Department of Cell Biology, University of Massachusetts Medical School,
Worcester, MA 01655, USA
* Present address: Central Research Institute of BodiTech, Inc. Chuncheon,
Kangwon-Do 200-160, South Korea
Present address: Department of Biological Sciences, Indiana University South
Bend, South Bend, IN 46634, USA
Present address: Department of Infection and Immunity, The Walter and Eliza
Hall Institute of Medical Research, VIC 3050, Australia
¶ Author for correspondence (e-mail: elizabeth.luna{at}umassmed.edu)
Accepted 11 February 2003
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Summary |
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Key words: Costamere, Sarcolemma, Membrane skeleton, C2C12 cells, 50MB-1 cells
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Introduction |
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The costamere-associated sarcolemmal membrane has been proposed to consist
of a mosaic of domains (Rahkila et al.,
2001; Williams and Bloch,
1999a
) containing dystrophin, integrins, spectrin and associated
proteins, and cholesterol-rich, low-density membrane domains called caveolae.
Caveolae are 50-100 nm flask-shaped membrane invaginations implicated in
signal transduction and endocytosis
(Anderson and Jacobson, 2002
).
Muscle caveolae are enriched in caveolin-3
(Tang et al., 1996
;
Way and Parton, 1996
), a
muscle-specific protein that cofractionates and coprecipitates with dystrophin
and associated proteins (Song et al.,
1996
; Sotgia et al.,
2000
). Although many component proteins have been identified and
interactions between costameric proteins have been described, additional
interactions between the lateral sarcolemma and the cytoskeleton are suggested
by the partial retention of costamere structure and/or function after loss of,
for instance, dystrophin or caveolin-3
(Galbiati et al., 2001
;
Williams and Bloch, 1999b
).
Thus, the complete composition and regulation of costameres during muscle
functioning have yet to be elucidated.
As part of our ongoing analyses of actin-based membrane skeletons in plasma
membranes of motile cells (Luna et al.,
1997), we have identified and isolated a neutrophil membrane
complex containing non-erythrocyte spectrin (fodrin), actin, non-muscle
myosin-IIA and a novel,
205 kDa membrane protein
(Nebl et al., 2002
;
Pestonjamasp et al., 1995
;
Pestonjamasp et al., 1997
).
This complex is enriched in cholesterol and contains associated signaling
proteins and integral proteins characteristic of low-density membrane domains
(Nebl et al., 2002
). The 205
kDa protein, named supervillin because of C-terminal sequence similarities to
the microvillar protein villin (Bretscher
and Weber, 1979
), binds directly to F-actin, promotes actin
filament bundling in vivo and resists extraction from neutrophil membranes
with high pH carbonate buffers (Nebl et
al., 2002
; Pestonjamasp et
al., 1995
; Pestonjamasp et
al., 1997
). Thus, supervillin is implicated as an actin-membrane
linkage protein in a membrane skeleton associated with cholesterol-rich,
low-density membrane domains.
Supervillin has been implicated in the direct or indirect control of cell
adhesion. In confluent, non-proliferating Madin-Darby bovine kidney (MDBK)
cells, supervillin localizes with E-cadherin at sites of cell-cell adhesion
and is internalized with adherens junctions proteins in ring-like structures
during EGTA-induced release of intercellular contacts
(Pestonjamasp et al., 1997).
Overexpression of full-length supervillin or N-terminal supervillin sequences
in COS7 and CV1 cells disrupts the integrity of focal adhesion plaques
(Wulfkuhle et al., 1999
), and
comparable levels of supervillin overexpression are found in several carcinoma
cell lines (Pope et al.,
1998
). Also, supervillin has been isolated as part of a protein
complex with laminin
3, integrin ß2, the P2X7 ATP-gated
ion channel, and receptor protein tyrosine phosphatase-ß
(Kim et al., 2001
), further
suggesting cross-communication with proteins involved in cell-substrate
attachment and/or motility.
Supervillin may also contribute to nuclear architecture and function.
Functional nuclear targeting sequences in the N-terminal and central domains
of supervillin target chimeric proteins into detergent-resistant structures
within the nuclei of COS7 cells (Wulfkuhle
et al., 1999). Cell-cycle-based control of one or more of these
nuclear targeting sequences is probably responsible for the localization of
endogenous supervillin to MDBK cell nuclei during active cell proliferation
(Pestonjamasp et al., 1997
).
Supervillin is also a transcriptional activator of the androgen receptor
(Ting et al., 2002
) and has
been implicated in the testosterone-mediated cessation of dermal papilla cell
proliferation (Pan et al.,
1999
), suggesting the possibility of other roles during cell
growth.
Quantification of supervillin message levels in human tissues indicate that
cross-hybridizing mRNAs are most abundant in human tissues rich in striated or
smooth muscle (Pope et al.,
1998). The highest message levels are found in skeletal muscle,
bladder, heart and aorta. In the current study, we have investigated the
nature of the supervillin-related protein in muscle by cloning and
characterizing a
250 kDa muscle isoform of supervillin from human and
mouse skeletal muscle. This protein is derived from the supervillin genomic
locus (SVIL) by differential splicing of five conserved exons, four of which
encode muscle-specific protein sequences distributed as two `inserts' within
the function-rich N-terminus of the protein. Because of the likelihood of
muscle-specific conserved functions, we suggest that the isoform of
supervillin found in muscle, the principal source of supervillin in the body,
be called `archvillin' (Latin, archi; Greek, árchos; `principal' or
`chief').
