Quantitative microscopic imaging of resonance energy transfer (RET) was applied for immunological high resolution proximity mapping of several cytoskeletal components of cell adhesions. To conduct this analysis, a microscopic system was developed, consisting of a highly stable field illuminator, computer-controlled filter wheels for rapid multiple-color imaging and a sensitive, high resolution CCD camera, enabling quantitative data recording and processing. Using this system, we have investigated the spatial inter-relationships and organization of four adhesion-associated proteins, namely vinculin, talin, alpha-actinin and actin. Cultured chick lens cells were double labeled for each of the junctional molecules, using fluorescein- and rhodamine-conjugated antibodies or phalloidin. RET images were acquired with fluorescein excitation and rhodamine emission filter setting, corrected for fluorescein and rhodamine fluorescence, and normalized to the fluorescein image. The results pointed to high local densities of vinculin, talin and F-actin in focal adhesions, manifested by mean RET values of 15%, 12% and 10%, respectively. On the other hand, relatively low values (less than 1%) were observed following double immunofluorescence labeling of the same cells for alpha-actinin. Double indirect labeling for pairs of these four proteins (using fluorophore-conjugated antibodies or phalloidin) resulted in RET values of 5% or lower, except for the pair alpha-actinin and actin, which yielded significantly higher values (13-15%). These results suggest that despite their overlapping staining patterns, at the level of resolution of the light microscope, the plaque proteins vinculin and talin are not homogeneously interspersed at the molecular level but form segregated clusters. alpha-Actinin, on the other hand, does not appear to form such clusters but, rather, closely interacts with actin. We discuss here the conceptual and applicative aspects of RET measurements and the implications of the results on the subcellular molecular organization of adherens-type junctions.

REFERENCES

Adams
S. R.
,
Harootunian
A. T.
,
Buechler
Y. J.
,
Taylor
S. S.
,
Tsien
R.
(
1991
).
Fluorescence ratio imaging of cyclic AMP in single cells.
Nature
349
,
694
697
Avnur
Z.
,
Small
J. V.
,
Geiger
B.
(
1983
).
Actin independent association of vinculin with the cytoplasmic aspect of the plasma membrane in cell-contact areas.
J. Cell Biol
96
,
1622
1630
Baron
M. D.
,
Davison
M. D.
,
Jones
P.
,
Critchly
D. R.
(
1987
).
The sequence of chick-actinin reveals homologies to spectrin and calmodulin.
J. Biol. Chem
262
,
17623
17629
Beechem
J. M.
,
Haas
E.
(
1989
).
Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements.
Biophys. J
55
,
1225
1236
Belkin
A. M.
,
Koteliansky
V. E.
(
1987
).
Interaction of iodinated vinculin, metavinculin and-actinin with cytoskletal proteins.
FEBS Lett
220
,
291
294
Bendori
R.
,
Salomon
D.
,
Geiger
B.
(
1989
).
Identification of two distinct functional domain on vinculin, involved in its association with focal contacts.
J. Cell Biol
108
,
2383
2393
Burridge
K.
,
Mangeat
P.
(
1984
).
An interaction between vinculin and talin,.
Nature
308
,
744
746
Burridge
K.
,
Fath
K.
,
Kelly
T.
,
Nuckolls
G.
,
Turner
C.
(
1988
).
Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton.
Annu. Rev. Cell Biol
4
,
487
525
Butler
J. A.
,
Lin
S.
(
1989
).
Actin capping activity of smooth muscle tensin.
J. Cell Biol
109
,
172
–.
Brands
R.
,
de Boer
A.
,
Feltkamp
C. A.
,
Roos
E.
(
1990
).
Disintegration of adhesion plaques in chicken embryo fibroblasts upon rous sarcoma virus-induced transformation: different dissociation rates for talin and vinculin.
Exp. Cell Res
186
,
138
148
Cardullo
R. A.
,
Agrawal
S.
,
Flores
C.
,
Zamecnik
P. C.
,
Wolf
D. E.
(
1988
).
Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.
