Jump to ContentJump to Main Navigation
Show Summary Details

Optofluidics, Microfluidics and Nanofluidics

formerly Optofluidics

Ed. by Sada, Cinzia

1 Issue per year

Emerging Science

Open Access
See all formats and pricing

Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

Cornelia Junghans
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany
/ Franz-Josef Schmitt
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany
/ Vladana Vukojević
  • Karolinska Institutet, Department of Clinical Neuroscience, Center for Molecular Medicine CMM L8:01, 17176 Stockholm, Sweden
/ Thomas Friedrich
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany
Published Online: 2015-12-31 | DOI: https://doi.org/10.1515/optof-2015-0001


Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time scale as the diffusion of the labeled complexes through the detection volume (1– 100 ms). Since the origin of this quenching process is currently unclear, care has to be taken when the Dreiklang label is intended to be used in FCS applications.

Keywords: Membrane protein diffusion; solvent viscosity; diffusion constants; lateral mobility; molecular crowding; HEK 293 cells; transient transfection; photoswitchable; fluorescence proteins


  • [1] T. Brakemann, A. C. Stiel, G. Weber, M. Andresen, I. Testa, T. Grotjohann, M. Leutenegger, U. Plessmann, H. Urlaub, C. Eggeling, M. C. Wahl, S. W. Hell, S. Jakobs, A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching, Nat. Biotechnol., 29, 2011, 942 [Crossref]

  • [2] O. Shimomura, F. H. Johnson, Y. and Saiga, Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea, J. Cell. Comp. Physiol., 59, 1962, 223 [Crossref]

  • [3] S. W. Hell, J. and Wichmann, Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy, Opt. Lett. 19, 1994, 780 [Crossref]

  • [4] T. A. Klar, S. W. and Hell, Subdiffraction resolution in far-field fluorescence microscopy, Opt. Lett. 24, 1999, 954 [Crossref]

  • [5] T. A. Klar, S. Jakobs, M. Dyba, A. Egner, S.W. Hell, Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission, Proc. Natl. Acad. Sci. U. S. A., 97, 2000, 8206 [Crossref]

  • [6] M. J. Rust, M. Bates, X. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM), Nat. Methods 3, 2006, 793

  • [7] S. T. Hess, T. P. Girirajan, M. D. Mason, Ultra-high resolution imaging by fluorescence photoactivation localization microscopy, Biophys. J. 91, 2006, 4258 [Crossref]

  • [8] O. Shimomura, Structure of the chromophore of Aequorea green fluorescent protein, FEBS Letters, 104, 1979, 220 [Crossref]

  • [9] C. D. Hu, Y. Chinenov, T. K. Kerppola, Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation, Mol. Cell, 9, 2002, 789 [Crossref]

  • [10] J. D. Pedelacq, S. Cabantous, T. Tran, T. C. Terwilliger, G. S. Waldo, Engineering and characterization of a superfolder green fluorescent protein, Nat. Biotechnol. 24, 2006, 79 [Crossref]

  • [11] S. Cabantous, T. C. Terwilliger, G. S.Waldo, Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein, Nat. Biotechnol. 23, 2005, 102 [Crossref]

  • [12] G. S. Baird, D. A. Zacharias, R. Y. Tsien, Circular permutation and receptor insertion within green fluorescent proteins, Proc. Natl. Acad. Sci. U. S. A., 96, 1999, 11241 [Crossref]

  • [13] J. Nakai, M. Ohkura, K. Imoto, A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein, Nat. Biotechnol., 19, 2001, 137 [Crossref]

  • [14] Y. Zhao, Y. Yang, Profiling metabolic states with genetically encoded fluorescent biosensors for NADH, Curr.Opin. Biotechnol., 31, 2015, 86 [Crossref]

  • [15] D.Magde,W.W. Webb, E. Elson, Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy, Phys. Rev. Lett., 29, 1972, 705 [Crossref]

  • [16] S. R. Aragón, R. Pecora, Fluorescence correlation spectroscopy as a probe ofmolecular dynamics, J. Chem. Phys., 64, 1976, 1791

