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Studying protein dynamics in living cells

Key Points

  • The cloning of green fluorescent protein (GFP), engineering of chimeric fusion proteins, and advances in fluorescence imaging methods have made it possible for researchers to follow the dynamics and interactions of proteins in living cells. In this review, we describe the application of biophysical microscopy-based techniques in combination with GFP-chimaeras to characterize protein dynamics in living cells.

  • The photobleaching technique FRAP (fluorescence recovery after photobleaching)) has been used since the mid-1970s to determine the diffusion constant of fluorescent antibody-labelled proteins on the plasma membrane of cells. Photobleaching of GFP-chimeric proteins localized throughout the cell has been recently exploited to determine the viscosities of different cellular environments and to reveal the diffusional mobilities of various proteins.

  • Variations of FRAP, including selective photobleaching and FLIP (fluorescence loss in photobleaching), can reveal the continuities and discontinuities of intracellular organelles and compartments. In addition, FLIP has been used to characterize the kinetics of protein binding and release in living cells.

  • FRET (fluorescence resonance energy transfer) is a property of certain pairs of fluorophores, in which a high energy fluorophore can excite a lower energy fluorophore when the two fluorophores are in extremely close proximity. FRET microscopy has been used to determine whether proteins that co-localize are physically interacting in fixed and living cells. We describe several recent applications and variations of FRET in the review.

  • The technique of FCS (fluorescence correlation spectroscopy) has recently become accessible to cell biologists with commercially available microscopes. FCS can be used to measure diffusion constants for multiple populations and ratios of bound and free proteins, allowing for sensitive measurements of protein–protein interactions in cells.

  • Ongoing development of GFP variants, unusual properties of GFP, alternatives to GFP in living cells, and new microscopy techniques hold much promise for future studies of protein dynamics in living cells.

Abstract

Since the advent of the green fluorescent protein, the subcellular localization, mobility, transport routes and binding interactions of proteins can be studied in living cells. Live cell imaging, in combination with photobleaching, energy transfer or fluorescence correlation spectroscopy are providing unprecedented insights into the movement of proteins and their interactions with cellular components. Remarkably, these powerful techniques are accessible to non-specialists using commercially available microscope systems.

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Figure 1: Fluorescence recovery after photobleaching.
Figure 2: Mechanisms that reduce the mobility of membrane proteins.
Figure 3: Fluorescence loss in photobleaching.
Figure 4: Principles of FRET.
Figure 5: Examples of recent applications of FRET microscopy.
Figure 6: Principles of fluorescence correlation spectroscopy.

References

  1. 1

    Matz, M. V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnol. 17, 969–973 (1999); erratum 17, 1227 (1999).

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Heim, R., Prasher, D. C. & Tsien, R. Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl Acad. Sci. USA 91, 12501–12504 (1994).

    CAS  PubMed  Article  Google Scholar 

  4. 4

    Swaminathan, R., Hoang, C. P. & Verkman, A. S. Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys. J. 72, 1900–1907 (1997).Characterizes the photobleaching properties of GFP in solution and in vivo in the cytoplasm.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5

    Lippincott–Schwartz, J. et al. in Green Fluorescent Proteins (eds Sullivan, K. & Kay, S.) 261–291 (Academic, San Diego, 1999).

    Google Scholar 

  6. 6

    Yang, F., Moss, L. G. & Phillips, G. N. J. The molecular structure of green fluorescent protein. Nature Biotechnol. 14, 1246–1251 (1996).

    CAS  Article  Google Scholar 

  7. 7

    Prendergast, F. G. Biophysics of the green fluorescent protein. Methods Cell Biol. 58, 1–18 (1999).

    CAS  PubMed  Google Scholar 

  8. 8

    Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E. & Webb, W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055–1069 (1976).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9

    White, J. & Stelzer, E. Photobleaching GFP reveals protein dynamics inside living cells. Trends Cell Biol. 9, 61–65 (1999).

    CAS  PubMed  Article  Google Scholar 

  10. 10

    Saxton, M. J. & Jacobsen, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).

    CAS  PubMed  Article  Google Scholar 

  11. 11

    Edidin, M. in Mobility and Proximity in Biological Membranes (eds Edidin, M., Szollosi, J. & Tron, L.) 109–135 (CRC Press, Boca Raton, Florida, 1994).