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Materials and Methods |
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Anti-pepA antibodies (-pepA)
Polyclonal antisera directed against amino acids 900-918 of bovine
supervillin were prepared as described
(Pestonjamasp et al.,
1997).
Cell culture and transfection
Helen Blau (Stanford University, Palo Alto, CA) kindly provided 50MB-1
human myoblasts (Webster et al.,
1988). Cells were grown at subconfluent densities in Ham's F-10
media supplemented with 20% fetal calf serum (FCS) and 1% v/v chick embryo
extract (60 Å ultrafiltrate; Gibco-BRL, Gaithersburg, MD), with medium
changes every other day. To induce differentiation, cultures were grown to
near confluence and then maintained in Dulbecco's modified Eagle's medium
(DMEM)-low glucose (Gibco-BRL), 5% horse serum, 0.3 µM insulin and 1 µM
dexamethasone (Sigma) without further medium changes until the appearance of
myotubes. Human diploid fibroblasts WI-38 and cervical carcinoma HeLa cells
(ATCC) were grown in DMEM-high glucose (DMEM/HG; Gibco-BRL) supplemented with
10% FCS and gentamycin.
The C2C12 mouse skeletal muscle cell line was a gift from Janet and Gary
Stein (University of Massachusetts Medical School, Worcester, MA). Cells were
maintained in DMEM/HG supplemented with 10% FCS and penicillin/streptomycin.
Differentiation was induced in 2% horse serum, DMEM/HG for 4 to 6 days
(Huang et al., 2000).
Myoblasts (50MB-1 and C2C12 cells) were transfected using the Effectene
Transfection kit (Qiagen, Valencia, CA) according to the manufacturer's
instructions. The effects of murine archvillin deletion proteins on
differentiation of C2C12 were measured as the percentage of the total number
of transfected cells present as myotubes after 6 days of incubation in
differentiation medium.
Muscle preparations and immunoprecipitations
Rabbit muscle fractions
Crude rabbit skeletal muscle plasma membranes were prepared from freshly
dissected or frozen back and leg muscles by flotation through 30% (w/v)
sucrose, as described (Ohlendieck et al.,
1991). The following protease inhibitors (Sigma Chemical Co., St
Louis, MO) were included in all buffers: 1 µg/ml aprotinin, 1 µM
pepstatin A, 0.5 µg/ml leupeptin, 1 mM benzamidine, 1 µM antipain, 1 mM
PMSF. Equivalent amounts of protein in the low-density fraction, which was
enriched in plasma membranes, and in the higher-density fraction, which was
enriched in sarcoplasmic reticulum and T-tubules, were analyzed by SDS
polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.
Muscle extracts
Murine hind leg muscles, or rabbit back and leg muscles, were ground under
liquid N2 and extracted twice with 1% SDS for 10 minutes at
70°C. Extracts either were denatured with sample buffer for SDS-PAGE or
were diluted 10-fold with 1% Triton X-100 in PBS for immunoprecipitations.
Triton X-100 extracts from 0.3 mg muscle were pre-cleared for 2 hours at
4°C with rabbit immunoglobulin (RIgG) bound to protein A/G beads (Pierce
Chemical Company, Rockford, IL) and incubated with 20 µg RIgG or
-H340 bound to protein A/G beads for 5 hours at 4°C. Beads were
washed extensively with PBS. Bound immunoprecipitated proteins were eluted by
heating for 5 minutes at 95°C in SDS sample buffer and analyzed by
-H340 immunoblot and F-actin blot overlay.
Immunoblots
Proteins were separated by SDS-PAGE
(Laemmli, 1970) and
electrotransferred to nitrocellulose (0.45 µm pore size) (Schleicher and
Schuell, Keene, NH). Nitrocellulose blots were blocked with 5% nonfat powdered
milk and probed with primary antibodies for 2 hours at room temperature or
overnight at 4°C. Concentrations of primary antibodies were as follows: 10
µg/ml affinity-purified
-H340, 20 µg/ml
-pepA, 5 µg/ml
anti-caveolin 3 (BD Transduction Laboratories, San Diego, CA), 1:20 dilution
of anti-dystrophin (Novocastra Lab, Burlingame, CA). Interacting polypeptides
were visualized using either 125I-labeled protein A or protein A
conjugated to horseradish peroxidase and an ECL substrate kit (KPL,
Gaithersburg, MD). Reactive polypeptides were detected by exposure to
Biomax-MS X-ray film (Eastman Kodak, Rochester, NY). For double labeling with
radioactively labeled F-actin, anti-rabbit antibody conjugated to alkaline
phosphatase was used with a BCIP/NBT substrate kit (KPL, Gaithersburg, MD) for
colorimetric detection.
