Author: Kelly Grooms

Live-Cell Imaging: It’s Time to See What Else Your Luminescence Assays Can Tell You

Posted on by Kelly Grooms
luminescent cells behind a molecular structure

Luminescent live-cell assays are powerful tools for cellular biology research. They offer both qualitative and quantitative insights into processes such as gene expression, cell viability, metabolic activity, protein and small molecule interactions, and targeted protein degradation. But what if you could go beyond the numbers and actually see what is happening in your cells? With luminescent imaging, you have the opportunity to uncover more dynamic data by visualizing what happens with your cells in real time.

Why Luminescent Imaging?

Bioluminescent reporters such as NanoLuc® luciferase are well-suited for bioluminescent imaging. NanoLuc, the smallest engineered luciferase available, is up to 150X brighter than firefly or Renilla luciferase, so exposure times can be shorter than they would be with other luminescent reporters. Its small size also makes it less likely to perturb normal biology or protein function.

Another benefit of bioluminescence for imaging is the inherent stability and sustainability of the bioluminescent signal, which does not require external excitation like fluorescent tags.  This allows direct visualization of protein dynamics in living cells without the need for repeated sample excitation. The lack of external excitation also reduces the risk of phototoxicity and photobleaching, common issues that can adversely affect cell viability and signal integrity over time.

Applications Across Cellular Research

Luminescent imaging complements traditional luminescence assays by adding spatial and temporal dimensions. With luminescent live-cell imaging, researchers can visualize NanoLuc® Luciferase assays to gain a deeper understanding of the real-time cellular processes occurring in each experiment. Applications include:

  • Determining which cells provide signal
  • Analyzing mixed cell populations
  • Identifying rare events
  • Monitoring protein:protein interactions
  • Identifying protein localization and translocation
  • Tracking protein degradation and stability over time
  • Visualizing ligand:protein interactions (target engagement)

Luminescent Imaging in Action

Targeted Protein Degradation

Selectively targeting proteins for removal from the cell—instead of inhibiting protein activity—is a newer approach with therapeutic potential. In this method, the protein is targeted for degradation using the cell’s natural ubiquitin proteasome system (UPS). The degradation process is initiated by compounds such as molecular glues and proteolysis targeting chimeras (PROTACs) linking the target protein to an E3 ligase. Once this linkage occurs, the cell’s UPS does the rest.

Luminescent substrates with increased signal stability, such as the Nano-Glo® Extended Live Cell Substrate, enable researchers to image targeted protein degradation in their cells in real time. In the example shown below, Nano-Glo® Vivazine™ Live Cell Substrate was used to image degradation of the GSPT1 protein by the CC-885 degrader over 5 hours.

gif showing luminescent signal disappearing with protein degradation

Targeted protein degradation over time. HEK293 cells expressing endogenous HiBiT-tagged GSPT1 and stably expressing LgBiT were treated with CC-885 degrader or DMSO control treatment. Assayed with Nano-Glo® Vivazine™ Live Cell Substrate and imaged over 5 hours using GloMax® Galaxy Bioluminescence Imager.

Combining Luminescent and Fluorescent Imaging to Detect Protein:Small Molecule Interactions

Using bioluminescence resonance energy transfer (BRET)-based assays such as NanoBRET® assays allows you to detect protein:protein interactions by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor. These assays can be used to monitor changes in protein interactions over time, making them a useful tool for small-molecule screening.

The schematic below illustrates how the NanoBRET® NanoGlo® Detection Systems can be used to visualize target engagement. The cells on the left are expressing a NanoLuc® fusion protein, resulting in a luminescent signal. Adding a fluorescent small tracer (center) results in energy transfer and a fluorescent signal (right). Using an imaging platform that has luminescence and fluorescence imaging capabilities will let you see this energy transfer in action.

schematic showing cells detected by luminescent and fluorescent imaging
Detecting protein:small molecule interactions with NanoBRET® NanoGlo® Detection Systems.  HCT116 cells expressing a PRMT5–NanoLuc® fusion were supplemented with a fluorescent small molecule tracer (center panel). Before tracer addition, luminescent signal indicates energy is present on the donor protein (left; 3-minute exposures for 15 minutes). Binding of fluorescent tracer results in energy transfer and fluorescent signal (right; 3-minute exposures for 60 minutes). Images were captured on the GloMax® Galaxy Bioluminescence Imager.

Bringing the Power of Luminescent Imaging to Your Lab

glomax galaxy imager and computer screen

The GloMax® Galaxy Bioluminescence Imager was built to visualize the NanoLuc®-based assays we engineered, in a benchtop instrument accessible to any lab. The Galaxy is a fully equipped microscope with luminescence, fluorescence, and brightfield imaging capabilities, giving researchers a way to see functional and dynamic cellular events across a cell population.

Conclusion

Luminescent imaging can enrich what we learn from live-cell assays and offers an unprecedented view into the dynamics of cellular processes. From monitoring drug responses to visualizing protein interactions, this technology delivers insights that go beyond the capabilities of traditional assays.

