Name: Video: Maintaining and Seeding HEK293T Cells for Split-Rluc Plasmid Transfection
Uploaded: 2024-10-11T13:10:00
Description: 164 Views. Maintaining and Seeding HEK293T Cells for Split-Rluc Plasmid Transfection

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

Maintaining and Seeding HEK293T Cells for Split-Rluc Plasmid Transfection

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

Research

JoVE Journal

Biochemistry

164 Views. Maintaining and Seeding HEK293T Cells for Split-Rluc Plasmid Transfection

Video: Maintaining and Seeding HEK293T Cells for Split-Rluc Plasmid Transfection

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 ...