MICROSCOPY & IMAGING
of protein translocation as readouts for biological events.
MOLECULAR INTERACTIONS Both protein complementation (NanoBiT) and BRET (NanoBRET) have been widely used for measurement of interactions between proteins and proteins and small molecules. BLI allows full spatio- temporal analysis
Fig. 5. Workflow of HiBiT translocation assay
which is critical for fully capturing the complexity of these interactions that govern protein function at a subcellular level.
POPULATION ANALYSIS Bioluminescence is widely used to measure key physiological measures, including cell proliferation, apoptosis and other aspects of cell health. Tis type of assay is usually performed in microwell plates for convenience and throughput. Capturing the functional responses on a cellular level provides valuable insights into the temporal distribution and amplitude of the response at the level of individual cells. Plate-based cytometry could be particularly valuable for the study of physiological changes in complex cell models that include different cell types. Tese examples illustrate
how bioluminescence provides a complementary approach to fluorescence for functional imaging.
Fig. 6. PKC - translocation to the plasma membrane. HeLa cells expressing PKCa-HiBiT and Membrane LocalBiT were treated with phorbol 12 myristate 13 acetate (PMA) which induced translocation of PKC - from the cytosol to the plasma membrane.
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TRANSLOCATION OF ENDOGENOUS PROTEINS Protein translocation is one of the principal mechanisms by which protein function and activity is controlled. Analysis of translocation is usually done by imaging, which, though undoubtedly providing valuable insights into protein movement, is a time- and resource-intensive method. Scientists recently showed proof of concept for a novel approach that would allow real-time measurement of translocation of endogenous protein in a plate-based format. Te technology consists of the protein of interest endogenously tagged with HiBiT in the cell line of choice and a co-expressed biosensor (LocalBiT) that comprises of a novel version of LgBiT with reduced affinity for HiBiT and a compartment targeting domain. Translocation of HiBit-tagged protein into the target compartment would result in complementation between HiBiT and LocalBiT, producing increased light output. Te underlying workflow is relatively straightforward (Fig. 5): a cell line expressing both LocalBiT and the HiBiT- tagged protein of interest are plated in a multi-well plate in the presence of NanoLuc substrate. After the addition of a translocation-inducing stimulus, the plate is placed in a plate reader to measure changes in light output. Fig. 6 shows an example for the translocation of endogenous protein kinase C alpha tagged with HiBiT from the cytosol to the plasma membrane following treatment with the phorbol ester
PMA. Te increase in signal following treatment is indicative of the kinetics and magnitude of the translocation event. Tis approach offers several advantages over standard imaging-based methods. First, the high sensitivity of HiBiT tagging allows detection at endogenous protein levels, which is important because preserving the original physiology of a cell is key for getting meaningful answers in biological inquiry. Second, it enables real-time analysis without limitations imposed by fluorescence microscopy, such as phototoxicity and photobleaching. Tird, it is suitable for both adherent and suspension cells. Lastly, the workflow is significantly easier to configure for automation and high throughput screening compared to microscopy.
CONCLUSION Since firefly luciferase was first used in life science around 30 years ago, bioluminescent technology has undergone tremendous advances that allowed its application in novel areas within life science, including drug discovery and diagnostics. Te examples shown in this article aim to further illustrate how continuous improvement and expansion of the bioluminescence toolbox enables novel applications in life science. For example, homogenous bioluminescent assays have been used to measure the interaction between SARS- CoV-2 spike protein receptor binding domain and human angiotensin-converting enzyme 2 (ACE2). In water testing, monitoring ATP in cooling water is relied on to help prevent Legionella outbreaks. Te future is certainly bright for bioluminescence and those who make use of it.
Thomas Machleidt, PhD is director, Research-Advanced Technologies, Promega.
www.promega.co.uk
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