All Aglow in the Ocean Deep

 

Fascinating bioluminescent creature floating on dark waters of the ocean. Polychaete tomopteris.

Today’s blog comes to you from the Promega North America Branch Office.

In nature, the ability to “glow” is actually quite common. Bioluminescence, the chemical reaction involving the molecule luciferin, is a useful adaptation for many lifeforms. Fireflies, mushrooms and creatures of the ocean deep use their internal lightshows to cope with a variety of situations. Used for hunting, communicating, ridding cells of oxygen, and simply surviving in the darkness of the ocean depths, bioluminescence is one of nature’s more flashy, and advantageous traits.

In new research published in April in the journal Scientific Reports, MBARI researchers Séverine Martini and Steve Haddock found that three-quarters of all sea animals make their own light.  The study reviewed 17 years of video from Monterey Bay, Calif in oceans that descended to 2.5 miles, to determine the commonality of bioluminescence in the deep waters.

Martini and Haddock’s observations concluded that 76 percent off all observed animals produced some light, including 97 to 99.7 cnidarians (jellyfish), half of fish, and most polychaetes (worms), cephalopods (squid), and crustaceans (shrimp).

Most of us are familiar with the fabled anglerfish, the menacing deep-sea creature known for attracting ignorant prey with a glowing lure attached to their head. As you descend below 200 meters, where light no longer penetrates, you will be surprised at the unexpected color display of the oceans’ sea life. Bioluminescence is not simply an exotic phenomenon, but an important ecological trait that the oceans’ sea creatures have wholeheartedly adopted to cope with complete darkness. Continue reading “All Aglow in the Ocean Deep”

Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events

A Bioluminescent Alternative

Fluorescence resonance energy transfer (FRET) probes or sensors are commonly used to measure cellular events. The probes typically have a matched pair of fluorescent proteins joined by a ligand-binding or responsive protein domain. Changes in the responsive domain are reflected in conformational changes that either bring the two fluorescent proteins together or drive them apart. The sensors are measured by hitting the most blue-shifted fluorescent protein with its excitation wavelength (donor). The resulting emission is transferred to the most red-shifted fluorescent protein in the pair, and the result is ultimately emission from the red-shifted protein (acceptor).

As pointed out by Aper, S.J.A. et al. below, FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic regulators to initiate the event you wish to monitor. Optogenetic regulators respond to specific wavelengths and initiate signaling. The challenge comes when the FRET donor excitation overlaps with the optogenetic initiation wavelengths. Researchers have sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent donor, making bioluminescence resonance energy transfer (BRET) probes. In the three papers described below, the authors chose NanoLuc® Luciferase as the BRET donor due to its extremely bright signal. Continue reading “Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events”

Probing RGS:Gα Protein Interactions with NanoBiT Assays

gpcr_in_membrane_on_white2When I was a post-doc at UT Southwestern, I was fortunate to interact with two Nobel prize winners, Johann Deisenhofer and Fred Gilman.  Johann once helped me move a -80°C freezer into his lab when we lost power in my building. I once replaced my boss at small faculty mixer with a guest speaker and had a drink with Fred Gilman and several other faculty members from around the university. Among the faculty, one professor had a cell phone on his belt, an odd sight in 1995. Fred Gilman asked him what it was and why he had it. It was so his lab could notify him of good results anytime of the day. Fred laughed and told him to get rid of it – if it’s good data, it will survive until morning.

I was reminded of this story when I read a recent paper by Bodle, C.R. et al (1) about the development of a NanoBiT® Complementation Assay (2) to measure interactions of Regulators of G Protein Signaling (RGS) with Gα proteins in cells. (Fred Gilman was the first to isolate a G protein and that led to him being a co-recipient of the Nobel Prize in 1994). The authors created over a dozen NanoBiT Gα:RGS domain pairs and found they could classify different RGS proteins by the speed of the interaction in a cellular context. The interactions were readily reversible with known inhibitors and were suitable for high-throughput screening due to Z’ factors exceeding 0.5. The study paves the way for future work to identify broad spectrum RGS domain:Gα inhibitors and even RGS domain-specific inhibitors. This is the second paper applying NanoBiT Technology to GPCR studies (3).

A Little Background…
A primary function of GPCRs is transmission of extracellular signals across the plasma membrane via coupling with intracellular heterotrimeric G proteins. Upon receptor stimulation, the Gα subunit dissociates from the βγ subunit, initiating the cascade of downstream second messenger pathways that alter transcription (4). The Gα subunits are actually slow GTPases that propagate signals when GTP is bound but shutdown and reassociate with the βγ subunit when GTP is cleaved to GDP. This activation process is known as the GTPase cycle. G proteins are extremely slow GTPases. Continue reading “Probing RGS:Gα Protein Interactions with NanoBiT Assays”

Two light stories for Friday

For this Friday blog, here’s a sampling of two recent papers highlighting use of the small, bright, NanoLuc luciferase in interesting ways.