    Google Scholar 

  12. 12

    Feder, T. J., Brust-Mascher, I., Slattery, J. P., Baird, B. & Webb, W. W. Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys. J. 70, 2767–2773 (1996).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13

    Ellenberg, J. et al. Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol. 138, 1193–1206 (1997).Shows the connectivity of the endoplasmic reticulum (ER) and nuclear envelope in vivo and provides a visual example of how a protein can be mobile in one domain of the cell and immobile in another domain.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14

    Edidin, M. in The Structure of Biological Membranes (ed. Yeagle, P.) 539–572 (CRC, Boca Raton, 1992).

    Google Scholar 

  15. 15

    Cole, N. B. et al. Diffusional mobility of Golgi proteins in membranes of living cells. Science 273, 797–801 (1996).First paper to use FRAP to measure the diffusion rate of a GFP chimaera in a cellular organelle, the Golgi. In addition, this paper introduces the FLIP method of photobleaching.

    CAS  PubMed  Article  Google Scholar 

  16. 16

    Sciaky, N. et al. Golgi tubule traffic and the effects of brefeldin A visualized in living cells. J. Cell Biol. 139, 1137–1155 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17

    Partikian, A., Olveczky, B., Swaminathan, R., Li, Y. & Verkman, A. S. Rapid diffusion of green fluorescent protein in the mitochondrial matrix. J. Cell Biol. 140, 821–829 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18

    Olveczky, B. P. & Verkman, A. S. Monte Carlo analysis of obstructed diffusion in three dimensions: application to molecular diffusion in organelles. Biophys. J. 74, 2722–2730 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19

    Siggia, E. D., Lippincott-Schwartz, J. & Bekiranov, S. Diffusion in an inhomogeneous media: theory and simulations applied to a whole cell photobleach recovery. Biophys. J. 79, 1761–1770 (2000).Describes a simulation program that can be used to calculate diffusion rates from FRAP data obtained by a confocal microscope.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20

    Dayel, M. J., Hom, E. F. Y. & Verkman, A. S. Diffusion of green fluorescent protein in the aqueous-phase lumen of endoplasmic reticulum. Biophys. J. 76, 2843–2851 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21

    Phair, R. D. & Misteli, T. High mobility of proteins in the mammalian cell nucleus. Nature 404, 604–609 (2000).The relatively slow diffusion rates of several nuclear GFP chimaeras are due to the high density of binding sites for the chimaeras throughout the nucleus. A kinetic model of the protein dynamics is used to calculate the on/off rates of chimaera binding.

    CAS  PubMed  Article  Google Scholar 

  22. 22

    Gordon, G. W., Chazotte, B., Wang, X. F. & Herman, B. Analysis of simulated and experimental fluorescence recovery after photobleaching. Data for two diffusing components. Biophys. J. 68, 766–778 (1995).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23

    Periasamy, N. & Verkman, A. S. Analysis of fluorophore diffusion by continous distributions of diffusion coefficients: application to photobleaching measurements of multicomponent and anomalous diffusion. Biophys. J. 75, 557–567 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24

    Kruhlak, M. J. et al. Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments. J. Cell Biol. 150, 41–51 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25

    Houtsmuller, A. B. et al. Action of DNA repair endonuclease ERCC1/XPF in living cells. Science 284, 958–961 (2000).

    Article  Google Scholar 

  26. 26

    Poo, M. M. & Cone, R. A. Lateral diffusion of rhodopsin in the photoreceptor membrane. Nature 247, 438–441 (1974).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27

    Marguet, D. et al. Lateral diffusion of GFP-tagged H2Ld molecules and of GFP–TAP1 reports on the assemby and retention of these molecules in the endoplasmic reticulum. Immunity 11, 231–240 (1999).Characterizes the effect of the formation of large protein complexes of TAP and MHC class I in the ER membrane on the diffusion rate, and calculates the relative contributions of several diffusing species to a single apparent diffusion coefficient.

    CAS  PubMed  Article  Google Scholar 

  28. 28

    Zaal, K. J. M. et al. Golgi membranes are absorbed into and reemerge from the ER during mitosis. Cell 99, 589–601 (1999).Uses FRAP to measure the mobilty of a Golgi membrane protein during interphase and metaphase to test whether the Golgi fragments or fuses with the endoplasmic reticulum during mitosis. In addition, selective photobleaching is used to calculate the relative rates of transport between the ER and Golgi in both directions.

