Abstract
“pHlash” is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Roos A, Boron WF (1981) Intracellular pH. Physiol Rev 61:296–434
Fricker MD, Plieth C, Knight H, Blancaflor E, Knight MR, White NS, Gilroy S (1999) Fluorescence and luminescence techniques to probe ion activities in living plant cells. In: Mayson WT (ed) Fluorescent and Luminescent Probes for Biological Activity. Academic Press, San Diego, pp 569–596
Miesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195
Kneen M, Farinas J, Li Y, Verkman AS (1998) Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys J 74:1591–1599
Schulte A, Lorenzen I, Böttcher M, Plieth C (2006) A novel fluorescent pH probe for expression in plants. Plant Methods 2:1–13
Ugarova NN, Maloshenok LG, Uporov IV, Koksharov MI (2005) Bioluminescence spectra of native and mutant firefly luciferases as a function of pH. Biochemistry (Moscow) 70:1534–1540
Matthews JC, Hori K, Cormier MJ (1977) Purification and properties of Renilla reniformis luciferase. Biochemistry 16:85–91
Verhaegen M, Christopoulos TK (2002) Recombinant Gaussia luciferase. Overexpression, purification and analytical application of a bioluminescent reporter for DNA hybridization. Anal Chem 74:4378–4385
Branchini BR, Ablamsky DM, Rosenberg JC (2010) Chemically modified firefly luciferase is an efficient source of near-infrared light. Bioconjugate Chem 21:2023–2030
Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848–1857
Loening AM, Fenn TD, Wu AM, Gambhir SS (2006) Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng Des Sel 19:391–400
Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A (2004) Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101:10554–10559
Xu Y, Piston D, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci U S A 96:151–156
Zhang Y, Xie Q, Roberson JB, Johnson CH (2012) pHlash: A new genetically encoded and ratiometric luminescence sensor of intracellular pH. PLoS One 7, e43072
Brett CL, Tukaye DN, Mukherjee S, Rao R (2005) The yeast endosomal Na+(K+)/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking. Mol Biol Cell 16:1396–1405
Xu X, Soutto M, Xie Q, Servick S, Subramanian C et al (2007) Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc Natl Acad Sci U S A 104:10264–10269
Krishnamoorthy A, Robertson JB (2015) Dual color monitoring overcomes limitations of single bioluminescent reporters in fast growing microbes and reveals phase-dependent protein productivity during metabolic rhythms of yeast. Appl Environ Microbiol 81:6484–6495
Xie Q, Soutto M, Xu X, Zhang Y, Johnson CH (2011) Bioluminescence resonance energy transfer (BRET) imaging in plant seedlings and mammalian cells. Methods Mol Biol 680:3–28
Roberson JB, Zhang Y, Johnson CH (2009) Light-emitting diode flashlights as effective and inexpensive light sources for fluorescence microscopy. J Microsc 236:1–4
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Zhang, Y., Robertson, J.B., Xie, Q., Johnson, C.H. (2016). Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells. In: Kim, S. (eds) Bioluminescence. Methods in Molecular Biology, vol 1461. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3813-1_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3813-1_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3811-7
Online ISBN: 978-1-4939-3813-1
eBook Packages: Springer Protocols