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Method Article

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

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DOI:

10.3791/67914

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August 7th, 2026

 ,  , 
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,

In This Article

Summary

We describe a method for measuring FRET values of inducible FRET biosensors targeting the ER (CFP-tagged) or mitochondria (YFP-tagged) in vitro to assess mitochondria-associated ER membranes (MAMs) tightness, which influences Aβ generation, and this method can be used for initial screening of synthetic or natural MAM-modulating agents as potential AD therapeutics.

Abstract

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.

Introduction

Mitochondria-associated endoplasmic reticulum contacts (MERCs), which are isolated biochemically as mitochondria-associated ER membranes (MAMs), are implicated in several neurodegenerative disorders, including Alzheimer's disease (AD)1. The recently proposed "MAM hypothesis" suggests that MAMs play a central role in the generation of amyloid β (Aβ), thereby initiating the pathogenic cascade of AD. This cascade includes the formation of neurofibrillary tangles (NFTs), calcium dyshomeostasis, and neuroinflammation2. MAMs consist of cholesterol-rich, lipid raft-like microdomains in the endoplasmic reticulum and the outer mitochondrial membrane (OMM), linked by specific tethering proteins that contribute to both their structural and functional diversity3. The stabilization of ER-mitochondria contacts influences various cellular processes, including ER calcium signaling and apoptosis4,5. It is estimated that 5%-20% of mitochondria form physical contacts with the ER to establish MAMs6. Several studies have indicated that the number, length, and gap width (thickness) between the ER and mitochondria at MAM sites are important determinants of their functional roles. Cryo-electron microscopy and in vivo analyses of mouse liver have demonstrated that the metabolic state of hepatocytes modulates both the thickness (ranging from 14 nm to 20 nm) and the length (ranging from 145 nm to 270 nm) of MAMs7.

MAMs are also emerging as potential therapeutic targets for various disorders, including cancer and metabolic disorders. MAM thickness or gap width regulates cell survival. The constitutive MAM stabilizers, MAM 1X and MAM 9X, designed to stabilize tight (6 nm ± 1 nm gap width) and loose MAMs (24 nm ± 3 nm gap width), respectively, demonstrated that the stabilization of tight MAMs dramatically increased Aβ generation in a novel 3D neural culture model of AD. In contrast, the stabilization of the loose MAMs had no effect8. Thus, converting the stabilization of tight MAMs to loose MAMs may be beneficial to lower amyloid pathology. The structural and functional complexities and the lack of precise quantitative analysis of the degree of MAM stabilization make targeting MAMs for drug discovery challenging. A novel live-cell imaging and kymography-based analysis of the axonal velocity of the ER-bound mitochondria demonstrated that the axonal velocity of mitochondria may be used as a metric to quantitatively measure MAM stabilization because tighter contact with the ER significantly reduced the mitochondrial axonal velocity compared to free mitochondria8,9. Using an inducible stabilizer of MAMs in hAPP-expressing Neuro 2A (N2AAPP) cells8, we found that tightening MAM gap widths increases Aβ generation in a dose-dependent manner8. The inducible MAM stabilizers are expression plasmids encoding the CFP (cyan fluorescence protein)-epitope-tagged ER-targeting and YFP (yellow fluorescence protein)-epitope-tagged mitochondria-targeting biosensors fused to the rapamycin-inducible FK506 rapamycin binding (FRB) or 12-kDa FK506-binding protein (FKBP) domains. The biosensors were denoted as ER-CFP and Mito-YFP, respectively. The CFP is a donor fluorophore that transfers energy to the acceptor fluorophore YFP when they are separated by >10 nm. Cells (N2AAPP) expressing the inducible biosensors increased Aβ generation upon rapamycin treatment8, suggesting that increasing tight MAMs increased Aβ generation. These methods will be beneficial for screening small molecule modulators of MAMs and their effective concentration required to achieve the stabilization of an optimal level of tight MAMs that will convert pathogenic ("Aβ increasing") to therapeutic ("Aβ lowering") MAMs without completely destabilizing the ER and mitochondria contacts, important for cell survival.

Experimentalmodel
This study used Neuro2A(N2A)cells constitutively expressing APP751 (N2AAPP). These cells release detectable levels of Aβ into the conditioned media (CM) compared to naïve N2A cells. N2AAPP cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamate supplemented with 200 µg/mL G418, as described previously10.