    CAS  PubMed  Article  Google Scholar 

  29. 29

    Saxton, M. Anomalous diffusion due to obstacles: a Monte Carlo study. Biophys. J. 66, 394–401 (1994).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30

    Saxton, M. Anomalous diffusion due to binding: a Monte Carlo study. Biophys. J. 70, 1250–1262 (1996).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31

    Adams, C. L., Chen, Y., Smith, S. J. & Nelson, W. J. Mechanisms of epithelial cell–cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin–green fluorescent-protein. J. Cell Biol. 142, 1105–1119 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32

    Nehls, S. et al. Dynamics and retention of misfolded proteins in native ER membranes. Nature Cell Biol. 2, 288–295 (2000).The diffusion rates and mobility of misfolded aggregated ER membrane proteins are compared to correctly folded proteins and are found to be similar. Only conditions that globally perturb folding in the ER were found to have an effect on protein mobility.

    CAS  PubMed  Article  Google Scholar 

  33. 33

    Subramanian, K. & Meyer, T. Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores. Cell 89, 963–971 (1997).

    CAS  PubMed  Article  Google Scholar 

  34. 34

    Terasaki, M., Jaffe, L. A., Hunnicutt, G. R. & Hammer, J. A. R. Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein. Dev. Biol. 179, 320–328 (1996).

    CAS  PubMed  Article  Google Scholar 

  35. 35

    Kohler, R. H., Cao, J., Zipfel, W. R., Webb, W. W. & Hanson, M. R. Exchange of protein molecules through connections between higher plastids. Science 276, 2039–2042 (1997).

    CAS  PubMed  Article  Google Scholar 

  36. 36

    Storrie, B. et al. Recycling of Golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering. J. Cell Biol. 143, 1505–1521 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37

    Presley, J. F., Miller, C., Zaal, K., Ellenberg, J. & Lippincott–Schwartz, J. In vivo dynamics of COPI. Mol. Biol. Cell 9, S746 (1998).

  38. 38

    Stephens, D. J., Lin-Marq, N., Pagano, A., Pepperkok, R. & Paccaud, J. P. COPI-coated ER-to-Golgi transport complexes segregate form COPII in close proximity to ER exit sites. J. Cell Sci. 113, 2177–2185 (2000).

    CAS  PubMed  Google Scholar 

  39. 39

    Vasudevan, C. et al. The distribution and translocation of the G protein ADP-ribosylation factor 1 in live cells is determined by its GTPase activity. J. Cell Sci. 111, 1277–1285 (1998).

    CAS  PubMed  Google Scholar 

  40. 40

    Oancea, E., Teruel, M. N., Quest, A. F. & Meyer, T. Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators of diacylglycerol signaling in living cells. J. Cell Biol. 140, 485–498 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41

    Reits, E. A., Benham, A. M., Plougastel, B., Neefjes, J. & Trowsdale, J. Dynamics of the proteasome distribution in living cells. EMBO J. 16, 6087–6094 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42

    Presley, J. F. et al. ER-to-Golgi transport visualized in living cells. Nature 389, 81–85 (1997).

    CAS  PubMed  Article  Google Scholar 

  43. 43

    Hirschberg, K. et al. Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells. J. Cell Biol. 143, 1485–1503 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44

    Nakata, T., Terada, S. & Hirokawa, N. Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons. J. Cell Biol. 140, 659–674 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45

    Wu, P. & Brand, L. Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13 (1994).

    CAS  PubMed  Article  Google Scholar 

  46. 46

    Clegg, R. M. Fluorescence resonance energy transfer. Curr. Opin. Biotechnol. 6, 103–110 (1995).

    CAS  PubMed  Article  Google Scholar 

  47. 47

    Patterson, G. H., Piston, D. W. & Barisas, B. G. Förster distances between green fluorescent protein pairs. Anal. Biochem. 284, 438–440 (2000).

    CAS  PubMed  Article  Google Scholar 

  48. 48

    Pollok, B. A. & Heim, R. Using GFP in FRET-based applications. Trends Cell Biol. 9, 57–60 (1999).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49

    Periasamy, A. & Day, R. N. Visualizing protein interactions in living cells using digitized GFP imaging and FRET microscopy. Methods Cell Biol. 58, 293–314 (1999).

    CAS  PubMed  Article  Google Scholar 

  50. 50

    Heim, R. & Tsien, R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182 (1996).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51

    Bastiaens, P. I. H. & Jovin, T. M. in Cell Biology: A Laboratory Handbook (ed. Celis, J. E.) 136–146 (Academic, New York, 1998).

    Google Scholar 

  52. 52

    Wouters, F. S. & Bastiaens, P. I. Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Curr. Biol. 9, 1127–1130 (1999).Describes a clever assay to evaluate the phosphorylation state of a protein using FRET.

