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

Tanmay A.M Bharat,Patrick C Hoffmann,Wanda Kukulski

doi:10.1016/j.str.2018.03.015

Tanmay A.M Bharat, Patrick C Hoffmann + Show 1 more

Open Access

https://doi.org/10.1016/j.str.2018.03.015

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Abstract

SummaryElectron 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.

Highlights

Biological macromolecules fulfill their tasks in crowded cellular environments
While large protein complexes, such as ribosomes or proteasomes, can be readily localized within the tomographic cell volume using template-matching algorithms (Asano et al, 2015; Pfeffer et al, 2015), smaller protein assemblies that lack such large characteristic shapes remain elusive. We have addressed both these limitations by implementing a high-precision correlative microscopy workflow that combines cryo-fluorescence microscopy with cryo-ET of vitreous sections
We found our correlative microscopy workflow ideal to study how membrane curvature generation and BAR protein assembly relate to each other in cells

Summary

Introduction

The organization of macromolecular functions in the cell often necessitates formation of transient complexes, such as proteins binding to membranes or to the cytoskeleton, or the self-association of proteins into oligomers Such interactions can generate short-lived or fragile higher-order assemblies, whose formation can depend on the local cellular environment, for example the composition of the organelle membrane at which they occur. Due to limitations imposed by the electron beam, unless thin areas of cells (

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