In this study we have examined for molecular heterogeneity of cell-matrix adhesions and the involvement of actomyosin contractility in the selective recruitment of different plaque proteins. For this purpose, we have developed a novel microscopic approach for molecular morphometry, based on automatic identification of matrix adhesions, followed by quantitative immunofluorescence and morphometric analysis. Particularly informative was fluorescence ratio imaging, comparing the local labeling intensities of different plaque molecules, including vinculin, paxillin, tensin and phosphotyrosine-containing proteins. Ratio imaging revealed considerable molecular heterogeneity between and within adhesion sites. Most striking were the differences between focal contacts, which are vinculin- and paxillin-rich and contain high levels of phosphotyrosine, and fibrillar adhesions, which are tensin-rich and contain little or no phosphotyrosine. Ratio imaging also revealed considerable variability in the molecular substructure of individual focal contacts, pointing to a non-uniform distribution of phosphotyrosine and the different plaque constituents. Studying the quantitative relationships between the various components of the submembrane plaque indicated that the levels of vinculin, paxillin and phosphotyrosine in adhesion sites are positively correlated with each other and negatively correlated with the levels of tensin. Tyrosine phosphorylation of focal contacts was highly sensitive to cellular contractility, and was diminished within 5 minutes after treatment with the kinase inhibitor H-7, an inhibitor of actomyosin contractility. This was followed by the loss of paxillin and vinculin from the focal adhesions. Tensin-rich fibrillar adhesions were relatively insensitive to H-7 treatment. These findings suggest a role for contractility in the generation of matrix adhesion diversity.

Abercrombie
M.
,
Heaysman
J. E.
,
Pegrum
S. M.
(
1971
).
The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella.
Exp. Cell Res
67
,
359
367
Abercrombie
M.
,
Dunn
G. A.
(
1975
).
Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy.
Exp. Cell Res
92
,
57
62
Ayalon
O.
,
Geiger
B.
(
1997
).
Cyclic changes in the organization of celladhesions and the associated cytoskeleton, induced by stimulation of tyrosine phosphorylation in bovine aortic endothelial cells.
J. Cell Sci
110
,
547
556
Bendory
R.
,
Salomon
D.
,
Geiger
B.
(
1989
).
Identification of two distinct functional domains on vinculin involoved in its association with focal contacts.
J. Cell Biol
108
,
2383
2393
Bershadsky
A.
,
Chausovsky
A.
,
Becker
E.
,
Lyubimova
A.
,
Geiger
B.
(
1996
).
Involvement of microtubules in the control of adhesion-dependent signal transduction.
Curr Biol
6
,
1279
1289
Burridge
K.
,
Fath
K.
(
1989
).
Focal contacts: transmembrane links between the extracellular matrix and the cytoskeleton.
BioEssays
10
,
104
108
Clark
E. A.
,
Brugge
J. S.
(
1995
).
Integrins and signal transduction pathways: the road taken.
Science
268
,
233
239
Cormack
B. K.
,
Valdivia
R. H.
,
Falkow
S.
(
1996
).
FACS-optimized mutants of the green fluorescent protein (GFP).
Gene
173
,
33
38
Geiger
B.
(
1979
).
A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells.
Cell
18
,
193
205
Geiger
B.
,
Yehuda-Levenberg
S.
,
Bershadsky
A. D.
(
1995
).
Molecular interactions in the submembrane plaque of cell-cell and cell-matrix adhesions.
Acta Anat. (Basel)
154
,
46
62
Gilmore
A. P.
,
Burridge
K.
(
1996
).
Molecular mechanisms for focal adhesion assembly through regulation of protein-protein interactions.
Structure
4
,
647
651
Heath
J. P.
,
Dunn
G. A.
(
1978
).
Cell to substratum contacts of chick fibroblasts and their relation to the microfilament system. A correlated interference-reflexion and high-voltage electron-microscope study.
J. Cell Sci
29
,
197
212
Hynes
R. O.
(
1987
).