    CAS  PubMed  Article  Google Scholar 

  53. 53

    Bastiaens, P. I. & Squire, A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9, 48–52 (1999).

    CAS  PubMed  Article  Google Scholar 

  54. 54

    Ng, T. et al. Imaging protein kinase Cα activation in cells. Science 283, 2085–2089 (1999).

    CAS  PubMed  Article  Google Scholar 

  55. 55

    Ng, T. et al. PKCα regulates β1 intergrin-dependent cell motility through association and control of integrin traffic. EMBO J. 18, 3909–3923 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. 56

    Emmanouilidou, E. et al. Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon. Curr. Biol. 9, 915–918 (1999).

    CAS  PubMed  Article  Google Scholar 

  57. 57

    Romoser, V. A., Hinkle, P. M. & Persechini, A. Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13270–13274 (1997).

    CAS  PubMed  Article  Google Scholar 

  58. 58

    Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59

    Zaccolo, M. et al. A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nature Cell Biol. 2, 25–29 (2000).

    CAS  PubMed  Article  Google Scholar 

  60. 60

    Honda, A. et al. Spatiotemporal dynamics of guanosine 3′,5′–cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc. Natl Acad. Sci. USA 98, 2437–2442 (2001).

    CAS  PubMed  Article  Google Scholar 

  61. 61

    Mitra, R. D., Silva, C. M. & Youvan, D. C. Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. Gene 173, 13–17 (1996).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  62. 62

    Mahajan, N. P., Harrison-Shostak, D. C., Michaux, J. & Herman, B. Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis. Chem. Biol. 6, 401–409 (1999).

    CAS  PubMed  Article  Google Scholar 

  63. 63

    Sagot, I., Bonneu, M., Balguerie, A. & Aigle, M. Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast. FEBS Lett. 447, 53–57 (1999).

    CAS  PubMed  Article  Google Scholar 

  64. 64

    Vanderklish, P. W. et al. Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer. Proc. Natl Acad. Sci. USA 97, 2253–2258 (2000).

    CAS  PubMed  Article  Google Scholar 

  65. 65

    Xu, X. et al. Detection of programmed cell death using fluorescence energy transfer. Nucleic Acids Res. 26, 2034–2035 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. 66

    Zlokarnik, G. et al. Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporter. Science 279, 84–88 (1998).

    CAS  PubMed  Article  Google Scholar 

  67. 67

    Xu, Y., Piston, D. W. & Johnson, C. H. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl Acad. Sci. USA 96, 151–156 (1999).

    CAS  PubMed  Article  Google Scholar 

  68. 68

    Siegel, R. M. et al. Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science 288, 2354–2357 (2000).

    CAS  PubMed  Article  Google Scholar 

  69. 69

    Chan, F. K.-M. et al. A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science 288, 2351–2354 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. 70

    Teruel, M. N. & Meyer, T. Translocation and reversible localization of signaling proteins: a dynamic future for signal transduction. Cell 103, 181–184 (2000).

    CAS  PubMed  Article  Google Scholar 

  71. 71

    Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    CAS  Article  PubMed  Google Scholar 

  72. 72

    Simons, K. & Toomre, D. Lipid rafts and signal transduction. Nature Rev. Mol. Cell Biol. 1, 31–39 (2000).

    CAS  Article  Google Scholar 

  73. 73

    Varma, R. & Mayor, S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394, 798–801 (1998).Uses a variation of FRET, homotransfer, to infer the structure of lipid rafts on the cell surface.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. 74

    Kenworthy, A. K. & Edidin, M. Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 Å using imaging fluorescence resonance energy transfer. J. Cell Biol. 142, 69–84 (1998).Uses acceptor photobleaching FRET to assess the organization of lipid rafts at the cell surface. Contains a detailed discussion of the theory for interpreting FRET from donors and acceptors constrained to membranes.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  75. 75

    Kenworthy, A. K., Petranova, N. & Edidin, M. High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol. Biol. Cell 11, 1645–1655 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  76. 76

    Rigler, R. & Elson, E. S. Fluorescence Correlation Spectroscopy (Springer, New York, 2001).

    Google Scholar 

  77. 77

    Maiti, S., Haupts, V. & Webb, W. W. Fluorescence correlation spectroscopy: diagnostics for sparse molecules. Proc. Natl Acad. Sci. USA 94, 11753–11757 (1997).