Whether you’re studying cancer biology, drug development or cellular signaling, luminescent imaging can help you uncover what’s hidden in your data and see your research in a whole new light.

Additional Resources

GloMax® Galaxy Luminescent Imager, NanoBRET® Nano-Glo® Detection Systems and Nano-Glo® Vivazine live Cell Substrate are for Research Use Only. Not for Use in Diagnostic Procedures.

Don’t Flush Your Kitty Litter! Toxoplasmosis Is a Growing Threat to Sea Otters and Other Marine Mammals

Posted on by Kelly Grooms
Sea otter in water with an overlay of Toxoplasma gondii oocysts.

Southern sea otters (Enhydra lutris nereis), endangered marine mammals along California’s coastlines, are facing an unexpected threat. The menace comes not from pollution, habitat loss or natural predators, but from a microscopic enemy—Toxoplasma gondii (T. gondii). This protozoan parasite, typically associated with domestic cats, has found its way into marine ecosystems with sometimes deadly consequences for sea otters. Recently, scientists identified transmission of virulent, atypical strains of T. gondii from terrestrial felids to sea otters along the southern California coast, with lethal consequences (1).

Understanding T. gondii and Its Hosts

T. gondii is a versatile parasite that can infect nearly all warm-blooded animals, including humans and marine mammals. However, the T. gondii lifecycle depends upon felids (e.g., domestic cats and their wild relatives) who serve as definitive hosts. It is in their intestines that the parasite completes its sexual reproductive stage. The resulting oocysts are excreted in the animals’ feces. T. gondii oocysts exhibit remarkable resilience, surviving in soil, freshwater and seawater for extended periods. They are even resistant to standard wastewater treatment processes, which means oocysts in cat waste disposed of by flushing will pass through the treatment plant and be discharged into the environment. ​(2,3).

Oocysts can also be washed from soil contaminated with cat waste and carried via storm drains and rivers into the ocean, dispersing them into coastal waters. Once there, the oocysts settle on kelp or in sediments where they can be picked up by marine invertebrates like snails, mussels and clams. Marine mammals such as sea otters become infected when they consume these contaminated invertebrates. Otters can also ingest oocysts during grooming sessions​ (1,3).

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Tardigrade Proteins Might Solve the Cold Chain Problem for Biologics

Posted on by Kelly Grooms
image depicting a microscopic tardigrade

Some of our most advanced medicines today rely on components derived from living organisms. These therapeutics, called biologics, include things like vaccines, blood products like Human Blood Clotting Factor VIII (FVIII), antibodies and stem cells. Biologics are incredibly temperature sensitive, which means they need to be kept cold during production, transport and storage, a process collectively called the cold chain. The stringent transport and storage temperature requirements for biologics create a barrier to accessing these lifesaving options; particularly for those in remote or underdeveloped regions, where maintaining a cold chain is logistically difficult and costly.

But what if we could break the cold chain? Inspired by one of the most resilient creatures on Earth – the tardigrade – scientists at the University of Wyoming are exploring ways to do just that.

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Mpox—The Latest Zoonotic Virus Making Headlines

Posted on by Kelly Grooms

Mpox (formerly known as Monkeypox; 1) has been making the news lately. The declaration by the WHO Director-General naming mpox a public health emergency of international concern (PHEIC; 2) has a lot of people wondering what it is, how it spreads and how concerned they should be. Understandably, we are all a little jumpy when we start hearing about a new viral disease, but the virus that causes mpox (monkeypox virus) isn’t new.

Artists rendering of monkeypox on a torso.

A member of the Poxviridae family, the monkeypox virus is closely related to the variola virus that causes smallpox; however, monkeypox causes milder symptoms and is less fatal (1). While the virus gained its unfortunate name from its discovery in monkeys in 1958 (3), the original source of the disease remains unknown. The virus exists in a wide range of mammals including rodents, anteaters, hedgehogs, prairie dogs, squirrels and shrews (4) and can spread to humans through close contact with an infected individual or animal. Symptoms can include fever, headache, muscle and back pain, swollen lymph nodes, chills and exhaustion (3). The most distinguishing symptom is the blister-like rash.

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The Battle of Shiloh’s Angel’s Glow: Fact, Civil War Legend or Modern Myth? 

Posted on by Kelly Grooms

It sounds like the script for a Hollywood movie. The story, first appearing in 2001, begins with a purported civil war legend from the Battle of Shiloh. The legend said that the wounds of some soldiers glowed (faintly) in the dark. Soldiers with these glowing wounds were more apt to survive, giving the phenomenon the name “Angels Glow”. The story ends with two curious teenagers solving the mystery using their science fair project. They identify infection by the bioluminescent bacteria Photorhabdus luminescens (formerly Xenorhabdus luminescens) as the likely cause of the glowing wounds. P. luminescens produces bacteriocins (antimicrobial peptides), which the teenagers attribute to helping keep other infections at bay, resulting in the improved survival rate for the soldiers whose wounds glowed.

The teenagers win. The mystery is solved. The credits roll. 