Proc. Nat. Acad. Sci. USA
85
,
8790
8794
Crawford
A. W.
,
Beckerle
M. C.
(
1991
).
Purification and characterization of zyxin, an 82,000 dalton component of adherens junctions.
J. Biol. Chem
266
,
5847
5853
Faulstich
H.
,
Trischmann
H.
,
Mayer
D.
(
1983
).
Preparation of Tetramethylrhodaminyl-phalloidin and uptake of the toxin into short-term cultured hepatocytes by endocytosis.
Exp. Cell Res
144
,
73
82
Feltkamp
C. A.
,
Pijnenburg
M. A. P.
,
Roos
E.
(
1991
).
Organization of talin and vinculin in adhesion plaques of wet-cleaved chicken embryo fibroblasts.
J. Cell Sci
100
,
579
587
Fernandez
S. M.
,
Berlin
R. D.
(
1976
).
Cell surface distribution of lectin receptors determined by resonance energy transfer.
Nature
264
,
411
415
Förster
T.
(
1946
).
Energiewanderung und Fluoreszenz.
Naturwissenschaften
33
,
166
175
Förster
Th
(
1959
).
Transfer mechanism of electronic excitation.
Disc. Farad. Soc
27
,
7
29
Geiger
B.
,
Ayalon
O.
(
1992
).
Cadherins.
Annu. Rev. Cell Biol
8
,
307
332
Geiger
B.
,
Volk
T.
,
Volberg
T.
(
1985
).
Molecular hetrogeneity of adherens junctions.
J. Cell Biol
101
,
1523
1531
Geiger
B.
,
Ginsberg
D.
,
Salomon
D.
,
Volberg
T.
(
1990
).
The molecular basis for the assembly and modulation of adherens-type junctions.
Cell Diff. Dev
32
,
243
354
Geiger
B.
,
Ginsberg
D.
(
1991
).
The cytoplasmic domain of adherens type junctions.
Cell Motil. Cytoskel
20
,
1
6
Gettins
P.
,
Beechem
J. M.
,
Crew
B. C.
,
Cummingham
L. W.
(
1990
).
Separation and localization of the four cysteine-949 residues in human alpha-2 macroglobulin using fluorescence energy transfer.
Biochemistry
29
,
7747
7753
Herman
B.
(
1989
).
Resonance energy transfer microscopy.
Meth. Cell Biol
30
,
219
243
Hiraoka
Y.
,
Sedat
J. W.
,
Agard
D. A.
(
1987
).
The use of charge-coupled device for quantitative optical microscopy of biological structures.
Science
238
,
36
41
Hiraoka
Y.
,
Swedlow
J. R.
,
Paddy
M. R.
,
Sedat
J. W.
,
Agard
D. A.
(
1991
).
Three-dimensional multi-wavelength fluorescence microscopy for the structural analysis of biological phenomena.
Semin. Cell Biol
2
,
153
165
Horwitz
A.
,
Duggan
K.
,
Buck
C.
,
Beckerle
M. C.
,
Burridge
K.
(
1986
).
Interaction of plasma memmbrane fibronectin receptor with talin- a transmemmbrane linkage.
Nature
320
,
531
533
Hynes
R. O.
(
1992
).
Integrins: Versatility, modulation and signaling in cell adhesion.
Cell
69
,
11
25
Ingber
D.
(
1993
).
The riddle of morphogenesis: a question of solution chemistry or molecular cell engineering.
Cell
75
,
1249
1253
Johnson
D. A.
,
Voet
J. G.
,
Taylor
P. J.
(
1984
).
Fluorescence energy transfer between cobra a-toxin molecules bound to the acetylcholine receptor.
J. Biol. Chem
259
,
5717
5725
Kabsch
W.
,
Mannherz
H. G.
,
Suck
D. S.
,
Pai
E. F.
,
Holmes
K. C.
(
1990
).
Atomic structure of the actin DNase I complex.
Nature
347
,
37
44
Kam
Z.
(
1987
).
Microscopic imaging of cells.
Q. Rev. Biophys
20
,
201
259
Kemler
R.
,
Ozawa
M.