    CAS  PubMed  Article  Google Scholar 

  78. 78

    Eigen, M. & Rigler, R. Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc. Natl Acad. Sci. USA 91, 5740–5747 (1994).

    CAS  PubMed  Article  Google Scholar 

  79. 79

    Van Craenenbroeck, E. & Engelborghs, Y. Fluorescence correlation spectroscopy: molecular recognition at the single molecule level. J. Mol. Recog. 13, 93–100 (2000).

    CAS  Article  Google Scholar 

  80. 80

    Cluzel, P., Surette, M. & Leibler, S. An ultrasensitive bacterial motor revealed by monitoring signaling proteins in single cells. Science 287, 1652–1655 (2000).

    CAS  Article  PubMed  Google Scholar 

  81. 81

    Heinze, K. G., Koltermann, A. & Schwille, P. Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis. Proc. Natl Acad. Sci. USA 97, 10377–10382 (2000).

    CAS  PubMed  Article  Google Scholar 

  82. 82

    Elowitz, M. B., Surette, M. G., Wolf, P. E., Stock, J. & Leibler, S. Photoactivation turns green fluorescent protein red. Curr. Biol. 7, 809–812 (1997).

    CAS  Article  PubMed  Google Scholar 

  83. 83

    Yokoe, H. & Meyer, T. Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nature Biotechnol. 14, 1252–1256 (1996).

    CAS  Article  Google Scholar 

  84. 84

    Creemers, T. H., Lock, A. J., Subramaniam, V., Jovin, T. M. & Volker, S. Photophysics and optical switching in green fluorescent protein mutants. Proc. Natl Acad. Sci. USA 97, 2974–2978 (2000).

    CAS  PubMed  Article  Google Scholar 

  85. 85

    Dickson, R. M., Cubitt, A. B., Tsien, R. Y. & Moerner, W. E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).

    CAS  Article  PubMed  Google Scholar 

  86. 86

    Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    CAS  Article  PubMed  Google Scholar 

  87. 87

    Farinas, J. & Verkman, A. S. Receptor-mediated targeting of fluorescent probes in living cells. J. Biol. Chem. 274, 7603–7606 (1999).

    CAS  PubMed  Article  Google Scholar 

  88. 88

    Sund, S. E. & Axelrod, D. Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching. Biophys. J. 79, 1655–1669 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  89. 89

    Toomre, D. K., Steyer, J. A., Almers, W. & Simons, K. Observing fusion of constitutive membrane traffic in real time by evanescent wave microscopy. J. Cell Biol. 149, 33–40 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  90. 90

    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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  91. 91

    Brown, E. B., Wu, E. S., Zipfel, W. & Webb, W. W. Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery. Biophys. J. 77, 2837–2849 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  92. 92

    Petersen, N. O. et al. Analysis of membrane protein cluster densities and sizes in situ by image correlation spectroscopy. Faraday Discuss. 289–305; discussion 331–343 (1998).

  93. 93

    Subramaniam, V., Kirsch, A. K. & Jovin, T. M. Cell biological applications of scanning near-field optical microscopy (SNOM). Cell. Mol. Biol. 44, 689–700 (1998).

    CAS  PubMed  Google Scholar 

  94. 94

    Klar, T., Jakobs, S., Dyba, M., Egner, A. & Hell, S. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000).

    CAS  PubMed  Article  Google Scholar 

  95. 95

    Nagorni, M. & Hell, S. W. 4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution. J. Struct. Biol. 123, 236–247 (1998).

    CAS  PubMed  Article  Google Scholar 

  96. 96

    Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R. & Piston, D. W. Use of green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  97. 97

    Baird, G. S., Zacharias, D. A. & Tsien, R. Y. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl Acad. Sci. USA 97, 11984–11989 (2000).

    CAS  PubMed  Article  Google Scholar 

  98. 98

    Dictenberg, J. B. et al. Pericentrin and γ-tubulin form a protein complex and are organized into a novel lattice at the centrosome. J. Cell Biol. 141, 163–174 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  99. 99

    Bastiaens, P. I., Majoul, I. V., Verveer, P. J., Soling, H. D. & Jovin, T. M. Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin. EMBO J. 15, 4246–4253 (1996).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  100. 100

    Damjanovich, S. et al. Structural hierarchy in the clustering of HLA class I molecules in the plasma membrane of human lymphoblastoid cells. Proc. Natl Acad. Sci. USA 92, 1122–1126 (1995).

