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

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.

Graphical abstract

Figure 1

Cryo-FM Allows Selection of Vitreous Section Areas that Are Suitable for Cryo-ET and Contain Fluorescent Signals of Interest (A) Grid overview in the green channel shows the distribution of vitreous sections appended to each other as ribbons. (B and C) Two planes from the same focal stack, separated by about 3.6 μm in z (direction of the light path), merge of red (shown in magenta) and green channels. Area of acquisition corresponds to the yellow dashed box in (A). The white box in (B) indicates a section area where the holey carbon film and the GFP signals in the section are in focus in the same plane. The magenta box indicates a section area where the carbon film is in focus in the plane shown in (B), whereas the GFP signals are in focus in a different plane shown in (C). (D) Intermediate-magnification EM image of the area indicated by the white dashed square in (B), magnified in (E). (E) The white circle marks a Pil1-GFP signal suitable for cryo-ET acquisition. The orange circles in (D and E) mark examples of fiducial markers. (F) Intermediate-magnification EM image of the area indicated by the magenta dashed square in (B and C), magnified in (G). (G) The white circle indicates a Pil1-GFP spot not suitable for cryo-ET acquisition. Scale bars represent 100 μm in (A), 10 μm in (B and C), 500 nm in (D and F).

Figure 2

Cryo-ET of Preselected Areas and Fiducial-Based Correlation to Identify Eisosomes by Pil1-GFP Localization (A) A single focal plane in cryo-FM, merge of green and red (shown in magenta) channels. White circle indicates a Pil1-GFP signal selected for cryo-ET. (B) Intermediate-magnification EM image. White dashed rectangle indicates the field of view imaged by cryo-ET (C). Yellow circles in (A and B) indicate fiducial markers for high-precision localization of the Pil1-GFP signal (center of green dashed circle). (C) Virtual slice through the tomographic volume. (D) Magnified view of the dashed rectangle shown in (C). Arrows in (C and D) indicate a furrow-like invagination. Scale bars represent 2 μm in (A and B), 100 nm in (C), and 25 nm in (D). See also Figure S2.

Figure 3

Sub-tomogram Averaging Analysis Reveals that Eisosomes with High Membrane Curvature Display a Dense Coat (A) Cryo-FM (green channel) of section areas over carbon film holes containing Pil1-GFP signals of interest (white circles). Panel V corresponds to Figure 1E. (B) Virtual slices through electron cryo-tomograms acquired at the corresponding positions. (C) 2D class averages of eisosome membranes marked by green dashed boxes in (B). In III–VI, the diameter (d) of the outer density layer is indicated by the red dashed circle segment. (D) 2D class averages of plasma membrane marked by yellow dashed boxes in (B). (E) Corresponding 3D eisosome shape modeled by segmentation of the plasma membrane. (F) Cartoon interpretation of coating and membrane curvature in presence of Pil1-GFP. Scale bars represent 500 nm in (A), 25 nm in (B and E), and 10 nm in (C and D).

Figure 4

Eisosomal Protein Coating Correlates with Membrane Curvature (A) Comparison of the relative membrane thickness of shallow (Figures 3I and 3II) and curved eisosomes (Figures 3III–3VI). ^∗p = 0.0211. The red lines indicate the mean and the standard deviation. (B) Model for the interdependence of BAR-domain scaffold formation and membrane curvature.