The authors are thankful to Dr. Jeffrey Golden (Cedars-Sinai Medical Center) for the critical review of the manuscript. This work was funded in part by the National Institute of Neurological Disease and Stroke (NINDS, R01NS113516).

1. Cell maintenance and seeding (Day 1)
<ol>
	<li>Maintain HEK293T cells in cell culture media containing Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (in 100 mm culture dishes) in a humidified incubator at 37 &#176;C with 5% CO2.</li>
	<li>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&#39;s phosphate-buffered saline (DPBS).</li>
	<li>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.</li>
	<li>Centrifuge at 300 &#215; g for 5 min at room temperature. Remove the media and resuspend the cell pellets with 1 mL of fresh media (DMEM + 10% FBS).</li>
	<li>Count the number of cells using a hemocytometer.
	<ol>
		<li>Ensure even distribution of cells by gently swirling the tube. Immediately take out 10 &#181;L of the cell suspension and place into a 1.7 mL microcentrifuge tube. Add 90 &#181;L of media to the same tube.</li>
		<li>After mixing the cells and media gently, take the cell suspension and apply 10 &#181;L to each of the chambers of the hemocytometer by gently pipetting underneath the coverslip.</li>
		<li>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.</li>
		<li>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&#39;s instructions.</li>
	</ol>
	</li>
	<li>Once cell counting is completed, prepare to plate cells in a 6-well plate at 7.5 &#215; 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.
	<ol>
		<li>Before plating, calculate the number of cells necessary for 7.5 &#215; 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.</li>
	</ol>
	</li>
	<li>After dispensing, tap the plate gently on all sides to spread the cells and then place it in a humidified incubator at 37 &#176;C with 5% CO2 overnight.</li>
</ol>
2. Polyethyleneimine (PEI)-mediated cell transfection and post transfection cell seeding (Day 2)
<ol>
	<li>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.</li>
	<li>Transfect the cells with split-Rluc plasmid DNA (pCAG-MitoRlucN-T2A-RlucCER-IRES-mCherry) dissolved in TE buffer (pH 8.0).
	<ol>
		<li>For each well, mix the DNA (750 ng) and PEI (ratio of DNA/PEI = 1:3) in 200 &#181;L 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 (&#60;3 s).</li>
		<li>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 &#176;C with 5% CO2 for 6 h.</li>
	</ol>
	</li>
	<li>While incubating, prepare a Poly-D-Lysine (PDL)-coated 96-well plate by first adding 70 &#181;L of PDL (50 &#181;g/mL in DPBS) to each well and incubating in a humidified incubator at 37 &#176;C with 5% CO2 for at least 1 h. Remove the PDL solution and then wash each well 2x with 100 &#181;L of DPBS. At the end of the second wash, ensure that all the remaining DPBS is removed.</li>
	<li>At 6 h post transfection, prepare to seed the transfected cells in a PDL-coated 96-well plate.
	<ol>
		<li>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 &#181;L 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.</li>
		<li>After transferring the cells along with media to a 50 mL tube, spin the tube at 300 &#215; 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).</li>
	</ol>
	</li>
	<li>Seed the cells in a PDL-coated 96-well plate at a density of 4 &#215; 104 cells/well in 100 &#181;L 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 &#181;L of the diluted cell suspension into each of the 96 wells. Incubate the cells for 18 h in a humidified incubator at 37 &#176;C with 5% CO2.
	<ol>
		<li>Calculate the number of cells necessary for 4 &#215; 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.</li>
	</ol>
	</li>
</ol>
3. Chemical treatment and luciferase assay in live cells (Day 3)
<ol>
	<li>In a 1.7 mL microfuge tube, prepare the three fresh solutions of 50 &#181;M Rhosin (50 mM stock in DMSO), 25 &#181;M Ehop-016 (25 mM stock in DMSO), and 50 &#181;M 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 &#181;L/well &#215; 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&#160;&#181;M. To make 0.5, 5, and 50 &#181;M ZCL278 solutions, perform serial dilutions using the initial 50 &#181;M 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.</li>
	<li>Remove the media from each well of the 96-well plate containing transfected cells and add 50 &#181;L of chemical and substrate media mixture to each well.</li>
	<li>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.
	<ol>
		<li>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 &#176;C by clicking Incubator, checking the temperature option, and typing in 37 &#176;C. Under Plate Layout, choose the option Unknown and specify the wells to measure by clicking and dragging.</li>
		<li>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 &#62;24 h after the addition of the live cell substrate.</li>
	</ol>
	</li>
	<li>Once the reading is complete, remove&#160;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 &#176;C, 5% CO2) until the next reading.</li>
	<li>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.</li>
</ol>
4. Validation of split-Rluc assay with other methods.
<ol>
	<li>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.</li>
</ol>

