We are studying how the cytoskeleton determines cell shape, using a simple model system, the marginal band of chicken erythrocytes. We previously identified a minor component of the marginal band by a monoclonal antibody, called 13H9 (Birgbauer and Solomon (1989). J. Cell Biol. 109, 1609–1620; Goslin et al. (1989). J. Cell Biol. 109, 1621–1631). mAb 13H9 also binds to the leading edges of fibroblasts and to neuronal growth cones and recognizes the cytoskeletal protein ezrin. In recent years, two proteins with a high degree of homology to ezrin were identified: moesin and radixin, together comprising the ERM protein family. We now show that the contiguous epitope sufficient for mAb 13H9 binding is a sequence present in each of the ERM proteins, as well as the product of the gene associated with neurofibromatosis 2, merlin or schwannomin. We used biochemical and immunological techniques, as well as PCR to characterize the expression and localization of the ERM proteins in chicken erythrocytes. The results demonstrate that radixin is the major ERM protein associated with the cytoskeleton. Both ezrin and radixin localize to the position of the marginal band. Our results suggest that the ERM proteins play functionally conserved roles in quite diverse organelles.

REFERENCES

Algrain
M.
,
Turunen
O.
,
Vaheri
A.
,
Louvard
D.
,
Arpin
M.
(
1993
).
Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker.
J. Cell Biol
120
,
129
139
Amieva
M. R.
,
Wilgenbus
K. K.
,
Furthmayr
H.
(
1994
).
Radixin is a component of hepatocyte microvilli in situ.
Exp. Cell Res
210
,
140
144
Anderson
R. A.
,
Marchesi
V. T.
(
1985
).
Regulation of the association of membrane skeletal protein 4. 1 with glycophorin by a polyphosphoinositide.
Nature
318
,
295
298
Andreoli
C.
,
Le Borgne
R.
,
Martin
M.
,
Mangeat
P.
(
1992
).
Ezrin-binding proteins from gastric tissue. Mol. Biol.
Cell
3
,
364
–.
Behnke
O.
(
1970
).
Microtubules in disk shaped blood cells.
Int. Rev. Exp. Pathol
9
,
1
92
Berryman
M.
,
Franck
Z.
,
Bretscher
A.
(
1993
).
Ezrin is concentrated in the apical microvilli of epithelial cells whereas moesin is found primarily in endothelial cells.
J. Cell Sci
105
,
1025
1032
Birgbauer
E.
,
Solomon
F.
(
1989
).
A marginal band-associated protein has properties of both microtubule-and microfilament-associated proteins.
J. Cell Biol
109
,
1609
1620
Birgbauer
E.
,
Dinsmore
J. H.
,
Winckler
B.
,
Lander
A. D.
,
Solomon
F.
(
1991
).
Association of ezrin isoforms with the neuronal cytoskeleton.
J. Neurosci. Res
30
,
232
241
Bond
J. F.
,
Fridovich-Keil
J. L.
,
Pillus
L.
,
Mulligan
R. C.
,
Solomon
F.
(
1986
).
A chicken-yeast chimeric-tubulin protein is incorporated into mouse microtubules in vivo.
Cell
44
,
461
468
Bretscher
A.
(
1983
).
Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells.
J. Cell Biol
97
,
425
532
Bretscher
A.
(
1986
).
Purification of the intestinal microvillus cytoskeletal proteins villin, fimbrin, and ezrin.
Meth. Enzymol
134
,
24
37
Bretscher
A.
(
1989
).
Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor.
J. Cell Biol
108
,
921
930
Fazioli
F.
,
Wong
W. T.
,
Ullrich
S. J.
,
Sakaguchi
K.
,
Appella
E.
,
Di Fiore
P. P.
(
1993
).
The ezrin-like family of tyrosine kinase substrates: receptor-specific pattern of tyrosine phosphorylation and relationship to malignant transformation.
Oncogene
8
,
1335
1345
Feick
P.
