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 a....

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 washin.......

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