Analyzing Endolysosome-Mitochondria Membrane Contact Sites by Thin Section TEM of Human Fibroblasts.

Membrane contact sites (MCS) are dynamic nanoregions of close apposition between two different organelles, functioning as discrete lipid or ion transfer sites. This new concept in cell biology involves unique proteins at both membrane sites, named tethers, and emerges in early observations by transmission electron microscopy (TEM). Currently, this technique still constitutes a valuable tool for MCS visualization and quantification. In the last decade, Lysosomal Storage Diseases (LSD) have been instrumental in studying the MCS between lysosomes (Ly), or endolysosomes (EL), and other organelles in close proximity such as mitochondria or the endoplasmic reticulum (ER). At present, the analysis of composition, functioning, and alterations/rewiring of MCS in health and disease represents an innovative area of research for designing therapeutic strategies in a variety of pathologies. Here, we describe procedures for chemical fixation using the Flat Embedding technique to characterize and quantify the MCS between LE/Lys and mitochondria in human fibroblasts by thin-section TEM.