where ip corresponds to the particle intensity, ib to the intensity of the background signal, Io to the averaged intensity for the first ten frames, and If to the averaged intensity for the last ten frames.
The graphs were classified according to the step-wise photobleaching of GFP subunits. Those samples that did not elicit step-wise photobleaching were discarded. To calculate the binomial distribution, we used the following equation:
where P is the probability that a GFP fluoresces (assuming P = 0.8), n is the estimated number of subunits, and K represents the expected number of fluorescent GFP molecules in each particle ranging from 1 to n. We calculate the binomial distribution for all possible values of K.
Results and discussion
We describe a simple method for photobleaching membrane proteins using a laser scanning confocal microscope (LSCM). Scanning confocal microscopy allows the observation of multiple distinct focal planes with a low background and better resolution (11) than standard epifluorescence microscopy (Figure 1A), although resolution is still limited in the z axis. In comparison, the axial resolution of TIRFM easily doubles what can be obtained with LSCM (11). This is because light coming from multiple out of focus planes is not efficiently eliminated by the pinhole, limiting the final resolution power.
A very efficient way to eliminate background signal is to remove the complete cell by sonication (Figure 1B). Once sonicated, pieces of plasma membrane left attached to the glass can be imaged to detect discrete punctae (Figure 2A and B). The membrane sheet preparation method was originally designed to provide access to intact synaptic vesicles (22); however, it can be also used to observe membrane proteins under nominally background-free conditions, allowing the observation of single molecules in native organization and the preservation of membrane associated functions. To demonstrate the combined methods, we set up a functional assay with membrane sheets from cells transfected with the pH-sensitive GFP pHluorin fused to the vesicular acetylcholine transporter (pHluorin-VAChT; Brauchi et al. 2008). Using this approach, we were able to monitor individual single vesicle fusions since each vesicle experiences a change in its intravesicular pH immediately after a fusion event (corresponding to the change from acid to neutral environment). When the membrane sheet is exposed to 2 mM Ca2 +, clear vesicular fusions can be detected (Supplementary movie s1), suggesting that vesicles and the associated machinery are intact and functional. We reasoned that docked vesicles are more delicate than proteins inserted in the plasma membrane; therefore, it is very likely that plasma membrane proteins preserve native stoichiometry after sonication.
To calibrate the experimental conditions of the technique, we transiently transfected HEK-293T cells with an EGF-tagged proton channel Hv1 (23). We choose Hv1 because independent studies strongly suggested that these channels form active dimers (18, 24, and 25). In order to estimate the number of channel subunits present on each particle, we defined ROIs over a number of bright spots and then performed SGP with the scanning laser (Figure 2B and C). Although the maximum number of bleaching steps observed was 4 (7%), the majority of the particles displayed only 2 bleaching steps (52%; Figure 2C and D). The distribution of bleaching steps showed that a significant population of spots also underwent only 1 step of bleaching (33%; Figure 2C and D). The bleaching profile observed in our samples was nearly identical under TIRF microscopy on intact cells (Figure 2E and F), and very similar to that previously reported for oocytes expressing Hv1 channels by Isacoff using TIRFM (18). We further analyzed the data set using binomial analysis. This approach allows us to compare our results with models of bleaching patterns generated for hypothetical channels of different numbers of subunits (1 to 6). The results from the binomial analysis clearly showed that the bleaching pattern obtained by TIRF or by using our cell-free assay corresponds to an arrangement of dimers,as previously described (18, 24, 25,; Figure 3).
The elimination of the cell when preparing the sheet of membrane also eliminates molecules located at different levels of the evanescent field. Thus, an advantage of our experimental approach is the prevention of bleaching steps of different size that are often observed in living cells under TIRF illumination. Another advantage is the ability to perfuse the sample, allowing the addition of chemicals to prevent bleaching. With this approach, trajectories would be larger and transitions much clearer. Finally, this method prevents the diffusion of molecules within the membrane. Although others have described lateral diffusion in PLBs (26), in our hands lateral diffusion at the time scale we sampled is minimal or imperceptible, making this a suitable technique for highly moving proteins such as Hv1 or TRPM8 channels (18, 19).
Several future applications can be envisioned for the assay. The intrinsic low noise of the sample makes this approach suitable for single molecule studies on plasma membrane proteins, not only with confocal microscopy but also with a regular epifluorescence microscope.
This work was funded by FONDECYT grants 1110906 (SB) and 1100871 (CBG). CT is a MECESUP fellow. We thank IS Ramsey for providing the Hv1 clone, and CA Toro for his assistance. The Brauchi Laboratory is part of CISNe-UACh and UACh Program in Cellular Dynamics and Microscopy.
The authors declare no competing interests.
