination
Structure. 2018 Jun 5;26(6):879-886.e3. doi: 10.1016/j.str.2018.03.015. Epub 2018 Apr 19.

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.

Author information

1
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK; Central Oxford Structural and Molecular Imaging Centre, South Parks Road, Oxford OX1 3RE, UK; Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
2
Cell Biology Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
3
Cell Biology Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: kukulski@mrc-lmb.cam.ac.uk.

Abstract

Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation.

KEYWORDS:

BAR domains; CEMOVIS; correlative microscopy; cryo-CLEM; cryo-EM; eisosomes; electron cryo-tomography; vitreous sections

PMID:
29681471
PMCID:
PMC5992340
DOI:
10.1016/j.str.2018.03.015
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center