Hemidesmosomes are complex macromolecular structures which integrate elements of the extracellular matrix and the cytoskeleton of epithelial cells. To characterize cell-matrix interactions in the hemidesmosome, we have made use of 804G cells which possess the unusual ability to assemble hemidesmosomes in vitro. During the course of our studies, we have raised a set of monoclonal antibodies against rat laminin-5, the major structural element comprising 804G matrix. One of these, termed CM6, recognizes the 150 kDa alpha chain of rat laminin-5 and binds the globular (G) domain of intact laminin-5 molecules as determined by rotary shadowing. CM6 antibodies perturb formed hemidesmosomes in 804G cells. In particular, within 1 hour of incubation of 804G cells with CM6 antibodies, colocalization of laminin-5 and alpha 6 beta 4 integrin is lost and by 2 hours, staining generated by hemidesmosomal antibodies appears primarily cytoplasmic in the perinuclear zone. Ultrastructurally, CM6 antibodies first appear to induce detachment of hemidesmosomes from the underlying matrix. Next, portions of the basal cell surface invaginate to form vesicles whose cytoplasmic-facing surface is coated with hemidesmosomes still associated with keratin intermediate filaments. Anchoring filaments extend into the inside compartment of the vesicles. We have also studied the impact of CM6 antibodies on a model system in which the matrix of 804G cells induces de novo assembly of hemidesmosomes in human keratinocytes. This process involves the plasma membrane reorganization of the hemidesmosome associated integrin alpha 6 beta 4 as well as a redistribution of other hemidesmosome components such as the 230 kDa bullous pemphigoid antigen. Pretreatment of 804G matrix with CM6 antibodies blocks such plasma membrane reorganization of hemidesmosome components and inhibits hemidesmosome formation. Our studies indicate a crucial role for the G domain of the alpha chain of laminin-5 in both nucleation of hemidesmosome assembly as well as maintenance of hemidesmosome structural integrity.
REFERENCES
Aberdam
D.
, Galliano
M. F.
, Mattei
M. G.
, Pisani-Spadafora
A.
, Ortonne
J. P.
, Meneguzzi
G.
(1994
). Assignment of mouse nicein genes to chromosomes 1 and 8.
Mamm. Genome
5
, 229
–233
Aberdam
D.
, Galliano
M.-F.
, Vailley
J.
, Pulkkinen
L.
, Bonifas
J.
, Christiano
A. M.
, Tryggvason
K.
, Uitto
J.
, Epstein
E. H.
Jr., Ortonne
J.-P.
, Meneguzzi
G.
(1994
). Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the2 subunit of nicein/kalinin (laminin-5).
Nature Genet
6
, 299
–304
Burgeson
R. E.
, Chiquet
M.
, Deutzmann
R.
, Ekblom
P.
, Engel
J.
, Kleinman
H.
, Martin
G. R.
, Meneguzzi
G.
, Paulsson
M.
, Sanes
J.
, Timpl
R.
, Tryggvason.
K.
, Yamada
Y.
, Yurchenco
P. D.
(1994
). A new nomenclature for the laminins.
Matrix Biol
14
, 209
–211
Carter
W. G.
, Ryan
M. C.
, Gahr
P. J.
(1991
). Epiligrin, a new cell adhesion ligand for integrinin epithelial basement membranes.
Cell
65
, 599
–610
Domloge-Hultsch
N.
, Gammon
W. R.
, Briggaman
R. A.
, Gil
S. G.
, Carter
W. G.
, Yancey
K. B.
(1992
). Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease.
J. Clin. Invest
90
, 1628
–1633
Eady
R. A. J.
, McGrath
J. A.
, McMillan
J. R.
(1994
). Ultrastructural clues to genetic disoders of skin: the dermal-epidermal junction.
J. Invest. Derm
103
, 13
–.Ehrig
K.
, Leivo
I.
, Argraves
W. S.
, Ruoslahti
E.
, Engvall
E.
(1990
). Merosin, a tissue-specific basement membrane protein, is a laminin-like protein.
Proc. Nat. Acad. Sci. USA
87
, 3264
–3268
Galliano
M. F.
, Aberdam
D.
, Aguzzi
A.
, Ortonne
J. P.
, Meneguzzi
G.
(1995
). Cloning and complete primary structure of the mouse laminin3 chain.
J. Biol. Chem
270
, 21820
–21826
Guo
L.
, Degenstein
L.
, Dowling
J.
, Yu
Q. C.
, Wollmann
R.
, Perman
B.
, Fuchs
E.
(1995
). Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration.
Cell
81
, 233
–243
Hashimoto
T.
, Amagai
M.
