Design-based stereology method for counting organelles
While the nature of these protein tethers has not been fully characterized and likely varies depending on cellular conditions and cell types, several MERCS tethering complexes have been identified. MERCS require proteins on both the ER and mitochondria to bridge the two organelles. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) play a central role in calcium signalling (Ca 2+), phospholipid synthesis and transfer, regulation of oxidative stress and inflammatory responses, mitochondrial dynamics, bioenergetic and cell survival ( Tubbs and Rieusset, 2017) ( Patergnani et al., 2011 Krols et al., 2016 Martinvalet, 2018 Missiroli et al., 2018 Perrone et al., 2020 Xu et al., 2020). In addition, a growing number of proteins have been shown to be required for these MCS ( Eisenberg-Bord et al., 2016 Simmen and Tagaya, 2017 Cohen et al., 2018 Scorrano et al., 2019). It is becoming clear that most, if not all, organelles interact via MCS. MCS are defined as areas of close apposition (typically less than 30 nm) between two organelles in the absence of membrane fusion. However, in recent years, the subject has undergone a revolution as we realised that cells use a network of contact sites between membranes of different organelles, termed membrane contact sites (MCS), to communicate and transfer metabolites ( Schrader et al., 2015 Cohen et al., 2018 Scorrano et al., 2019). For decades, organelle research has centred on identifying each compartment and their distinct properties with the thought that transfer of material between organelles occurred through diffusion of soluble metabolites or vesicular trafficking ( Dennis and Kennedy, 1972). Organelles are responsible for many of the anabolic and catabolic processes required for the proper functioning of eukaryotic cells. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. While these two techniques allow the detection of endogenous proteins, preventing some problems associated with techniques relying on overexpression (FRET, split fluorescence probes), they come with their own issues. Among these techniques, fluorescence-based imaging is widely used, including analysing signal overlap between two organelles and more selective techniques such as in-situ proximity ligation assay (PLA). A number of biochemical and imaging approaches have been established to address these questions, resulting in the identification of a number of molecular tethers between mitochondria and the ER. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. This occurs through the formation of close connections through membrane contact sites. Organelles cooperate with each other to regulate vital cellular homoeostatic functions. 2Centre D'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois, Université du Québec à Montréal, Montréal, QC, Canada.1Groupe de Recherche en Signalisation Cellulaire and Département de Biologie Médicale, Université Du Québec à Trois-Rivières, Trois-Rivières, QC, Canada.Sara Benhammouda 1,2 †, Anjali Vishwakarma 1,2 †, Priya Gatti 1,2 and Marc Germain 1,2*