F-actin blot overlay
For F-actin overlays, 125I-labeled actin was polymerized in the
presence of rabbit gelsolin, stabilized with phalloidin and used at a final
concentration of 50 µg/ml in 5% nonfat powdered milk
(Luna, 1998). In some
experiments, actin was labeled with [
-32P]ATP
(Mackay et al., 1997
), using 1
mg of actin and 1 mCi of [
-32P]ATP. Nitrocellulose blots
were exposed to film for 5 days at 80°C or to an imaging screen for
2 hours. Signal was visualized with a Phosphor Imager SITM optical
scanner and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Human and murine archvillin cDNAs
Human archvillin
Oligonucleotide primers (H96-2R, HU5P-GAP)
(Table 1) were designed from
human supervillin cDNA sequences (AF051850, AF051851) and used with the
Clontech AP1 primer and Marathon-ReadyTM human skeletal muscle cDNA in
5'-RACE (rapid amplification of cDNA ends) reactions with Advantage
KlenTaq polymerase (Clontech, Palo Alto, CA). Clones (HSK02, HSK03, HSK16,
HSK21, HSK31, HSK43, HSK61 and HSK69) encoding the 5'-end of the
archvillin cDNA were obtained by cloning into pGEM-T (Promega). Colonies were
identified by screening with a randomly-primed 32P-labeled fragment
of the human supervillin sequence corresponding to nt 12 to 1015
(Pope et al., 1998).
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The rest of the archvillin coding sequence was generated using sets of non-degenerate primers (HSKP-1F and HSKP-1R; HSKP-2F and HSKP-2R) (Table 1) designed from human supervillin sequences and from the 5'-RACE archvillin products, above. These primers were used with the Marathon-ReadyTM human skeletal muscle cDNA (Clontech) and ExpandTM long-template polymerase (Roche Molecular Biochemicals, Indianapolis, IN) in touchdown thermal reactions to generate clones containing full-length human archvillin (FAV), or a smaller clone (SAV). FAV was completely sequenced in both directions, and SAV was completely sequenced in one direction and in both directions in the regions of muscle-specific sequence. An additional 419 bp of 3'-untranslated region (UTR) sequence was identified as the consensus of expressed sequence tags (ESTs) AA136154, AA149325, Z28958, AA442438, AW020040 and AA442798.
Murine archvillin
Murine archvillin cDNA also was cloned by PCR-RACE. The upstream primer
(MSV-F1) (Table 1) corresponded
to a consensus sequence from human and bovine supervillin 5'-UTRs
(accession nos AF051850, AF025996) and rat EST AI549127. The reverse primer
(MSV-R1) was designed using Primer Premier (Premier Biosoft International,
Palo Alto, CA) from a consensus of 41 mouse ESTs homologous to the
3'-ends of the human and bovine supervillin coding sequences. Additional
upstream sequences were obtained in nested 5'-RACE reactions with the
Clontech AP1 and AP2 primers and reverse primers complementary to nt 281-312
(MAV-R296) and nt 121-144 (MAV-R121) within the murine archvillin coding
sequence, respectively. A forward (CRATY-F) primer for 3'-RACE with the
Clontech AP1 primer was chosen from mouse EST #AA048260, which encodes a
sequence homologous to the highly conserved amino acids 1089-1184 of bovine
supervillin and amino acids 1085-1180 of human supervillin.
Marathon-ReadyTM skeletal muscle cDNA (Clontech) and the HerculaseTM
polymerase blend (Stratagene, La Jolla, CA) were included in touchdown thermal
reactions to generate clones containing nearly full-length murine archvillin
(clones M02, M03, M08), 5'-RACE products (clones M04U, M08U, M09U, M15U,
M20U) and 3'-RACE products (clones M09, M23, M26, M23B).
Enhanced green fluorescent protein (EGFP)-tagged murine
archvillin
The full-length coding region of the murine archvillin cDNA was generated
by assembling consensus-matching regions of clones M02 and M09. This construct
was then used as a template to generate by PCR cDNAs from forward primers with
a 5' XhoI site and reverse primers with a 5'
SacII site (Table 1).
PCR products were generated using HerculaseTM polymerase and ligated into
TOPO-TA vectors (Invitrogen, Carlsbad, CA), sequenced, recovered by digestion
with XhoI and SacII, and subcloned into pEGFP-C1 (Clontech).
The expression of the resulting pEGFP-MAV chimeras was confirmed by western
blotting of lysates from transfected C2C12 cells using -H340 and an
antibody against GFP (Roche Molecular Biochemicals, Indianapolis, IN).
DNA sequencing and structural analyses
All clones except SAV were fully sequenced in both directions by primer
walking at the Iowa State University DNA Sequencing and Synthesis Facility
(Ames, IA) or at the University of Massachusetts DNA Sequencing Facility
(Worcester, MA). Consensus cDNA sequences for human and murine archvillins
were constructed using Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI)
and deposited in GenBank (accession nos AF109135 and AF317422, respectively).
The sequence of the potential alternatively spliced murine archvillin is
available as AF317423. Optimized sequence alignments with CLUSTALW 1.8
(Jeanmougin et al., 1998) were
performed at
http://clustalw.genome.ad.jp/.
Protein compositional analyses were carried out using Web sites available at
http://www3.ncbi.nlm.nih.gov/,
http://www.up.univ-mrs.fr/~wabim/d_abim/compo-p.html,
http://us.expasy.org/cgi-bin/protscale.pl,
http://www.cbs.dtu.dk/services/Netphos/,
and
http://psort.nibb.ac.jp/.
Human multiple tissue northern
A human multiple tissue northern blot of poly(A)+ RNA (Clontech)
was hybridized overnight at 65°C in 7% SDS, 0.25 M
Na2PO4, 10 mM EDTA, pH 7.3
(Church and Gilbert, 1984),
with a 32P-labeled random-primed probe prepared from a 697-bp
XhoI/SmaI fragment corresponding to nt 4382-5079 from the
human consensus coding region. The blot was washed three times at 65°C for
20 minutes in 2X SSC (0.3 M NaCl, 30 mM sodium citrate), 1.0% SDS, and exposed
to film. The blot was stripped for reprobing by boiling for 10 minutes in 0.5%
SDS in RNase-free water and then probed as above with a 187 bp XbaI
fragment from the archvillin-specific region of the HSK61 clone (nt
1227-1414). Finally, the blot was probed with a 32P-labeled
random-primed ß-actin control cDNA, washed as above, and washed twice
more at 68°C for 30 minutes in 0.1X SSC, 0.1% SDS.
In situ hybridization
Detection of RNA from supervillin and/or archvillin genes was performed
using nick-translated human supervillin cDNA from clone H09
(Pope et al., 1998). Detection
of RNA from the ß-cardiac myosin heavy chain gene (cMyHC) was performed
using clone HM-1, a
32 kb ß-cMyHC specific genomic probe obtained
from Choong-Chin Liew (University of Toronto, Ontario, Canada)
(Yamauchi-Takihara et al.,
1989
). The methods used here, including procedures for
non-isotopic probe preparation and fluorescence in situ hybridization, have
been published in detail (Carter et al.,
1991
; Johnson et al.,
1991
). Images were captured using a Photometrics P-250 cooled CCD
camera and the MetaMorph (Universal Imaging Corp., West Chester, PA)
image-processing package. The microscope was a Zeiss Axioplan with a
100x Plan-Apo 1.4 objective and a triple band-pass filter set (63000,
Chroma, Brattleboro, VT).
Immunofluorescence microscopy
Hamster thigh muscles were the generous gift of Thomas Schoenfeld
(University of Massachusetts Medical School, Worcester, MA). Muscle sections
of 5-7 µm were cut at 20°C on a Microm HM 500 OM microtome
cryostat (Carl Zeiss, Walldorf, Germany). Before fixation, cultured myogenic
cells or cryosectioned hamster muscle were washed at room temperature in
sterile Dulbecco's phosphate buffered saline (DPBS), immediately fixed in
either 4% paraformaldehyde in PBS, pH 7.4, or in 20°C methanol for
10 minutes, rinsed three times for 15 minutes in PBS, and then blocked for 30
minutes in blocking solution (10% horse serum, 1% BSA, 0.02% sodium azide,
PBS). Cells and muscles were stained overnight at 4°C with 10 µg/ml
affinity-purified anti-H340 in blocking solution. Rabbit polyclonal antibody
against non-muscle myosin II heavy chain (Biomedical Technologies, Stoughton,
MA) was used at a dilution of 1:30. Primary monoclonal antibodies were diluted
with blocking solution as follows: anti-dystrophin (Novocastra Lab,
Burlingame, CA), 1:20; anti-lamin A/C (Novocastra Lab, Burlingame, CA), 1:25;
and anti-vinculin (Sigma Chemical Co.), 1:200. Nuclear DNA was visualized with
ethidium homodimer-1 (Molecular Probes, Inc., Eugene, OR). F-actin was
visualized with Alexa 594TM-phalloidin (Molecular Probes). Samples
were incubated for 1 hour at room temperature with a 1:2000 dilution of the
appropriate secondary antibody (goat anti-rabbit Alexa 488TM or goat
anti-mouse Alexa 594TM; Molecular Probes), washed three times for 15
minutes in PBS, mounted in a Vecta mounting medium (Vector Laboratories,
Burlingame, CA) and sealed with nail polish. Slides were analyzed on a Zeiss
Axioskop fluorescence microscope or a Bio-Rad MRC 1024 laser scanning confocal
microscope (Bio-Rad Laboratories, Hercules, CA) equipped with LaserSharp
Version 3.2 software.
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Results |
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Cloning of the muscle-specific supervillin isoform
To determine the molecular nature of the muscle protein and the extent of
its conservation across species, we used PCR-based strategies to clone
supervillin-related cDNAs from human and murine skeletal muscle (accession nos
AF109135 and AF317422, respectively). Consensus cDNA sequences of 7586 bp and
8138 bp were assembled from multiple human and mouse clones, respectively
(Fig. 2A). BLASTN 2.1.1
searches of the dbest database (Altschul et
al., 1997) suggested the presence of an additional 419 nt in the
3'-untranslated region (UTR) of the human sequence, indicating that the
mRNAs are each
8 kb long, exclusive of a poly(A) tail. The human cDNA is
almost identical to that deduced from human genomic sequences (XM_030478),
except for single basepair differences at position 3 within codons and changes
at nucleotides 1319, 2017 and 4456, which would mostly result in conservative
amino acid changes (Ala-189
Val, Ile-422
Val, Ala-1233
Pro).
Thus, these differences may represent polymorphisms. The 3740 nt at the
3'-end of the cloned murine cDNA is virtually identical to a partial
cDNA predicted from murine genomic sequences (XM_128880), with only three
divergent base pairs in noncoding sequence.
|
Northern blots from several human tissues confirmed the presence of an
abundant larger message in muscle (Fig.
2B-D). The 7.5-kb message encoding supervillin (SV) was
readily detected in placenta, lung, kidney and pancreas
(Fig. 2B, lanes 3, 4, 7 and 8,
arrowhead) and was present at lower levels in brain and liver
(Fig. 2B, lanes 2 and 5). As
predicted from previous northern dot blot analyses
(Pope et al., 1998
),
cross-hybridizing mRNAs were most abundant in cardiac and skeletal muscle
(Fig. 2B, lanes 1 and 6).
Shorter exposures to film showed that the human muscle mRNAs were
8.5 kb
(Fig. 2C, asterisk), a size
consistent with a 6645 bp coding region, 756 bp 5'-UTR, 903 bp
3'-UTR, and a
200 bp poly(A) tail. Hybridization with a probe
against sequences present only in the skeletal muscle cDNA
(Fig. 2A, probe 1; see below)
showed that the
8.5 kb message was essentially absent from the non-muscle
tissues analyzed (Fig. 2D,
asterisk). Hybridization with ß-actin sequences served as a control for
mRNA loading and integrity (Fig.
2E).
Predicted proteins and message structure
The consensus human and mouse cDNA sequences were predicted to encode
homologous proteins of 2214 and 2170 amino acids, respectively
(Fig. 3). The human muscle
protein was predicted to exhibit a molecular mass of 247,706.29 Da and an
isoelectric point (pI) of 6.55; the mouse protein was predicted to be
243,161.63 Da with a pI of 6.44. Overall, these proteins were 80.7% identical
and 90.2% similar, with the highest homology (97%) in the C-termini. Most
sequence predicted for the human muscle protein was virtually identical to
sequences in human (accession no. AF051850) and bovine (accession no.
AF025996) supervillins. In particular, the villin-gelsolin homology region and
sequences in the central part of the protein that have been shown to promote
targeting of EGFP-chimeras into Triton-resistant nuclear aggregates
(Wulfkuhle et al., 1999) were
present in the muscle cDNA. This nuclear localization was apparently mediated
by nuclear localization sequences (Fig.
3A, dark boxes) and a predicted coiled-coil sequence
(Fig. 3A, hatched box). All of
these structural features were found across species and in both muscle and
non-muscle proteins.
|
In addition to sequences found in supervillin, three conserved
muscle-specific sequences were identified. These sequences were encoded by
exons that appeared to be differentially expressed in muscle. Two of the
muscle-specific insert sequences altered the nature of the N-termini of the
muscle proteins (Fig. 3A, boxed
sequences). The first conserved insert sequence of 394 (human) or 372 (murine)
residues is encoded by three muscle-specific exons on human chromosome 10
(NT_008609) and murine chromosome 18 (NW_000134). The human and murine amino
acid sequences are 63% identical and 73% similar to each other, overall, with
regions of sequence conservation that are 90% identical. The first
muscle-specific inserts of the human and murine proteins were predicted to
contain two conserved nuclear targeting sequences
(Fig. 3A, bold type) and to be
enriched in arginine, glutamic acid, proline and serine residues. Expression
of insert 1 as a chimeric protein with EGFP greatly enhanced the amount of
fluorescent EGFP in the nucleus (Fig.
3B), supporting its predicted nuclear targeting capability. In
both human and mouse, the second muscle-specific insert of 32 amino acids was
separated from the first by 79 residues encoded by three exons also expressed
in non-muscle supervillin. The second muscle-specific inserts were 90%
identical and 94% similar between species and were relatively rich in
arginine, proline and serine residues. Consequently, several sites fitting
consensus sequences for serine/threonine protein kinases were predicted within
each insert (Fig. 3A,
underlines). Taken together, the two inserts encode muscle-specific sequences
of 47.0 kDa (human) and 44.6 kDa (mouse) that are absent from non-muscle
supervillin. To denote both the identical regions and the differences between
the muscle and non-muscle proteins and to facilitate comparisons between the
two, we propose to call the second, muscle-specific isoform of supervillin
`archvillin'.
The third conserved muscle-specific sequence (exon M-3 in
Fig. 3C) represented the
5'-ends of leader sequences (5'-UTRs) of at least 756 nt and 770
nt in human and mouse archvillin cDNAs, respectively. Archvillin 5'-UTRs
were much longer than the 20-100 nt leader sequences of most vertebrate mRNAs
(Kozak, 1987) and contained
six (human) or eight (murine) AUG codons upstream of the consensus start site.
Despite their locations of
90 kb (human) and
52 kb (mouse) upstream
of the first protein-coding exon, the human and murine M-3 exons were 74%
identical to each other overall and contained sequences of 254 nt and 34 nt
that were 88% and 94% identical across species
(Fig. 3C, double arrows). The
first conserved upstream open reading frame (uORF) in exon M-3 potentially
encoded a peptide of 49 residues; the second conserved uORF potentially
encoded peptides of 4 (human, cow) or 17 (mouse) residues
(Fig. 3C, thick bars). Other
uORFs were present in both human (HAV) and murine (MAV) archvillin
5'-UTRs. Such sequences are found in only
9% of all vertebrate
mRNAs, but are present in two thirds of messages encoding oncogenes and mRNAs
that are regulated post-transcriptionally near the site of protein synthesis
(reviewed by Hellen and Sarnow,
2001
; Kozak, 1987
;
Morris and Geballe, 2000
).
The archvillin 3'-UTR was much less remarkable. As is true for most
messages (Hawkins, 1988), the
entire 3'-UTR sequence (Pope et al.,
1998
) is found within the last coding exon. All known supervillin
and archvillin cDNAs and ESTs from both muscle and non-muscle sources contain
this sequence, suggesting a lack of alternative splicing in the 3'-UTR.
The 3'-UTR sequences diverged across species, except for a conserved
sequence of 131-142 nt located
302-317 nt downstream of the stop codon
(not shown). This conserved 3' sequence contained motifs characteristic
of binding sites for the ELAV/Hu family of message stabilizing and targeting
proteins (reviewed by Antic and Keene,
1997
; Brennan and Steitz,
2001
; Keene,
2001
). The presence of ELAV/Hu motifs suggested that archvillin
and supervillin messages may be stabilized against degradation (reviewed by
Jacobson and Peltz, 1996
) and
raised the possibility of mRNA targeting
(Antic and Keene, 1998
;
Gao and Keene, 1996
).
Message localization
To test the possibility that supervillin and/or archvillin messages can be
targeted within the cytoplasm, we examined non-muscle and muscle cells by in
situ hybridization with a nick-translated human cDNA probe to sequences found
in both mRNAs (Fig. 4).
Specificity of this probe for the single SVIL locus on chromosome 10 was shown
previously (Pope et al.,
1998). Sequences hybridizing with SVIL exons were found throughout
the cytoplasm, as well as within the nuclei, of migrating non-muscle WI-38
fibroblasts (Fig. 4A) and in
the cytoplasm of elongated, but undifferentiated, 50MB-1 myoblasts
(Fig. 4B). By contrast,
prominent clusters of hybridizing species presumably archvillin mRNAs
because 50MB-1 cells do not express detectable amounts of supervillin
(Fig. 1) were observed
within the elongated processes of differentiating myotubes
(Fig. 4C,D,
Fig. 5A). These clusters
appeared as large granules in fluorescence optics.
|
|
Although significant amounts of diffusely distributed archvillin messages
were also observed in myotube cytoplasm, most of the granular clusters were
highly polarized, especially compared with messages for the myofibrillar
protein, ß-cardiac myosin heavy chain
(Fig. 5). The myosin heavy
chain mRNA exhibited a relatively uniform distribution throughout the myotube
(Fig. 5B, green in
Fig. 5C) when hybridized in the
same experiment under conditions identical to those used for the archvillin
mRNA (Fig. 5A, red in
Fig. 5C). The more highly
polarized localization of the archvillin message was independent of the label
used, with similar results observed in experiments in which the labels for the
two probes were reversed (data not shown). Thus, the enrichment of
hybridization signal for archvillin mRNA at the tips of myotubes was specific
for this message and was not an artifact of fixation, permeability or some
other technical condition. Given that similar clusters of RNA-containing
granules are characteristic of translocated mRNP complexes in oocytes, budding
yeast and neurons (reviewed by Mohr and
Richter, 2001), our results are consistent with increased mRNP
granule formation and/or polarized targeting of archvillin messages during
myoblast differentiation and fusion into myotubes.
Archvillin protein localization in muscle
Archvillin was predominantly localized at the cell peripheries in dissected
skeletal muscle (Fig. 6). In
optical and oblique cross-sections obtained by epifluorescence
(Fig. 6B) and confocal
(Fig. 6G) microscopy with
affinity-purified -H340 antibody, archvillin appeared as `arches' along
the sarcolemma (Fig. 6, double
arrowheads). These structures were revealed as alternating thick and thin
bands in en face confocal sections (Fig.
