Abstract
Living cells employ complex and highly dynamic signaling networks and transcriptional circuits to maintain homeostasis and respond appropriately to constantly changing environments. These networks enable cells to maintain tight control on intracellular concentrations of ions, metabolites, proteins, and other biomolecules and ensure a careful balance between a cell’s energetic needs and catabolic processes required for growth. Establishing molecular mechanisms of genetic and pharmacological perturbations remains challenging, due to the interconnected nature of these networks and the extreme sensitivity of cellular systems to their external environment. Live cell imaging with genetically encoded fluorescent biosensors provides a powerful new modality for nondestructive spatiotemporal tracking of ions, small molecules, enzymatic activities, and molecular interactions in living systems, from cells, tissues, and even living organisms. By deploying large panels of cell lines, each with distinct biosensors, many critical biochemical pathways can be monitored in a highly parallel and high-throughput fashion to identify pharmacological vulnerabilities and combination therapies unique to a given cell type or genetic background. Here we describe the experimental and analytical methods required to conduct multiplexed parallel fluorescence microscopy experiments on live cells expressing stable transgenic synthetic protein biosensors.
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References
Terai T, Nagano T (2013) Small-molecule fluorophores and fluorescent probes for bioimaging. Pflugers Arch 465:347–359
Mohsin M, Ahmad A, Iqbal M (2015) FRET-based genetically-encoded sensors for quantitative monitoring of metabolites. Biotechnol Lett 37:1919–1928
Greer LF 3rd, Szalay AA (2002) Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17:43–74
Padilla-Parra S, Tramier M (2012) FRET microscopy in the living cell: different approaches, strengths and weaknesses. BioEssays 34:369–376
Sanford L, Palmer A (2017) Recent advances in development of genetically encoded fluorescent sensors. Methods Enzymol 589:1–49
Harvey CD, Ehrhardt AG, Cellurale C et al (2008) A genetically encoded fluorescent sensor of ERK activity. Proc Natl Acad Sci U S A 105:19264–19269
Chapnick DA, Bunker E, Liu X (2015) A biosensor for the activity of the "sheddase" TACE (ADAM17) reveals novel and cell type-specific mechanisms of TACE activation. Sci Signal 8:rs1
Komatsu N, Aoki K, Yamada M et al (2011) Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol Biol Cell 22:4647–4656
Zhou X, Clister TL, Lowry PR et al (2015) Dynamic visualization of mTORC1 activity in living cells. Cell Rep 10:1767–1777
Tsou P, Zheng B, Hsu CH et al (2011) A fluorescent reporter of AMPK activity and cellular energy stress. Cell Metab 13:476–486
Ravier MA, Cheng-Xue R, Palmer AE et al (2010) Subplasmalemmal Ca(2+) measurements in mouse pancreatic beta cells support the existence of an amplifying effect of glucose on insulin secretion. Diabetologia 53:1947–1957
Yoshizaki H, Ohba Y, Kurokawa K et al (2003) Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J Cell Biol 162:223–232
San Martin A, Ceballo S, Ruminot I et al (2013) A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells. PLoS One 8:e57712
Imamura H, Nhat KP, Togawa H et al (2009) Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc Natl Acad Sci U S A 106:15651–15656
Kunkel MT, Toker A, Tsien RY et al (2007) Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter. J Biol Chem 282:6733–6742
Palmer AE, Jin C, Reed JC et al (2004) Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A 101:17404–17409
Lam AJ, St-Pierre F, Gong Y et al (2012) Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9:1005–1012
Takanaga H, Frommer WB (2010) Facilitative plasma membrane transporters function during ER transit. FASEB J 24:2849–2858
Gruenwald K, Holland JT, Stromberg V et al (2012) Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors. PLoS One 7:e38591
Ma Y, Yamamoto Y, Nicovich PR et al (2017) A FRET sensor enables quantitative measurements of membrane charges in live cells. Nat Biotechnol 35:363–370
San Martin A, Ceballo S, Baeza-Lehnert F et al (2014) Imaging mitochondrial flux in single cells with a FRET sensor for pyruvate. PLoS One 9:e85780
Acknowledgments
This work was supported by NIH grant R01GM113141 and the DARPA cooperative agreement W911NF-14-2-0019.
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Chapnick, D.A., Bunker, E., Liu, X., Old, W.M. (2019). Temporal Metabolite, Ion, and Enzyme Activity Profiling Using Fluorescence Microscopy and Genetically Encoded Biosensors. In: D'Alessandro, A. (eds) High-Throughput Metabolomics. Methods in Molecular Biology, vol 1978. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9236-2_21
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DOI: https://doi.org/10.1007/978-1-4939-9236-2_21
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