A Simple Assay to Measure ER-Mitochondria Contacts for High-Throughput Screens

<ol><li>Aoyama-Ishiwatari, S., Hirabayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endoplasmic+reticulum-mitochondria+contact+sites-emerging+intracellular+signaling+hubs%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Endoplasmic reticulum-mitochondria contact sites-emerging intracellular signaling hubs.</a> Front Cell Dev Biol. 9, 653828(2021).</li>
<li>Sassano, M. L., Felipe-Abrio, B., Agostinis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ER-mitochondria+contact+sites%3B+a+multifaceted+factory+for+Ca2%2B+signaling+and+lipid+transport%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">ER-mitochondria contact sites; a multifaceted factory for Ca2+ signaling and lipid transport.</a> Front Cell Dev Biol. 10, 988014(2022).</li>
<li>Scorrano, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coming+together+to+define+membrane+contact+sites%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Coming together to define membrane contact sites.</a> Nat Commun. 10 (1), 1287(2019).</li>
<li>Voeltz, G. K., Sawyer, E. M., Hajnóczky, G., Prinz, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+the+connection%3A+How+membrane+contact+sites+have+changed+our+view+of+organelle+biology%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Making the connection: How membrane contact sites have changed our view of organelle biology.</a> Cell. 187 (2), 257-270 (2024).</li>
<li>Paillusson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((There%27s+something+wrong+with+my+MAM%3B+the+ER-mitochondria+axis+and+neurodegenerative+diseases%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">There's something wrong with my MAM; the ER-mitochondria axis and neurodegenerative diseases.</a> Trends Neurosci. 39 (3), 146-157 (2016).</li>
<li>Sasi, U. S. S., Sindhu, G., Raj, P. S., Raghu, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+associated+membranes+%28MAMs%29%3A+emerging+drug+targets+for+diabetes%5BTitle%5D)+AND+%22Curr+Med+Chem%22%5BJournal%5D)">Mitochondria associated membranes (MAMs): emerging drug targets for diabetes.</a> Curr Med Chem. 27 (20), 3362-3385 (2019).</li>
<li>Joshi, A. U., Kornfeld, O. S., Mochly-Rosen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+entangled+ER-mitochondrial+axis+as+a+potential+therapeutic+strategy+in+neurodegeneration%3A+A+tangled+duo+unchained%5BTitle%5D)+AND+%22Cell+Calcium%22%5BJournal%5D)">The entangled ER-mitochondrial axis as a potential therapeutic strategy in neurodegeneration: A tangled duo unchained.</a> Cell Calcium. 60 (3), 218-234 (2016).</li>
<li>Moltedo, O., Remondelli, P., Amodio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mitochondria-endoplasmic+reticulum+contacts+and+their+critical+role+in+aging+and+age-associated+diseases%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">The mitochondria-endoplasmic reticulum contacts and their critical role in aging and age-associated diseases.</a> Front Cell Dev Biol. 7, 172(2019).</li>
<li>Rieusset, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+endoplasmic+reticulum-mitochondria+contact+sites+in+the+control+of+glucose+homeostasis%3A+an+update%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">The role of endoplasmic reticulum-mitochondria contact sites in the control of glucose homeostasis: an update.</a> Cell Death Dis. 9 (3), 388(2018).</li>
<li>Bouguerra, M. D., Lalli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ER-mitochondria+interactions%3A+Both+strength+and+weakness+within+cancer+cells%5BTitle%5D)+AND+%22Biochim+Biophys+Acta+Mol+Cell+Res%22%5BJournal%5D)">ER-mitochondria interactions: Both strength and weakness within cancer cells.</a> Biochim Biophys Acta Mol Cell Res. 1866 (4), 650-662 (2019).</li>
<li>Tepikin, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+junctions+with+cellular+organelles%3A+Ca2%2B+signalling+perspective%5BTitle%5D)+AND+%22Pfl%C3%BCgers+Arch%22%5BJournal%5D)">Mitochondrial junctions with cellular organelles: Ca2+ signalling perspective.</a> Pflügers Arch. 470 (8), 1181-1192 (2018).</li>
<li>Masson, G. L., Przedborski, S., Abbott, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+computational+model+of+motor+neuron+degeneration%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A computational model of motor neuron degeneration.</a> Neuron. 83 (4), 975-988 (2014).</li>
<li>Giamogante, F., Barazzuol, L., Brini, M., Calì, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ER-mitochondria+contact+sites+reporters%3A+strengths+and+weaknesses+of+the+available+approaches%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">ER-mitochondria contact sites reporters: strengths and weaknesses of the available approaches.</a> Int J Mol Sci. 21 (21), 8157(2020).</li>