Bioluminescence-based hormone:receptor binding studies

A review by Ya-Li Liu and Zhan-Yun Guo, published this week in Amino Acids summarizes recent work of the authors and others using NanoLuc luciferase labeled protein/peptide hormones in receptor binding assays. Typically, studies assessing binding of hormones to receptors have used radioactive tracers. The brightness of NanoLuc luciferase makes bioluminescence an attractive alternative as a sensitive and safer option. Because cell membrane receptors are difficult to purify in quantity, the amounts available for experiments are usually limited. Therefore, tracers used in binding assays need to have a high affinity for the receptor, must not interfere with binding, and must be highly sensitive. Continue reading “Two light stories for Friday”

It’s the Little Things: Celebrating NanoLuc and Other Small Things

NanoLuc dual reporters
Illustration showing NanoLuc and firefly luciferase reporters.

This month we are celebrating a small thing—NanoLuc® luciferase, an enzyme whose tiny size and bright luminescence enable much more sensitive detection of intracellular events than other bioluminescent reporters. Scaling down the size of the luciferase protein makes it “fit” in situations where larger reporters do not, and also makes it less likely to interfere with natural biology than larger proteins. Scaling up the brightness allows you to detect the reporter at low abundance, enhancing sensitivity and allowing detection of small changes in gene expression at concentrations closer to physiological levels.

These properties of Nanoluc® luciferase allow its bioluminescence to be used to detect intracellular events in ways not possible before. One example of how this small size and intense brightness are being applied to help solve biological problems is the insertion of NanoLuc® luciferase into influenza viruses, where the genome size is small and does not tolerate large insertions. Unlike viruses incorporating larger luciferases, the influenza reporters incorporating NanoLuc® luciferase are stable, retain pathogenicity and are bright enough to track at low doses during the early stages of infection (1). You can find out more about the many applications of NanoLuc® luciferase here.

In the spirit of “celebrating small things” we will be sharing plenty of information about NanoLuc® applications over the next few weeks, and will also be highlighting other examples where “small” can be a beautiful thing. By definition, molecular biology is a study of the smallest building blocks of life, so it’s not hard to find an abundance of small things to talk about. We hope you will participate with us by commenting, liking or sharing posts, or by contributing your own ideas on little things that make a difference, or that just make you smile.

Here’s a list for starters—it includes examples from history and from last week where going smaller turned out to be both smarter and better for overcoming a variety of biological challenges. Continue reading “It’s the Little Things: Celebrating NanoLuc and Other Small Things”

A Little Bioluminescence for the International Year of Light

22159-2268-CR-WC-NanolucWebNavImage280x1402015 is the International Year of Light, and activities around the globe are planned to celebrate light in nature, the scientists who have helped us understand the nature of light and the engineers who have developed countless tools and technologies harnessing the power of light. At Promega, our favorite kind of light in nature is bioluminescence. So your Promega Connections bloggers thought we would share this incredible National Geographic video of ocean bioluminescence. In this video, starlight cameras capture the bioluminescence of the ocean, revealing an amazingly beautiful lightscape that is invisible to the unaided human eye. Enjoy!

Interested in Learning More? Check out these Bioluminescence-Related Blog Posts:

Angel’s Glow: Bioluminescence on the Battlefield

Bioluminescence from the Sky to the Ocean’s Depths

Tracking the Progression of the Plague Using Bioluminescence

6 + 1 Ways Dual-Reporter Assays Can Save Your Data

Dual-Reporter-Assay

Transient transfection is often used to perform reporter assays.  We have advocated using a dual-reporter system for decades to normalize the data obtained and gain a clearer understanding of your results.  The experimental reporter should vary with treatment and the control reporter should vary little with treatment. The control reporter thus serves as a marker to help you understand the relative activity of your experimental reporter. Here are seven ways in which dual-reporter assays help you avoid misinterpreting results.

Simply comparing the ratio of the experimental to the control reporter can resolve differences in:

  1. Number of Cells/Well: When manually pipeting cells into a 96-well plate, there is always a chance of having variable numbers of cells in each well. This variation is cell number will affect the experimental and control reporters equally, so the ratio of experimental:control reporter activity will eliminate false interpretation of the experimental data–whether it affects an entire row or column on the plate or individual wells.
  2. Transfection Efficiency: The variations in transfection efficiency will equally affect both the experimental and control reporters so the ratio will normalize the data.
  3. Cell Viability: Often, reporter assays look at the dose response curve of a particular compound with regard to gene expression. Ideally, if a compound causes a change in the experimental reporter the control reporter will demonstrate little effect. However, if the compound is toxic, both the experimental and control will be altered and the ratio will tell you whether the compound truly affects reporter activity or just kills the cells.
  4. Lysis Efficiency: When lysing a plate of cells, you could encounter situations where rows or columns lyse differently, especially if you are using manual disruption or get interrupted mid-plate. The difference is lysis will affect the experimental and control equally so the ratio will remove the variation.
  5. Temperature: Ideally, a plate should be equillibrated to ambient room temperature before proceeding to the reporter assay. Plates can cool at different rates or researchers anxious to record data may read the data early. Temperature variations will affect both reporters so the ratio will limit the affect on the data.
  6. Measurement Time: Repetition of data is a hallmark of good science. You are often called upon to repeat experiments sometimes days or weeks apart. Let’s say you repeat your experiment one week after the initial experiment. The first time you measured the response, you waited 10 minutes after reagent addition to read, this week you waited 30 minutes. This will affect both reporters equally and therefore the ratio will allow you to more easily compare the data from this week and last week.

Bonus Benefit from Dual-Luciferase®, Dual-Glo® and the NanoGlo® Dual Luciferase Reporter Systems: Lysate Splitting: Promega dual-reporter assays are designed for same-well multiplexing so there is no chance of variations creeping into your data due to unequal splitting of the cellular lysate to measure two separate reporter activities.

Further Reading:
Normalizing Genetic Reporter Assays Approaches and Considerations for Increasing Consistency and Statistical Significance

Protein Kinase Inhibitors Show Promise in Malaria Study

Life cycle of the Malaria parasite.
Life cycle of the Malaria parasite.
A paper published in on August 8 in ChemBioChem has identified a number of small molecule kinase inhibitors that may have potential as antimalarial drugs. The authors, Derbyshire et al from Duke University, used a panel of human kinase inhibitors to screen for activity against malaria parasites. Using a high-throughput screening approach, they were able to identify several potential drug targets among the kinases of Plasmodium sp.,—most of which were effective against the parasite during both it’s blood-borne and liver-based life cycle stages.

Liver and blood-stage malaria parasites have different gene expression profiles and infect different host cells. The authors exploited these differences to try to specifically identify compounds that were active against the parasite while it was still in the liver, the idea being that any drug-based prevention strategy needs to be effective against the parasites in the liver in order to eradicate infection.

The authors screened a library of over 1300 kinase inhibitors that included several compounds already being used in clinical trials for anti-cancer activity. Initial screening was performed in human liver-derived HepG2 cells infected with Plasmodium berghei expressing a luciferase reporter. Compounds that decreased parasite load by more than 95% were further characterized in dose-response experiments, and promising hits were tested in using luminescent and fluorescent cell based assays to identify compounds that were not toxic to liver cells. Continue reading “Protein Kinase Inhibitors Show Promise in Malaria Study”

Dual-Luciferase Imaging in vivo

22159-2268-CR-WC-NanolucWebNavImage280x140We are used to seeing multicolored fluorescence images labeling either specific events or structures within cells. When compared to imaging with fluorescent methods, bioluminescence imaging methods provide the advantages of low background and subsequent higher signal to noise ratio—enhancing sensitivity. A key prerequisite for dual-imaging experiments is the ability to distinguish the signal from each event separately and clearly. However, compared to the large number of available fluorescent compounds (many spectrally distinct fluorescent proteins and dyes), there are not many different luciferases to choose from. This lack of variety has limited the capabilities of bioluminescence for imaging multiple molecular events in the same sample. Therefore, there is a need for new luciferases with substrates and emission spectra that are different from the beetle luciferases currently in widespread use.

A paper published in Molecular Imaging in October 2013 describes use of firefly and the new NanoLuc® Luciferase to image cell signaling events in cultured cells and in a mouse model system. The paper, authored by Stacer et al. of the University of Michigan, details a proof-of-concept experiment using firefly and NanoLuc luciferases to image two distinct events in the TGF-beta1 signaling pathway. Continue reading “Dual-Luciferase Imaging in vivo”

NanoLuc® Luciferase: A Good Thing for Small Packages

influenza virusesA paper published on October 2 in the Journal of Virology describes an exciting development in the world of influenza research—the construction of a luciferase reporter virus that does not affect virulence and can be used to track development and spread of infection in mice.

Insertion of luciferase reporter genes into viruses has been accomplished before for several viruses, but has not been successful for influenza. Construction of influenza reporter viruses is complicated because the viral genome is small and all the viral genes are critical for infection. Therefore, replacement of an existing gene with a reporter gene or insertion of additional reporter sequences without affecting the virus’s ability to replicate and cause infection has proven difficult. To be successful, a reporter gene needs to be small enough to insert into the viral genome without eliminating any other vital functionality. Continue reading “NanoLuc® Luciferase: A Good Thing for Small Packages”