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The Single Molecule Imaging Approach to Membrane Protein Stoichiometry

Published online by Cambridge University Press:  26 July 2012

Richard Hallworth  and
Michael G. Nichols
Richard Hallworth*
Affiliation:
Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA
Michael G. Nichols
Affiliation:
Department of Physics, Creighton University, Omaha, NE 68178, USA
*
Corresponding author. E-mail: [email protected]
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Abstract

Recent technical advances have enabled the imaging of single fluorescent molecules. The application of single molecule visualization techniques has opened up new avenues of experimentation in biology at the molecular level. In this article, we review the application of single fluorescent molecule visualization and analysis to an important problem, that of subunit stoichiometry in membrane proteins, with particular emphasis on our approach. Single fluorescent molecules, coupled to fluorescent proteins, are localized in the membranes of cells. The molecules are then exposed to continuous low-level excitation until their fluorescent emissions reach background levels. The high sensitivity of modern instrumentation has enabled direct observations of discrete step decreases in the fluorescence of single molecules, which represent the bleaching of single fluorophores. By counting the number of steps over a large number of single molecules, an average step count is determined from which the stoichiometry is deduced using a binomial model. We examined the stoichiometry of a protein, prestin, that is central to mammalian hearing. We discuss how we prepared, identified, and imaged single molecules of prestin. The methodological considerations behind our approach are described and compared to similar procedures in other laboratories.

Keywords

prestinmembrane proteinsingle molecule imaginghair cell
Type
Special Section: Seventh Omaha Imaging Symposium
Information
Microscopy and Microanalysis , Volume 18 , Issue 4 , August 2012 , pp. 771 - 780
Copyright
Copyright © Microscopy Society of America 2012

