iMEM: Isolation of Plasma Membrane for Cryoelectron Microscopy.
The plasma membrane and the cell cortex are essential parts of the eukaryotic cell. The plasma membrane delimitates the cell and mediates communication with the outside. The cell cortex is the submembrane cytoskeleton shaping the cell and is able to reorganize for the passage of material. To study events at and near the plasma membrane, cryoelectron microscopy (cryo-EM) may be used. Most intact cells are too thick for direct cryo-EM imaging. Generating cell-free membrane patches could be a means to study features at the plasma membrane. Here we present an unroofing method, termed iMEM (isolation of membrane patches for cryo-EM) where the plasma membrane is isolated directly on an EM grid. The in situ isolation of membrane patches has several advantages: it is a one-step procedure providing a higher throughput than focused-ion beam cryomilling. It enables the time-precise control over biochemical events before cryofixation.
Experimental Morphology, Institute of Anatomy, University of Bern, Baltzerstrasse 2, Bern 3012, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern 3012, Switzerland.
Camille Françoise
CF
Peitsch
Experimental Morphology, Institute of Anatomy, University of Bern, Baltzerstrasse 2, Bern 3012, Switzerland.
Sven
S
Beckmann
Experimental Morphology, Institute of Anatomy, University of Bern, Baltzerstrasse 2, Bern 3012, Switzerland. Electronic address: benoit.zuber@ana.unibe.ch.
Benoît
B
Zuber
D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods; D016716:PC12 Cells; D051381:Rats
actin cortex; blotting; cell-free system; cryoelectron tomography; electron microscopy; endocytosis; exocytosis; filter paper; large dense core vesicles; live fluorescence imaging; plasma membrane