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

In my lab, we study interorganelle communication that takes the form of membrane contact and their roles in maintaining cellular health, as well as in disease pathogenesis. We are particularly interested in membrane contacts between mitochondria and the endoplasmic reticulum and trying to understand how they are regulated and how their dysregulation can lead to neurodegeneration. Although we know that ER mitochondrial contacts are important for cell health and that their dysregulation is associated with many diseases, we do not know much about how their level is regulated.

The first step to address this is to establish a simple, reliable protocol to measure the level of ER mitochondrial contacts in cells. Our split luciferase assay offers an easier and faster way to quantify ER mitochondrial contacts in live cells compared to most assays. We take advantage of two split halves of renilla luciferase, one half targeted to ER, and the other, to mitochondria.

They can be reassembled together only where the ER and mitochondrial membranes have close contact, which results in reconstituted luciferase with full enzymatic activity. This method is simple, suitable for high-throughput screening, and easily accessible for many labs. We hope that it will advance the neurodegenerative disease field by identifying molecular and genetic modulators of ER mitochondrial contacts that can be further developed into therapeutic agents.

Our results have paved the way for important questions, such as how do different drugs or drug combinations affect ER mitochondrial contacts, and what are the important molecular players behind these contacts? From these questions, we can gain a better understanding of the mechanism of ER mitochondrial contacts and find ways to combat related diseases. To begin, maintain HEK293T cells in culture media containing DMEM with 10%FBS in a humidified incubator.

Check the plate's confluency under a microscope. When cells reach approximately 80 to 90%confluency, remove the media and wash them with 10 milliliters of DPBS. Then remove the DPBS from the culture dish.

Treat the cells with one milliliter of 0.05%trypsin-EDTA, phenol red, and incubate the cells for three minutes at 37 degrees Celsius. After removing the plate from the incubator, add nine milliliters of culture media to stop the trypsin reaction and mix by pipetting to dislodge any remaining cells. Now, transfer the cell suspension to a 50-milliliter tube.

Centrifuge at 300g for five minutes at room temperature. Once the media is removed, re-suspend the cell pellets in one milliliter of fresh media. To plate cells in a six well plate, combine the necessary volume of cells with 13 milliliters of media in a 50 milliliter tube.

Then dispense two milliliters of the diluted cell suspension into each of the six wells, and gently tap the plate on all sides to spread the cells evenly. Afterward, place the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide overnight. To begin, take cultured HEK293T from the incubator.

Aspirate out the existing culture media from each well, and add two milliliters of fresh media per well. For transecting the cells with split R loop plasma DNA, mix the DNA and PEI in 200 microliters of DMEM in a 1.7 milliliter micro centrifuge tube for each well. Quickly vortex the DNA and PEI at level eight for approximately two seconds to ensure they mix well to form a complex, then spin it down briefly in a mini-centrifuge.

After incubation, drop the mixture onto the surface of the culture media in the six well plate containing cells. Tap the plate gently to evenly distribute the mixture, and incubate it in a humidified incubator at 37 degrees Celsius, with 5%carbon dioxide for six hours. Afterward, remove the six well plate from the incubator.

Aspirate out the media and wash each well with two milliliters of DPBS. Once the DPBS is removed, add 350 microliters of 0.05%trypsin-EDTA phenol red. At the end of the incubation, add 1.7 milliliters of media to each well to stop the trypsin reaction.

Now, transfer the cells along with media to a 50-milliliter tube and spin at 300g for five minutes in a tabletop centrifuge. Aspirate the media and re-suspend the cell pellet with one milliliter of fresh media. To seed the cells in a Poly-D-lysine coated 96 well plate, add the necessary volume of cells in a 50 milliliter tube, along with the appropriate volume of media.

Using a multi-channel pipette, dispense 100 microliters of the diluted cell suspension into each of the 96 wells. Incubate the cells for 18 hours at 37 degrees Celsius with 5%carbon dioxide. To begin, seed split R-loop transfected HEK293T cells in a Poly-D-lysine coated 96 well plate.

Next, prepare three fresh solutions of rhosin, Ehop-016 and ZCL278 in a 1.7 milliliter microfuge 2. Add live cell substrate for vanilla luciferase, diluted 1 to 2, 000 to a final concentration of 30 millimolar to each solution. To make 0.5, 5, and 50 micromolar ZCL278 solutions, perform serial dilution using the initial 50 micromolar ZCL278 in culture media.

Then remove the media from each well of the 96 well plate containing transfected cells. Add 50 microliters of the chemical and substrate media mixture to each well. After incubating the cells for one hour, two hours, or five hours, load the plate onto the luminescence plate reader and measure luminescence.

Ensure the plate reader is turned on before starting. Access the Microplate Reader Software and click New Session to create a new file. Click Incubator to set the plate reader's temperature to 37 degrees Celsius.

Check the temperature option and type 37 degrees Celsius. Under plate layout, choose the option Unknown, and click and drag the well to specify the wells to measure. Then go to Protocol, click Luminescence, and keep the default settings.

Once the setup is complete, load the plate with the lid off into the plate reader, and choose Run plate in. Click Start and a Save File window will appear. Rename the file and click Save.

Once the reading is complete, remove the plate from the plate reader and then click the Run Plate in Option to put the reader back in. Once completed, place the plate back in the humidified incubator until the next reading. At one, two, and five hours of the treatment, rhosin-treated cells showed no significant changes in luciferase activity compared to control DMSO-treated cells.

The Rac GTPase inhibitor EHop-016 showed significantly lower luciferase activity than DMSO. ZCL278-treated cells had significantly lower luciferase activity than the control DMSO at all three time points. Luciferase activities measured at one, two, and five hours post-treatment showed a dose-dependent response.

Higher concentrations of ZCL278 still showed significant changes compared to the control DMSO.