Integrins: a family of cell surface receptors.
Cell
48
,
549
554
Hynes
R. O.
(
1992
).
Integrins: versatility, modulation, and signaling in cell adhesion.
Cell
69
,
11
25
Izzard
C. S.
,
Lochner
L. R.
(
1976
).
Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique.
J. Cell Sci
21
,
129
159
Jockusch
B. M.
,
Bubeck
P.
,
Giehl
K.
,
Kroemker
M.
,
Moschner
J.
,
Rothkegel
M.
,
Rudiger
M.
,
Schluter
K.
,
Stanke
G.
,
Winkler
J.
(
1995
).
The molecular architecture of focal adhesions. Annu. Rev. Cell.
Dev. Biol
11
,
379
416
Kam
Z.
,
Volberg
T.
,
Geiger
B.
(
1995
).
Mapping of adherens junction components using microscopic resonance energy transfer imaging.
J. Cell Sci
108
,
1051
1062
Kioka
N.
,
Sakata
S.
,
Kawauchi
T.
,
Amachi
T.
,
Akiyama
S. K.
,
Okazaki
K.
,
Yaen
C.
,
Yamada
K. M.
,
Aota
S. I.
(
1999
).
Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization.
J. Cell Biol
144
,
56
69
Miyamoto
S.
,
Teramoto
H.
,
Coso
O. A.
,
Gutkind
J. S.
,
Burbelo
P. D.
,
Akiyama
S. K.
,
Yamada
K. M.
(
1995
).
Integrin function: molecular hierarchies of cytoskeletal and signaling molecules.
J. Cell Biol
131
,
791
805
Price
G. L.
,
Jones
P.
,
Davison
M. D.
,
Patel
B.
,
Bendori
R.
,
Geiger
B.
,
Critchley
D. R.
(
1989
).
Primary sequence and domain structure of chicken vinculin.
Biochem. J
259
,
453
461
Schwartz
M. A.
,
Schaller
M. D.
,
Ginsberg
M. H.
(
1995
).
Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell.
Dev. Biol
11
,
549
599
Tian
B.
,
Millar
C.
,
Kaufman
P. L.
,
Bershadsky
A.
,
Becker
E.
,
Geiger
B.
(
1998
).
Effects of H-7 on the iris and ciliary muscle in monkeys.
Arch. Ophthalmol
116
,
1070
1077
Turner
C. E.
,
Glenney
J. R.
Jr.
,
Burridge
K.
(
1990
).
Paxillin: a new vinculin-binding protein present in focal adhesions.
J. Cell Biol
111
,
1059
1068
Usson
Y.
,
Guignandon
A.
,
Laroche
N.
,
Marie-Helene
L. P.
,
Vico
L.
(
1997
).
Quantitation of cell-matrix adhesion using confocal image analysis of focal contact associated proteins and interference reflection microscopy.
Cytometry
28
,
298
304
Volberg
T.
,
Zick
Y.
,
Dror
R.
,
Sabanay
I.
,
Gilon
C.
,
Levitzki
A.
,
Geiger
B.
(
1992
).
The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions.
EMBO J
11
,
1733
1742
Volberg
T.
,
Geiger
B.
,
Citi
S.
,
Bershadsky
A. D.
(
1994
).
Effect of protein kinase inhibitor H-7 on the contractility, integrity, and membrane anchorage of the microfilament system.
Cell Motil Cytoskel
29
,
321
338
Yamada
K. M.
(
1997
).
Integrin signaling.
Matrix Biol
16
,
137
141
Yamada
K. M.
,
Geiger
B.
(
1997
).
Molecular interactions in cell adhesion complexes.
Curr. Opin. Cell Biol
9
,
76
85
Yamada
K. M.
,
Miyamoto
S.
(
1995
).
Integrin transmembrane signaling and cytoskeletal control.
Curr. Opin. Cell Biol
7
,
681
689
This content is only available via PDF.