Protocol for measuring interorganelle contact sites in primary cells using a modified proximity ligation assay.

Interorganelle contact sites regulate lipid metabolism, organelle dynamics and positioning, as well as apoptosis and autophagy. Here, we present a proximity ligation assay (PLA) protocol for measuring the association of two organelles in fixed cells. We describe steps for primary cell culture, primary cell transfection, and the assay itself. We then detail procedures for manual and image J-based analysis of PLA foci. This protocol optimizes the use of assay products and improves the identification of PLA foci labeling actual contact sites. For complete details on the use and execution of this protocol, please refer to Ilamathi et al. (2023).1.

Contact sites between endoplasmic reticulum sheets and mitochondria regulate mitochondrial DNA replication and segregation.

Mitochondria are multifaceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) replication is a spatially regulated process essential for the maintenance of mitochondrial function, its defect causing mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is affected by mitochondrial dynamics: The absence of mitochondrial fusion is associated with mtDNA depletion whereas loss of mitochondrial fission causes the aggregation of mtDNA within abnormal structures termed mitobulbs. Here, we show that contact sites between mitochondria and ER sheets, the ER structure associated with protein synthesis, regulate mtDNA replication and distribution within mitochondrial networks. DRP1 loss or mutation leads to modified ER sheets and alters the interaction between ER sheets and mitochondria, disrupting RRBP1-SYNJ2BP interaction. Importantly, mtDNA distribution and replication were rescued by promoting ER sheets-mitochondria contact sites. Our work identifies the role of ER sheet-mitochondria contact sites in regulating mtDNA replication and distribution.

Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction.

Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. 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. 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). 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. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins.