The surface distribution of the alpha 2/delta subunit of the 1,4-dihydropyridine receptor and its topographical relationship with the neural cell adhesion molecule (N-CAM) were investigated during early myogenesis in vitro, by double immunocytochemical labeling with the monoclonal antibody 3007 and an anti-N-CAM polyclonal antiserum. The monoclonal antibody 3007 has been previously shown to immunoprecipitate dihydropyridine receptor from skeletal muscle T-tubules. In further immunoprecipitation experiments on such preparations and muscle cell cultures, it was demonstrated here that the monoclonal antibody 3007 exclusively recognizes the alpha 2/delta subunit of the 1,4-dihydropyridine receptor. In rabbit muscle cell cultures, the labeling for both alpha 2/delta and N-CAM was first detected on myoblasts, in the form of spots on the membrane and perinuclear patches. Spots of various sizes organized in aggregates were then found on the membrane of myotubes. At fusion (T0), aggregates of N-CAM spots alone were found at the junction between fusing cells. At T6 and later stages, all alpha 2/delta aggregates present on myotubes co-localized with N-CAM, while less than 3% of N-CAM aggregates did not co-localize with alpha 2/delta. A uniform N-CAM staining also made its appearance. At T12, when myotubes showed prominent contractility, alpha 2/delta-N-CAM aggregates diminished in size. Dispersed alpha 2/delta spots of a small regular size spread over the whole surface of the myotubes and alignments of these spots became visible. Corresponding N-CAM spots were now occasionally seen, and uniform N-CAM staining was prominent. These results show that alpha 2/delta and N-CAM are co-localized and that their distributions undergo concomitant changes during early myogenesis until the T-tubule network starts to be organized. This suggest that these two proteins might jointly participate in morphogenetic events preceding the formation of T-tubules.
REFERENCES
Almers
W.
, Fink
R.
, Palade
P. T.
(1981
). Calcium depletion in frog muscle tubules: The decline of calcium current under maintained depolarization.
J. Physiol
312
, 177
–207
Barhanin
J.
, Coppola
T.
, Schmid
A.
, Borsotto
M.
, Lazdunski
M.
(1987
). The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization.
Eur. J. Biochem
164
, 525
–531
Beam
K. G.
, Adams
B. A.
, Niidome
T.
, Numa
S.
, Tanabe
T.
(1992
). Function of a truncated dihydropyridine receptor as both voltage sensor and calcium channel.
Nature
360
, 169
–171
Bloch
R. J.
(1992
). Clusters of neural cell adhesion molecule at sites of cell-cell contact.
J. Cell Biol
116
, 449
–463
Brawley
R. M.
, Hosey
M. M.
(1992
). Identification of two distinct proteins that are immunologically related to the1subunit of the skeletal muscle dihydropyridine-sensitive calcium channel.
J. Biol. Chem
267
, 18218
–18223
Brust
P.F.
, Simerson
S.
, McCue
A.
, Deal
C.R.
, Schoonmaker
S.
, Williams
M.
, Veliçelebi
G.
, Johnson
E.C.
, Harpold
M.M.
, Ellis
S.B.
(1993
). Human neuronal voltage-dependant calcium channels: studies on subunit structure and role in channel assembly.
Neuropharmacology
32
, 1089
–1102
Campbell
K. P.
, Leung
A. T.
, Sharp
A. H.
(1988
). The biochemistryand molecular biology of the dihydropyridine-sensitive calcium channel.
Trends Neurosci
11
, 425
–430
Catterall
W. A.
, Seagar
M. J.
, Takahashi
M.
(1988
). Molecular properties of dihydropyridine-sensitive calcium channels in skeletal muscle.
J. Biol. Chem
263
, 3535
–3538
Catterall
W. A.
(1991
). Structure and function of voltage-gated sodium and calcium channels.
Curr. Opin. Neurobiol
1
, 5
–13
Chan
C. Z.
, Sato
K.
, Shimada
Y.
(1990
). Three-dimensional electron microscopy of the sarcoplasmic reticulum and T-system in embryonic chick skeletal muscle cells in vitro.
Protoplasma
154
, 112
–121
Chaudhari
N.
, Beam
K. G.
(1993
). mRNA for cardiac calcium channel is expressed during development of skeletal muscle.
