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Imaging single events at the cell membrane

Nature Chemical Biology volume 3, pages 92–98 (2007)Cite this article

Abstract

The ability to sense and respond to the environment is a hallmark of living systems. These processes occur at the levels of the organism, cells and individual molecules. Sensing of extracellular changes could result in a structural or chemical alteration in a molecule, which could in turn trigger a cascade of intracellular signals or regulated trafficking of molecules at the cell surface. These and other such processes allow cells to sense and respond to environmental changes. Often, these changes and the responses to them are spatially and/or temporally localized, and visualization of such events necessitates the use of high-resolution imaging approaches. Here we discuss optical imaging approaches and tools for imaging individual events at the cell surface with improved speed and resolution.

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Figure 1: Resolution of various optical imaging approaches.
Figure 2: Comparison of wide-field and TIRF imaging.
Figure 3: Use of TIRFM to study behavior of individual vesicles near the cell membrane.
Figure 4: Myosin V processive run with heads labeled with QDs of different colors.

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References

  1. Neher, E. & Sakmann, B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260, 799–802 (1976).

    Article  CAS  Google Scholar 

  2. Sakmann, B. & Neher, E. Patch clamp techniques for studying ionic channels in excitable membranes. Annu. Rev. Physiol. 46, 455–472 (1984).

    Article  CAS  Google Scholar 

  3. Sakmann, B. Elementary steps in synaptic transmission revealed by currents through single ion channels. Science 256, 503–512 (1992).

    Article  CAS  Google Scholar 

  4. Angleson, J.K. & Betz, W.J. Monitoring secretion in real time: capacitance, amperometry and fluorescence compared. Trends Neurosci. 20, 281–287 (1997).

    Article  CAS  Google Scholar 

  5. Mosharov, E.V. & Sulzer, D. Analysis of exocytotic events recorded by amperometry. Nat. Methods 2, 651–658 (2005).

    Article  CAS  Google Scholar 

  6. Ruta, V., Chen, J. & MacKinnon, R. Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel. Cell 123, 463–475 (2005).

    Article  CAS  Google Scholar 

  7. White, S.H., Ladokhin, A.S., Jayasinghe, S. & Hristova, K. How membranes shape protein structure. J. Biol. Chem. 276, 32395–32398 (2001).

    Article  CAS  Google Scholar 

  8. Gandhavadi, M., Allende, D., Vidal, A., Simon, S.A. & McIntosh, T.J. Structure, composition, and peptide binding properties of detergent soluble bilayers and detergent resistant rafts. Biophys. J. 82, 1469–1482 (2002).

    Article  CAS  Google Scholar 

  9. Sekar, R.B. & Periasamy, A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 160, 629–633 (2003).

    Article  CAS  Google Scholar 

  10. Rust, M., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  11. Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  CAS  Google Scholar 

  12. Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T. & Kasai, H. Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet. Science 297, 1349–1352 (2002).

    Article  CAS  Google Scholar 

  13. Webb, W.W. Applications of fluorescence correlation spectroscopy. Q. Rev. Biophys. 9, 49–68 (1976).

    Article  CAS  Google Scholar 

  14. Fahey, P.F. et al. Lateral diffusion in planar lipid bilayers. Science 195, 305–306 (1977).

    Article  CAS  Google Scholar 

  15. Schwille, P., Korlach, J. & Webb, W.W. Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes. Cytometry 36, 176–182 (1999).

    Article  CAS  Google Scholar 

  16. Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774 (2001).

    Article  CAS  Google Scholar 

  17. Axelrod, D. Cell-substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89, 141–145 (1981).

    Article  CAS  Google Scholar 

  18. Axelrod, D., Burghardt, T.P. & Thompson, N.L. Total internal reflection fluorescence. Annu. Rev. Biophys. Bioeng. 13, 247–268 (1984).

    Article  CAS  Google Scholar 

  19. Kawano, Y. et al. High-numerical-aperture objective lenses and optical system improved objective type total internal reflection fluorescence microscopy. Proc. SPIE 4098, 142–151 (2000).

    Article  CAS  Google Scholar 

  20. Axelrod, D. Selective imaging of surface fluorescence with very high aperture microscope objectives. J. Biomed. Opt. 6, 6–13 (2001).

    Article  CAS  Google Scholar 

  21. Schneckenburger, H. Total internal reflection fluorescence microscopy: technical innovations and novel applications. Curr. Opin. Biotechnol. 16, 13–18 (2005).

    Article  CAS  Google Scholar 

  22. Kittel, R.J. et al. Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312, 1051–1054 (2006).

    Article  CAS  Google Scholar 

  23. Willig, K.I., Rizzoli, S.O., Westphal, V., Jahn, R. & Hell, S.W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006).

    Article  CAS  Google Scholar 

  24. Sieber, J.J., Willig, K.I., Heintzmann, R., Hell, S.W. & Lang, T. The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane. Biophys. J. 90, 2843–2851 (2006).

