A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping

Electrophysiologists are talented scientists/artists who see into the events of the cell with amazing detail.
Electrophysiology experiments provide a view into the cell with amazing detail. The paper reviewed here describes a molecular reporter biosensor (NanoBRET) that can offer the same kind of temporal and spatial resolution traditionally reserved for extremely labor-intensive experiments like patch clamp analysis.

I confess that I struggled through biophysics, and my Bertil Hille textbook Ion Channels of Excitable Membranes lies neglected somewhere in a box in my basement (I have not tossed it into the recycle bin—I can’t bear too, I spent too much time bonding with that book in graduate school).

My struggles in that graduate class and my attendance at the seminars of my grad school colleagues who were conducting electrophysiological studies left me with a sincere awe and appreciation of both the genius and the artistry required to produce nice electrophysiology data. The people who are good at these experiments are artists—they have the golden touch when it comes to generating that megaohm seal between a piece of cell membrane and a finely pulled glass pipette. And, they are brilliant scientists, they really understand the physics, the chemistry and the biology of the cells they study from a perspective that very few scientists ever develop.

Electrophysiology data, which often demonstrate the gating of a single channel protein in response to a single stimulus in real time–ions crossing a membrane through a single protein–are amazing for their ability, unlike virtually any other experimental data for the story they can tell about what is going on in a cell in real time under physiological conditions.

So when I read the paper recently published by Mashuo et al. in Science SignalingDistinct profiles of functional discrimination among G proteins determine the action of G protein-coupled receptors”, this sentence really caught my attention:

When constructs were ectopically expressed in HEK 293T/17 cells, we obtained very similar kinetics for the GPCR-driven responses between NanoBRET™ biosensors and the patch clamp recordings.

They continue:

Indeed, the activation rates that we observed were very similar to those of GPCR-stimulated GIRKs [G protein-coupled, inwardly rectifying K+ channel] in native cells, suggesting that the conditions of this assay closely match the in vivo setting. This finding further demonstrates the ability of the system to resolve the fast, physiological relevant kinetics of GPCR signaling.

A reporter biosensor that can resolve events similarly to patch clamping?!  Amazing. Continue reading “A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping”

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) . Continue reading “Targeting MYC: The Need to Study Protein:Protein Interactions in Cells”

Piecing the Puzzle Together: Using Multiple Assays to Better Understand What Is Happening with Your Cells

You often need several pieces of information to really understand what is happening within a cell or population of cells. If your cells are not proliferating, are they dying? Or, are you seeing cytostasis? If they are dying, what is the mechanism? Is it apoptosis or necrosis? If you are seeing apoptosis, what is the pathway: intrinsic or extrinsic?

If you are measuring expression of a reporter gene and you see a decrease in expression, is that decrease due to transfection inefficiencies, cytotoxicity, or true down regulation of your reporter gene?

To investigate these multiple parameters, you can run assays in parallel, but that requires more sample, and sample isn’t always abundant.

Multiplexing assays allows you to obtain information about multiple parameters or events (e.g., reporter gene expression and cell viability; caspase-3 activity and cell viability) from a single sample. Multiplexing saves sample, saves time and gives you a more complete picture of the biology that is happening with your experimental sample.

What information do you need about your cells to complete the picture?
What information do you need about your cells to complete the picture?

Multiplexing assay reagents to measure biomarkers in the same sample has often been considered an application only accomplished with antibodies or dyes and sophisticated detection instrumentation. However, Promega has developed microwell plate based assays for cells in culture that allow multiplexed detection of biomarkers in the same sample well using standard multimode multiwell plate readers. Continue reading “Piecing the Puzzle Together: Using Multiple Assays to Better Understand What Is Happening with Your Cells”

In Which I Tour a Cell Sorting Lab

Close up shot of the head of a FACS machine.
I recently had the chance to visit the core Flow Cytometry facility at the University of Alberta’s Faculty of Medicine and Dentistry, and to see a real FACS (Fluorescence Activated Cell Sorting) machine in action. Saturated as we are in technology that would have seemed miraculous even a decade ago, it’s sometimes hard for me to appreciate the sheer ingenuity of modern scientific apparatus. But somehow, stepping out of my familiar stomping grounds of smartphone programming, and seeing what we can now do with things in the real world instead of just ones and zeros, snapped me out of my complacency.

The facility supervisor – Dorothy Kratochwil-Otto, who kindly demonstrated the inner workings of one of the FACS machines as she spoke – explained to me in relative layman’s terms how the FACS technology works. Continue reading “In Which I Tour a Cell Sorting Lab”

Spinning Wheels Go Round and Round: Classic Experiments on the Cell Cycle

While  working on a cell cycle lecture for the Education Resources web at Promega.com, I reread some classic papers describing classic cell-cycle experiments. Two of these papers describe the experiments by Murray and Kirschner showing that cyclin B synthesis and degradation are required for cycling in Xenopus oocyte extracts. When I took my first graduate-level cell biology course in 1989, these papers had just been published. I remember the instructors of the course being particularly excited about this work. (I also remember getting the events of Xenopus oocyte activation and fertilization mixed up on one of the tests for this course and realizing it about two minutes before I had to hand in my test, but I digress.)

Looking at these papers now with the eyes of someone who has followed cell biology for more years than I care to admit, with the eyes of an educator, and with the eyes of someone who now truly “gets” that science is an iterative process, I understand the instructors’ excitement. Continue reading “Spinning Wheels Go Round and Round: Classic Experiments on the Cell Cycle”

Apoptosis in Normal and Cancer Cell Biology Webinar

webinarCurious about the role that apoptosis plays in normal and cancer cell biology? Do you need to monitor apoptosis in cells? Do you need to know what apoptosis biomarkers to follow for a particular study? Register today for the Science/AAAS Webinar: Apoptotic Signaling in Normal and Cancer Cell Biology (sponsored by Promega).