Dev. Biol
155
, 507
–515
Chen
C.
, Corbley
M. J.
, Roberts
T. M.
, Hess
P.
(1988
). Voltage-sensitive calcium channels in normal and transformed fibroblasts.
Science
239
, 1024
–1026
Connolly
J. A.
(1984
). Role of the cytoskeleton in the formation, stabilization, and removal of acetylcholine receptor clusters in cultured muscle cells.
J. Cell Biol
99
, 148
–154
Covault
J.
, Sanes
J. R.
(1986
). Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle.
J. Cell Biol
102
, 716
–730
Covault
J.
, Merlie
J.P.
, Goridis
C.
, Sanes
J.R.
) (1986
). Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle.
J. Cell Biol
102
, 731
–739
Cunningham
B. A.
, Hemperly
J. J.
, Murray
B. A.
, Prediger
E. A.
, Brackenbury
R.
, Edelman
G. M.
(1987
). Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation and alternative RNA splicing.
Science
236
, 799
–806
De Jongh
K. S.
, Merrick
D. K.
, Catterall
W. A.
(1989
). Subunits of purified calcium channels: a 212-kDa form of1and partial amino acid sequence of a phosphorylation site of an independent subunit.
Proc. Nat. Acad. Sci. USA
86
, 8585
–8589
De Jongh
K. S.
, Warner
C.
, Catterall
W. A.
(1990
). Subunits of purified calcium channels.2and are encoded by the same gene.
J. Biol. Chem
265
, 14738
–14741
De Jongh
K. S.
, Warner
C.
, Colvin
A. A.
, Catterall
W. A.
(1991
). Characterization of the two size forms of the1subunit of skeletal muscle L-type calcium channels.
Proc. Nat. Acad. Sci. USA
88
, 10778
–10782
Dickson
G.
, Peck
D.
, Moore
S. E.
, Barton
C. H.
, Walsh
F. S.
(1990
). Enhanced myogenesis in N-CAM-transfected mouse myoblasts.
Nature
344
, 348
–351
Doherty
P.
, Cohen
J.
, Walsh
F. S.
(1990
). Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid.
Neuron
5
, 209
–219
Doherty
P.
, Ashton
S. V.
, Moore
S. E.
, Walsh
F. S.
(1991
). Morpho-regulatory activities of N-CAM and N-cadherin can be accounted for by G protein-dependent activation of L-and N-type neuronal Ca2+channels.
Cell
67
, 21
–33
Edelman
G. M.
(1986
). Cell adhesion molecules in the regulation of animal form and tissue pattern.
Annu. Rev. Cell Biol
2
, 81
–116
Edelman
G. M.
(1987
). CAMs and Igs: Cell adhesion and the evolutionary origins of immunity.
Immunol. Rev.
100
, 11
–45
Ellis
S. B.
, Williams
M. E.
, Ways
N. R.
, Brenner
R.
, Sharp
A. H.
, Leung
A. T.
, Campbell
K. P.
, McKenna
E.
, Koch
W. J.
, Hui
A.
, Schwartz
A.
, Harpold
M. M.
(1988
). Sequence and expression of mRNAs encoding the1and 2subunits of a DHP-sensitive calcium channel.
Science
241
, 1661
–1664
Fischbach
G. D.
, Cohen
S. A.
(1973
). The distribution of acetylcholine sensitivity over uninnervated muscle fibers grown in cell culture.
Dev. Biol
31
, 147
–162
Flucher
B. E.
, Morton
M. E.
, Froehner
S. C.
, Daniels
M. P.
(1990
). Localization of the1and 2subunits of the dihydropyridine receptor and ankyrin in skeletal muscle triads.
Neuron
5
, 339
–351
Flucher
B. E.
, Phillips
J. L.
, Powell
J. A.
(1991
). Dihydropyridine receptor-subunits in normal and dysgenic muscle in vitro- expression of 1is required for proper targeting and distribution of2.
J. Cell Biol
115
, 1345
–1356
Flucher
B. E.
(1992
). Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum and the triad.
Dev. Biol
154
, 245
–260
Franzini-Armstrong
C.
, Porter
K. R.
(1964
). Sarcolemmal invaginations constituting the T system in fish muscle fibers.
J. Cell Biol
22
, 675
–696
Galizzi
J. P.
, Fosset
M.
, Lazdunski
M.
(1984
). Properties of receptors ofthe Ca2+blocker verapamil in transverse-tubule membranes of skeletal muscle.
Eur. J. Biochem
144
, 211
–215
He
H. T.
, Barbet
J.
, Chaix
J. C.
, Goridis
C.
(1986
). Phosphatidylinositol is involved in the membrane attachment of N-CAM-120, the smallest component of the neural cell adhesion molecule.
EMBO J
5
, 2489
–2494
Hosey
M. M.
, Barhanin
J.
, Schmid
A.
, Vandaele
S.
, Ptasienski
J.
, O'Callahan
C.
, Cooper
C.
, Lazdunski
M.
(1987
). Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine sensitive calcium channels.
Biochem. Biophys. Res. Commun
147
, 1137
–1145
Hosey
M. M.
, Lazdunski
M.
(1988
). Calcium channels: molecular pharmacology, structure and regulation.
J. Membr. Biol
104
, 81
–105
Huxley
H. E.
(1964
). Evidence for continuity between the central elements of the triads and the extracellular space in frog sartorius muscle.
Nature
202
, 1067
–1071
Ishikawa
H.
(1968
). Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation.
J. Cell Biol
38
, 51
–66
Jay
S. D.
, Ellis
S. B.
, McCue
A. F.
, Williams
M. E.
, Vedvick
T. S.
, Harpold
M. M.
, Campbell
K. P.
(1990
). Primary structure of the gamma subunit of the DPH-sensitive calcium channel from skeletal muscle.
Science
248
, 490
–492
Jay
S. D.
, Sharp
A. H.
, Kahl
S. D.
, Vedvick
T. S.
, Harpold
M. M.
, Campbell
K. P.
(1991
). Structural characterization of the dihydropyridine-sensitive calcium channel2-subunit and the associated peptides.
J. Biol. Chem
266
, 3287
–3293
Jorgensen
A. O.
, Shen
A. C.-Y.
, Arnold
W.
, Leung
A. T.
, Campbell
K. P.
(1989
). Subcellular distribution of the 1,4-dihydropyridine receptor in rabbit skeletal muscle in situ: an immunofluorescence and immunocolloidal gold-labeled study.
J. Cell Biol
109
, 135
–147
Kelly
A. M.
(1971
). Sarcoplasmic reticulum and T tubules in differentiating rat skeletal muscle.
J. Cell Biol
49
, 335
–344
Knudsen
K. A.
(1990
). Cell adhesion molecules in myogenesis.
Curr. Opin. Cell Biol
2
, 902
–906
Knudsen
K. A.
, McElwee
S. A.
, Myers
L.
(1990
). A role for the neural cell adhesion molecule, N-CAM, in myoblast interaction during myogenesis.
Dev. Biol
138
, 159
–68
Knudsen
K. A.
, Myers
L.
, McElwee
S. A.
(1990
). A role for the Ca2+-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis.
Exp. Cell Res
188
, 175
–84
Laemmli
U. K.
(1970
). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
, 680
–685
Lai
Y.
, Seagar
M. J.
, Takahashi
M.
, Catterall
W. A.
(1990
). Cyclic AMP-dependent phosphorylation of two size forms of1subunits of L-type calcium channels in rat skeletal muscle cells.
J. Biol. Chem
265
, 20839
–20848
Leung
A. T.
, Imagawa
T.
, Block
B.
, Franzini-Armstrong
C.
, Campbell
K. P.
(1988
). Biochemical and ultrastructural characterization of the 1,4-dihydropyridine receptor from rabbit skeletal muscle.
J. Biol. Chem
263
, 994
–1001
Malouf
N. N.
, McMahon
D. K.
, Hainsworth
C. N.
, Kay
B. K.
(1992
). A two-motif isoform of the major calcium channel subunit in skeletal muscle.
Neuron
8
, 899
–906
Mege
R. M.
, Goudou
D.
, Diaz
C.
, Nicolet
M.
, Garcia
L.
, Geraud
G.
, Rieger
F.