<li>Rizzuto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Close+contacts+with+the+endoplasmic+reticulum+as+determinants+of+mitochondrial+Ca2%2B+responses%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses.</a> Science. 280 (5370), 1763-1766 (1998).</li>
<li>Valm, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applying+systems-level+spectral+imaging+and+analysis+to+reveal+the+organelle+interactome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Applying systems-level spectral imaging and analysis to reveal the organelle interactome.</a> Nature. 546 (7656), 162-167 (2017).</li>
<li>Csordás, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+interorganelle+contacts+and+local+calcium+dynamics+at+the+ER-mitochondrial+interface%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface.</a> Mol Cell. 39 (1), 121-132 (2010).</li>
<li>Cieri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SPLICS%3A+a+split+green+fluorescent+protein-based+contact+site+sensor+for+narrow+and+wide+heterotypic+organelle+juxtaposition%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition.</a> Cell Death Differ. 25 (6), 1131-1145 (2018).</li>
<li>Kakimoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+multiple+inter-organelle+contact+sites+using+the+organelle-+targeted+split-GFP+system%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Visualizing multiple inter-organelle contact sites using the organelle- targeted split-GFP system.</a> Sci Rep. 8 (1), 6175(2018).</li>
<li>Wu, M. M., Covington, E. D., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+analysis+of+diffusion+and+trapping+of+STIM1+and+Orai1+at+ER-plasma+membrane+junctions%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at ER-plasma membrane junctions.</a> Mol Biol Cell. 25 (22), Mac.E14-06-1107 (2014).</li>
<li>Csordas, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+functional+features+and+significance+of+the+physical+linkage+between+ER+and+mitochondria%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Structural and functional features and significance of the physical linkage between ER and mitochondria.</a> J Cell Biol. 174 (7), 915-921 (2006).</li>
<li>de Brito, O. M., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitofusin+2+tethers+endoplasmic+reticulum+to+mitochondria%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mitofusin 2 tethers endoplasmic reticulum to mitochondria.</a> Nature. 456 (7222), 605-610 (2008).</li>
<li>Kremer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developing+3D+SEM+in+a+broad+biological+context%5BTitle%5D)+AND+%22J+Microsc%22%5BJournal%5D)">Developing 3D SEM in a broad biological context.</a> J Microsc. 259 (2), 80-96 (2015).</li>
<li>Söderberg, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nat Methods. 3 (12), 995-1000 (2006).</li>
<li>Lim, Y., Cho, I. -T., Rennke, H. G., Cho, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B22-adrenergic+receptor+regulates+ER-mitochondria+contacts%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">β2-adrenergic receptor regulates ER-mitochondria contacts.</a> Sci Rep. 11 (1), 21477(2021).</li>
<li>Lam, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+evolution+of+APEX2+for+electron+microscopy+and+proximity+labeling%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Directed evolution of APEX2 for electron microscopy and proximity labeling.</a> Nat Methods. 12 (1), 51-54 (2014).</li>
<li>Cho, I. -T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ascorbate+peroxidase+proximity+labeling+coupled+with+biochemical+fractionation+identifies+promoters+of+endoplasmic+reticulum-mitochondrial+contacts%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum-mitochondrial contacts.</a> J Biol Chem. 292 (39), 16382-16392 (2017).</li>
<li>Lim, Y., Cho, I. -T., Schoel, L. J., Cho, G., Golden, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hereditary+spastic+paraplegia-linked+REEP1+modulates+endoplasmic+reticulum%2Fmitochondria+contacts%5BTitle%5D)+AND+%22Ann+Neurol%22%5BJournal%5D)">Hereditary spastic paraplegia-linked REEP1 modulates endoplasmic reticulum/mitochondria contacts.</a> Ann Neurol. 78 (5), 679-696 (2015).</li>
<li>Arnold, T. R., Stephenson, R. E., Miller, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rho+GTPases+and+actomyosin%3A+Partners+in+regulating+epithelial+cell-cell+junction+structure+and+function%5BTitle%5D)+AND+%22Exp+Cell+Res%22%5BJournal%5D)">Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function.</a> Exp Cell Res. 358 (1), 20-30 (2017).</li>
</ol>

The authors declare no conflicts of interest.

