Using immuno-EM, we have studied the distribution of the beta 1 integrin subunit in chicken embryo fibroblasts allowed to adhere and spread for 3 hours on a fibronectin-coated surface in serum-free medium. The cells were wet-cleaved, which removed most of the cell body, yielding ventral plasma membranes with little, and sometimes virtually no, associated cytoskeleton. The beta 1 integrin subunit was detected with antibodies against the cytoplasmic domain. In immune fluorescence, it colocalized with adhesion plaques, in a punctate staining pattern, and often seemed to be at the periphery of the plaque. By immuno-EM, beta 1 was in fact found in discrete clusters, not throughout the plaque. In deep-cleaved cells from which virtually all cytoskeleton was removed, clusters could often be seen to be located on fibronectin fibrils. Furthermore, beta 1 was present in clusters at the cell margins, and isolated or in small groups at the very edge of the cell. When fibronectin synthesis, and consequently fibril formation, was inhibited by cycloheximide, large adhesion plaque-like structures were formed at the cell margin. This phenotype was reversed by addition of soluble fibronectin, which was incorporated into fibrils. As in normal plaques, talin and vinculin were present, the plasma membrane was very close (10-20 nm) to the substratum and the fibronectin layer underneath was removed. These plaques did contain beta 1 integrins but they were not in clusters. These observations indicate that the talin-vinculin network of an adhesion plaque is normally anchored to the substratum at discrete beta 1 integrin clusters that may be located on fibronectin fibrils, and that elsewhere the plaque is not necessarily attached to the substratum by interaction of integrins with matrix proteins. In the absence of fibronectin fibrils, adhesion plaque-like structures can be formed, but these are aberrant in size, location and fine structure.

REFERENCES

Avnur
Z.
,
Geiger
B.
(
1981
).
The removal of extracellular fibronectin from areas of cell-substratum contact.
Cell
25
,
121
132
Bendori
R.
,
Salomon
D.
,
Geiger
B.
(
1989
).
Identification of two distinct functional domains on vinculin involved in its association with focal contacts.
J. Cell Biol
108
,
2383
2393
Birchmeier
C.
,
Kreis
T. E.
,
Eppenberger
H. M.
,
Winterhalter
K.
,
Birchmeier
W.
(
1980
).
Corrugated attachment membrane in WI-38 fibroblasts: Alternating fibronectin fibers and actin-containing focal contacts.
Proc. Nat. Acad. Sci. USA
77
,
4108
4112
Brands
R.
,
Feltkamp
C. A.
(
1988
).
Wet cleaving of cells: a method to introduce macromolecules into the cytoplasm.
Exp. Cell Res
176
,
309
318
Brands
R.
,
De Boer
A.
,
Feltkamp
C.A.
,
Roos
E.
(
1990
).
Desintegration 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
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
Charo
I. F.
,
Nannizzi
L.
,
Smith
J. W.
,
Cheresh
D.A.
) (
1990
).
The vitronectin receptorV 3binds fibronectin and acts in concert with5 1in promoting cellular attachment and spreading on fibronectin.
J. Cell Biol
111
,
2795
2800
Chen
W.-T.
,
Singer
S. J.
(
1982
).
Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.
J. Cell Biol
95
,
205
222
Damsky
C. H.
,
Knudsen
K. A.
,
Bradley
D.
,
Buck
C. A.
,
Horwitz
A. F.
(
1985
).
Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture.
J. Cell Biol
100
,
1528
1539
Dzamba
B.J.
,
Peters
D.M.
) (
1991
).
Arrangement of cellular fibronectin in noncollagenous fibrils in human fibroblast cultures.
J. Cell Sci
100
,
605
612
Fath
K. R.
,
Edgell
C.-J. S.
,
Burridge
K.
(
1989
).
The distribution of distinct integrins in focal contacts is determined by the substratum composition.
J. Cell Sci
92
,
67
75
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
Geiger
B.
(
1989
).
Cytoskeleton-associated cell contacts.
Curr. Opin. Cell Biol
1
,
103
109
Geiger
B.
,
Salomon
D.
,
Takeichi
M.
,
Hynes
R. O.
(
1992
).
A chimeric N-cadherin/1-integrin receptor which localizes to both cell-cell and cell-matrix adhesions.
J. Cell Sci
103
,
943
951
Horwitz
A.
,
Duggan
K.
,
Buck
C.
,
Beckerle
M.C.
,
Burridge
K.
(
1986
).
Interaction of plasma membrane fibronectin receptor with talin, a transmembrane linkage.
Nature
320
,
531
533
Hynes
R. O.
(
1992
).
Integrins: versatility, modulation, and signaling in cell adhesion.
Cell
69
,
11
25
Kelly
T.
,
Molony
L.
,
Burridge
K.
(
1987
).
Purification of two smooth muscle glycoproteins related to integrin.
J. Biol. Chem
262
,
17189
17199
La Flamme
S. E.
,
Akiyama
S. K.
,
Yamada
K. M.
(
1992
).
Regulation of fibronectin receptor distribution.
J. Cell Biol
117
,
437
447
Marcantonio
E. E.
,
Hynes
R. O.
(
1988
).
Antibodies to the conserved cytoplasmic domain of the integrin1subunit react with proteins in vertrebrates, invertebrates, and fungi.
J. Cell Biol
106
,
1765
1772
McDonald
J. A.
(
1988
).
Extracellular matrix assembly.
Annu. Rev. Cell Biol
4
,
183
207
McKeown-Longo
P. J.
,
Mosher
D. F.
(
1983
).
Binding of plasma fibronectin to cell layers of human skin fibroblasts.
J. Cell Biol
97
,
466
472
Otey
C. A.
,
Pavalko
F. M.
,
Burridge
K.
(
1990
).
An interaction between-actinin and the 1integrin subunit in vitro.
J. Cell Biol
111
,
721
729
Pesciotta Peters
D. M.
,
Portz
L. M.
,
Fullenwider
J.
,
Mosher
D. F.
(
1990
).
Co-assembly of plasma and cellular fibronectins into fibrils in human fibroblast cultures.
J. Cell Biol
111
,
249
256
Pytela
R.
,
Piersbacher
M. D.
,
Ruoslahti
E.
(
1985
).
A 125/115 kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin.
Proc. Nat. Acad. Sci. USA
82
,
5766
5770
Slot
J. W.
,
Geuze
H. J.
(
1985
).
A new method of preparing gold probes for multiple-labeling cytochemistry.
Eur. J. Cell Biol
38
,
87
93
Takada
Y.
,
Ylänne
J.
,
Mandelman
D.
,
Puzon
W.
,
Ginsberg
M. H.
(
1992
).
A point mutation of integrin1subunit blocks binding of 51to fibronectin and invasin but not recruitment to adhesion plaques.
J. Cell Biol
119
,
913
921
Vogel
B. E.
,
Tarone
G.
,
Giancotti
F. G.
,
Gailit
J.
,
Ruoslahti
E.
(
1990
).
A novel fibronectin receptor with an unexpected subunit composition (V 1).
J. Biol. Chem
265
,
5934
5937
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