Detection of Mitochondria-Associated Endoplasmic Reticulum Membrane Tightness Using Inducible FRET Biosensors.

Multiple studies have demonstrated that the number, length, and gap width (thickness) of ER-mitochondria contacts, or mitochondria-associated membranes (MAMs), influence their biological roles. Our previous work showed that the stabilization of tight MAMs, characterized by a gap width of approximately 7 nm, leads to an increase in amyloid β (Aβ) levels, whereas the presence of loose MAMs, with a gap width of around 40 nm, reduces Aβ production in a three-dimensional (3D) neural model of Alzheimer's disease (AD). To investigate the effects of MAMs with different gap widths, ER- and mitochondria-targeted FRET (Förster Resonance Energy Transfer) biosensors-ER-CFP (cyan fluorescent protein) and Mito-YFP (yellow fluorescent protein), respectively-were developed to quantify tight MAMs (<10 nm gap width) in contrast to loose or non-MAMs. FRET occurs when the donor fluorophore (CFP) and the acceptor fluorophore (YFP) are within 10 nm of each other, making this system suitable for assessing MAM proximity. This protocol outlines the use of spectral ratiometric FRET to measure the extent of tight MAM formation.