Address correspondence to Sebastian Brauchi, Instituto de Fisiología, Facultad de Medicina, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile. E-mail: [email protected]
1.) Ali, M.H., and B. Imperiali. 2005. Protein oligomerization: how and why. Bioorg. Med. Chem. 13:5013-5020. 2.) Weiss, A., and J. Schlessinger. 1998. Switching signals on or off by receptor dimerization. Cell 94:277-280. 3.) Terrillon, S., and M. Bouvier. 2004. Roles of G-protein-coupled receptor dimerization. EMBO Rep. 5:30-34. 4.) Zheng, J., M.C. Trudeau, and W.N. Zagotta. 2002. Rod Cyclic Nucleotide-Gated Channels Have a Stoichiometry of Three CNGA1 Subunits and One CNGB1 Subunit. Neuron 36:891-896. 5.) Kroeger, K.M., A.C. Hanyaloglu, R.M. Seeber, L.E. Miles, and K.A. Eidne. 2001. Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer. J. Biol. Chem. 276:12736-12743. 6.) Quinn, D.J., N.V. Mcferran, J. Nelson, and W.P. Duprex. 2012. Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology. Biosci. Rep. 32:333-343. 7.) Sergeev, M., A.G. Godin, L. Kao, N. Abuladze, P.W. Wiseman, and I. Kurtz. 2012. Determination of Membrane Protein Transporter Oligomerization in Native Tissue Using Spatial Fluorescence Intensity Fluctuation Analysis. PLoS ONE 7:e36215. 8.) Kasai, R.S., K.G.N. Suzuki, E.R. Prossnitz, I. Koyama-Honda, Ch. Nakada, T.K. Fujiwara, and A. Kusumi. 2011. Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging. J. Cell Biol. 192:463-480. 9.) Madl, J., J. Weghuber, R. Fritsch, I. Derler, M. Fahrner, I. Frischauf, B. Lackner, Ch. Romanin, and G.J. Schütz. 2010. Resting State Orai1 Diffuses as Homotetramer in the Plasma Membrane of Live Mammalian Cells. J. Biol. Chem. 285:41135-41142. 10.) Iino, R., I. Koyama, and A. Kusumi. 2001. Single Molecule Imaging of Green Fluorescent Proteins in Living Cells: E-Cadherin Forms Oligomers on the Free Cell Surface. Biophys. J. 80:2667-2677. 11.) Steyer, J.A., and W. Almers. 2001. A real-time view of life within 100nm of the plasma membrane. Nat. Rev. Mol. Cell Biol. 2:268-275. 12.) Ulbrich, M.H., and E.Y. Isacoff. 2007. Subunit counting in membrane-bound proteins. Nat. Methods 4:319-321. 13.) Demuro, A., A. Penna, O. Safrina, A.V. Yeromin, A. Amcheslavsky, M.D. Cahalan, and I. Parker. 2011. Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states. Proc. Natl. Acad. Sci. USA 108:17832-17837. 14.) Reiner, A., R.J. Arant, and E.Y. Isacoff. 2012. Assembly Stoichiometry of the GluK2/GluK5 Kainate Receptor Complex. Cell Reports 1:234-240. 15.) Haggie, P.M., and A.S. Verkman. 2008. Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging. J. Biol. Chem. 283:23510-23513. 16.) Tajima, M., J.M. Crane, and A.S. Verkman. 2010. Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras. J. Biol. Chem. 285:8163-8170. 17.) Harms, G.S., L. Cognet, P.H.M. Lommerse, G.A. Blab, H. Kahr, R. Gamsjäger, H.P. Spaink, N.M. Soldatov. 2001. Single-Molecule Imaging of L-Type Ca2+ Channels in Live Cells. Biophys. J. 81:2639-2646. 18.) Tombola, F., M.H. Ulbrich, and E.Y. Isacoff. 2008. The voltage-gated proton channel Hv1 has two pores each controlled by one voltage sensor. Neuron 58:546-556. 19.) Veliz, L.A., C.A. Toro, J.P. Vivar, L.A. Arias, J. Villegas, M.A. Castro, and S. Brauchi. 2010. Near-membrane dynamics and capture of TRPM8 channels within transient confinement domains. PLoS ONE 5:e13290. 20.) Latorre, R., and O. Alvarez. 1981. Voltage-dependent channels in planar lipid bilayer membranes. Physiol. Rev. 61:77-150. 21.) Poulos, J.L., S.A. Portonovo, H. Bang, and J.J. Schmidt. 2010. Automatable lipid bilayer formation and ion channel measurement using sessile droplets. J. Phys.: Condens. Matter. 22:454105. 22.) Barszczewski, M., J.J. Chua, A. Stein, U. Winter, R. Heintzmann, F.E. Zilly, D. Fasshauer, T. Lang, and R. Jahn. 2008. A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion. Mol. Biol. Cell 19:776-784. 23.) Ramsey, I.S., M.M. Moran, J.A. Chong, and D.E. Clapham. 2006. A voltage-gated proton-selective channel lacking the pore domain. Nature 440:1213-1216. 24.) Koch, H.P., T. Kurokawa, Y. Okochi, M. Sasaki, Y. Okamura, and H.P. Larsson. 2008. Multimeric nature of voltage-gated proton channels. Proc. Natl. Acad. Sci. USA 105:9111-9116. 25.) Lee, S.Y., J.A. Letts, and R. Mackinnon. 2008. Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1. Proc. Natl. Acad. Sci. USA 105:7692-7695. 26.) Ladha, S., A.R. Mackie, L.J. Harvey, D.C. Clark, E.J. Lea, M. Brullemans, and H. Duclohier. 1996. Lateral diffusion in planar lipid bilayers: a fluorescence recovery after photobleaching investigation of its modulation by lipid composition, cholesterol, or alamethicin content and divalent cations. Biophys. J. 71:1364-1373.