(1992
). N-cadherin and N-CAM in myoblast fusion: compared localization and effect of blockade by peptides and antibodies.
J. Cell Sci
103
, 897
–906
Moore
S. E.
, Thompson
J.
, Kirkness
V.
, Dickson
J. G.
, Walsh
F. S.
(1987
). Skeletal muscle neural cell adhesion molecule (N-CAM): changes in protein and mRNA species during myogenesis of muscle cell lines.
J. Cell Biol
105
, 1377
–1386
Morton
M. E.
, Froehner
S. C.
(1987
). Monoclonal antibody identifies a 200-kDa subunit of the dihydropyridine-sensitive calcium channel.
J. Biol. Chem
262
, 11904
–11907
Morton
M. E.
, Froehner
S. C.
(1989
). The1and 2polypeptides of the dihydropyridine-sensitive calcium channel differ in developmental expression and tissue distribution.
Neuron
2
, 1499
–1506
Olek
A. J.
, Ling
A.
, Daniels
M. P.
(1986
). Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes.
J. Neurosci
6
, 487
–497
Rieger
F.
, Grumet
M.
, Edelman
G. M.
(1985
). N-CAM at the vertebrate neuromuscular junction.
J. Cell Biol
101
, 285
–293
Rios
E.
, Brum
G.
(1987
). Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle.
Nature
325
, 717
–720
Rios
E.
, Pizarro
G.
, Stefani
E.
(1992
). Charge movement and the nature of signal transduction in skeletal muscle excitation-contraction coupling.
Annu. Rev. Physiol
54
, 109
–133
Romey
G.
, Garcia
L.
, Dimitriadou
V.
, Pinçon-Raymond
M.
, Rieger
F.
, Lazdunski
M.
(1989
). Ontogenesis and localization of Ca2+channels in mammalian skeletal muscle in culture and role in excitation-contraction coupling.
Proc. Nat. Acad. Sci. USA
86
, 2933
–2937
Rotman
E. I.
, De Jongh
K. S.
, Florio
V.
, Lai
Y.
, Catterall
W. A.
(1992
). Specific phosphorylation of a COOH-terminal site on the full-length form of the1-subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase.
J. Biol. Chem
267
, 16100
–16105
Rotzier
S.
, Schramek
H.
, Brenner
H. R.
(1992
). Metabolic stabilization of endplate acetylcholine receptors regulated by Ca2+influx associated with muscle activity.
Nature
349
, 337
–339
Ruth
P.
, Röhrkasten
A.
, Biel
M.
, Bosse
E.
, Regulla
S.
, Meyer
H.
, Flockerzi
V.
, Hofmann
F.
(1989
). Primary structure of thesubunit of the DHP-sensitive calcium channel from skeletal muscle.
Science
245
, 1115
–1118
Rutishauser
U.
, Acheson
A.
, Hall
A. K.
, Mann
D. M.
, Sunshine
J.
(1988
). The neural cell adhesion molecule (N-CAM) as a regulator of cell-cell interactions.
Science
240
, 53
–57
Sanchez
J. A.
, Stefani
E.
(1978
). Inward calcium current in twitch muscles of the frog.
J. Physiol
283
, 197
–216
Sanes
J.R.
, Schachner
M.
, Covault
J.
(1986
). Expression of several adhesion macromolecules (N-CAM, L1, J1, uvomorulin, laminin, and a heparan sulfate proteoglycan) in embryonic, adult and denervated adult skeletal muscle.
J. Cell Biol
102
, 420
–431
Schiaffino
S.
, Cantini
M.
, Sartore
S.
(1977
). T-system formation in cultured rat skeletal tissue.
Tissue & Cell
9
, 437
–446
Sieber
M.
, Nastainczyk
W.
, Zubor
V.
, Wernet
W.
, Hofmann
F.
(1987
). The 165-kDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel.
Eur. J. Biochem
167
, 117
–122
Singer
D.
, Biel
M.
, Lotan
I.
, Flockerzi
V.
, Hofmann
F.
, Dascal
N.
(1991
). The roles of the subunits in the function of the calcium channel.
Science
253
, 1553
–1557
Singer
L. D.
, Lotan
I.
, Itagaki
K.
, Schwartz
A.
, Dascal
N.
