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. 2001 Jun;80(6):2667–2677. doi: 10.1016/S0006-3495(01)76236-4

Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.

R Iino 1, I Koyama 1, A Kusumi 1
PMCID: PMC1301454  PMID: 11371443

Abstract

Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions).

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams C. L., Chen Y. T., 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. 1998 Aug 24;142(4):1105–1119. doi: 10.1083/jcb.142.4.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams C. L., Nelson W. J. Cytomechanics of cadherin-mediated cell-cell adhesion. Curr Opin Cell Biol. 1998 Oct;10(5):572–577. doi: 10.1016/s0955-0674(98)80031-8. [DOI] [PubMed] [Google Scholar]
  3. Axelrod D., Burghardt T. P., Thompson N. L. Total internal reflection fluorescence. Annu Rev Biophys Bioeng. 1984;13:247–268. doi: 10.1146/annurev.bb.13.060184.001335. [DOI] [PubMed] [Google Scholar]
  4. Brieher W. M., Yap A. S., Gumbiner B. M. Lateral dimerization is required for the homophilic binding activity of C-cadherin. J Cell Biol. 1996 Oct;135(2):487–496. doi: 10.1083/jcb.135.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Colman D. R. Neuronal polarity and the epithelial metaphor. Neuron. 1999 Aug;23(4):649–651. doi: 10.1016/s0896-6273(01)80024-6. [DOI] [PubMed] [Google Scholar]
  6. De Angelis D. A., Miesenböck G., Zemelman B. V., Rothman J. E. PRIM: proximity imaging of green fluorescent protein-tagged polypeptides. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12312–12316. doi: 10.1073/pnas.95.21.12312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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. 1997 Jul 24;388(6640):355–358. doi: 10.1038/41048. [DOI] [PubMed] [Google Scholar]
  8. Funatsu T., Harada Y., Tokunaga M., Saito K., Yanagida T. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature. 1995 Apr 6;374(6522):555–559. doi: 10.1038/374555a0. [DOI] [PubMed] [Google Scholar]
  9. Gelles J., Schnapp B. J., Sheetz M. P. Tracking kinesin-driven movements with nanometre-scale precision. Nature. 1988 Feb 4;331(6155):450–453. doi: 10.1038/331450a0. [DOI] [PubMed] [Google Scholar]
  10. Gumbiner B. M. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell. 1996 Feb 9;84(3):345–357. doi: 10.1016/s0092-8674(00)81279-9. [DOI] [PubMed] [Google Scholar]
  11. Gumbiner B. M. Regulation of cadherin adhesive activity. J Cell Biol. 2000 Feb 7;148(3):399–404. doi: 10.1083/jcb.148.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Huber O., Kemler R., Langosch D. Mutations affecting transmembrane segment interactions impair adhesiveness of E-cadherin. J Cell Sci. 1999 Dec;112(Pt 23):4415–4423. doi: 10.1242/jcs.112.23.4415. [DOI] [PubMed] [Google Scholar]
  13. Iwane A. H., Funatsu T., Harada Y., Tokunaga M., Ohara O., Morimoto S., Yanagida T. Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro. FEBS Lett. 1997 Apr 28;407(2):235–238. doi: 10.1016/s0014-5793(97)00359-1. [DOI] [PubMed] [Google Scholar]
  14. Kubitscheck U., Kückmann O., Kues T., Peters R. Imaging and tracking of single GFP molecules in solution. Biophys J. 2000 Apr;78(4):2170–2179. doi: 10.1016/S0006-3495(00)76764-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kusumi A., Sako Y. Cell surface organization by the membrane skeleton. Curr Opin Cell Biol. 1996 Aug;8(4):566–574. doi: 10.1016/s0955-0674(96)80036-6. [DOI] [PubMed] [Google Scholar]
  16. Kusumi A., Sako Y., Fujiwara T., Tomishige M. Application of laser tweezers to studies of the fences and tethers of the membrane skeleton that regulate the movements of plasma membrane proteins. Methods Cell Biol. 1998;55:173–194. doi: 10.1016/s0091-679x(08)60408-2. [DOI] [PubMed] [Google Scholar]
  17. Kusumi A., Sako Y., Yamamoto M. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J. 1993 Nov;65(5):2021–2040. doi: 10.1016/S0006-3495(93)81253-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kusumi A., Suzuki K., Koyasako K. Mobility and cytoskeletal interactions of cell adhesion receptors. Curr Opin Cell Biol. 1999 Oct;11(5):582–590. doi: 10.1016/s0955-0674(99)00020-4. [DOI] [PubMed] [Google Scholar]