, Ebihara
T.
, Gamou
S.
, Shimizu
N.
, Tsubata
T.
, Hasegawa
A.
, Miki
K.
, Nishikawa
T.
(1993
). Further analyses of epitopes for human monoclonal anti-basement membrane zone antibodies produced by stable human hybridoma cell lines constructed with Epstein-Barr virus transformants.
J. Invest. Dermatol
100
, 310
–315
Hopkinson
S. B.
, Baker
S. E.
, Jones
J. C. R.
(1995
). Molecular genetic studies of a human epidermal autoantigen (the 180-kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and evidence for an interaction between BP180 and alpha 6 integrin.
J. Cell Biol
130
, 117
–125
Hormia
M.
, Falk-Marzillier
J.
, Plopper
G.
, Tamura
R. N.
, Jones
J. C. R.
, Quaranta
V.
(1995
). Rapid spreading and mature hemidesmosome formation in HaCaT keratinocytes induced by incubation with soluble laminin-5r.
J. Invest. Dermatol
105
, 557
–561
Hunzicker-Dunn
M.
, Cutler
R. E.
, Maizels
E. T.
, DeManno
D. A.
, Lamm
M. L. G.
, Erlichman
J.
, Sanwal
B. D.
, LaBarbera
A. R.
(1991
). Isozymes of cAMP-dependent protein kinase present in the rat coprus luteum.
J. Biol. Chem
266
, 7166
–7175
Jones
J. C. R.
, Kurpakus
M. A.
, Cooper
H. M.
, Quaranta
V.
(1991
). A function for the integrin alpha 6 beta 4 in the hemidesmosome.
Cell Reg
2
, 427
–438
Jones
J. C. R.
, Asmuth
J.
, Baker
S. E.
, Langhofer
M.
, Roth
S. I.
, Hopkinson
S. B.
(1994
). Hemidesmosomes: extracellular matrix/intermediate filament connectors.
Exp. Cell Res
213
, 1
–11
Kallunki
P.
, Saino
K.
, Eddy
R.
, Byers
M.
, Kallunki
T.
, Sariola
H.
, Beck
K.
, Hirvonen
H.
, Shows
T. B.
, Tryggvason
K.
(1992
). A truncated laminin chain homologous to the B2 chain: structure, spatial expression and chromosomal assignment.
J. Cell Biol
109
, 3377
–3390
Kivirikko
S.
, McGrath
J. A.
, Baudoin
C.
, Aberdam
D.
, Ciatti
S.
, Dunnill
M. G.
, McMillan
J. R.
, Eady
R. A.
, Ortonne
J. P.
, Meneguzzi
G.
, Uitto
J.
, Christiano
A. M.
(1995
). A homozygous nonsense mutation in thechain gene of laminin 5 (LAM) in lethal (Herlitz) junctional epidermolysis bullosa.
Hum. Mol. Genet
4
, 959
–962
Kurpakus
M. A.
, Quaranta
V.
, Jones
J. C. R.
(1991
). Surface relocation of alphbeta4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing.
J. Cell Biol
115
, 1737
–1750
Langhofer
M.
, Hopkinson
S. B.
, Jones
J. C. R.
(1993
). The matrix secreted by 804G cells contains laminin-related components that participate in hemidesmosome assembly in vitro.
J. Cell Sci
105
, 753
–764
Liu
Z.
, Diaz
L. A.
, Troy
J. L.
, Taylor
A. F.
, Emery
D. J.
, Fairley
J. A.
, Giudice
G. J.
(1993
). A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid using antibodies generated against the hemidesmosomal antigen, BP180.
J. Clin. Invest
92
, 2480
–2488
Mainiero
F.
, Pepe
A.
, Wary
K. K.
, Spinardi
L.
, Mohammadi
M.
, Schlessinger
J.
, Giancotti
F. G.
(1995
). Signal transduction by theintegrin: distinct subunit sites mediate recruitment of Shc/Grb2 andassociation with the cytoskeleton of hemidesmosomes.
EMBO J
14
, 4470
–4481
Marinkovich
M. P.
, Lunstrum
G. P.
, Burgeson
R. E.
(1992
). The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.
J. Biol. Chem
267
, 17900
–17906
Matter
M. L.
, Laurie
G. W.
(1994
). A novel laminin E8 cell adhesion site required for lung alveolar formation in vitro.
J. Cell Biol
124
, 1083
–1090
Niessen
C. M.
, Hogervorst
F.
, Jaspars
L. H.
, De Melker
A. A.
, Delwel
G. O.
, Hulsman
E. H. M.
, Kuikman
I.
, Sonnenberg
A.