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-MitoRlucN-T2A-RlucCER-IRES-mCherry, encoding each split-Rluc component (MitoRlucN and RlucCER) and a self-cleaving peptide 2A sequence from Thosea asigna virus to ensure the expression of the same amount of each split half of luciferase. As a substrate for the Renilla luciferase, instead of coelenterazine, a modified live cell substrate (protected derivative of coelenterazine; see the Table of Materials) was used for the following reasons. First, unlike coelenterazine which generates autoluminescence in an aqueous environment (especially worse in serum), this protected derivative generates very low autoluminescence (often not detectable) in medium containing 10% serum, thus its signal-to-background ratio is higher than that generated by coelenterazine. Second, differently from coelenterazine, where luminescence is the strongest shortly after adding the substrate but begins to diminish very quickly, luminescence produced by the modified live substrate arrives at its highest point attainable approximately 1.5 h after substrate addition and stays&#160;continuous for more than 24 h. These changes in reaction kinetics make it easier to measure luminescence in a multi-well plate, which is very useful for high-throughput screening. Furthermore, the luminescence produced by the protected substrate remaining constant in the long run allows for the measuring of a single set of samples at multiple time points, as opposed to measuring multiple sets of samples used at various time points (single point per sample).
Using this updated version of the split-Rluc construct, we tested if RHO, RAC, and CDC42 GTPases participate in ER-Mito contact formation. We added GTPase inhibitors, Rhosin (RHO inhibitor), Ehop-016 (RAC inhibitor), and ZCL278 (CDC42 inhibitor), to HEK293T cells expressing split-Rluc components. Our assay results indicate that ZCL278 is the most potent inhibitor of ER-mitochondria contact formation and Ehop-016 is the next significant inhibitor, while Rhosin did not affect their contacts. These data suggest that CDC42-mediated actin polymerization (as well as RAC-mediated one to a lesser extent) is required to maintain ER-mitochondria contacts in the cell, while RHO-mediated actin polymerization is probably not important.
To ensure maximum efficiency, there are many critical steps that must be considered for this protocol. First and foremost, the amount of construct transfected must be tested as too much DNA can have a toxic effect on the cells. Second, as the number of cells can have a significant impact on the luciferase levels measured, keeping the density of cells as close as what was stated in the protocol is crucial in ensuring the ideal and consistent 80% confluency of cells by the time of the assay. There may be a reduction in luminescence if the cells reach confluency during the chemical exposure time, as confluent cells make less protein in general. Third, to ensure the cells do not slough off during the addition of chemicals and substrate, we use a 96-well plate pre-coated with Poly-D-Lysine. Fourth, for the compounds (including the substrate) used in this protocol, it is important to keep them at -20 &#176;C or lower (-70 &#176;C) in aliquots and add them to the cells immediately thawed. Lastly, due to possible temperature fluctuations that can affect luciferase activity, it may be best to choose to use the innermost wells of the plate and ignore the wells lining the outside of the plate.
The split-Rluc8 assay offers an easy yet robust method to determine ER-mitochondria contacts, ideal for high-throughput screening; however, there are some limitations that need to be noted. For cell physiology, there is the issue of changes in cell behavior due to the constant overexpression of the construct DNA. Currently, we are working on a construct with an inducible promoter to combat the potential changes in cell physiology due to constant overexpression. Further, when this assay is used for a screening purpose, it should be noted that false positive results can arise from any indirect effect of the drugs on i) expression levels, ii) organelle-targeting efficiencies of the said protein fragments, or iii) enzymatic activities of the reconstituted luciferases. Due to these limitations, this assay must be tested in conjunction with other methods to further validate results. Table 1 refers to various methods of testing these contact sites along with their advantages and disadvantages. We have taken advantage of other methods, including but not limited to proximity ligation assays (PLA), which detect proximity between proteins, and transmission electron microscopy (TEM), which allows for clear visualization of organelle contacts, in conjunction with the split-Rluc assay to validate that the resulting changes in RLU are indeed due to changes in ER-mitochondria contact sites (Figure 4 and Lim et al.24). We have also shown a correlation between alteration in ER-mitochondria contacts and functional parameters such as calcium transfer and mitochondrial fission (Figure 4 and Lim et al.24). To ensure the effect of the drugs on split-Rluc activity is not due to increased expression of split-Rluc gene, a transcription or translation inhibitor can be added together with the drugs as we have done previously24.
For the application of the split-Rluc system in differentiated neurons in culture where the transfection rate is low, viral infection (with lentivirus expressing split-Rluc components) would be an alternative approach. Its use can be extended to create a screening system where the cells (e.g., human induced pluripotent stem cells) are engineered to stably express each component of the split-Rluc, MitoRlucN and RlucCER, either by viral infection or by CRSPR-mediated gene editing. However, for a stable expression system, one must be aware that there could be a selection pressure for cells tolerating the split-Rluc system better, which might not be representative of the normal physiological state. This adaptation or selection could introduce bias in the experimental outcomes.
In summary, despite some of the limitations discussed above, split-Rluc is a fast, straightforward, and powerful assay for high-throughput screening for small molecules or genes regulating the inter-organelle contacts in the cell, as it has been shown in our previous drug screening study24.

