Genetically Encoded Biosensors for Research and Drug Discovery

Mitochondrial integrity and function are pivotal to cellular energy production, and mitochondrial dysfunction has been shown to alter the cell cycle, metabolism, cell viability, gene regulation, and other critical aspects of cellular growth and survival.

Mitochondrial Dysfunction – Disease and Cytotoxicity

Mitochondrial dysfunction is associated with a broad spectrum of diseases. For example, in cancer, glycolysis persists to continuously supply ATP for tumor growth while bypassing the need for healthy mitochondria in a phenomenon known as the Warburg Effect (1). Although the underlying genetic reasons for the links between aerobic glycolysis, tumor growth, and hypoxia are not fully understood, the available evidence supports a link between the ability of cancer cells to bypass normal cellular metabolic pathways and mitochondrial dysfunction. Elsewhere, research into neurodegenerative disorders (e.g., Alzheimer’s, amyotrophic lateral sclerosis (ALS), Huntington’s, and Parkinson’s) has revealed the essentiality of mitochondria for neuronal survival, cellular metabolism, and reactive oxygen species production (ROS). Neurons depend on oxidative phosphorylation as a critical source of energy and are very sensitive to intracellular ROS. Consequently, mitochondrial biogenesis and dysfunction are associated with neurodegeneration and aging.

Cellular metabolism may be measured using a variety of compounds, molecules, and ions as byproducts of complex pathways that are essential for cellular survival and growth, for example, lipid metabolism, proteasomal degradation, and carbohydrate metabolism. ATP and oxygen are critical indicators of aerobic cellular respiration and oxidative phosphorylation, and since the aerobic pathway depends on mitochondria, mitochondrial dysfunction is a vital indicator of cytotoxicity. The mitochondrial retention of ATP only constitutes ~25% of overall cellular respiration and leaked oxygen due to ROS-induced stress in the cytosol, and accordingly, cytosolic measurements of ATP and O2 can reveal clues about the impact of internal or external factors on cytotoxicity in many cases. However, specific measurements in the mitochondrial inner membrane may be more useful under certain circumstances (2).

The Need for Biosensors in Research and Drug Discovery

Given the undisputed role of mitochondria in cellular health, and its usefulness as an indicator of cytotoxicity, the ability to carefully monitor and measure various parameters of mitochondrial function has gained significant attention in basic research aimed at understanding mitochondrial dysfunction as well as preclinical drug discovery programs, including those designed to improve mitochondrial function to treat associated diseases.

Conventional methods to monitor and measure cellular activity include the use of synthetic chemical dyes such as Fura-2 and related chemical dyes such as Rhod-4 and Fluo-4, which fluoresce differentially upon binding to Ca2+ and have been a mainstay in mitochondrial research for many years (reviewed in 3). Despite their usefulness, methods that rely on chemical sensors have their shortcomings. For instance, it is often difficult to target specific cellular organelles using these approaches because the chemicals tend to compartmentalize randomly, and they eventually get eliminated from the cell during lengthy recording experiments. Another shortfall associated with chemical dyes is that they may alter cellular functions or exert toxic effects during drug discovery and development, as recently seen in cardiomyocytes (4).

Genetically Encoded Biosensors

While conventional chemical sensors are still used, their use has been largely replaced following the development of genetically encoded biosensors over the last few years.

Genetically encoded biosensors (also known as genetically encoded fluorophores) build on fluorescent protein-based approaches to visualize proteins or cellular compartments, such as approaches based on green fluorescent protein (GFP). The biosensors are synthetic proteins with functional and fluorescent domains. They are relatively easily transfected into live cells, tissues, or even whole organisms, thus facilitating the localization and dynamics of the protein of interest through the inherent fluorescence of the FP. Since a gene encoding a FP can theoretically be fused with the encoding gene for any protein of interest, the versatility of this approach is huge, and the exact setup may be optimized to monitor gene expression levels, visualize and monitor intracellular protein dynamics, and label specific subcellular compartments, all in a highly controllable manner since the FP will only be synthesized in the cells that harbor the recombinant construct. Genetically encoded biosensors are an extension of the aforementioned FP strategies, whereby the chromophore (the color moiety) of the FP is modified by targeted mutagenesis so that it becomes sensitive and responsive to new analytes of interest (reviewed in 5).

Unlike chemical sensors, genetically encoded biosensors are made by the cell and become functional without further intervention by the researcher, thus simplifying assay procedures. Genetically encoded biosensors address all of the drawbacks associated with their traditional counterparts, as outlined below.

Advantages of genetically encoded biosensors over chemical sensors:

  • Can readily by customized to meet experimental needs, as they vary in fluorescence range, kinetics, and cellular compartmental targeting.
  • Can be readily incorporated into a variety of cell types to support research across diverse physiological systems and processes.
  • Genetically based biosensors can be fine-tuned and specifically targeted to particular cellular compartments by taking advantage of molecular biology. For example, Tempo Bioscience’s TempoMito™ was developed to specifically monitor mitochondrial calcium flux and cytotoxicity in the mitochondria using a highly sensitive fluorescent-based biosensor as a reporter.
  • They bypass the host cell toxicity concerns that are associated with traditional sensors. We know that experimental biases and “wrong results” gleaned from traditional sensors have had major consequences, for example, drug discovery and development using cardiomyocytes has been challenging because many methods (i.e., traditional dyes) lead to aberrant results in this cell type.

New Genetic Encoded Biosensors for Research and Drug Discovery

Tempo Bioscience recently developed  a range of biosensors for cytotoxicity and cellular metabolism studies; TempoATP and TempoO2. These biosensors can be readily introduced into immortalized cancer cell lines, tumors, human inducible pluripotent stem cells (iPSCs), and a range of iPSC-derived cell types. Additionally, TempoCal and TempoVol are a set of calcium and voltage biosensors respectively, that target to different cellular compartments.

You can read about Tempo Bioscience’s genetically encoded biosensors here, and please don’t hesitate to get in touch with us if you want to know more!

References

  1. M. V. Liberti, J. W. Locasale, The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci 41, 211-218 (2016).
  2. S. Sakamuru, M.S. Attene-Ramos, M. Xia, Mitochondrial Membrane Potential Assay. Methods Mol. Biol. 1473, 17-22 (2016).
  3. R. M. Paredes, J. C. Etzler, L. T. Watts, W. Zheng, J. D. Lechleiter, Chemical calcium indicators. Methods 46, 143-151 (2008).
  4. Y. Chang, C. N. Broyles, F. A. Brook, M. J. Davies, C. W. Turtle, T. Nagai, M. J. Daniels, Non-invasive phenotyping and drug testing in single cardiomyocytes or beta-cells by calcium imaging and optogenetics. PLoS One. 12(4),  e017418 (2017).
  5. J. R. Enterina, L. Wu, R. E. Campbell, Emerging fluorescent protein technologies. Curr Opin Chem Biol 27, 10-17 (2015).

Article by Karen O’Hanlon Cohrt PhD. Contact Karen at karen@tempobioscience.com.

Karen O’Hanlon Cohrt is a Science Writer with a PhD in biotechnology from Maynooth University, Ireland (2011). After her PhD, Karen moved to Denmark and held postdoctoral positions in mycology and later in human cell cycle regulation, before moving to the world of drug discovery. Her broad research background provides the technical know-how to support scientists in diverse areas, and this in combination with her passion for writing helps her to keep abreast of exciting research developments as they unfold. Follow Karen on Twitter @KarenOHCohrt. Karen has been a science writer since 2014; you can find her other work on her portfolio.

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