Except life (and science) is rarely as simple as a summer block buster. 

Cannon at sunset on a civil war battlefield
The Battle of Shiloh took place in Hardin County Tennessee on April 6th and 7th, 1862.
Continue reading “The Battle of Shiloh’s Angel’s Glow: Fact, Civil War Legend or Modern Myth? “

Mind Control, Mutilation and Death. The Fungal Fate That Lurks in Waiting for Emerging Periodical Cicadas

Posted on by Kelly Grooms

For the first time since Thomas Jefferson was president, broods of 13- and 17-year periodical cicadas are emerging from the ground at the same time. The fate that awaits some of these periodic cicadas—a fungal infection that hijacks their behavior and destroys their genitalia — sounds like the script of a bad zombie horror film. The culprit (or villain) is the entomopathogenic fungus Massospora cicadina.  

An adult red eyed 17-year periodical cicada sits on a leaf

While most entomopathogens kill their host before releasing their infectious spores, M. cicadina is one of the few species that increase spore dispersal by hijacking their host’s behavior and keeping them alive while sporulating (1). The manner it uses to do this is both gruesome and fascinating. If you can stomach some details of insect sex and dismemberment, read on.

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Discovery of Protein Involved in TDP-43 Cytoplasmic Re-Localization Points to Potential Gene Therapy for ALS and FTD

Posted on by Kelly Grooms
A mouse stands on test tubes next to graphic of DNA double helix.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal and rapidly progress as neurodegenerative diseases. While inherited mutations can cause both conditions, they mostly appear sporadically in individuals without a known family history. Despite affecting different neurons, both diseases share a common hallmark: the pathogenic buildup of abnormal nuclear TAR-binding protein 43 (TDP-43) in the cytoplasm of affected motor neuron cells. Current theories propose that this cytoplasmic re-localization triggers toxic phosphorylation and fragmentation of TDP-43. Concurrently, a decrease of TDP-43 in the nucleus diminishes TDP-43-related physiological nuclear functions, contributing to the diseases’ progression (1).

Although this cytoplasmic accumulation of TDP-43 plays a significant role in the pathogenesis of ALS and FTD, the cellular mechanisms involved in the re-localization of TDP-43 to the cytoplasm is not known (2). A team of Australian neuroscientists led by Dr. Lars Ittner believe that they have found part of the answer for sporadic forms of the diseases. They identified novel interactions between pathogenic or dysfunctional forms of TDP-43 and the 14.3.3ɵ isoform of the cytoplasmic protein 14-3-3. By targeting this interaction with an AAV-based gene therapy vector, they were able to block and even partially reverse neurodegeneration in ALS/FTD mouse models.  

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Dynein Motor Proteins Could Be the Moving Power Behind Cancer Metastasis

Posted on by Kelly Grooms
3D Cancer Cell

“The cancer has spread.” are perhaps some of the most frightening words for anyone touched by cancer. It means that cancer cells have migrated away from the primary tumor, invaded health tissues and firmed secondary tumors. Called metastasis, this event is the deadliest feature of any type of cancer (1). The cellular mechanisms that play a role in metastasis could serve as powerful therapeutic targets. Unfortunately, understanding of these mechanisms is limited. However, some studies have suggested a link between the dysregulation of microtubule motors and cancer progression. A new study by a team from Penn State has revealed that the motor protein dynein plays a pivotal role in the movement of metastatic breast cancer cells through two model systems simulating soft tissues (1).

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Cyanobacteria Identified as Cause of Elephant Mass Mortality Event

Posted on by Kelly Grooms

The largest contiguous population of elephants in Africa lives in the Kavango-Zambezi Trans Frontier Conservation Area (KAZA TFCA) which encompasses parts of Botswana Zimbabwe, Zambia, Angola and Namibia. Within KAZA, nearly 90% of the elephant population is concentrated in Botswana (58%) and Zimbabwe (29%). In June of 2020, over 300 elephants were found dead in Botswana under mysterious circumstances. Less than two months later—in a span of only 27 days—34 more elephant deaths were reported in neighboring Zimbabwe. The news of these mass mortality events was both notable and concerning given the importance of the KAZA elephant metapopulation to species conservation.

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Uncovering the Neuroscience of Imagination Using a Virtual Reality World for Rats

Posted on by Kelly Grooms

Imagination is often considered a uniquely human trait. Simply put, it is what allows us to think about things that aren’t happening in that moment, and it plays an integral part in our day-to-day lives. We use it when we think through our calendar for the day, consider restaurant options for dinner, or visualize the best route. It turns out this trait might not be as unique to humans as we thought. In fact, a study published in Science suggests that we might share this ability with rats (1).

Rats are the most divisive of rodents. Some people see disease-carrying scourges; some see intelligent, affectionate creatures with larger-than-life personalities; and still others simply can’t get past their bare tails and small eyes. Love them or hate them, science has shown that there is more to these creatures than meets the eye. They are intelligent, ticklish and empathetic; and the study in Science suggests, imaginative.

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