6C), with occasional longitudinal strands (arrow in
Fig. 6C, inset). This
appearance is characteristic of the myofibril-to-sarcolemmal attachment sites
called costameres (Craig and Pardo,
1983
; Pardo et al.,
1983
; Williams and Bloch,
1999a
). In fact, the `arched'
-H340 signal colocalized with
the costameric protein dystrophin at the sarcolemma
(Fig. 6D-F,G-I).
|
Enhanced -H340 signal was also observed in larger structures at the
myofiber periphery (Fig. 6J,M;
green in Fig. 6L,O). These
larger peripheral signals often colocalized with the DNA marker, ethidium
homodimer-1 (Fig. 6K,N; red in
Fig. 6L,O; overlap in
yellow/orange), suggesting that archvillin is also associated with
peripherally located myonuclei and/or the nuclei of satellite cells. Such
presumptive nuclear localizations were more easily seen in longitudinally
sectioned muscle (Fig. 6J-L,
arrows) than within cross-sections (Fig.
6M-O). Additional minor
-H340 staining within the interiors
of the muscle cells (Fig. 6M,O,
asterisks) was difficult to localize, but preliminary observations suggested
that small amounts of archvillin may also be found in the vicinity of
t-tubules and/or the spaces between the Z-bands of adjacent myofibrils (not
shown).
Early myogenesis
The apparent amounts and localizations of archvillin changed during
differentiation along the myogenic pathway (Figs
7,
8,
9). In undifferentiated human
50MB-1 (Fig. 1C,
Fig. 7) and murine C2C12
(Fig. 8) myoblasts, the
-H340 signal was largely nuclear with some cytoplasmic staining
(Fig. 1C,
Fig. 7A,D,G,J,
Fig. 8A). The nuclear signal
was punctate and distributed throughout the nucleoplasm, but was excluded from
nucleoli (Fig. 7A-C,
Fig. 8A). Cytoplasmic staining
consisted of both diffuse and punctate signals, with preferential localization
of immunoreactive punctae along the plasma membrane
(Fig. 7D-L).
Membrane-associated punctae often colocalized along the sides and at the ends
of microfilament bundles (Fig.
7D-I, arrows). Partial colocalization of archvillin punctae with
vinculin also was observed (Fig.
7J-L, arrows), suggesting occasional overlap with
vinculin-containing focal contacts (Zamir
and Beiger, 2001
).
|
|
|
Myogenic cells expressing low levels of EGFP-tagged full-length murine
archvillin exhibited a similar pattern of staining
(Fig. 8). Signal in
undifferentiated myoblasts was mostly nuclear
(Fig. 8B,C, arrows), and the
relative amount of cytoplasmic staining, as detected by both EGFP and the
-H340 antibody, increased in cells expressing higher levels of
transfected protein (Fig. 8B,C, arrowheads). Confocal sections of basal membrane surfaces exhibited punctate
and fibrillar archvillin distributions that colocalized well with F-actin
(Fig. 8D-F) and non-muscle
myosin II (Fig. 8G-I).
Endogenous levels of archvillin increased during differentiation into
myotubes; essentially all of this increase was due to enhanced staining of
cytoplasm and/or membrane structures (Fig.
9A,B). Polarized clusters of archvillin protein were observed both
in untransfected C2C12 myotubes
(Fig.9A,B) and in myotubes
transfected with low levels of EGFP-tagged archvillin
(Fig. 9C-F). The polarized
localization of EGFP-tagged archvillin from a construct lacking archvillin
5'- and 3'-UTR sequences indicated that these sequences were not
required for protein targeting during early myogenesis. This distribution
presaged the appearance of dystrophin at myotube tips because dystrophin is
not expressed at appreciable levels until 10-11 days after the induction of
differentiation (Belkin and Burridge,
1995; Kobayashi et al.,
1995
).
As an initial assay for functional involvement in myogenesis, C2C12 cells were transiently transfected with a series of murine archvillin deletion proteins that were tagged with EGFP at their N-termini. After six days in differentiation medium, the percentages of total fluorescent cells present as multinucleated myotubes were scored as a measure of the relative efficiency of differentiation (Fig. 10). By this method, about half of the cells in differentiation medium that were expressing either EGFP alone or EGFP-tagged full-length archvillin (amino acids 1-2170), archvillin C-terminus (aa 1353-2170) or the first coding muscle-specific archvillin sequence (aa 257-629, insert 1) were recovered as myotubes (Fig. 10), despite a range of transfection efficiencies (not shown). Because each myotube may contain nuclei from several transfected myoblasts, these percentages are probably underestimates of the overall differentiation efficiencies. By contrast, cells expressing either archvillin N-terminal sequences (aa 1-1353, aa 1-739) or the N-terminal region containing both muscle-specific coding sequences (aa 257-739) were far less efficient at forming myotubes under identical conditions in four separate experiments (Fig. 10). These results are consistent with a dominant-negative effect of archvillin N-terminal sequences during early myogenesis.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Archvillin binds directly to F-actin, co-isolates with dystrophin and caveolin-3 in low-density sarcolemmal membranes, and colocalizes with dystrophin at costameres in skeletal muscle. In myoblasts, archvillin localizes primarily within nuclei but is also concentrated at the plasma membrane with F-actin, non-muscle myosin II and vinculin. A role for archvillin during early myogenesis is suggested by the striking localization of archvillin protein and message at the ends of differentiating myotubes and by the apparent dominant-negative inhibition of myotube formation on overexpression of chimeric proteins containing N-terminal archvillin sequences.
