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Updated: Aug 20, 2026
FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
Detection of Mitochondria-Associated Endoplasmic Reticulum Membrane Tightness Using Inducible FRET Biosensors
Jacob C Zellmer1, Marina B Tarantino1, Raja Bhattacharyya2
1Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Henry and Allison McCance Center for Brain Health, Department of Neurology, Massachusetts General Hospital, Harvard Medical School.
None:
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.