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References

REFERENCES

Aitken, C.E., Marshall, R.A. & Puglisi, J.D. (2008). An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94(5), 1826–1835.CrossRefGoogle ScholarPubMed
Ashmore, J.F. (2008). Cochlear outer hair cell motility. Physiol Rev 88(1), 173–210.CrossRefGoogle ScholarPubMed
Bats, C., Groc, L. & Choquet, D. (2007). The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking. Neuron 53(5), 719–734.CrossRefGoogle ScholarPubMed
Bonigk, W., Altenhofen, W., Muller, F., Dose, A., Illing, M., Molday, R.S. & Kaupp, U.B. (1993). Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels. Neuron 10(5), 865–877.CrossRefGoogle ScholarPubMed
Chung, S.H. & Kennedy, R.A. (1991). Forward-backward non-linear filtering technique for extracting small biological signals from noise. J Neurosci Methods 40(1), 71–86.CrossRefGoogle ScholarPubMed
Dallos, P., Wu, X., Cheatham, M.A., Gao, J., Zheng, J., Anderson, C.T., Jia, S., Wang, X., Cheng, W.H., Sengupta, S., He, D.Z. & Zuo, J. (2008). Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification. Neuron 58(3), 333–339.CrossRefGoogle ScholarPubMed
Das, S.K., Darshi, M., Cheley, S., Wallace, M.I. & Bayley, H. (2007). Membrane protein stoichiometry determined from the step-wise photobleaching of dye-labeled subunits. Chembiochem 8(9), 994–999.CrossRefGoogle Scholar
Detro-Dassen, S., Schanzler, M., Lauks, H., Martin, I., zu Berstenhorst, S.M., Nothmann, D., Torres-Salazar, D., Hidalgo, P., Schmalzing, G. & Fahlke, C. (2008). Conserved dimeric subunit stoichiometry of SLC26 multifunctional anion exchangers. J Biol Chem 283(7), 4177–4188.CrossRefGoogle ScholarPubMed
Ehlers, M.D., Heine, M., Groc, L., Lee, M.C. & Choquet, D. (2007). Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity. Neuron 54(3), 447–460.CrossRefGoogle ScholarPubMed
Ehrensperger, M.V., Hanus, C., Vannier, C., Triller, A. & Dahan, M. (2007). Multiple association states between glycine receptors and gephyrin identified by SPT analysis. Biophys J 92(10), 3706–3718.CrossRefGoogle ScholarPubMed
Hallworth, R. & Nichols, M.G. (2012). Prestin in HEK cells is an obligate tetramer. J Neurophysiol 107(1), 5–11.CrossRefGoogle ScholarPubMed
Ji, W., Xu, P., Li, Z., Lu, J., Liu, L., Zhan, Y., Chen, Y., Hille, B., Xu, T. & Chen, L. (2008). Functional stoichiometry of the unitary calcium-release-activated calcium channel. Proc Natl Acad Sci USA 105(36), 13668–13673.CrossRefGoogle ScholarPubMed
Jiang, Y., Douglas, N.R., Conley, N.R., Miller, E.J., Frydman, J. & Moerner, W.E. (2011). Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution. Proc Natl Acad Sci USA 108(41), 16962–16967.CrossRefGoogle ScholarPubMed
Joo, C., McKinney, S.A., Nakamura, M., Rasnik, I., Myong, S. & Ha, T. (2006). Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126(3), 515–527.CrossRefGoogle ScholarPubMed
Karymov, M.A., Krasnoslobodtsev, A.V. & Lyubchenko, Y.L. (2007). Dynamics of synaptic SfiI-DNA complex: Single-molecule fluorescence analysis. Biophys J 92(9), 3241–3250.CrossRefGoogle ScholarPubMed
Kaupp, U.B., Niidome, T., Tanabe, T., Terada, S., Bonigk, W., Stuhmer, W., Cook, N.J., Kangawa, K., Matsuo, H., Hirose, T., Miyata, T. & Numa, S. (1989). Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature 342(6251), 762–766.CrossRefGoogle ScholarPubMed
Kozuka, J., Yokota, H., Arai, Y., Ishii, Y. & Yanagida, T. (2006). Dynamic polymorphism of single actin molecules in the actin filament. Nat Chem Biol 2(2), 83–86.CrossRefGoogle ScholarPubMed
Leake, M.C., Chandler, J.H., Wadhams, G.H., Bai, F., Berry, R.M. & Armitage, J.P. (2006). Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 443(7109), 355–358.CrossRefGoogle ScholarPubMed
Liberman, M.C., Gao, J., He, D.Z., Wu, X., Jia, S. & Zuo, J. (2002). Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 419(6904), 300–304.CrossRefGoogle ScholarPubMed
Liu, D.T., Tibbs, G.R. & Siegelbaum, S.A. (1996). Subunit stoichiometry of cyclic nucleotide-gated channels and effects of subunit order on channel function. Neuron 16(5), 983–990.CrossRefGoogle ScholarPubMed
Liu, S., Bokinsky, G., Walter, N.G. & Zhuang, X. (2007). Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic “fingerprinting.” Proc Natl Acad Sci USA 104(31), 12634–12639.CrossRefGoogle ScholarPubMed
Madl, J., Weghuber, J., Fritsch, R., Derler, I., Fahrner, M., Frischauf, I., Lackner, B., Romanin, C. & Schutz, G.J. (2011). Resting state Orai1 diffuses as homotetramer in the plasma membrane of live mammalian cells. J Biol Chem 285(52), 41135–41142.CrossRefGoogle Scholar
McKinney, S.A., Joo, C. & Ha, T. (2006). Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys J 91(5), 1941–1951.CrossRefGoogle ScholarPubMed
Mio, K., Kubo, Y., Ogura, T., Yamamoto, T., Arisaka, F. & Sato, C. (2008). The motor protein prestin is a bullet-shaped molecule with inner cavities. J Biol Chem 283(2), 1137–1345.CrossRefGoogle ScholarPubMed
Murakoshi, M., Gomi, T., Iida, K., Kumano, S., Tsumoto, K., Kumagai, I., Ikeda, K., Kobayashi, T. & Wada, H. (2006). Imaging by atomic force microscopy of the plasma membrane of prestin-transfected Chinese hamster ovary cells. J Assoc Res Otolaryngol 7(3), 267–278.CrossRefGoogle ScholarPubMed
Myong, S., Rasnik, I., Joo, C., Lohman, T.M. & Ha, T. (2005). Repetitive shuttling of a motor protein on DNA. Nature 437(7063), 1321–1325.CrossRefGoogle Scholar
Penna, A., Demuro, A., Yeromin, A.V., Zhang, S.L., Safrina, O., Parker, I. & Cahalan, M.D. (2008). The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers. Nature 456(7218), 116–120.CrossRefGoogle ScholarPubMed
Schuler, B. & Eaton, W.A. (2008). Protein folding studied by single-molecule FRET. Curr Opin Struct Biol 18(1), 16–26.CrossRefGoogle ScholarPubMed
Tombola, F., Ulbrich, M.H., Kohout, S.C. & Isacoff, E.Y. (2010). The opening of the two pores of the Hv1 voltage-gated proton channel is tuned by cooperativity. Nat Struct Mol Biol 17(1), 44–50.CrossRefGoogle ScholarPubMed
Ulbrich, M.H. & Isacoff, E.Y. (2007). Subunit counting in membrane-bound proteins. Nat Methods 4(4), 319–321.CrossRefGoogle ScholarPubMed
Wang, X., Yang, S., Jia, S. & He, D.Z. (2010). Prestin forms oligomer with four mechanically independent subunits. Brain Res 1333, 28–35.CrossRefGoogle ScholarPubMed
Watkins, L.P. & Yang, H. (2005). Detection of intensity change points in time-resolved single-molecule measurements. J Phys Chem B 109, 617–628.CrossRefGoogle ScholarPubMed
Yildiz, A., Forkey, J.N., McKinney, S.A., Ha, T., Goldman, Y.E. & Selvin, P.R. (2003). Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization. Science 300(5628), 2061–2065.CrossRefGoogle Scholar
Zheng, J., Du, G.G., Anderson, C.T., Keller, J.P., Orem, A., Dallos, P. & Cheatham, M. (2006). Analysis of the oligomeric structure of the motor protein prestin. J Biol Chem 281, 19916–19924.CrossRefGoogle ScholarPubMed
Zheng, J., Shen, W., He, D.Z., Long, K.B., Madison, L.D. & Dallos, P. (2000). Prestin is the motor protein of cochlear outer hair cells. Nature 405(6783), 149–155.CrossRefGoogle ScholarPubMed
Ziegler, U., Vinckier, A., Kernen, P., Zeisel, D., Biber, J., Semenza, G., Murer, H. & Groscurth, P. (1998). Preparation of basal cell membranes for scanning probe microscopy. FEBS Lett 436(2), 179–184.CrossRefGoogle ScholarPubMed