A logical hypothesis is that archvillin serves as an additional linkage
between actin filaments and the plasma membrane at costameres. Although
dystrophin is required for sarcolemmal integrity, loss of muscle function
develops only over time, suggesting the existence of partially redundant
proteins (Blake et al., 2002;
Hoffman et al., 1987
). One
such protein is the dystrophin-related protein, utrophin, but even mice
lacking both dystrophin and utrophin exhibit superficially normal muscle
function until about four weeks of age
(Grady et al., 1997
). Other
possible actin-membrane linkages at costameres include protein complexes that
contain spectrin or focal adhesion proteins, including integrin, vinculin and
-actinin (Berthier and Blaineau,
1997
; Stromer,
1995
). In support of the idea of at least partial functional
redundancy, costameres containing ß-spectrin and vinculin are retained,
although frequently disarranged, in sarcolemma of the mdx mouse
(Williams and Bloch, 1999b
).
Conversely, loss of
- or ß-spectrin in nematodes leads to muscle
dysfunction (Hammarlund et al.,
2000
; Moorthy et al.,
2000
), suggesting cross-talk among muscle membrane skeleton
proteins. Because archvillin contains all the sequences found in supervillin,
a nonmuscle protein that interacts with vinculin-containing focal adhesions
(Wulfkuhle et al., 1999
) and
co-isolates with spectrin, non-muscle myosin II and
-actinin
(Nebl et al., 2002
;
Pestonjamasp et al., 1997
),
archvillin is likely to participate in the cross-talk between the spectrin-
and focal adhesion-based membrane skeletons at costameres in muscle.
Similarities between signal transduction pathways and membrane-cytoskeletal
attachments in muscle and nonmuscle cells are reasonable in the context of our
current understanding of myogenesis (Perry
and Rudnick, 2000; Taylor,
2002
). Myogenic cell migration from embryonic somites
(Perry and Rudnick, 2000
) and
the recruitment of fusion-competent myoblasts to the vicinity of founder cells
(Taylor, 2002
) are
morphologically reminiscent of chemotactic behaviors exhibited by neutrophils
and macrophages (reviewed by Weiner,
2002
). Furthermore, both muscle and non-muscle cells must be able
to regulate cytoskeletal organization and cell adhesion to the extracellular
matrix during changes in tensile forces. However, there are clear differences
in scale between muscle and non-muscle cells, and muscle-specific expression
of protein isoforms is the rule, rather than the exception. Thus, both
similarities and differences are observed in membrane skeleton proteins, such
as archvillin and supervillin, that participate in the regulation of motile
cell architecture.
Archvillin may also play a role within myonuclei during early myogenesis.
This possibility is consistent with its strong nuclear immunolocalization
before and during muscle differentiation and by the ability of supervillin
sequences that are also present in archvillin to bind and transactivate the
androgen receptor in prostate and muscle cells
(Ting et al., 2002). Although
a link between androgen receptor activation and increased muscle mass is
widely recognized (Sheffield-Moore,
2000
; Tindall,
2000
), the mechanism is unknown. One possibility is enhanced
expression of a regulatory factor, such as myogenin
(Lee, 2002
), that potentiates
myoblast differentiation. But non-genotropic effects of androgens on
membrane-associated signal transduction pathways are also recognized
(Benten et al., 1999
;
Kousteni et al., 2001
). Thus,
interference with either type of signaling pathway could reasonably account
for the apparent dominant-negative effects of EGFP-tagged archvillin
N-terminal sequences.
Another possibility, suggested by the colocalization of archvillin with
F-actin, vinculin and non-muscle myosin II at the membrane in myoblasts, is
that archvillin acts in the early stages of muscle-specific membrane
specializations. For instance, the first step during myofibril formation is
the assembly of punctae containing F-actin, non-muscle myosin II and vinculin
at the plasma membrane (LoRusso et al.,
1997; Rhee et al.,
1994
; Sanger et al.,
2000
). During the maturation of these premyofibrils into
contractile myofibrils, non-muscle myosin II is replaced by muscle-specific
myosin II that assembles into thick filaments with the dimensions of muscle
A-bands (LoRusso et al., 1997
;
Rhee et al., 1994
).
Finally, the localizations of archvillin message and protein at the ends of
myotubes suggest a contribution to the formation and/or stability of
myotendinous junctions. These structures, which transduce longitudinal forces
during muscle contraction, are enriched in several types of membrane
skeletons, including dystrophin/dystroglycan- and integrin-based transmembrane
attachments (Benjamin and Ralphs,
2000; Small et al.,
1992
; Tidball,
1991
). The conserved uORFs in the long 5' leader sequence of
the targeted archvillin mRNA hint at the possibility that archvillin protein
synthesis may be under local control at the ends of myotubes and/or
myotendinous junctions. Post-transcriptional control of key regulatory
proteins is well documented in oocytes and neurons
(Mohr and Richter, 2001
), and
it is intriguing to note that the membrane skeleton composition and
architecture at this important region of the sarcolemma may be capable of
relatively rapid remodeling in response to local stimuli.
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Acknowledgments |
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