The interactions between the ER and the mitochondria are vital for cellular homeostasis and survival1,2,3,4. 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 cancer5,6,7,8,9,10. 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&#39;s disease5,11. Similarly, reduced ER-mitochondria contacts can result in a decrease in ATP production&#160;and impairment of Ca2+ intake, as seen in models of amyotrophic lateral sclerosis5,11,12. 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 sites3,4,13. The approaches to monitor ER-mitochondria coupling include fluorescence marker-based imaging14,15, FRET-reporter-based imaging16, and split-fluorescence-probe-based imaging17,18, 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 approaches20,21,22. However, these EM-based methods are a very low-throughput technique that can also be affected by chemical &#64257;xation 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 proximities23,24, while a revised version of the ascorbate peroxidase (APEX) assay has been utilized in identifying new contact-site proteins25,26. 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&#39; 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&#39; 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.

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

split-luciferase reassembly assay to measure endoplasmic reticulum-mitochondria contacts in live cells

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&#39;s disease, Parkinson&#39;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&#160;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.

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&#160;when activated&#160;and are inhibited by ZCL278, Rhosin, and Ehop-016, respectively24. HEK293T cells transfected with split-Rluc were treated with DMSO (control), ZCL278 (50 &#181;M), Rhosin (50 &#181;M), or Ehop-016 (25 &#181;M) and incubated for 1 h, 2 h, and 5 h. Using a plate reader, we measured split-Rluc activity at defined time points (Figure 2).
Rhosin (RHO GTPase inhibitor)-treated cells showed no significant changes in luciferase activity compared to the control DMSO-treated cells at 1 h, 2 h, and 5 h of the treatment (p &#62; 0.9999, p= 0.6956, p &#62; 0.9999) (Figure 2). These results demonstrate that Rhosin does not affect ER-mitochondria contacts, suggesting no involvement of RHO GTPase activity in regulating the contacts between the two organelles.
In contrast, the RAC GTPase inhibitor Ehop-016 showed significantly lower luciferase activity than DMSO at 1 h, 2 h, and 5 h of the treatment (p = 0.0106, p = 0.0009, p = 0.0024) (Figure 2). These data indicate that RAC GTPase activity is required for maintaining normal ER-mitochondria interaction in the cell.
Finally, CDC42 inhibitor ZCL278 showed the most drastic changes in luciferase activity (Figure 2). ZCL278-treated cells showed a significant decrease in luciferase activity compared to the control DMSO and had the lowest p-values through all three time points, maintaining a p &#60; 0.0001 throughout. Our data demonstrate a strong inhibition of ER-mitochondria coupling when CDC42 GTPase activity is blocked by ZCL278, consistent with our previous report using the original split-Rluc assay performed in cells co-transfected with two vectors encoding each half of the split-luciferase separately24.
Given the striking results with ZCL278, especially when compared to the other GTPase inhibitors, ZCL278 activity was further tested at various concentrations [0.5 &#181;M, 5 &#181;M, 50 &#181;M] against a DMSO control. Luciferase activities measured at 1 h, 2 h, and 5 h post treatment showed a dose-dependent response; 0.5 &#181;M of ZCL278 resulted in the weakest effect on luciferase activity, while 5 &#181;M showed stronger, and 50 &#181;M showed the strongest reduction in reconstituted enzyme activity (Figure 3). Together, our results show that ZCL278 downregulates split-Rluc activity in a dose-dependent manner and, as time progresses, higher concentrations of ZCL278 (CDC42 inhibitor) still show a significant change relative to the control DMSO as compared to lower concentrations.
As a way to validate our split-Rluc assay, well-established independent assays, such as proximity ligation assay (PLA), transmission electron microscopy (TEM), and mitochondrial calcium uptake monitoring, can be performed (Figure 4 and data not shown)24. Isoproterenol, a &#946;2 adrenergic receptor agonist, which is one of the compounds identified in our screen for ER-mitochondria contact promoters (Figure 4A), was used to confirm that the luciferase activity of the split-Rluc upon isoproterenol treatment was increased due to enhanced ER-mitochondria contacts as demonstrated by changes in PLA signal intensity as well as mitochondrial calcium uptake (Figure 4B-E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66862/66862fig01.jpg" /> 
