Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Summary

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

Abstract

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca²⁺ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

Introduction

The interactions between the ER and the mitochondria are vital for cellular homeostasis and survival^1,2,3,4. Previous evidence indicates that any type of disruption or dysregulation in ER-mitochondria contact sites can contribute to several neurodegenerative, metabolic, and cardiovascular diseases, as well as cancer^5,6,7,8,9,10. For example, an abnormal increase of Ca²⁺ uptake into the mitochondria can lead to cell death by opening mitochondria permeability transition pores, which are commonly seen in some models of Alzheimer's disease^5,11. Similarly, reduced ER-mitochondria contacts can result in a decrease in ATP production and impairment of Ca²⁺ intake, as seen in models of amyotrophic lateral sclerosis^5,11,12. As more studies are being conducted in the realm of ER-mitochondria contacts, additional disease-related proteins and genes that could affect these contacts are being discovered. Despite current knowledge and evidence showcasing the role of ER-mitochondria contact sites, much work is still needed to elucidate how these contacts could lead to loss of cellular function and ultimately cell death.

There have been various methods developed to evaluate the proximity of the two membranes, structural morphology, and the distance between the two organelle contact sites^3,4,13. The approaches to monitor ER-mitochondria coupling include fluorescence marker-based imaging^14,15, FRET-reporter-based imaging¹⁶, and split-fluorescence-probe-based imaging^17,18, which use epifluorescence and confocal microscopy. Super-resolution and atomic resolution microscopy are also powerful tools for accurately visualizing inter-organelle contacts, although their utilization in contact site analysis is still limited since they require highly dedicated microscopes and technical expertise¹⁹. In addition, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and other EM techniques such as electron tomography (ET) and cryo-electron microscopy, are commonly used as they provide high-resolution ultrastructural imaging of the contact sites, which are often impossible to explore using other experimental approaches^20,21,22. However, these EM-based methods are a very low-throughput technique that can also be affected by chemical fixation procedures. More recently, proximity labeling-based methods have been used to detect contact sites as well as to identify new contact-site proteins. For example, proximity ligation assay (PLA) has been used to quantify organelle proximities^23,24, while a revised version of the ascorbate peroxidase (APEX) assay has been utilized in identifying new contact-site proteins^25,26. It is important to recognize that all these methods described above have strengths and intrinsic limitations in detecting the contacts between the organelles. Thus, the pairing of different techniques is required to obtain a thorough interpretation of the organelle contact sites.

Previously we have established the split-Renilla luciferase 8 reassembly assay (split-Rluc assay) to monitor the level of ER-mitochondria membrane contacts (Figure 1A)^24,26,27. Briefly, each split half of Renilla luciferase is conjugated with an ER- or mitochondria- targeting sequence. When transfected together, each split half of the enzyme is expressed either in the ER or mitochondrial membrane. When the ER and mitochondria are positioned in close proximity to each other, the split halves come together and reconstitute the whole enzyme with luciferase activity. For the split-Rluc construct, we used Renilla luciferase 8 (Rluc8) in pBAD/Myc-His²⁷ for the initial template. The split site (between amino acids 91 and 92) was determined based on previous reports²⁷. For the N-terminal half of the Rluc8, DNA sequences for amino acids 1-91 of the Rluc8 were fused to the 3' end of the FLAG tag and the mouse AKAP1 mitochondrial-targeting sequence in pcDNA3.1 TOPO vector by PCR²⁷. For the C-terminal half targeted to the ER, DNA sequences that encode amino acids 92-311 were fused to the 5' end of the myc tag and the yeast UBC6 ER localization sequence. Here, we have upgraded the split-Rluc plasmid construct such that split halves of the Renilla luciferase are expressed in a single vector (pCAG) under the same promoter and subsequently cleaved into two fragments as T2A, a self-cleaving peptide 2A sequence from Thosea asigna virus, is inserted in between the two split halves (Figure 1B). The plasmid DNA map and sequences are provided in Supplemental File 1 and Supplemental Figure S1. Using this system, we have measured the effects of three chemicals (inhibiting GTPases involved in actin polymerization) on ER-mitochondria contacts. This split-Rluc assay is a simple but robust assay system for high-throughput screening for inter-organelle contact modulators²⁴.