  • [17] G. Bonnet, O. Krichevsky, A. Libchaber, Kinetics of conformational fluctuations in DNA hairpin-loops, Proc. Natl. Acad. Sci. U.S.A., 95, 1998, 8602 [Crossref]

  • [18] M. Ehrenberg, R. Rigler, Rotational Brownian motion and fluorescence intensity fluctuations, Chem. Phys., 4, 1974, 390 [Crossref]

  • [19] U. Haupts, S. Maiti, P. Schwille, W. W. Webb, Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy, Proc. Natl. Acad. Sci. U.S.A., 95, 19098, 13573

  • [20] P. Kask, P. Piksarv, M. Pooga, Ü. Mets, E. Lippmaa, Separation of the rotational contribution in fluorescence correlation experiments, Biophys. J., 55, 1989, 213 [Crossref]

  • [21] D.Magde, Chemical kinetics and fluorescence correlation spectroscopy, Quart. Rev. Biophys., 9, 1976, 35 [Crossref]

  • [22] B. Rauer, E. Neumann, J.Widengren, R. Rigler, Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor, Biophys. Chem., 58, 1996, 3 [Crossref]

  • [23] J. Widengren, Ü. Mets, R. Rigler, Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study, J. Phys. Chem., 99, 1995, 13368

  • [24] J. Widengren, R. Rigler, Ü. Mets, Triplet-state monitoring by fluorescence correlation spectroscopy, J. Fluoresc., 4, 1994, 255 [Crossref]

  • [25] E. Haustein, P. Schwille, Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy, Methods, 29, 2003, 153 [Crossref]

  • [26] C. Eggeling, S. Berger, L. Brand, J. R. Fries, J. Schaffer, A. Volkmer, C. A. Seidel, Data registration and selective singlemolecule analysis using multi-parameter fluorescence detection, J. Biotechnol., 86, 2001, 163

  • [27] B. Lounis, H. A. Bechtel, D. Gerion, P. Alivisatos, W. E. Moerner, Photon antibunching in single CdSe/ZnS quantum dot fluorescence, Chem. Phys. Letters, 329, 2000, 399

  • [28] S. J. Singer, G. L. Nicolson, The fluid mosaic model of the structure of cell membranes, Science, 175, 1972, 720

  • [29] A. D. Dupuy, D. M. Engelman, Protein area occupancy at the center of the red blood cell membrane, Proc. Natl. Acad. Sci. U. S. A., 105, 2008, 2848 [Crossref]

  • [30] E. Zinser, C. D. Sperka-Gottlieb, E. V. Fasch, S. D. Kohlwein, F. Paltauf, F., G. Daum, Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae, J. Bacteriol., 173, 1991, 2026

  • [31] P. G. Saffman, M. Delbrück, Brownian motion in biological membranes, Proc. Natl. Acad. Sci. U. S. A., 72, 1975, 3111 [Crossref]

  • [32] Y. Gambin, R. Lopez-Esparza, M. Reffay, E. Sierecki, N. S. Gov, M. Genest, R. S. Hodges, W. Urbach, Lateral mobility of proteins in liquid membranes revisited, Proc. Natl. Acad. Sci. U. S. A., 103, 2006, 2098 [Crossref]

  • [33] M. Khalid, F. Cornelius, R. J. Clarke, Dual mechanisms of allosteric acceleration of the Na(+),K(+)-ATPase by ATP, Biophys. J., 98, 2010, 2290 [Crossref]

  • [34] M. De Fusco, R. Marconi, L. Silvestri, L. Atorino, L. Rampoldi, L. Morgante, A. Ballabio, P. Aridon, G. Casari, Haploinsuflciency of ATP1A2 encoding the Na+/K+ pump alpha2 subunit associated with familial hemiplegic migraine type 2, Nat. Genet. 33, 2003, 192

  • [35] J. P. Morth, H. Poulsen, M. S. Toustrup-Jensen, V. R. Schack, J. Egebjerg, J. P. Andersen, B. Vilsen, P. Nissen, The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations, Philos. Trans. R. Soc. Lond. B Biol. Sci., 364, 2009, 217

  • [36] N. N. Tavraz, K. L. Dürr, J. B. Koenderink, T. Freilinger, E. Bamberg, M. Dichgans, T. Friedrich, Impaired plasma membrane targeting or protein stability by certain ATP1A2 mutations identified in sporadic or familial hemiplegic migraine, Channels (Austin), 3, 2009, 82

