Observing “unroofed” cells by metal-replica TEM
Agathe Franck1, Jeanne Lainé1, Marc Bitoun1, Ghislaine Frébourg2, Michaël Trichet1, Stéphane Vassilopoulos1
1 Institut de Myologie, Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, Paris, France
2 Institut de Biologie Paris-Seine (IBPS), Service de microscopie électronique, Sorbonne universités, UPMC Univ Paris 06, CNRS, Paris, France
Keywords: metal-replica, TEM, unroofing
The “unroofing” technique has been successfully used to observe the cytoplasmic side of the plasma membrane (PM) using either light or electron microscopy. Combined to transmission electron microscopy (TEM), it is an invaluable method to reveal the composition of the PM and to directly observe macromolecular complexes including the cytoskeleton and endocytic membrane invaginations. This method has been optimized over decades to preserve membranes close to their native states by the combination of quick freezing, followed by deep etching and rotary replication (the so-called “QF-DE-RR” technique). However, a serious setback in implementing unroofing combined with QF-DE-RR stems from the necessity to use complicated apparatus, such as quick freezing and freeze-fracturing devices, along with strong expertise to handle them. Here, I will present a simple and straightforward protocol for observation of the cytoplasmic side of plasma membrane which only requires chemical treatment of samples prior to replication. This method has been optimized towards sample preparation and is easily amenable to higher throughput. We used TEM analysis of unroofed membranes from adherent muscle cells and show the advantages of this technique towards visualization of the cytoskeleton and different endocytic structures such as clathrin coated pits and caveolae.