  19. Le T. L., Yap A. S., Stow J. L. Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. J Cell Biol. 1999 Jul 12;146(1):219–232. [PMC free article] [PubMed] [Google Scholar]
  20. Lu H. P., Xun L., Xie X. S. Single-molecule enzymatic dynamics. Science. 1998 Dec 4;282(5395):1877–1882. doi: 10.1126/science.282.5395.1877. [DOI] [PubMed] [Google Scholar]
  21. Nagar B., Overduin M., Ikura M., Rini J. M. Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature. 1996 Mar 28;380(6572):360–364. doi: 10.1038/380360a0. [DOI] [PubMed] [Google Scholar]
  22. Nie S., Chiu D. T., Zare R. N. Probing individual molecules with confocal fluorescence microscopy. Science. 1994 Nov 11;266(5187):1018–1021. doi: 10.1126/science.7973650. [DOI] [PubMed] [Google Scholar]
  23. Nose A., Nagafuchi A., Takeichi M. Expressed recombinant cadherins mediate cell sorting in model systems. Cell. 1988 Sep 23;54(7):993–1001. doi: 10.1016/0092-8674(88)90114-6. [DOI] [PubMed] [Google Scholar]
  24. Okada Y., Hirokawa N. A processive single-headed motor: kinesin superfamily protein KIF1A. Science. 1999 Feb 19;283(5405):1152–1157. doi: 10.1126/science.283.5405.1152. [DOI] [PubMed] [Google Scholar]
  25. Overduin M., Harvey T. S., Bagby S., Tong K. I., Yau P., Takeichi M., Ikura M. Solution structure of the epithelial cadherin domain responsible for selective cell adhesion. Science. 1995 Jan 20;267(5196):386–389. doi: 10.1126/science.7824937. [DOI] [PubMed] [Google Scholar]
  26. Ozawa M., Kemler R. The membrane-proximal region of the E-cadherin cytoplasmic domain prevents dimerization and negatively regulates adhesion activity. J Cell Biol. 1998 Sep 21;142(6):1605–1613. doi: 10.1083/jcb.142.6.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pertz O., Bozic D., Koch A. W., Fauser C., Brancaccio A., Engel J. A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation. EMBO J. 1999 Apr 1;18(7):1738–1747. doi: 10.1093/emboj/18.7.1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pierce D. W., Hom-Booher N., Vale R. D. Imaging individual green fluorescent proteins. Nature. 1997 Jul 24;388(6640):338–338. doi: 10.1038/41009. [DOI] [PubMed] [Google Scholar]
  29. Pierce D. W., Vale R. D. Single-molecule fluorescence detection of green fluorescence protein and application to single-protein dynamics. Methods Cell Biol. 1999;58:49–73. doi: 10.1016/s0091-679x(08)61948-2. [DOI] [PubMed] [Google Scholar]
  30. Romberg L., Pierce D. W., Vale R. D. Role of the kinesin neck region in processive microtubule-based motility. J Cell Biol. 1998 Mar 23;140(6):1407–1416. doi: 10.1083/jcb.140.6.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Saffman P. G., Delbrück M. Brownian motion in biological membranes. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3111–3113. doi: 10.1073/pnas.72.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sako Y., Kusumi A. Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether. J Cell Biol. 1995 Jun;129(6):1559–1574. doi: 10.1083/jcb.129.6.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sako Y., Kusumi A. Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis. J Cell Biol. 1994 Jun;125(6):1251–1264. doi: 10.1083/jcb.125.6.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sako Y., Minoghchi S., Yanagida T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol. 2000 Mar;2(3):168–172. doi: 10.1038/35004044. [DOI] [PubMed] [Google Scholar]