Protocol

1. Cell maintenance and seeding (Day 1)

1. Maintain HEK293T cells in cell culture media containing Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (in 100 mm culture dishes) in a humidified incubator at 37 °C with 5% CO₂.
2. Before starting, check the confluency of the plate by viewing under the microscope. When the cells reach approximately 80-90% confluency, prepare to seed the cells in a 6-well culture plate by removing the media and washing with 10 mL of Dulbecco's phosphate-buffered saline (DPBS).
3. Remove the DPBS from the culture dish and treat the cells with 1 mL of 0.05% Trypsin-EDTA phenol red. Incubate for 3 min, then take out the plate from the incubator and add 9 mL of culture media (DMEM with 10% FBS) to stop the trypsin reaction. Slowly pipette up and down to ensure any cells still stuck to the plate are dislodged. Transfer the cell suspension to a 50 mL tube.
4. Centrifuge at 300 × g for 5 min at room temperature. Remove the media and resuspend the cell pellets with 1 mL of fresh media (DMEM + 10% FBS).
5. Count the number of cells using a hemocytometer.
   1. Ensure even distribution of cells by gently swirling the tube. Immediately take out 10 µL of the cell suspension and place into a 1.7 mL microcentrifuge tube. Add 90 µL of media to the same tube.
   2. After mixing the cells and media gently, take the cell suspension and apply 10 µL to each of the chambers of the hemocytometer by gently pipetting underneath the coverslip.
   3. Using a 10x objective, focus the grid lines of the hemocytometer under a microscope. Once focused, count the number of cells in one set of 16 squares (4 x 4 squares) using a hand tally counter. Repeat until all four sets of 16 squares are counted.
   4. Calculate the total number of cells/mL by taking the average cell count (from each of 16 corner squares) and multiplying that by 10⁵.
      NOTE: Instead of using a hemocytometer to count cells, an automated cell counter can be used according to the manufacturer's instructions.
6. Once cell counting is completed, prepare to plate cells in a 6-well plate at 7.5 × 10⁵ cells/well density with 2 mL of media/well. In a 50 mL tube, add the necessary volume of cells along with 13 mL of media. Dispense 2 mL of the diluted cell suspension into each of the 6 wells.
   1. Before plating, calculate the number of cells necessary for 7.5 × 10⁵ cells/well using C₁V₁ = C₂V₂ where C₁ is the initial cell count (cell number/mL), V₁ is the volume needed from the initial cell suspension, C₂ is the desired target cell density (cell number/mL), and V₂ is the final volume needed for cell seeding.
7. After dispensing, tap the plate gently on all sides to spread the cells and then place it in a humidified incubator at 37 °C with 5% CO₂ overnight.