    Article  CAS  Google Scholar 

  25. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    Article  CAS  Google Scholar 

  26. Giepmans, B.N., Adams, S.R., Ellisman, M.H. & Tsien, R.Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    Article  CAS  Google Scholar 

  27. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  28. Griesbeck, O. Fluorescent proteins as sensors for cellular functions. Curr. Opin. Neurobiol. 14, 636–641 (2004).

    Article  CAS  Google Scholar 

  29. Lippincott-Schwartz, J. & Smith, C.L. Insights into secretory and endocytic membrane traffic using green fluorescent protein chimeras. Curr. Opin. Neurobiol. 7, 631–639 (1997).

    Article  CAS  Google Scholar 

  30. Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha, V.V. Innovation: photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6, 885–891 (2005).

    Article  CAS  Google Scholar 

  31. Lippincott-Schwartz, J., Altan-Bonnet, N. & Patterson, G.H. Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. Suppl, S7–S14 (2003).

  32. Chudakov, D.M. & Lukyanov, K.A. Use of green fluorescent protein (GFP) and its homologs for in vivo protein motility studies. Biochemistry (Mosc.) 68, 952–957 (2003).

    Article  CAS  Google Scholar 

  33. Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S.W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl. Acad. Sci. USA 102, 17565–17569 (2005).

    Article  CAS  Google Scholar 

  34. Betzig, et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  35. Jaiswal, J.K., Goldman, E.R., Mattoussi, H. & Simon, S.M. Use of quantum dots for live cell imaging. Nat. Methods 1, 73–78 (2004).

    Article  Google Scholar 

  36. Jaiswal, J.K. & Simon, S.M. Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol. 14, 497–504 (2004).

    Article  CAS  Google Scholar 

  37. Gao, X. et al. In vivo molecular and cellular imaging with quantum dots. Curr. Opin. Biotechnol. 16, 63–72 (2005).

    Article  CAS  Google Scholar 

  38. Michalet, X. et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307, 538–544 (2005).

    Article  CAS  Google Scholar 

  39. Uyeda, H.T., Medintz, I.L., Jaiswal, J.K., Simon, S.M. & Mattoussi, H. Synthesis of compact multidentate ligands to prepare stable hydrophilic quantum dot fluorophores. J. Am. Chem. Soc. 127, 3870–3878 (2005).

    Article  CAS  Google Scholar 

  40. An, S. & Zenisek, D. Regulation of exocytosis in neurons and neuroendocrine cells. Curr. Opin. Neurobiol. 14, 522–530 (2004).

    Article  CAS  Google Scholar 

  41. Palfrey, H.C. & Artalejo, C.R. Secretion: kiss and run caught on film. Curr. Biol. 13, R397–R399 (2003).

    Article  CAS  Google Scholar 

  42. Jaiswal, J.K., Chakrabarti, S., Andrews, N.W. & Simon, S.M. Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis. PLoS Biol. 2, E233 (2004).

    Article  Google Scholar 

  43. Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005).

    Article  CAS  Google Scholar 

  44. Lampson, M.A., Schmoranzer, J., Zeigerer, A., Simon, S.M. & McGraw, T.E. Insulin-regulated release from the endosomal recycling compartment is regulated by budding of specialized vesicles. Mol. Biol. Cell 12, 3489–3501 (2001).

    Article  CAS  Google Scholar 

  45. Oheim, M., Loerke, D., Stuhmer, W. & Chow, R.H. The last few milliseconds in the life of a secretory granule. Docking, dynamics and fusion visualized by total internal reflection fluorescence microscopy (TIRFM). Eur. Biophys. J. 27, 83–98 (1998).

    Article  CAS  Google Scholar 

  46. Steyer, J.A., Horstmann, H. & Almers, W. Transport, docking and exocytosis of single secretory granules in live chromaffin cells. Nature 388, 474–478 (1997).

    Article  CAS  Google Scholar 

  47. Kreitzer, G. et al. Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells. Nat. Cell Biol. 5, 126–136 (2003).

    Article  CAS  Google Scholar 

  48. Ma, L. et al. Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion. Proc. Natl. Acad. Sci. USA 101, 9266–9271 (2004).

    Article  CAS  Google Scholar 

  49. Ehrlich, M. et al. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118, 591–605 (2004).

    Article  CAS  Google Scholar 

  50. Ohara-Imaizumi, M. et al. Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy. Biochem. J. 363, 73–80 (2002).

    Article  CAS  Google Scholar 

  51. O'Connell, K.M. & Tamkun, M.M. Targeting of voltage-gated potassium channel isoforms to distinct cell surface microdomains. J. Cell Sci. 118, 2155–2166 (2005).

    Article  CAS  Google Scholar 

  52. Massol, R.H., Larsen, J.E. & Kirchhausen, T. Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking. Exp. Cell Res. 306, 142–149 (2005).