    CAS  PubMed  Article  Google Scholar 

  101. 101

    Jovin, T. M. & Arndt-Jovin, D. J. in Cell Structure and Function by Microspectrofluorimetry (eds Kohen, E., Ploem, J. S. & Hirschberg, J. G.) 99–117 (Academic, Orlando, Florida,1989).

    Google Scholar 

  102. 102

    Nagai, Y. et al. A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo. Nature Biotechnol. 18, 313–316 (2000).

    CAS  Article  Google Scholar 

  103. 103

    Kam, Z., Volberg, T. & Geiger, B. Mapping of adherens junction components using microscopic resonance energy transfer imaging. J. Cell Sci. 108, 1051–1062 (1995).An elegant FRET microscopy study using immunofluorescence labelling to probe the distribution of proteins in adherens junctions.

    CAS  PubMed  Google Scholar 

  104. 104

    Xia, Z., Zhou, Q., Lin, J. & Liu, Y. Stable SNARE complex prior to evoked synaptic vesicle fusion revealed by fluorescence resonance energy transfer. J. Biol. Chem. 276, 1766–1771 (2001).

    CAS  PubMed  Article  Google Scholar 

  105. 105

    Wouters, F. S., Bastiaens, P. I. H., Wirtz, K. W. A. & Jovin, T. M. FRET microscopy demonstrates molecular assocation of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO J. 17, 7179–7189 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  106. 106

    Bacsó, Z., Bene, L., Bodnár, A., Matkó, J. & Damjanovich, S. A photobleaching energy transfer analysis of CD8/MHC-I and LFA-1/ICAM-1 interactions in CTL-target cell conjugates. Immunol. Lett. 54, 151–156 (1996).

    PubMed  Article  Google Scholar 

  107. 107

    Gadella, T. W. Jr & Jovin, T. M. Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. J. Cell Biol. 129, 1543–1558 (1995).

    CAS  PubMed  Article  Google Scholar 

  108. 108

    Sako, Y., Minoghchi, S. & Yanagida, T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nature Cell Biol. 2, 168–172 (2000).Combines single-molecule fluorescence techniques, total internal reflection microscopy, and FRET to follow the dimerization of the EGFR upon EGF binding.

    CAS  PubMed  Article  Google Scholar 

  109. 109

    Angers, S. et al. Detection of β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl Acad. Sci. USA 97, 3684–3689 (2000).

    CAS  PubMed  Google Scholar 

  110. 110

    Schütz, G. J., Kada, G., Pastushenko, V. P. & Schindler, H. Propeties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 19, 892–901 (2000).

    PubMed  PubMed Central  Article  Google Scholar 

  111. 111

    Mahajan, N. P. et al. Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer. Nature Biotechnol. 16, 547–552 (1998).

    CAS  Article  Google Scholar 

  112. 112

    Ruehr, M. L., Zakhary, D. R., Damron, D. S. & Bond, M. Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins. J. Biol. Chem. 274, 33092–33096 (1999).

    CAS  PubMed  Article  Google Scholar 

  113. 113

    Kindzelskii, A. L., Yang, Z., Nabel, G. J., Todd, R. F. R. & Petty, H. R. Ebola virus secretory glycoprotein (sGP) diminishes Fcγ RIIIB-to-CR3 proximity on neutrophils. J. Immunol. 164, 953–958 (2000).

    CAS  PubMed  Article  Google Scholar 

  114. 114

    Damelin, M. & Silver, P. A. Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex. Mol. Cell 5, 133–140 (2000).Novel interactions between nuclear transport factors and nucleoporins are revealed in a FRET-based protein–protein interaction screen in yeast.

    CAS  Article  PubMed  Google Scholar 

  115. 115

    Day, R. N. Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy. Mol. Endocrinol. 12, 1410–1419 (1998).

    CAS  PubMed  Article  Google Scholar 

  116. 116

    Schmid, J. A. et al. Dynamics of NF-κB and IκBα studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNA by fluorescence resonance energy transfer. J. Biol. Chem. 275, 17035–17042 (2000).

    CAS  PubMed  Article  Google Scholar 

  117. 117

    Llopis, J. et al. Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription. Proc. Natl Acad. Sci. USA 97, 4363–4368 (2000); erratum 97, 9819 (2000).

    CAS  PubMed  Article  Google Scholar 

  118. 118

    Prufer, K., Racz, A., Lin, G. C. & Barsony, J. Dimerization with retinoid X receptors promotes nuclear localization and subnuclear targeting of vitamin D receptors. J. Biol. Chem. 275, 41114–41123 (2000).