  • [37] N. N. Tavraz, T. Friedrich, K. L. Dürr, J. B. Koenderink, E. Bamberg, T. Freilinger, M. Dichgans, Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type, J. Biol. Chem., 283, 2008, 31097

  • [38] D. P. Calderon, R. Fremont, F. Kraenzlin, K. Khodakhah, The neural substrates of rapid-onset Dystonia-Parkinsonism, Nat. Neurosci., 14, 2011, 357

  • [39] P. de Carvalho Aguiar, K. J. Sweadner, J. T. Penniston, J. Zaremba, L. Liu, M. Caton, G. Linazasoro, M. Borg, M. A. Tijssen, S. B. Bressman, W. B. Dobyns, A. Brashear, L. J. Ozelius, Mutations in the Na+/K+ -ATPase alpha3 gene ATP1A3 are associated with rapid-onset dystonia parkinsonism, Neuron, 43, 2004, 169

  • [40] E. L. Heinzen, A. Arzimanoglou, A. Brashear, S. J. Clapcote, F. Gurrieri, D. B. Goldstein, S. H. Johannesson, M. A. Mikati, B. Neville, S. Nicole, L. J. Ozelius, H. Poulsen, T. Schyns, K. J. Sweadner, A. van den Maagdenberg, B. Vilsen, Distinct neurological disorders with ATP1A3 mutations, Lancet Neurol. 13, 2014, 503

  • [41] M. K. Demos, C. D. van Karnebeek, C. J. Ross, S. Adam, Y. Shen, S. H. Zhan, C. Shyr, G. Horvath, M. Suri, A. Fryer, S. J. Jones, J. M. Friedman, A novel recurrent mutation in ATP1A3 causes CAPOS syndrome, Orphanet J. Rare Dis., 9, 2014, 15

  • [42] X. Liu, Z. Spicarova, S. Rydholm, J. Li, H. Brismar, A. Aperia, Ankyrin B modulates the function of Na,K-ATPase/inositol 1,4,5- trisphosphate receptor signaling microdomain, J. Biol. Chem., 283, 2008, 11461

  • [43] P. J. Mohler, J. J. Schott, A. O. Gramolini, K. W. Dilly, S. Guatimosim, W. H. duBell, L, S. Song, K. Haurogne, F. Kyndt, M. E. Ali, T. B. Rogers, W. J. Lederer, D. Escande, H. Le Marec, V. Bennett, Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death, Nature, 421, 2003, 634

  • [44] V. Vukojevic, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, R. Rigler, Quantitative single-molecule imaging by confocal laser scanning microscopy, Proc. Natl. Acad. Sci. U. S. A., 105, 2008, 18176 [Crossref]

  • [45] V. Vukojevic, Y. Ming, C. D’Addario, M. Hansen, U. Langel, R. Schulz, B. Johansson, R. Rigler, L. Terenius, Mu-opioid receptor activation in live cells, FASEB J., 22, 2008, 3537 [Crossref]

  • [46] J. R. Lakowicz, Principles of fluorescence spectroscopy, 3rd ed. , 2006, Springer, New York

  • [47] D. Magde, E. L. Elson, W. W. Webb, Fluorescence correlation spectroscopy. II. An experimental realization, Biopolymers, 13, 1974, 29 [Crossref]

  • [48] G.Majer, J. P. Melchior, Characterization of the fluorescence correlation spectroscopy (FCS) standard rhodamine 6G and calibration of its diffusion coeflcient in aqueous solutions, J. Chem. Phys., 140, 2014, 094201

  • [49] R. Rigler, P. Grasselli, M. Ehrenberg, Fluorescence Correlation Spectroscopy and Application to the Study of Brownian Motion of Biopolymers, Phys. Scr., 19, 1979, 486

  • [50] P. Kapusta, Absolute Diffusion Coeflcients: Compilation of Reference Data for FCS Calibration, Picoquant GmbH, 2010, http://www.picoquant.com/images/uploads/page/files/ 7353/appnote_diffusioncoeflcients.pdf, accessed on June 12, 2015.