Figure 1: The split-Rluc reassembly assay. (A) Schematic representation of the Split-RLuc assay. Where there are no membrane contacts between the mitochondria and the endoplasmic reticulum, the Mito-RlucN and the RlucC-ER fail to interact and thus there is no Rluc8 activity. However, interaction between the Mito-RlucN and the RlucC-ER, where the ER and mitochondria membranes come into contact, results in full enzymatic activity, which is detected by the conversion of the substrate to luminescence. (B) Schematic map of the split-Rluc DNA construct (pCAG-MitoRlucN-T2A-RlucCER-IRES-mCherry). (C) Diagram of workflow for split-Rluc assay in HEK293T cells. Abbreviations: Mito = mitochondria; ER = endoplasmic reticulum; IRES, internal ribosomal entry site; rb_glob pA, rabbit beta-globin poly adenylation signal; T2A, a self-cleaving peptide 2A sequence from Thosea asigna virus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66862/66862fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66862/66862fig02.jpg" /> 
Figure 2: The split-Rluc activities corresponding to the levels of ER-Mito contacts in the cells treated with GTPase inhibitors. (A-C) Reconstituted luciferase activities at (A) 1 h, (B) 2 h, or (C) 5 h after DMSO (control), Rhosin [50 &#181;M], Ehop-016 [25 &#181;M], or ZCL278 [50 &#181;M] treatment (n = 12 for each group). All data were analyzed using one-way ANOVA with multiple comparisons. P values are reported as p &#62; 0.05 (ns), p &#8804; 0.05 (*), p &#8804; 0.01 (**), p &#8804; 0.001 (***), p &#8804; 0.0001 (****). Error bars indicate mean &#177; standard error of measurement. Abbreviations: Mito = mitochondria; ER = endoplasmic reticulum; RLU = relative light unit. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66862/66862fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66862/66862fig03.jpg" /> 
Figure 3: The split-Rluc activities measuring the levels of ER-Mito contacts in the cells treated with ZCL278. (A-C) Reconstituted luciferase activities at (A) 1 h, (B) 2 h, or (C) 5 h after DMSO (control) or ZCL278 at [0.5 &#181;M], [5 &#181;M], or [50 &#181;M] concentration (n = 12 for each group). All data were analyzed using one-way ANOVA with multiple comparisons. P values are reported as p &#62; 0.05 (ns), p &#8804; 0.05 (*), p &#8804; 0.01 (**), p &#8804; 0.001 (***), p &#8804; 0.0001 (****). Error bars indicate mean &#177; standard error of measurement. Abbreviations: Mito = mitochondria; ER = endoplasmic reticulum; RLU = relative light unit. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66862/66862fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66862/66862fig04.jpg" /> 
Figure 4: Validation of split-Rluc assay. (A) An example of quantification of split-Rluc activity after treating HEK293T cells with 11 drugs, including isoproterenol, identified in a compound screen for ER-mitochondria contact promoters. (B) An example of proximity ligation assay in HeLa cells that were treated with DMSO or isoproterenol (1 &#181;M). PLA signal (red) indicates a close contact between ER and Mito. Sec61-GFP (green): to label ER; DAPI (blue): nucleus. (C) Quantification of PLA signal in A (n = 4 experiments with 77-97 cells each; Mann-Whitney test). (D) Mitochondrial Calcium uptake in response to histamine (100 &#181;M). Mito-R-GECO1 fluorescent intensity change (average was taken over multiple HeLa cells) plotted every 2.5 s in DMSO- or isoproterenol-treated cells. Arrow indicates histamine addition. (E) Left: Maximum peak of mitochondrial calcium uptake in A. Unpaired two-tailed t-test (n = 10, DMOS; 14, Isoproterenol). Right: Mitochondrial calcium uptake rate (as in A; 7.5 to 12.5 s). Mann-Whitney test (n = 12, DMSO; 11, Isoproterenol). This figure is&#160;modified from Lim et al.24. Abbreviations: RLU = relative light unit; PLA = proximity ligation assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66862/66862fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Summary of current techniques for ER-mitochondria contacts. List of advantages and limitations for each method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66862/66862_R2_updated_Table1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental File 1: Zip file containing DNA sequence (PDF and GBK formats) of pCAG-MitoRlucN-T2A-RlucCER-IRES-mCherry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66862/66862_R3_plasmidDNAsequence.zip" target="_blank">Please click here to download this File.</a>
Supplemental Figure S1: Map of pCAG-MitoRlucN-T2A-RlucCER-IRES-mCherry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66862/66862_Supplemental Figure 1_PlasmidMap_PrimerSequence_.zip" target="_blank">Please click here to download this File.</a>