Expression plasmid
An expression plasmid was generated by fusing the YFP (yellow fluorescent protein)-tagged mitochondrial targeting sequence of the mammalian A-kinase anchor protein 1 [AKAP1(34-63)] and CFP (cyan fluorescent protein)-tagged ER-targeting sequence of phosphatidylinositol-3-phosphatase Sac1 phosphatase [Sac1(521-587)] with a self-cleaving Tav2A sequence encoding the EGRGSLLTCGDVEENPGP peptide sequence, denoted as pYFP-AKAP1-Tav2A-Sac1-CFP8. The Tav2A-fused expression plasmid is also flanked with nucleotide sequences encoding the FK506 rapamycin binding (FRB) domain [EMWHEGLEEASRLYFGERNVKGMFEVLEPL HAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNV (K2095P)DLTQAWDLYYHVFRRISKQ] or the 12-kDa FK506-binding protein (FKBP) domain (MGVEKQVIRPGNGPKPAPGQTVTVHCTGFGKD GDLSQKFWSTKDEGQKPFSFQIGKGAVIKGWDEGVIGMQ IGEVARLRCSSDYAYGAGGFPAWGIQPNSVLDFEIEVLSVQ). The fusion plasmid FRB-YFP-AKAP1(34-63)-Tav2A-Sac1(521-587)-CFP-FKBP, denoted here as MAM-Tav2A, was generated to obtain equimolar expression levels of FRB-YFP-AKAP1(34-63) and Sac1(521-587)-CFP-FKBP forthe ratiometric FRET analysis. The Sac1(521-587)-CFP-FKBP, denoted as ER-CFP, is the donor fluorophore, whereas Mito-YFP-FRB, denoted as, Mito-YFP, is the acceptor fluorophore that generates FRET when separated by ~10 nm8. Thus, the FRET-based biosensors detect tight MAMs (6-10 nm, gap width). The FRB and FKBP were introduced to induce the tightening of MAM gap widths upon the addition of rapamycin in a dose-dependent manner that could be quantified by measuring the increased FRET signal8.

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Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1.Transfection

  1. Transfect N2AAPP cells with MAM-Tav2A expression plasmid (14-18 h).
    1. Before beginning, plate 1 × 106 N2AAPPcellsper well of 6-well plates containing glass coverslips and maintain the cells in the growth media for 12-16 h.
    2. Transfect 1-3 μg MAM-Tav2A plasmid (Supplementary File 1) using Lipofectamine transfection reagent following the manufacturer's protocol.
    3. After 12-16 h of transfection, perform drug treatment.
      NOTE: Always have cells transfected with an expression plasmid encoding only the donor (ER-CFP) or the acceptor (Mito-YFP) to be used as the FRET background.

2. Drug treatment

  1. Treat the MAM-Tav2A-expressing N2AAPP cells with Rapamycin (Rapa) or Rapalog (Log) (3-6 h).
    1. Before treatment, prepare stock solutions (10 mM) of Rapa and Log.
    2. Change the media with fresh media containing 0 nM, 100 nM, or 250 nM Rapa or Log.
    3. After 3 h of treatment, add 3.3% paraformaldehyde (PFA) to fix the cells.
    4. Prepare the slides for fluorescence confocal microscopy by placing one drop of antifade reagent.
    5. Mount the coverslips over the droplet and gently press with the forceps.
      NOTE: The glass coverslips are fragile and require caution. As an alternative to glass coverslips, use glass bottom plates. The major shortcoming of using glass-bottom plates is that the antifade reagent cannot be used, which makes imaging difficult due to bleaching of the fluorophores. DO NOT use antifade reagent containing DAPI (4′,6-diamidino-2-phenylindole) because its blue fluorescence will interfere with FRET between CFP and YFP.

3. Confocal microscopy for Fluorescence Resonance Energy Transfer (FRET)

  1. Place the slides under an inverted microscope and adjust the focus of the microscope until the cells become visible.
  2. Excite the fluorophore using a 440 nm laser and capture images using 468-503 nm emission for the donor CFP (FKBP-CFP-ER) or 525-565 nm for the acceptor YFP (FRB-YFP-Mito).
  3. Adjust the signal intensity using the built-in fluorescent filter until the background signal has dissipated (should be nearly solid black).
    NOTE: A confocal microscope was used to capture fluorescent images using NIS Element AR software. 60x magnification was used at a resolution of 512 pixels.
  4. Save the images as .nds files.
  5. Perform spectral ratiometric FRET analysis.