(1994
). Theintegrin is a receptor of both laminin and kalinin.
Exp. Cell Res
211
, 360
–367
Pulkkinen
L.
, Christiano
A. M.
, Airenne
T.
, Haakana
H.
, Tryggvason
K.
, Uitto
J.
(1994
). Mutations in the2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa.
Nature Genet
6
, 293
–298
Pulkkinen
L.
, Christiano
A. M.
, Gerecke
D.
, Wagman
D. W.
, Burgeson
R. E.
, Pittelkow
M. R.
, Uitto
J.
(1994
). A homozygous nonsense mutation in the3 chain gene of laminin 5 (LAMB3) in Herlitz junctional epidermolysis bullosa.
Genomics
24
, 357
–360
Riddelle
K. S.
, Green
K. J.
, Jones
J. C. R.
(1991
). Formation of hemidesmosomes in vitro by a transformed rat bladder cell.
J. Cell Biol
112
, 159
–168
Rousselle
P.
, Lunstrum
G. P.
, Keene
D. R.
, Burgeson
R. E.
(1991
). Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.
J. Cell Biol
114
, 567
–576
Rousselle
P.
, Aumailley
M.
(1994
). Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.
J. Cell Biol
125
, 205
–214
Ryan
M. C.
, Tizard
R.
, VanDevanter
D. R.
, Carter
W. G.
(1994
). Cloning of the lamA3 gene coding the3 chain of the adhesive ligand epiligrin.
J. Biol. Chem
269
, 22779
–22787
Sanger
F.
, Nicklen
S.
, Coulson
A. R.
(1977
). DNA sequencing with chain terminating inhibitors.
Proc. Nat. Acad. Sci. USA
74
, 5463
–5467
Spinardi
L.
, Einheber
S.
, Cullen
T.
, Milner
T. A.
, Giancotti
F. G.
(1995
). A recombinant tail-less integrin4 subunit disrupts hemidesmosomes, but does not suppress -mediated cell adhesion to laminins.
J. Cell Biol
129
, 473
–487
Skubitz
A. P.
, Letourneau
P. C.
, Wayner
E.
, Furcht
L. T.
(1991
). Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit.
J. Cell Biol
115
, 1137
–1148
Sonnenberg
A.
, Calafat
J.
, Janssen
H.
, Daams
H.
, van der Raaj
H.
, Hemler
M. E.
, Falcioni
R.
, Kennel
S. J.
, Aplin
J. D.
, Baker
J.
, Loizidou
M.
, Garrod
D.
(1991
). Integrincomplex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion.
J. Cell Biol
113
, 907
–917
Stepp
M. A.
, Spurr-Michaud
S.
, Tisdale
A.
, Elwell
J.
, Gipson
I. K.
(1990
). integrin heterodimer is a component of hemidesmosomes.
Proc. Nat. Acad. Sci. USA
87
, 8970
–8974
Streuli
C. H.
, Schmidhauser
C.
, Bailey
N.
, Yurchenco
P.
, Scubitz
A. P. N.
, Roskelley
C.
, Bissell
M. J.
(1995
). Laminin mediates tissue-specific gene expression in mammary epithelia.
J. Cell Biol
129
, 591
–603
Tamura
R. N.
, Rozzo
C.
, Starr
L.
, Chambers
J.
, Reichardt
L. F.
, Cooper
H. M.
, Quaranta
V.
(1990
). Epithelial integrin: complete primary structure of and variant forms of.
J. Cell Biol
111
, 1593
–1604
Tryggvason
K.
(1993
). The laminin family.
Curr. Opin. Cell Biol
5
, 877
–882
Verrando
P.
, Hsi
B.-L.
, Yeh
C.-J.
, Pisani
A.
, Serieys
N.
, Ortonne
J.-P.
(1987
). Monoclonal antibody GB3, a new probe fo the study of human basement membranes and hemidesmosomes.
Exp. Cell Res
170
, 116
–128
Von Heijne
G.
(1986
). A new method for predicting signal sequence cleavage sites.
Nucl. Acids Res
14
, 4683
–4690
Yurchenco
P. D.
, Sung
U.
, Ward
M. D.
, Yamada
Y.
, O'Rear
J. J.
(1993
). Recombinant laminin G domain mediates myoblast adhesion and heparin binding.
J. Biol. Chem
268
, 8356
–8365
This content is only available via PDF.
© 1996 by Company of Biologists
1996
-2023JournalMeeting.png?versionId=4316)
-PolarityCFP.png?versionId=4316)
-ECRpage.jpg?versionId=4316)
-CSTW.png?versionId=4316)
-GameteFusion.jpeg?versionId=4316)