    CAS  PubMed  Article  Google Scholar 

  119. 119

    Brock, R., Vamosi, G., Vereb, G. & Jovin, T. M. Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy. Proc. Natl Acad. Sci. USA 96, 10123–10128 (1999).Provides a concise overview of how FCS and confocal microscopy can be linked to study the diffusional mobility of GFP-tagged proteins in cells.

    CAS  PubMed  Article  Google Scholar 

  120. 120

    Brock, R. & Jovin, T. M. Fluorescence correlation microscopy (FCM)-fluorescence correlation spectroscopy (FCS) taken into the cell. Cell Mol. Biol. 44, 847–856 (1998).

    CAS  PubMed  Google Scholar 

  121. 121

    Brock, R., Hink, M. A. & Jovin, T. M. Fluorescence correlation microscopy of cells in the presence of autofluorescence. Biophys. J. 75, 2547–2557 (1998).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  122. 122

    Wachsmuth, M., Waldeck, W. & Langowski, J. Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy. J. Mol. Biol. 298, 677–689 (2000).

    CAS  PubMed  Article  Google Scholar 

  123. 123

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

    CAS  PubMed  Article  Google Scholar 

  124. 124

    Politz, J. C., Browne, E. S., Wolf, D. E. & Pederson, T. Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells. Proc. Natl Acad. Sci. USA 95, 6043–6048 (1998).

    CAS  PubMed  Article  Google Scholar 

  125. 125

    Köhler, R. H., Schwille, P., Webb, W. W. & Hanson, M. R. Active protein transport through plastid tubles: velocity quantified by fluorescence correlation spectroscopy. J. Cell Sci. 113, 3921–3930 (2000).

    PubMed  Google Scholar 

  126. 126

    Terada, S., Kinjo, M. & Hirokawa, N. Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons. Cell 103, 141–155 (2000).

    CAS  PubMed  Article  Google Scholar 

  127. 127

    Rigler, R. et al. Specific binding of proinsulin C-peptide to human cell membranes. Proc. Natl Acad. Sci. USA 96, 13318–13323 (1999).

    CAS  PubMed  Article  Google Scholar 

  128. 128

    Trier, U., Olah, Z., Kleuser, B. & Schafer-Korting, M. Fusion of the binding domain of Raf-1 kinase with green fluorescent protein for activated Ras detection by fluorescence correlation spectroscopy. Pharmazie 54, 263–268 (1999).

    CAS  PubMed  Google Scholar 

  129. 129

    Terry, B. R., Matthews, E. K. & Haseloff, J. Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy. Biochem. Biophys. Res. Commun. 217, 21–27 (1995).

    CAS  PubMed  Article  Google Scholar 

  130. 130

    Haupts, U., Maiti, S., Schwille, P. & Webb, W. W. Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc. Natl Acad. Sci. USA 95, 13573–13578 (1998).

    CAS  PubMed  Article  Google Scholar 

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Acknowledgements

We thank members of the Lippincott-Schwartz laboratory for helpful comments on the manuscript. We also thank Gregoire Bonnet for useful discussions on fluorescence correlation spectroscopy. Anne Kenworthy was supported by a National Research Council–NICHD Research Associateship. Erik Snapp was supported by a PRAT Fellowship.

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DATABASE LINKS

high mobility group 17

ASF

fibrillarin

protein kinase Cα

Fas

FasL

tumour necrosis factor receptor 1

tumour necrosis factor receptor 2

EGFR

Arf1

Sec13

FURTHER INFORMATION

Compact barrel-like structure of GFP

Lippincott-Schwartz lab

ENCYCLOPEDIA OF LIFE SCIENCES

Green fluorescent protein

Fluorescence microscopy

Fluorescence resonance energy transfer

Glossary

GREEN FLUORESCENT PROTEIN

Fluorescent protein cloned from the jellyfish Aequoria victoria. The most frequently used mutant, EGFP, is excited at 488 nm and has an emission maximum at 510 nm.

RED FLUORESCENT PROTEIN

Fluorescent protein cloned from the sea anemone Discosoma striata with an excitation maximum of 558 nm and emission maximum at 583 nm.

ULTRAVIOLET-LIGHT-INDUCED DNA DAMAGE

Ultraviolet light promotes a covalent linkage of two adjacent pyrimidine bases (most often two thymines) in DNA.

NUCLEAR ENVELOPE

Double membrane that surrounds the nucleus. The outer nuclear membrane is continuous with the endoplasmic reticulum. The outer nuclear membrane is connected to the inner nuclear membrane at nuclear pores.