,
Foisner
R.
,
Wiche
G.
(
1991
).
Immunolocalization and molecular properties of a high molecular weight microtubule-bundling protein (syncolin) from chicken erythrocytes.
J. Cell Biol
112
,
689
699
Franck
Z.
,
Gary
R.
,
Bretscher
A.
(
1993
).
Moesin, like ezrin, colocalizes with actin in the cortical cytoskeleton in cultured cells, but its expression is more variable.
J. Cell Sci
105
,
219
231
Funayama
A.
,
Nagafuchi
A.
,
Sato
N.
,
Tsukita
S.
,
Tsukita
S.
(
1991
).
Radixin is a novel member of the band 4.1 family.
J. Cell Biol
115
,
1039
1048
Gary
R.
,
Franck
Z.
,
Bretscher
A.
(
1992
).
Ezrin binds moesin in vivo and in vitro. Mol. Biol.
Cell
3
,
364
–.
Goslin
K.
,
Birgbauer
E.
,
Banker
G.
,
Solomon
F.
(
1989
).
The role of cytoskeleton in organizing growth cones: a microfilament-associated growth cone component depends upon microtubules for its localization.
J. Cell Biol
109
,
1621
1631
Gould
K. L.
,
Bretscher
A.
,
Esch
F. S.
,
Hunter
T.
(
1989
).
cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1.
EMBO J
8
,
4133
4142
Kim
S.
,
Magendantz
M.
,
Katz
W.
,
Solomon
F.
(
1987
).
Development ofa differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo.
J. Cell Biol
104
,
51
59
Koerner
T. J.
,
Hill
J. E.
,
Myers
A. M.
,
Tzagoloff
A.
(
1991
).
High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors.
Meth. Enzymol
194
,
477
490
Kosik
K. S.
,
Orecchio
L. D.
,
Bakalis
S.
,
Duffy
L.
,
Neve
R. L.
(
1988
).
Partial sequence of MAP2 in the region of a shared epitope with Alzheimer neurofibrillary tangles.
J. Neurochem
51
,
587
598
Kosik
K. S.
,
Orecchio
L. D.
,
Binder
L.
,
Trojanowski
J. Q.
,
Lee
V. M.-Y.
,
Lee
G.
(
1988
).
Epitopes that span the tau molecule are shared with paired helical filaments.
Neuron
1
,
817
825
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Lankes
W. T.
,
Furthmayr
H.
(
1991
).
Moesin: A member of the protein 4.1-talin-ezrin family of proteins.
Proc. Nat. Acad. Sci. USA
88
,
8297
8301
Luna
E. J.
(
1991
).
Molecular links between the cytoskeleton and membranes.
Curr. Opin. Cell Biol
3
,
120
126
Matsudaira
P.
(
1991
).
Modular organization of actin crosslinking proteins.
Trends Biochem. Sci
16
,
87
90
Matus
A.
(
1988
).
Microtubule-associated proteins: their role in determining neuronal morphology.
Annu. Rev. Neurosci
11
,
29
44
Mehra
V. D.
,
Sweetser
D.
,
Young
R. A.
(
1986
).
Efficient mapping of protein antigenic determinants.
Proc. Nat. Acad. Sci. USA
83
,
7013
7017
Miller
M.
,
Solomon
F.
(
1984
).
Kinetics and intermediates of marginal band reformation: evidence for peripheral determinants of microtubule organization.
J. Cell Biol
99
,
70
–.
Murphy
D. B.
,
Wallis
K. T.
(
1985
).
Erythrocyte microtubule assembly in vitro.
J. Biol. Chem
260
,
12293
–.
Olmsted
J. B.
(
1981
).
Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples.
J. Biol. Chem
256
,
11955
11957
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
Rees
D. J. G.
,
Ades
S. A.
,
Singer
S. J.
,
Hynes
R. O.
(
1990
).
Sequence and domain structure of talin.