Author Spotlight: Regulation and Dysregulation of ER-Mitochondria Contacts &#8212; Implications for Neurodegenerative Disease Pathogenesis

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.

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

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

split-luciferase-reassembly-assay-to-measure-endoplasmic-reticulum

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.3K Views.  Cedars-Sinai Medical Center. In mijn lab bestuderen we interorganische communicatie die een vorm van membraancontact aanneemt en hun rol bij het handhaven van de cellulaire gezondheid, evenals bij de pathogenese van ziekten. We zijn vooral geïnteresseerd in membraancontacten tussen mitochondriën en endoplasmatisch reticulum en proberen te begrijpen hoe ze worden gereguleerd en hoe hun ontregeling kan leiden tot neurodegeneratie. Hoewel we weten dat contacten met ER-mitochondriën belan...

Video: Auteur in de schijnwerpers: regulatie en ontregeling van ER-mitochondriëncontacten - implicaties voor de pathogenese van neurodegeneratieve ziekten

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Biology

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

CRAF-14-3-3 Interaction Analysis in Live Cells Using a BRET-Based Approach

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous complexes formed by nucleotide sugar transporters (NSTs). These ER- and Golgi-resident multitransmembrane proteins carry the cytoplasmically synthesized nucleotide sugars across organelle membranes to supply enzymes that mediate glycosylation with their substrates. NSTs exist as dimers and/or higher oligomers. Heterologous interactions between&#160;different NSTs have also been reported. To verify whether the technique is suitable for studying the phenomenon of NST heteromerization, we tested it against a combination of the two Golgi-resident NSTs that have been previously shown to associate by several other means. The&#160;luciferase complementation assay appears to be particularly suitable for studying interactions between Golgi-resident membrane proteins, as it does not require high expression levels, which often trigger protein mislocalization and increase the risk of false positives.

The&#160;protocol presented here is intended to demonstrate the occurrence of heterologous interactions between Golgi-resident type III membrane proteins with cytoplasmically exposed N- and/or C-termini in live mammalian cells using the most recent variant of the split luciferase complementation assay.

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (&gt;40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

Split Luciferase Complementation Assay to Identify Specific Protein-Protein Interactions

Source: Wiertelak, W. et.al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61669">Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay.</a> J. Vis. Exp. (2020)
This video demonstrates the split luciferase complementation assay to detect protein-protein interactions. In this assay, the proteins of interest are tagged to small and large fragments of the luciferase enzyme. When the proteins interact, the large and small fragments combine to form an active enzyme complex, which in the presence of a specific substrate, releases bright luminescence that can be measured.

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

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