NUCLEAR LAMINA

Electron-dense layer lying on the nucleoplasmic side of the inner membrane of a nucleus.

TUNICAMYCIN

An antibiotic that inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins.

ARF1

Small GTPase that regulates the assembly of coats and vesicle budding.

ɛCOP

One of seven subunits of the COPI coatomer complex.

SEC13

Component of the COPII coat complex.

PROTEASOMES

Large multisubunit protease complex that selectively degrades intracellular proteins. Targeting to proteasomes most often occurs through attachment of multi-ubiquitin tags.

CYAN FLUORESCENT PROTEIN

S65A, Y66W, S72A, N1461I, M153T, V163A mutant of green fluorescent protein with excitation peak of 434 nm and an emission maximum at 477 nm.

YELLOW FLUORESCENT PROTEIN

S65G, V68L, S72A, T203Y mutant of green fluorescent protein with an excitation peak of 514 nm and an emission maximum at 527 nm.

QUANTUM YIELD

The probability of luminescence occurring in given conditions, expressed by the ratio of the number of photons (the quanta of light) emitted by the luminescing species to the number absorbed.

FITC

Fluorescent dye with an excitation maximum of 492 nm and an emission maximum of 520 nm.

RHODAMINE

Fluorescent dye with an excitation maximum at 550 nm and an emission maximum at 590 nm.

CY3

Fluorescent cynanine dye with an excitation maximum at 550 nm and an emission maximum at 570 nm.

CY5

Fluorescent cynanine dye with an excitation maximum at 650 nm and an emission maximum at 670 nm.

REPORTER CONSTRUCTS

Artificial proteins engineered to act as intracellular sensors. Often consist of a pair of GFP mutants that act as a FRET pair linked by a peptide that undergoes conformational changes or is physically altered in response to the intracellular environment or enzyme activity.

CY3.5

Fluorescent cynanine dye with an excitation maximum at 580 nm and an emission maximum at 590 nm.

AUTOCORRELATION FUNCTION

Mathematical function that is used to extract statistical properties of time-dependent noise. Used to analyse time-dependent fluctuations of fluorescence intensity in an FCS experiment to find similarities within the signal — for example, a correlation time reflecting diffusion of a fluorescent protein through a sample volume.

TWO-PHOTON MICROSCOPY

A form of multiphoton microscopy.

FLASH

A membrane-permeable fluorophore (fluorescein arsenical helix binder) that specifically, non-covalently, and reversibly binds a recombinant protein motif containing four cysteines at the i, i+1, i+4, and i+5 positions.

SINGLE-CHAIN ANTIBODIES

Peptides derived from immunoglobulins (which usually consist of two heavy chains and two light chains). These peptides do not oligomerize and have specific affinity for an antigen.

TOTAL INTERNAL REFLECTION MICROSCOPY

Fluorescence microscopy technique with significant depth discrimination, that can selectively excite only those fluorescent molecules within 100 nm of the interface between a cell and a coverslip.

MULTIPHOTON MICROSCOPY

Microscopy technique that uses the simultaneous absorbance of two or more photons of low energy (long wavelength) to excite fluorophores normally excited with single photons of shorter wavelengths. The technique reduces photodamage and permits imaging of much thicker samples.

IMAGE CORRELATION SPECTROSCOPY

Technique that measures the density and degree of aggregation of fluorescent particles using autocorrelation analysis of images from laser scanning confocal microscopy. Can be used, for example, to measure quantitatively the state of aggregation of receptors on the cell surface.

ATOMIC FORCE MICROSCOPY

A microscope that nondestructively measures the forces (at the atomic level) between a sharp probing tip (which is attached to a cantilever spring) and a sample surface. The microscope images structures at the resolution of individual atoms.

4-PI MICROSCOPE

A microscope that combines the wavefronts produced by two opposed high-aperture lenses and a two-photon excitation laser to allow three-dimensional imaging of transparent biological specimens with an axial resolution in the 100–140-nm range.

STIMULATED EMISSION MICROSCOPE

(Also referred to as ultrafast-dynamics microscope). A light microscope that increases the spatial resolution of a fluorescent sample by exciting the fluorophore with a femtosecond laser pulse followed by a quenching time-delayed red-shifted femtosecond laser pulse that depletes fluorescence at the focal rim surrounding the focal volume.

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Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2, 444–456 (2001). https://doi.org/10.1038/35073068

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