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

176 visualizações. Para começar, mantenha HEK293T células em meios de cultura contendo DMEM com 10% de FBS em uma incubadora umidificada. Verifique a confluência da placa ao microscópio. Quando as células atingirem aproximadamente 80 a 90% de confluência, remova o meio e lave-os com 10 mililitros de DPBS.Em seguida, remova o DPBS da placa de cultura. Trate as células com um mililitro de vermelho de fenol 0,05% tripsina-EDTA e incube as células por três minutos a...

Vídeo: Manutenção e semeadura de células HEK293T para transfecção de plasmídeo Split-Rluc

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 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.

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

Polyethyleneimine (PEI)-Mediated HEK293T Cell Transfection and Post-Transfection Cell Seeding

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 transfecting the cells with split R-loop plasmid DNA, mix the DNA and PEI in 200 microliters of DMEM in a 1.7-milliliter microcentrifuge 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 300 G for five minutes in a tabletop centrifuge. Aspirate the media and resuspend 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 multichannel 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.

Chemical Treatment and Luciferase Assay to Measure the Level of ER-Mitochondria Contacts in Live Cells

To begin, seed split our loop transfected HEK293 T 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 II.Add live cell substrate for renilla luciferase, diluted 1 to 2, 000, to a final concentration of 30 millimolar to each solution. To make 0.55 and 50 micromolar ZCL278 solutions, perform serial dilutions 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.