Thomas Machleidt on exploring new applications for bioluminescence
Over the past three decades, bioluminescence has emerged as one of the most broadly used reporter technologies in life science research. For the first two decades, the use of bioluminescence was largely dominated by ATP-dependent luciferase, such as firefly luciferase. The remarkable breadth of tools derived from this family of luciferases was predicated on its biophysical and chemical properties and can be divided in four principal assay categories: (a) transcriptional reporters for pathway analysis (b) viability detectors based on measurement of cellular ATP levels (c) pro-luciferins for analysis of enzyme activity (d) luciferase based biosensors for measuring physiological processes (Fig. 1).
Despite the tremendous value firefly luciferase brought to life science, its utility proved limited by some of its intrinsic features, including the relatively low specific signal, large size, complex structure and ATP dependency.
To overcome these liabilities, Promega developed a novel luciferase system called NanoLuc, which was originally derived from a bright, hetero-tetrameric luciferase isolated from the deep-sea shrimp Oplophorus gracilirostris. Within the enzyme complex, the 19kD sub-unit was found to be associated with luciferase activity. Though rather dim and unstable in its wildtype form, scientists were able to improve specific activity over two million-fold by synergistically combining directed evolution and substrate development. The resulting luciferase produces a glow-type luminescence with a specific activity 100 times higher than firefly luciferase. Furthermore, the enzyme is small and thermostable up to 55°C and shows no selective partitioning or post-translational modifications in mammalian cells. Although NanoLuc was originally conceived as an improved transcriptional reporter, it soon became evident that it provided the foundation for expanding bioluminescence into entirely new applications in life sciences. Building on the strength of the system, scientists further engineered two binary reporter systems, NanoBiT and HiBiT. Both systems consist of a small peptide (SmBiT or HiBiT) that bind to LgBiT, a truncated version of NanoLuc. NanoBiT, a low-affinity complementation system, has proved valuable for the analysis of protein-protein interactions (PPI). In contrast, HiBiT represents a high-affinity complementation system that allows sensitive detection of tagged proteins and is widely used in the field of targeted protein degradation. Furthermore, combining NanoLuc with suitable fluorophores enables development of bioluminescence resonance energy transfer (BRET)-based assay for quantitative analysis of PPI and protein-specific small molecule target engagement in living cells.
The utility of NanoLuc technologies is illustrated here using imaging and protein translocation as examples.
Bioluminescence is a well-established imaging modality for small animal imaging, but it has not been used widely in the past for microscopy for several reasons. First and foremost, low light imaging requires long exposure time and/or acquisition strategies such as image stacking, which leads to comparatively poor temporal and spatial resolution, especially in living cells. In addition, the demands of low light detection require the use of specialised and costly equipment.
On the other hand, using bioluminescence for microscopy provides significant benefits. The absence of an exogenous light source eliminates issues with phototoxicity and photobleaching. Bioluminescence is also mostly devoid of sample-associated background, which translates into large dynamic range and high sensitivity. These are critical features for detection and analysis of low-expressing endogenously tagged proteins.
In this context, it should be emphasised that bioluminescence imaging (BLI) should be viewed as complementary to fluorescence microscopy. Although it cannot provide the same spatial or temporal resolution, it offers the opportunity to make full use of the toolbox of bioluminescence technologies and assays for functional imaging. It is also anticipated that advances in detector technologies are poised to make BLI more accessible, as demonstrated by the recent development of photon counting gCMOS cameras. The following examples illustrate how BLI can be applied in discovery and assay development.
CRISPR-mediated tagging of endogenous proteins with peptide tags (e.g. HiBiT) or reporters (e.g. NanoLuc) has proven extremely valuable for the interrogation of proteins in the appropriate physiological context. However, addition of peptide tags or reporters has the potential to impact protein function. Subcellular localisation is one of the indicators of protein function that can be rapidly assessed for HiBiT- or NanoLuc-tagged protein in living cells by BLI without the need for protein-specific antibodies and labour-intensive staining protocols. Furthermore, the ability to conduct BLI in living cells allows analysis of protein translocation as readouts for biological events.
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 which is critical for fully capturing the complexity of these interactions that govern protein function at a subcellular level.
Bioluminescence is widely used to measure key physiological measures, including cell proliferation, apoptosis and other aspects of cell health. This 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.
These examples illustrate how bioluminescence provides a complementary approach to fluorescence for functional imaging.
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. The 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.
The 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. The increase in signal following treatment is indicative of the kinetics and magnitude of the translocation event. This 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. Third, 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.
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. The 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. The future is certainly bright for bioluminescence and those who make use of it.
Thomas Machleidt, PhD is director Research-Advanced Technologies, Promega