iMEM: Isolation of Plasma Membrane for Cryoelectron Microscopy Camille Françoise Peitsch, Sven Beckmann, Benoît Zuber  Structure  Volume 24, Issue 12, Pages 2198-2206 (December 2016) DOI: 10.1016/j.str.2016.09.016 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 2198-2206DOI: (10.1016/j.str.2016.09.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Schematic Patch Production (A) Cells (green/black) are grown on EM grids (brown dotted line). A drop of cytosol-like buffer is added to the cells (blue half circle). Blotting with a filter paper (salmon) can be done directly from the cell side (CS), from the EM grid side (GS), or from both sides (purple arrows). (B) The EM grid is approached and gently dropped on the filter paper. The absorption of the cytosol-like buffer is carried on until only a thin layer remains in order to keep the sample hydrated. (C) Membrane patches (green curved lines) are produced by blotting. A thin layer of buffer remains (blue line over the cell patches). Structure 2016 24, 2198-2206DOI: (10.1016/j.str.2016.09.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Isolated Plasma Membrane Patches with LDCVs from PC12 Cells (A) Overview of the unroofed PC12 cells blotted twice from the cell side with 12/N filter paper (white arrows show a few patches). Inset: magnified view showing three patches (black arrows). (B) Overview and magnified view (inset) of PC12 cell blotted once from the cell side, once from the grid side, and once from the cell side again with 12/N filter paper showing isolation of the cell cortex with entrapped LDCVs (white stars). See also Figure S1. (C) Cross-sectioned PC12 membrane patch (black arrow) with docked LDCVs (white stars) on the carbon film (white triangle) blotted twice from the cell side with filter paper 12/N. Scale bars, 100 μm (A), 500 nm (B), and 400 nm (C). Structure 2016 24, 2198-2206DOI: (10.1016/j.str.2016.09.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Time-Lapse Fluorescence Microscopy of Calcium-Dependent Fusion in PC12 Membrane Patches Isolated with Filter Paper 12/N (A) Sample carrier set-up. Membrane patches are facing the glow-discharged glass coverslip. (B) Normalized intensities of untreated samples, MgCl2-treated samples, and CaCl2-treated samples. Error bars represent 1.96× SE to the mean. From 2 min on, the CaCl2-treated group is significantly different to both control groups (p < 0.001). (C) Maximum intensity projection of an untreated control patch at the first and the last time points. Colored circles designate vesicles throughout the time series. (D) Maximum intensity projection of a CaCl2-treated sample at selected time points. Colored circles designate vesicles throughout the time series. The green circle shows a few vesicles that fused and the blue circle shows a vesicle that did not respond to calcium stimulation. Scale bars, 2 μm. Structure 2016 24, 2198-2206DOI: (10.1016/j.str.2016.09.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Cryo-EM and -ET on PC12-Isolated Membrane Patches with Filter Paper 12/N (A) Low-magnification overview of several membrane patches. (B) Medium-magnification image showing many vesicles of various diameters. (C) Spherical LDCVs (stars) in an untreated state. (D) Pear-shaped LDCVs after 5 min incubation with 3 mM CaCl2 (stars). (E) Ellipticity distribution for vesicles in untreated and stimulated samples. (F–H) Clathrin assemblies (F), clathrin-coated pit (G), and clathrin-coated vesicle (H) in samples incubated for 10 min with 3 mM CaCl2 (arrowheads). Scale bars, 2 μm (A) and (B), 200 nm (C) and (D), and 100 nm (F–H). Structure 2016 24, 2198-2206DOI: (10.1016/j.str.2016.09.016) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Negative Staining of Membrane Patches Isolated from HEK293, 3T3 Mouse Fibroblasts, and HU Smooth Muscle Cells (A) Overview of HEK293 patches (arrows). (B) The dotted line delineates a zoom-in on a single HEK293 membrane patch. (C–E) 3T3 mouse fibroblasts. Overview of unroofed cells (arrows) (C). Membrane patch (closed arrowhead). Inset: Magnified view of the lower part of the image showing proteins (open arrowheads) (D). Membrane (closed arrowhead) and actin filaments (light arrowhead) (E). (F) Tomographic slice through an HU cell membrane patch showing actin filaments. Inset: Fourier transform with a 5.8-nm repeat corresponding to an actin helical pitch. Scale bars, 50 μm (A) and (C), 10 μm (B), 500 nm (D), and 200 nm (E and F). Structure 2016 24, 2198-2206DOI: (10.1016/j.str.2016.09.016) Copyright © 2016 Elsevier Ltd Terms and Conditions