(1992
). Evidence for the existence of RNA of Ca2+-channel2/ subunit in Xenopus oocytes.
Biochim. Biophys. Acta
1137
, 39
–44
Snutch
T. P.
, Reiner
P. B.
(1992
). Ca2+channels: diversity of form and function.
Curr. Opin. Neurobiol
2
, 247
–253
Striessnig
J.
, Moosburger
K.
, Goll
A.
, Ferry
D. R.
, Glossmann
H.
(1986
). Stereoselective photoaffinity labelling of the purified 1,4-dihydropyridine receptor of the voltage-dependent calcium channel.
Eur. J. Biochem
161
, 603
–609
Stya
M.
, Axelrod
D.
(1983
). Diffusely distributed acetylcholine receptors can participate in cluster formation on cultured rat myotubes.
Proc. Nat. Acad. Sci. USA
80
, 449
–453
Sytkowski
A. J.
, Vogel
Z.
, Nirenberg
M. W.
(1973
). Development of acetylcholine receptor clusters on cultured muscle cells.
Proc. Nat. Acad. Sci. USA
70
, 270
–274
Takahashi
M.
, Seagar
M. J.
, Jones
J. F.
, Reber
B. F.
, Catterall
W. A.
(1987
). Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle.
Proc. Nat. Acad. Sci. USA
84
, 5478
–5482
Tanabe
T.
, Takeshima
H.
, Mikami
A.
, Flockerzi
V.
, Takahashi
H.
, Kangawa
K.
, Kojima
M.
, Matsuo
H.
, Hirose
T.
, Numa
S.
(1987
). Primary structure of the receptor for calcium channel blockers from skeletal muscle.
Nature
328
, 313
–318
Tanabe
T.
, Beam
K. G.
, Powell
J. A.
, Numa
S.
(1988
). Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA.
Nature
336
, 134
–139
Tassin
A. M.
, Mege
R. M.
, Goudou
D.
, Edelman
G. M.
, Rieger
F.
(1991
). Modulation of expression and cell surface distribution of N-CAM during myogenesis in vitro.
Neurochem. Int
18
, 97
–106
Thornhill
W. B.
, Levinson
S. R.
(1992
). Biosynthesis of ion channels in cell-free and metabolically labeled cell systems.
Meth. Enzymol
207
, 659
–670
Toutant
M.
, Gabrion
J.
, Vandaele
S.
, Peraldi-Roux
S.
, Barhanin
J.
, Bockaert
J.
, Rouot
B.
(1990
). Cellular distribution and biochemical characterization of G-protein in skeletal muscle: comparative location with voltage-dependent calcium channels.
EMBO J
9
, 363
–369
Vandaele
S.
, Fosset
M.
, Galizzi
J. P.
, Lazdunski
M.
(1987
). Monoclonal antibodies that co-immunoprecipitate the 1,4-dihydropyridine and phenylalkylamine components associated with the voltage-dependant calcium channel from skeletal muscle.
Biochemistry
26
, 5
–9
Varadi
G.
, Orlowski
J.
, Schwartz
A.
(1989
). Developmental regulation of expression of the1and 2subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line.
FEBS Lett
250
, 515
–518
Varadi
G.
, Lory
P.
, Schultz
D.
, Varadi
M.
, Schwartz
A.
(1991
). Acceleration of activation and inactivation by thesubunit of the skeletal muscle calcium channel.
Nature
352
, 159
–162
Woscholski
R.
, Marme
D.
(1992
). Dihydropyridine binding of the calcium channel complex from skeletal muscle is modulated by subunit interaction.
Cell. Signal
4
, 209
–218
Yaffe
D.
(1968
). Retention of differentiation potentialities during prolonged cultivation of myogenic cells.
Proc. Nat. Acad. Sci. USA
61
, 477
–483
Yuan
S.
, Arnold
W.
, Jorgensen
A. O.
(1991
). Biogenesis of transverse tubules and triads: immunolocalization of the 1,4-dihydropyridine receptor, TS28, and the ryanodine receptor in rabbit skeletal muscle developing in situ.
J. Cell Biol
112
, 289
–301
This content is only available via PDF.
© 1994 by Company of Biologists
1994
-PolarityCFP.png?versionId=5216)
