TABLE 1.
Advantages and disadvantages of the various experimental approaches to study endoplasmic reticulum–mitochondria contact sites (ERMCSs).
| Approaches | Advantages | Disadvantages | |||
| Study of ERMCS morphology and structure | |||||
| Electron microscopy | Transmission electron microscopy (TEM) | • Golden standard | • Provides static, high-resolution ultrastructure information | • Suitable for samples with a large amount of contact sites | • Fixation may introduce artifacts |
| Electron tomography (ET) | • Three-dimensional view of the subcellular structures | • Technically challenging | • “missing wedge” artifacts | ||
| Scanning electron microscopy (SEM) | • Provides high-resolution 3D image | • Overcome the “missing wedge” artifacts | • Needs powerful computer to process large datasets | ||
| Epifluorescence and confocal microscopy | Super-resolution microscopy | • Suitable for both static and live-cell imaging | • Enable the high-resolution observation of ERMCS dynamics | • Optical diffraction limit | • Fixation may introduce artifacts |
| FRET-based reporter | • Provides temporal quantitative measurements of contact distance | • Prolonged drug treatments can introduce artifacts | |||
| Split green fluorescent protein (GFP) | • Different probes could be used to examine the narrow and wide contacts | • Less responsive to the subtle changes in the contacts | |||
| Light-inducible ER–mitochondria tethering (LIT) system | • Temporally regulate the contacts | • Avoid side effects caused by continuous ER–mitochondria tethering | • Needs careful control | ||
| Split Rluc8 | • Easy technique | • Needs careful control | |||
| Proximity ligation assay (PLA) | • Mainly used to detect the proximity between the two proteins | • Requires antibodies to the proteins of interest | |||
| Detection of resident proteins in ERMCSs | |||||
| Cell fractionation | • Major technique to isolate the fraction of ERMCS and identify its protein components | • Purity is hard to guarantee | |||
| Ascorbate peroxidase (APEX) Tagging | • Identify new contact-site proteins | • Combining it with biochemical cell fractionation will reach a better purity | • Technically challenging | • Needs careful control | |