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

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).

[1]  O. Ohara,et al.  Single molecular assay of individual ATP turnover by a myosin‐GFP fusion protein expressed in vitro , 1997, FEBS letters.

[2]  Akihiro Kusumi,et al.  Development of time-resolved microfluorimetry and its application to studies of cellular membranes , 1990, Photonics West - Lasers and Applications in Science and Engineering.

[3]  W. Nelson,et al.  Biosynthesis of the cell adhesion molecule uvomorulin (E-cadherin) in Madin-Darby canine kidney epithelial cells. , 1991, The Journal of biological chemistry.

[4]  T. Yanagida,et al.  Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy. , 1997, Biochemical and biophysical research communications.

[5]  A. Nose,et al.  N-linked oligosaccharides are not involved in the function of a cell-cell binding glycoprotein E-cadherin. , 1986, Cell structure and function.

[6]  K. Sullivan,et al.  Green Fluorescent Proteins , 1999 .

[7]  S. Hirohashi,et al.  E-cadherin functions as a cis-dimer at the cell–cell adhesive interface in vivo , 1999, Nature Structural Biology.

[8]  Ronald D. Vale,et al.  Imaging individual green fluorescent proteins , 1997, Nature.

[9]  H Schindler,et al.  Imaging of single molecule diffusion. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Cynthia L. Adams,et al.  Mechanisms of Epithelial Cell–Cell Adhesion and Cell Compaction Revealed by High-resolution Tracking of E-Cadherin– Green Fluorescent Protein , 1998, The Journal of cell biology.

[11]  C. Yeaman,et al.  New perspectives on mechanisms involved in generating epithelial cell polarity. , 1999, Physiological reviews.

[12]  A Kusumi,et al.  Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis , 1994, The Journal of cell biology.

[13]  P. Saffman,et al.  Brownian motion in biological membranes. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Akinao Nose,et al.  Expressed recombinant cadherins mediate cell sorting in model systems , 1988, Cell.

[15]  Joseph R. Lakowicz,et al.  Time-resolved laser spectroscopy in biochemistry II , 1990 .

[16]  S. Weiss Fluorescence spectroscopy of single biomolecules. , 1999, Science.

[17]  B. Gumbiner,et al.  Lateral clustering of the adhesive ectodomain: a fundamental determinant of cadherin function , 1997, Current Biology.

[18]  M. Sheetz,et al.  Tracking kinesin-driven movements with nanometre-scale precision , 1988, Nature.

[19]  I. Sase,et al.  Real time imaging of single fluorophores on moving actin with an epifluorescence microscope. , 1995, Biophysical journal.

[20]  B. Gumbiner,et al.  Molecular and functional analysis of cadherin-based adherens junctions. , 1997, Annual review of cell and developmental biology.

[21]  Peter D. Kwong,et al.  Structural basis of cell-cell adhesion by cadherins , 1995, Nature.

[22]  M. Takeichi,et al.  Cadherin cell adhesion receptors as a morphogenetic regulator. , 1991, Science.

[23]  A. Kusumi,et al.  Compartmentalization of the erythrocyte membrane by the membrane skeleton: intercompartmental hop diffusion of band 3. , 1999, Molecular biology of the cell.

[24]  Wayne A. Hendrickson,et al.  Structure-Function Analysis of Cell Adhesion by Neural (N-) Cadherin , 1998, Neuron.

[25]  R. Vale,et al.  Single-molecule fluorescence detection of green fluorescence protein and application to single-protein dynamics. , 1999 .

[26]  Toshio Yanagida,et al.  Single-molecule imaging of EGFR signalling on the surface of living cells , 2000, Nature Cell Biology.

[27]  R N Zare,et al.  Probing individual molecules with confocal fluorescence microscopy. , 1994, Science.

[28]  R. Tsien,et al.  On/off blinking and switching behaviour of single molecules of green fluorescent protein , 1997, Nature.

[29]  David R. Colman,et al.  The Diversity of Cadherins and Implications for a Synaptic Adhesive Code in the CNS , 1999, Neuron.

[30]  A Kusumi,et al.  Cell surface organization by the membrane skeleton. , 1996, Current opinion in cell biology.

[31]  B. Gumbiner,et al.  Cell Adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis , 1996, Cell.

[32]  A Kusumi,et al.  Mobility and cytoskeletal interactions of cell adhesion receptors. , 1999, Current opinion in cell biology.

[33]  B. Gumbiner,et al.  Regulation of Cadherin Adhesive Activity , 2000, The Journal of cell biology.

[34]  T. Kues,et al.  Imaging and tracking of single GFP molecules in solution. , 2000, Biophysical journal.

[35]  M. Ikura,et al.  Structural basis of calcium-induced E-cadherin rigidification and dimerization , 1996, Nature.

[36]  D. Colman Neuronal Polarity and the Epithelial Metaphor , 1999, Neuron.

[37]  Gerald Kada,et al.  Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy , 2000, The EMBO journal.

[38]  N. Hirokawa,et al.  A processive single-headed motor: kinesin superfamily protein KIF1A. , 1999, Science.

[39]  Kiwamu Saito,et al.  Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution , 1995, Nature.

[40]  X. Xie,et al.  Single-molecule enzymatic dynamics. , 1998, Science.

[41]  Ronald D. Vale,et al.  Role of the Kinesin Neck Region in Processive Microtubule-based Motility , 1998, The Journal of cell biology.

[42]  T. Fujiwara,et al.  Application of laser tweezers to studies of the fences and tethers of the membrane skeleton that regulate the movements of plasma membrane proteins. , 1998, Methods in cell biology.

[43]  A. Kusumi,et al.  Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. , 1993, Biophysical journal.

[44]  C. L. Adams,et al.  Cytomechanics of cadherin-mediated cell-cell adhesion. , 1998, Current opinion in cell biology.

[45]  Akihiro Kusumi,et al.  Regulation Mechanism of the Lateral Diffusion of Band 3 in Erythrocyte Membranes by the Membrane Skeleton , 1998, The Journal of cell biology.

[46]  J. Stow,et al.  Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. , 1999 .

[47]  N. Thompson,et al.  Total internal reflection fluorescence. , 1984, Annual review of biophysics and bioengineering.

[48]  B. Gumbiner,et al.  Lateral dimerization is required for the homophilic binding activity of C-cadherin , 1996, The Journal of cell biology.

[49]  D. Langosch,et al.  Mutations affecting transmembrane segment interactions impair adhesiveness of E-cadherin. , 1999, Journal of cell science.

[50]  B. Zemelman,et al.  PRIM: proximity imaging of green fluorescent protein-tagged polypeptides. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Akihiro Kusumi,et al.  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 , 1998, The Journal of cell biology.

[52]  S. Troyanovsky,et al.  Mechanism of cell-cell adhesion complex assembly. , 1999, Current opinion in cell biology.

[53]  M. Ikura,et al.  Solution structure of the epithelial cadherin domain responsible for selective cell adhesion , 1995, Science.

[54]  Michael P. Sheetz,et al.  Laser tweezers in cell biology , 1998 .

[55]  O. Pertz,et al.  A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E‐cadherin homoassociation , 1999, The EMBO journal.

[56]  R. Kemler,et al.  The Membrane-proximal Region of the E-Cadherin Cytoplasmic Domain Prevents Dimerization and Negatively Regulates Adhesion Activity , 1998, The Journal of cell biology.

[57]  A Kusumi,et al.  Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether , 1995, The Journal of cell biology.

[58]  E. Elson,et al.  Weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion. , 1999, Biophysical journal.