  35. Sako Y., Nagafuchi A., Tsukita S., Takeichi M., Kusumi A. Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton. J Cell Biol. 1998 Mar 9;140(5):1227–1240. doi: 10.1083/jcb.140.5.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sase I., Miyata H., Corrie J. E., Craik J. S., Kinosita K., Jr Real time imaging of single fluorophores on moving actin with an epifluorescence microscope. Biophys J. 1995 Aug;69(2):323–328. doi: 10.1016/S0006-3495(95)79937-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schmidt T., Schütz G. J., Baumgartner W., Gruber H. J., Schindler H. Imaging of single molecule diffusion. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2926–2929. doi: 10.1073/pnas.93.7.2926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schütz G. J., Kada G., Pastushenko V. P., Schindler H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 2000 Mar 1;19(5):892–901. doi: 10.1093/emboj/19.5.892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shapiro L., Colman D. R. The diversity of cadherins and implications for a synaptic adhesive code in the CNS. Neuron. 1999 Jul;23(3):427–430. doi: 10.1016/s0896-6273(00)80796-5. [DOI] [PubMed] [Google Scholar]
  40. Shapiro L., Fannon A. M., Kwong P. D., Thompson A., Lehmann M. S., Grübel G., Legrand J. F., Als-Nielsen J., Colman D. R., Hendrickson W. A. Structural basis of cell-cell adhesion by cadherins. Nature. 1995 Mar 23;374(6520):327–337. doi: 10.1038/374327a0. [DOI] [PubMed] [Google Scholar]
  41. Shirayoshi Y., Nose A., Iwasaki K., Takeichi M. N-linked oligosaccharides are not involved in the function of a cell-cell binding glycoprotein E-cadherin. Cell Struct Funct. 1986 Sep;11(3):245–252. doi: 10.1247/csf.11.245. [DOI] [PubMed] [Google Scholar]
  42. Shore E. M., Nelson W. J. Biosynthesis of the cell adhesion molecule uvomorulin (E-cadherin) in Madin-Darby canine kidney epithelial cells. J Biol Chem. 1991 Oct 15;266(29):19672–19680. [PubMed] [Google Scholar]
  43. Takeda H., Shimoyama Y., Nagafuchi A., Hirohashi S. E-cadherin functions as a cis-dimer at the cell-cell adhesive interface in vivo. Nat Struct Biol. 1999 Apr;6(4):310–312. doi: 10.1038/7542. [DOI] [PubMed] [Google Scholar]
  44. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991 Mar 22;251(5000):1451–1455. doi: 10.1126/science.2006419. [DOI] [PubMed] [Google Scholar]
  45. Tamura K., Shan W. S., Hendrickson W. A., Colman D. R., Shapiro L. Structure-function analysis of cell adhesion by neural (N-) cadherin. Neuron. 1998 Jun;20(6):1153–1163. doi: 10.1016/s0896-6273(00)80496-1. [DOI] [PubMed] [Google Scholar]
  46. Tokunaga M., Kitamura K., Saito K., Iwane A. H., Yanagida T. Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy. Biochem Biophys Res Commun. 1997 Jun 9;235(1):47–53. doi: 10.1006/bbrc.1997.6732. [DOI] [PubMed] [Google Scholar]
  47. Tomishige M., Kusumi A. Compartmentalization of the erythrocyte membrane by the membrane skeleton: intercompartmental hop diffusion of band 3. Mol Biol Cell. 1999 Aug;10(8):2475–2479. doi: 10.1091/mbc.10.8.2475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tomishige M., Sako Y., Kusumi A. Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton. J Cell Biol. 1998 Aug 24;142(4):989–1000. doi: 10.1083/jcb.142.4.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Troyanovsky S. M. Mechanism of cell-cell adhesion complex assembly. Curr Opin Cell Biol. 1999 Oct;11(5):561–566. doi: 10.1016/s0955-0674(99)00021-6. [DOI] [PubMed] [Google Scholar]
  50. Weiss S. Fluorescence spectroscopy of single biomolecules. Science. 1999 Mar 12;283(5408):1676–1683. doi: 10.1126/science.283.5408.1676. [DOI] [PubMed] [Google Scholar]
  51. Yap A. S., Brieher W. M., Gumbiner B. M. Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol. 1997;13:119–146. doi: 10.1146/annurev.cellbio.13.1.119. [DOI] [PubMed] [Google Scholar]
  52. Yap A. S., Brieher W. M., Pruschy M., Gumbiner B. M. Lateral clustering of the adhesive ectodomain: a fundamental determinant of cadherin function. Curr Biol. 1997 May 1;7(5):308–315. doi: 10.1016/s0960-9822(06)00154-0. [DOI] [PubMed] [Google Scholar]
  53. Yeaman C., Grindstaff K. K., Nelson W. J. New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol Rev. 1999 Jan;79(1):73–98. doi: 10.1152/physrev.1999.79.1.73. [DOI] [PubMed] [Google Scholar]

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