4. Ratiometric FRET analysis (6 - 12 h)

NOTE: Fluorescence emission from the donor ER-CFP (468-503 nm) and the acceptor Mito-YFP (525-565 nm) was detected using a confocal microscope. ImageJ software was used to quantify the average pixel fluorescence intensity following background subtraction. The ratio of YFP to CFP emission intensity was calculated and used as the readout for spectral FRET analysis.

  1. Open Image J (Fiji).
  2. Either drag the .nds file onto Image J, or press File -> Open and select the Image.
  3. On the following screen, select the Split Channels box.
  4. Click on the C = 0 panel.
  5. Click on Analyze -> Measure.
    NOTE: Write down the Mean Value for later calculation.
  6. Click on Process -> Math -> Subtract.
  7. Enter the Mean Value.
  8. Repeat step 4 for the C = 1 panel.
  9. Click on the Freehand selection tool.
  10. Circle a cell of interest, then press Ctrl+T to record it to the ROI.
  11. Click on Show All at the bottom of the ROI to show what you have already selected.
  12. Select all selected cells and click on Measure, Record these values into Excel.
  13. Click on the C = 1 panel and click on Show All.
  14. Select all cells and click on Measure again, and record these values into a separate column in Excel.
  15. In Excel, create a function to put the values of the donor signal over the recipient (C0/C1). This is the FRET ratio.

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Results

Ratiometric FRET analysis of FACS-enriched N2AAPP cells expressing MAM-Tav2A demonstrated a dose-dependent increase in Aβ40 levels following rapamycin treatment8. Rapamycin acts by inhibiting the mammalian target of rapamycin (mTOR) signaling pathway, which in turn downregulates TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) expression in microglia, leading to reduced Aβ plaque clearance in 5XFAD mouse models of Alzheimer's disease

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Discussion

The modulation of MAMs is an emerging area of research with potential implications for various disorders, including cancer, metabolic disorders, and neurodegenerative diseases13. Many pharmacological agents, including the dynein-antagonist LDC-3/Dynarrestin, antidiabetic drug metformin, and anti-cancer agent sulforaphane, disrupt MAMs and are currently under pre-clinical and clinical trials for cancer and metabolic disorders. These pharmacological agents modulate the ER-mitochondria contacts or MA...

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Acknowledgements

We sincerely thank Dr. Rudolph E. Tanzi, Professor at Massachusetts General Hospital (MGH), for his insightful comments on the manuscript. We thank Dr. Masato Maesako, Assistant Professor at MGH, for sharing his expertise in FLIM assays. We thank Dr. Gyorgy Hajnoczky, Professor, Thomas Jefferson University, Philadelphia, for generously providing us with expression plasmids encoding ER-CFP or Mito-YFP. This study was supported by the Cure Alzheimer's Fund to RB.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
BSAFisher Scientific501781532
Confocal microscope Olympus FV3000RS (Tokyo, Japan)N/A
DMEM with GlutaMAX supplementGibco/Thermo Fisher Scientific10564011
EDTA  Life Technologies41116134
Falcon 6 Well Plates VWR International41122107
GAPDH Polyclonal AntibodyThermo Fisher ScientificPA1-988
Gelatin VWR International9000-70-8
Geneticin (G418 Sulfate)Gibco/Thermo Fisher Scientific10131035
GFP monoclonal AntibodyInvitrogenMA5-15256
Graphpad Prism N/APrism 9, version 9.5.0N/A
HeparinSigma-Aldrich H0200000
ImageJ Software ImageJ 1.53aN/A
Inverted confocal microscope Nikon (C2 Eclipse Ti2)N/A
Lipofectamine2000Invitrogen11668027
MS Excel  Microsoft Excel, version 2302N/A
NaCl  Fisher Scientific7647145
NuPAGE 4–12% Bis-Tris gel  InvitrogenNP0321BOX
Penicillin/Streptomycin/Amphotericin B Lonza 17-745E
PhotoshopAdobe Photoshop CC 20.0.10 N/A
Tris-HCL, pH 7.6  Boston BioProducts42000000
Triton X-100  Sigma-AldrichT8787
Trypsin-EDTAGibco/Thermo Fisher Scientific25200056
Tween 20Fisher Scientific501657287

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