Nature
347
,
685
689
Rouleau
G. A.
and 20 others
(
1993
).
Alteration in a new gene encoding a putative membrane-organizing protein causes neurofibromatosis type 2.
Nature
363
,
515
521
Sato
N.
,
Yonemura
S.
,
Obinata
T.
,
Tsukita
S.
,
Tsukita
S.
(
1991
).
Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis.
J. Cell Biol
113
,
321
330
Sato
N.
,
Funayama
N.
,
Nagafuchi
A.
,
Yonemura
S.
,
Tsukita
S.
,
Tsukita
S.
(
1992
).
A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites.
J. Cell Sci
103
,
131
143
Solomon
F.
,
Magendantz
M.
,
Salzman
A.
(
1979
).
Identification withcellular microtubules of one of the coassembling microtubule-associated proteins.
Cell
18
,
431
438
Stetzkowski-Marden
F.
,
Deprette
C.
,
Cassoly
R.
(
1991
).
Identification of tubulin-binding proteins of the chicken erythrocyte plasma membrane skeleton which co-localize with the microtubular marginal band.
Eur. J. Cell Biol
54
,
102
–.
Stone
K. L.
,
Elliott
J. I.
,
Peterson
G.
,
McMurray
W.
,
Williams
K. R.
(
1990
).
Reversed-phase high-performance liquid chromatography for fractionation of enzymatic digests and chemical cleavage products of proteins.
Meth. Enzymol
193
,
389
412
Stossel
T. P.
(
1993
).
On the crawling of animal cells.
Science
260
,
1086
1094
Tam
J. P.
(
1988
).
Synthetic peptide vaccine design: Synthesis and properties of a high-density multiple antigenic peptide system.
Proc. Nat. Acad. Sci. USA
85
,
5409
5413
Tobin
H.
,
Staehelin
T.
,
Jordon
J.
(
1979
).
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications.
Proc. Nat. Acad. Sci. USA
76
,
4350
4354
Trofatter
J. A.
,
MacCollin
M. M.
,
Rutter
J. L.
,
Murrell
J. R.
,
Duyao
M. P.
,
Parry
D. M.
,
Eldridge
R.
,
Kley
N.
,
Menon
A. G.
,
Pulaski
K.
,
Haase
V. H.
,
Ambrose
C. M.
,
Munroe
D.
,
Bove
C.
,
Haines
J. L.
,
Martuza
R. L.
,
MacDonald
M. E.
,
Seizinger
B. R.
,
Short
M. P.
,
Buckler
A. J.
,
Gusella
J. F.
(
1993
).
A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor.
Cell
72
,
791
800
Tsukita
S.
,
Hieda
Y.
,
Tsukita
S.
(
1989
).
A new 82-kD barbed end-capping protein (radixin) localized in the cell-to-cell adherens junction: Purification and characterization.
J. Cell Biol
108
,
2369
2382
Tucker
R. P.
(
1990
).
The roles of microtubule-associated proteins in brain morphogenesis: a review.
Brain Res
15
,
101
20
Turunen
O.
,
Winqvist
R.
,
Pakkanen
R.
,
Grzeschik
K.-H.
,
Wahlstrom
T.
,
Vaheri
A.
(
1989
).
Cytovillin, a microvillar M r75, 000 protein: cDNA sequence, prokaryotic expression, and chromosomal localization.
J. Biol. Chem
264
,
16727
16732
Wiche
G.
(
1989
).
High M rmicrotubule-associated proteins: properties and functions.
Biochem. J
259
,
1
12
Wiche
G.
,
Oberkanins
C.
,
Himmler
A.
(
1991
).
Molecular structure and function of microtubule-associated proteins.
Int. Rev. Cytol
124
,
217
273
Winckler
B.
,
Solomon
F.
(
1991
).
A role for microtubule bundles in the morphogenesis of chicken erythrocytes.
Proc. Nat. Acad. Sci. USA
88
,
6033
6037
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