2. Polyethyleneimine (PEI)-mediated cell transfection and post transfection cell seeding (Day 2)

1. Remove the 6-well plate from the incubator and aspirate out the existing culture media from each well. Add 2 mL of fresh media per well.
2. Transfect the cells with split-Rluc plasmid DNA (pCAG-MitoRluc^N-T2A-Rluc^CER-IRES-mCherry) dissolved in TE buffer (pH 8.0).
   1. For each well, mix the DNA (750 ng) and PEI (ratio of DNA/PEI = 1:3) in 200 µL of DMEM in a 1.7 mL microcentrifuge tube. Quickly vortex the DNA/PEI at level 8 for approximately 2 s to ensure they mix well together to form complex. After that, spin it down in a mini centrifuge briefly (<3 s).
   2. Let the mixture of DNA/PEI incubate at room temperature for 15 min and then add the mixture (by dropping it) to the surface of the culture media in the 6-well plate containing cells. Tap the plate gently (to evenly distribute the mixture) and incubate it in a humidified incubator at 37 °C with 5% CO₂ for 6 h.
3. While incubating, prepare a Poly-D-Lysine (PDL)-coated 96-well plate by first adding 70 µL of PDL (50 µg/mL in DPBS) to each well and incubating in a humidified incubator at 37 °C with 5% CO₂ for at least 1 h. Remove the PDL solution and then wash each well 2x with 100 µL of DPBS. At the end of the second wash, ensure that all the remaining DPBS is removed.
4. At 6 h post transfection, prepare to seed the transfected cells in a PDL-coated 96-well plate.
   1. Take out the 6-well plate containing transfected cells from the incubator, aspirate out the media, and wash each well with 2 mL of DPBS. After removing the DPBS, add 350 µL of 0.05% Trypsin-EDTA phenol red and incubate for 1-2 min. To stop the trypsin reaction, add 1.7 mL of media to each well.
   2. After transferring the cells along with media to a 50 mL tube, spin the tube at 300 × g for 5 min in a tabletop centrifuge. Aspirate the media and resuspend the cell pellet with 1 mL of fresh media. Count the number of cells using a hemocytometer (as described in step 1.5).
5. Seed the cells in a PDL-coated 96-well plate at a density of 4 × 10⁴ cells/well in 100 µL of culture media. For this, in a 50 mL tube, add the necessary volume of cells (see below for calculation) along with appropriate volume of media (12 mL total volume). Using a multichannel pipette, dispense 100 µL of the diluted cell suspension into each of the 96 wells. Incubate the cells for 18 h in a humidified incubator at 37 °C with 5% CO₂.
   1. Calculate the number of cells necessary for 4 × 10⁴ cells/well using C₁V₁ = C₂V₂ where C₁ is the initial cell count (cell number/mL), V₁ is the volume needed from the initial cell suspension, C₂ is the desired target cell density (cell number/mL), and V₂ is the final volume needed for cell seeding.

3. Chemical treatment and luciferase assay in live cells (Day 3)

1. In a 1.7 mL microfuge tube, prepare the three fresh solutions of 50 µM Rhosin (50 mM stock in DMSO), 25 µM Ehop-016 (25 mM stock in DMSO), and 50 µM ZCL278 (50 mM stock in DMSO) by diluting each stock solution (50 mM Rhosin, 25 mM Ehop-016, and 50 mL ZCL278; all in DMSO) 1:1,000 in the required amount (50 µL/well × number of wells) of culture media. To each solution, add live cell substrate (50 mM stock in DMSO) for Renilla luciferase diluted 1:2,000 to a final concentration of 25 µM. To make 0.5, 5, and 50 µM ZCL278 solutions, perform serial dilutions using the initial 50 µM ZCL278 in culture media.
   NOTE: If the chemical incubation time is desired for less than 1-1.5 h, the cells can be preincubated with live cell substrate (for Renilla luciferase) for 1-1.5 h before they are treated with each chemical. We add chemicals and live cell substrate simultaneously to optimize the protocol for high-throughput drug screening.
2. Remove the media from each well of the 96-well plate containing transfected cells and add 50 µL of chemical and substrate media mixture to each well.
3. After incubating the cells for 1 h, 2 h, or 5 h, take out the culture plate from the incubator, load it onto the luminescence plate reader, and measure luminescence.
   1. Before starting, ensure that the plate reader is turned on. Access the microplate reader software and create a new file by clicking New Session. Set the temperature of the plate reader to 37 °C by clicking Incubator, checking the temperature option, and typing in 37 °C. Under Plate Layout, choose the option Unknown and specify the wells to measure by clicking and dragging.
   2. Under Protocol, click Luminescence and keep to the default settings. Once finished with the setup, load the plate with the lid off into the plate reader and choose Run Plate In. Click Start and save file window will appear. Save the file to your desktop by renaming it and clicking Save.
      NOTE: The luminescence will continue to be produced for >24 h after the addition of the live cell substrate.
4. Once the reading is complete, remove the plate from the plate reader and then click Run Plate In to put the reader back in. Once completed, place the plate back in the humidified incubator (37 °C, 5% CO₂) until the next reading.
5. When finished, transfer the luminescence data to a graphing program to plot relative luminescence unit (RLU) (y-axis) for each variable (e.g., chemicals or concentrations) (x-axis) and analyze the data.

4. Validation of split-Rluc assay with other methods.

1. Perform other assays such as proximity ligation assay (PLA), transmission electron microscopy, and mitochondrial calcium uptake monitoring to validate the results obtained from split-Rluc assay as described previously²⁴.