(
1989
).
Uvomorulin- catenin complex: Cytoplasmic anchorage of a Ca2+-dependent cell adhesion molecule.
BioEssays
11
,
88
91
Kries
T. E.
,
Geiger
B.
,
Schlessinger
J.
(
1982
).
Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery.
Cell
29
,
835
845
Kubitscheck
U.
,
Kircheis
M.
,
Schweitzer-Stenner
R.
,
Dreybrodt
W.
,
Jovin
T.
,
Pecht
I.
(
1991
).
Fluorescence resonance energy transfer on single living cells: Application to binding of monovalent haptens to cell-bound immunoglobulin.
E. Biophys. J
60
,
307
318
Ludwig
M.
,
Hensel
N. F.
,
Hartzman
R. J.
(
1992
).
Calibration of a resonance energy transfer imaging system.
Biophys. J
61
,
845
857
Milam
L. M.
(
1985
).
Electron microscopy of rotary shaddowed vinculin and vinculin complexes.
J. Mol. Biol
184
,
543
545
Molony
L.
,
Burridge
K.
(
1985
).
Molecular shape and self association of vinculin and metavinculin.
J. Cell. Biochem
29
,
31
36
Otey
C. A.
,
Pavalko
F. M.
,
Burridge
K.
(
1990
).
An interaction between-actinin and the 1 integrin subunit in vitro.
J. Cell Biol
111
,
721
729
Pollard
T. D.
,
Cooper
J. A.
(
1986
).
Actin and actin binding proteins. A critical evaluation of mechanisms and functions.
Annu. Rev. Biochem
55
,
987
1035
Stryer
L.
,
Haugland
R. P.
(
1967
).
Energy transfer: a spectroscopic ruler.
Proc. Nat. Acad. Sci. USA
58
,
719
726
Stryer
L.
(
1978
).
Fluorescence energy transfer as a spectroscopic ruler.
Annu. Rev. Biochem
47
,
819
847
Stryer
L.
,
Thomas
D. D.
,
Meares
C. F.
(
1982
).
Diffusion-enhanced fluorescence energy transfer.
Annu. Rev. Biophys. Bioeng
11
,
203
222
Taylor
D. L.
,
Reidler
J.
,
Spudich
J.
,
Stryer
L.
(
1981
).
Detection of actin assembly by fluorescence energy transfer.
J. Cell Biol
89
,
362
367
Tron
L.
,
Szöllösi
J.
,
Damjanovich
S.
,
Helliwell
S. H.
,
Arndt-Jovin
D. J.
,
Jovin
T. M.
(
1984
).
Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces: Quantitative evaluation of the transfer efficiency on a cell-by-cell basis.
Biophys. J
45
,
939
946
Tsien
R. Y.
(
1989
).
Fluorescent indicators of ion concentrations.
Meth. Cell Biol
30
,
127
156
Turner
C. E.
,
Glenny
J. R.
Jr.
,
Burridge
K.
(
1990
).
Paxillin: A new vinculin-binding protein present in focal adhesions.
J. Cell Biol
111
,
1059
1068
Uster
P. S.
,
Pagano
R. E.
(
1986
).
Resonance energy transfer microscopy: Observation of membrane-bound fluorescence probes in model membranes and in living cells.
J. Cell Biol
103
,
1221
1234
Volberg
T.
,
Sabanay
H.
,
Geiger
B.
(
1986
).
Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle.
Differentiation
32
,
34
43
Volk
T.
,
Geiger
B.
(
1984
).
A 135 Kd membrane protein of intercellular adherens junctions.
EMBO J
3
,
2249
2260
Wulf
E.
,
Deboren
A.
,
Bautz
F. A.
,
Faulstich
H.
,
Wieland
Th
(
1979
).
Fluorescent phalloidin, a tool for the visualization if cellular actin.
Proc. Nat. Acad. Sci. USA
76
,
4498
4502
Yguerabide
J.
,
Förester
M.
(
1981
).
Fluorescence spectroscopy of biological membranes.
Mol. Biol. Biochem. Biophys
31
,
199
269
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