    Article  CAS  Google Scholar 

  53. Lidke, D.S., Lidke, K.A., Rieger, B., Jovin, T.M. & Arndt-Jovin, D.J. Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors. J. Cell Biol. 170, 619–626 (2005).

    Article  CAS  Google Scholar 

  54. Warshaw, D.M. et al. Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity. Biophys. J. 88, L30–L32 (2005).

    Article  CAS  Google Scholar 

  55. Dahan, M. et al. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302, 442–445 (2003).

    Article  CAS  Google Scholar 

  56. Courty, S., Luccardini, C., Bellaiche, Y., Cappello, G. & Dahan, M. Tracking individual kinesin motors in living cells using single quantum-dot imaging. Nano Lett. 6, 1491–1495 (2006).

    Article  CAS  Google Scholar 

  57. Pramanik, A. & Rigler, R. Ligand-receptor interactions in the membrane of cultured cells monitored by fluorescence correlation spectroscopy. Biol. Chem. 382, 371–378 (2001).

    Article  CAS  Google Scholar 

  58. Lieto, A.M., Cush, R.C. & Thompson, N.L. Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy. Biophys. J. 85, 3294–3302 (2003).

    Article  CAS  Google Scholar 

  59. Bacia, K., Scherfeld, D., Kahya, N. & Schwille, P. Fluorescence correlation spectroscopy relates rafts in model and native membranes. Biophys. J. 87, 1034–1043 (2004).

    Article  CAS  Google Scholar 

  60. Ohsugi, Y., Saito, K., Tamura, M. & Kinjo, M. Lateral mobility of membrane-binding proteins in living cells measured by total internal reflection fluorescence correlation spectroscopy. Biophys. J. 91, 3456–3464 (2006).

    Article  CAS  Google Scholar 

  61. Gustafsson, M.G. Extended resolution fluorescence microscopy. Curr. Opin. Struct. Biol. 9, 627–634 (1999).

    Article  CAS  Google Scholar 

  62. Jaiswal, J.K., Andrews, N.W. & Simon, S.M. Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells. J. Cell Biol. 159, 625–635 (2002).

    Article  CAS  Google Scholar 

  63. Schmoranzer, J., Goulian, M., Axelrod, D. & Simon, S.M. Imaging constitutive exocytosis with total internal reflection fluorescence microscopy. J. Cell Biol. 149, 23–32 (2000).

    Article  CAS  Google Scholar 

  64. Johns, L.M., Levitan, E.S., Shelden, E.A., Holz, R.W. & Axelrod, D. Restriction of secretory granule motion near the plasma membrane of chromaffin cells. J. Cell Biol. 153, 177–190 (2001).

    Article  CAS  Google Scholar 

  65. Allersma, M.W., Bittner, M.A., Axelrod, D. & Holz, R.W. Motion matters: secretory granule motion adjacent to the plasma membrane and exocytosis. Mol. Biol. Cell 17, 2424–2438 (2006).

    Article  CAS  Google Scholar 

  66. Rappoport, J.Z., Taha, B.W. & Simon, S.M. Movement of plasma-membrane-associated clathrin spots along the microtubule cytoskeleton. Traffic 4, 460–467 (2003).

    Article  CAS  Google Scholar 

  67. Merrifield, C.J., Feldman, M.E., Wan, L. & Almers, W. Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat. Cell Biol. 4, 691–698 (2002).

    Article  CAS  Google Scholar 

  68. Merrifield, C.J., Perrais, D. & Zenisek, D. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593–606 (2005).

    Article  CAS  Google Scholar 

  69. Rappoport, J.Z., Benmerah, A. & Simon, S.M. Analysis of the AP-2 adaptor complex and cargo during clathrin-mediated endocytosis. Traffic 6, 539–547 (2005).

    Article  CAS  Google Scholar 

  70. Habuchi, S. et al. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc. Natl. Acad. Sci. USA 102, 9511–9516 (2005).

    Article  CAS  Google Scholar 

  71. Sosa, H., Peterman, E.J., Moerner, W.E. & Goldstein, L.S. ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy. Nat. Struct. Biol. 8, 540–544 (2001).

    Article  CAS  Google Scholar 

  72. Basche, T., Moerner, W.E., Orrit, M. & Talon, H. Photon antibunching in the fluorescence of a single dye molecule trapped in a solid. Phys. Rev. Lett. 69, 1516–1519 (1992).

    Article  CAS  Google Scholar 

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Acknowledgements

Work in our laboratory is supported by grants from the National Science Foundation (NSF FEB 00520813) and the National Institutes of Health (P20 GM072015 and GM072015 to S.M.S.). We thank P. Coffino for helpful comments on the manuscript.

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    Jyoti K Jaiswal & Sanford M Simon

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Correspondence to Sanford M Simon.

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Jaiswal, J., Simon, S. Imaging single events at the cell membrane. Nat Chem Biol 3, 92–98 (2007). https://doi.org/10.1038/nchembio855

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