maintaining and seeding hek293t cells for split-rluc plasmid transfection

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

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.

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

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

In my lab, we study interorganal communication that takes a form of membrane contact and their roles in maintaining cellular health, as well as in disease pathogenesis. We're particularly interested in membrane contacts between mitochondria and endoplasmic reticulum and trying to understand how they're regulated and how their dysregulation can lead to neurodegeneration. Although we know that ER mitochondria contacts are important for cell health and 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 mitochondria contacts in cells. Our split luciferase assay offers an easier and faster way to quantify ER mitochondria 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 mitochondria membranes have close contact, which results in reconstituted luciferase with a full enzyme activity. This method is simple, suitable for high throughput screenings, and easily accessible for many labs. We hope that it will advance the neurodegenerative disease field by identifying molecular and genetic modulators of ER mitochondria 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 mitochondria 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 mitochondria contacts, and find ways to combat related diseases.

Maintaining and Seeding HEK293T Cells for Split-Rluc Plasmid Transfection

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, resuspend 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.

maintaining-seeding-hek293t-cells-for-split-rluc-plasmid

Research

JoVE Journal

Biochemistry

33 Views. 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...