Fig. 6: Fluorescence-based techniques to visualise MERCS.

a PLA: the sample is fixed and incubated with primary antibodies targeting proteins in MERCS (usually MERCS tethers) and a modified secondary antibody (which is attached to an oligonucleotide strand). If the two proteins are in close proximity, as they are in MERCS, the addition of a third oligonucleotide results in complementary base pairing between the three oligonucleotides and the formation of circular DNA. The circular DNA can undergo rolling circle amplification, creating a long strand of DNA and multiple copies of the circular DNA. Fluorescent probes are designed to be complementary to a sequencing within the circular DNA. The probes hybridise in multiple regions, allowing the visualisation of the fluorescent signal and MERCS. b BiFC also utilises proximity to visualise MERCS. Two non-fluorescence fragments of a fluorescence protein, commonly GFP or Venus, are fused to transmembrane domains, or whole proteins, found in the ER or mitochondrial membrane. If MERCS does not form the BiFc, constructs remain separate and do not fluoresce, but when MERCS forms, the ER and mitochondria membrane come into close proximity bringing with them the two non-fluorescent fragments, allowing them refold into whole GFP or Venus protein whose fluorescence can be visualised and measured. c Similarly, the ddGFP system is fused to protein fragments or whole proteins found in ER and mitochondria membrane; however, each protein is fused to non-fluorescence ddGFP monomers. One monomer contains a chromophore that is destabilised and quenched, while the other completely lacks a chromophore. When MERCS forms, these ddGFP monomers come into close proximity, heterodimerise and complement each other, allowing fluorescent detection. As there is no protein folding, this process is reversible. d splitFAST utilises the 14- kda protein (FAST), which is split into N- and C-terminal fragments. These fragments can be attached to two interacting proteins or membrane fragments of a protein. For the study of MERCS, these interacting proteins would be in the ER or mitochondrial membrane. When MERCS forms, the N and C fragments combine, and upon the addition of HBR, they fluoresce allowing visualisation of MERCS. This is reversible, as HBR can be added or removed, but the FAST protein can also dissociate when the ER and mitochondria membrane are not in close proximity. e FRET: a FRET donor (CFP) and FRET acceptor (YFP) are fused to resident ER or mitochondrial proteins or transmembrane protein fragments. When ER or mitochondrial membranes are not in close proximity, a light source illuminates the CFP donor but the YFP acceptor is not close enough for FRET to occur and so blue light emitted. When MERCS forms, the FRET donor (CFP) and acceptor (YFP) are in close proximity, so FRET can occur, and blue light is transferred to YFP and yellow fluorescence is emitted. This is reversible as no protein folding or contact occurs between FRET pairs, f BRET also employs FRET to visualise MERCS; however, rather than CFP, the resident ER, mitochondria protein or transmembrane fragments are fused to a luciferase enzyme that acts as a light source. When MERCS forms, the luciferase enzyme and FRET acceptor are in close proximity, and so energy transfer can occur from the luciferase enzyme and YFP, which is emitted as yellow florescence. This is reversible as no protein folding occurs.