  • [51] J. Widengren, Ü. Mets, R. Rigler, Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy, Chem. Phys., 250, 1999, 171

  • [52] R.Swaminathan, C. P. Hoang, A. S. Verkman, Photobleaching recovery and anisotropy decay of green fluorescent protein GFPS65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion, Biophys. J., 72, 1997, 1900

  • [53] M. A. Hink, R. A. Griep, J. W. Borst, A. van Hoek, M. H. Eppink, A. Schots, A. J. Visser, Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein, J. Biol. Chem. 275, 2000, 17556

  • [54] Glycerine Producers’ Association, Physical Properties of Glycerine and Its Solutions, New York, 1963, http://www.aciscience.org/docs/physical_properties_of_ glycerine_and_its_solutions.pdf.

  • [55] D. A. Zacharias, J. D. Violin, A. C. Newton, R. Y. Tsien, Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells, Science, 296, 2002, 913

  • [56] M. S. Paller, Lateral mobility of Na,K-ATPase and membrane lipids in renal cells. Importance of cytoskeletal integrity, J. Membr. Biol. 142, 1994, 127

  • [57] K. Weiss, A. Neef, Q. Van, S. Kramer, I. Gregor, J. Enderlein, Quantifying the diffusion of membrane proteins and peptides in black lipid membranes with 2-focus fluorescence correlation spectroscopy, Biophys. J., 105, 2013, 455

  • [58] S. Ramadurai, A. Holt, V. Krasnikov, G. van den Bogaart, J. A. Killian, B. Poolman, Lateral diffusion of membrane proteins, J. Am. Chem. Soc. 131, 2009, 12650

  • [59] A. Naji, A. J. Levine, P. A. Pincus, Corrections to the Saffman- Delbruck mobility for membrane bound proteins, Biophys. J., 93, 2007, L49

  • [60] T. Shinoda, H. Ogawa, F. Cornelius, C. Toyoshima, Crystal structure of the sodium-potassiumpump at 2.4 Å resolution, Nature, 459, 2009, 446

  • [61] P. Cicuta, S. L. Keller, S. L. Veatch, Diffusion of liquid domains in lipid bilayer membranes, J. Phys. Chem. B, 111, 2007, 3328

  • [62] W. L. C. Vaz, F. Goodsaid-Zalduondo, K. Jacobson, Lateral diffusion of lipids and proteins in bilayer membranes, FEBS Letters, 174, 1984, 199

  • [63] C. Jordan, B. Puschel, R. Koob, D. Drenckhahn, Identification of a binding motif for ankyrin on the alpha-subunit of Na+,K+- ATPase, J. Biol. Chem., 270, 1995, 29971

  • [64] T. Cai, H. Wang, Y. Chen, L. Liu, W. T. Gunning, L. E. Quintas, Z. J. Xie, Regulation of caveolin-1 membrane traflcking by the Na/KATPase, J. Cell Biol. 182, 2008, 1153

  • [65] T. Friedrich, E. Bamberg, G. Nagel, Na+,K+-ATPase pump currents in giant excised patches activated by an ATP concentration jump, Biophys. J., 71, 1996, 2486

  • [66] J. Kockskämper, G. Gisselmann, H. G. Glitsch, Comparison of ouabain-sensitive and -insensitive Na/K pumps in HEK293 cells, Biochim. Biophys. Acta, 1325, 1997, 197

  • [67] N. A. Jensen, J. G. Danzl, K. I. Willig, F. Lavoie-Cardinal, T. Brakemann, S. W. Hell, S. Jakobs, Coordinate-targeted and coordinate-stochastic super-resolution microscopy with the reversibly switchable fluorescent protein Dreiklang, ChemPhysChem 15, 2014, 756 [Crossref]

Received: 2015-10-06

Accepted: 2015-10-20

Published Online: 2015-12-31

Citation Information: Optofluidics, Microfluidics and Nanofluidics. Volume 2, Issue 1, Pages 1–14, ISSN (Online) 2300-7435, DOI: https://doi.org/10.1515/optof-2015-0001, December 2015

© 2015 C. Junghans et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. (CC BY-NC-ND 3.0)

Comments (0)

Please log in or register to comment.