Results

We have used the protocol described above to measure the level of ER-mitochondria contacts upon the addition of three compounds known to inhibit specific GTPases. CDC42, RHO, and RAC are GTPases that promote actin polymerization²⁸ when activated and are inhibited by ZCL278, Rhosin, and Ehop-016, respectively²⁴. HEK293T cells transfected with split-Rluc were treated with DMSO (control), ZCL278 (50 µM), Rhosin (50 µM), or Ehop-016 (25 µM) and...

Discussion

We have used a split-Renilla luciferase 8 reassembly assay (split-Rluc assay) to quantify the level of ER-mitochondria couplings. In this study, we have modified the original split-Rluc construct²⁴ by generating a single vector, pCAG-MitoRluc^N-T2A-Rluc^CER-IRES-mCherry, encoding each split-Rluc component (MitoRluc^N and Rluc^CER) and a self-cleaving peptide 2A sequence from Thosea a...

Materials

Name	Company	Catalog Number	Comments
1.7 mL SafeSeal Microcentrifuge Tube	Sorenson	16070
6-well plate TC Treated	USA Scientific	CC7682-7506
10 mL Pipette Tips OneTip	USA Scientific	1110-3700
10 μL pipette tips OneTip	USA Scientific	1110-3700
20-200 μL Beveled tips OneTip	USA Scientific	1111-1210
50 mL Polypropylene Conical Tube	Falcon	352070
96-Well Flipper Microtube Racks	ThermoFisher Scientific	8770-11
96-well plate TC Treated	USA Scientific	CC7682-7596
100 mm x 20 mm TC Treated Dish	USA Scientific	CC7682-3394
1250 μL Tips OneTip	USA Scientific	1112-1720
Centrifuge 5910 Ri - Refrigerated Centrifuge	Eppendorf	5943000131
Dimethyl sulfoxide, anhydrous, ≥99.9%	Sigma-Aldrich	276855-100ML
DMEM, high glucose	ThermoFisher Scientific	11965092
DPBS, no calcium, no magnesium	ThermoFisher Scientific	14190144
EHop 016	Bio-Techne Tocris	6248	Dissolve in DMSO; store at -70 °C
EnduRen Live Cell Substrate	Promega	E6481	Store aliquots at -70 °C
Eppendorf 2-20 μL pipette	Eppendorf	3123000039
Eppendorf Research plus 100-1000 μL pipette	Eppendorf	3123000063
Eppendorf Research Plus 1-10 µL pipette	Eppendorf	3123000020
Eppendorf Research plus 12-channel	Eppendorf	3125000028
Eppendorf Research plus 200 μL pipette	Eppendorf	3123000055
Fetal Bovine Serum, qualified, USDA-approved regions	ThermoFisher Scientific	10437028
Forma Steri-Cycle CO2 Incubator, 184 L, Polished Stainless Steel	ThermoFisher Scientific	381
Hand tally counter	Sigma-Aldrich	HS6594
HEK 293T Cells	ATCC	CRL-3216
Hemacytometer - Neubauer Bright Line, Double-Counting Chamber	LW Scientific	CTL-HEMM-GLDR
Invitrogen TE Buffer	ThermoFisher Scientific	8019005
Microscope	Zeiss	Axiovert 25 CFL
Mini centrifuge	Benchmark Scientific	C1012
Multi Tube Rack For 50ml Conical, 15ml Conical, And Microcentrifuge Tubes	Boekel Scientific	120008
PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)	Polysciences	24765-100MG
Pipet-Aid XP	USA Scientific	4440-0101
Poly-D-lysine hydrobromide	Sigma-Aldrich	P6407-5MG
Rhosin hydrochloride	Bio-Techne Tocris	5003	Dissolve in DMSO; store at -70 °C
Trypsin-EDTA (0.05%), phenol red	ThermoFisher Scientific	25300054
Varioskan LUX multimode microplate reader	ThermoFisher Scientific	VL0000D0
Vortex	ThermoFisher Scientific	2215365	level 8
VWR Vacuum Aspiration System	VWR	75870-734
ZCL 278	Bio-Techne Tocris	4794	Dissolve in DMSO; store at -70 °C