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INTRODUCTION |
One of the most spectacular examples of cytoskeletal architecture
found in cells is the sarcomere, the contractile unit of striated
muscle. Antiparallel polarized actin filaments (thin filaments),
bipolar myosin filaments (thick filaments), titin, and nebulin
filaments interdigitate and interact with each other to create
sarcomeres, which support the contraction of muscle cells (1). Despite
the similarity in the basic organization of the filaments in all
sarcomeres, a closer examination reveals some important and interesting
variations in sarcomeric architecture in different types of muscle.
Most strikingly, while thick filament lengths are remarkably constant
among different vertebrate muscles (2-4), thin filament lengths
exhibit considerable variability among physiologically distinct types
of muscles (for review, see Ref. 5). For example, in the rabbit psoas
muscle, a fast twitch skeletal muscle, thin filament lengths fall into
a narrow range of 1.11 ± 0.03 µm (6). In contrast, in rat
cardiac atrial muscle, thin filament lengths vary from 0.6 to 1.1 µm
(7). Differences in thin filament length between fast and slow skeletal
muscles have also been observed in the fish Perca fluviaris
(8). This variation in thin filament length can be an important
determinant of the length-tension relationship during contraction and
thus can influence the physiological properties of the muscles
(i.e. cardiac, fast skeletal, and slow skeletal) (7, 8).
Nevertheless, the mechanisms underlying the regulation of variations in
thin filament lengths are not yet understood.
An excellent candidate for regulating the length of the thin filaments
is tropomodulin, the capping protein for the thin filament pointed ends
(5, 9). When embryonic chick cardiac myocytes are microinjected with an
antibody that blocks tropomodulin's ability to cap actin filament
pointed ends in vitro, actin elongates from the thin
filament pointed ends (10). Furthermore, the cells are no longer able
to beat, demonstrating that maintenance of thin filament length by
tropomodulin is critical for normal contraction (10). The levels of
tropomodulin also appear to be important for the stabilization and
proper organization of thin filaments in myofibrils, based on results
from studies employing sense and antisense erythrocyte tropomodulin
(E-Tmod)1 mRNAs in
adenovirus vectors in neonatal rat cardiac myocytes (11). In transgenic
mice, overexpression of tropomodulin in the heart after birth results
in dilated cardiomyopathy, presumably due to alterations in normal
myofibril organization (12).
Many myofibrillar components exist as families of multiple isoforms,
which are differentially expressed in distinct types of muscle (for
review, see Ref. 13). The variable expression of protein isoforms among
muscles of different types is a major determinant of the contractile
properties of each muscle. Our previous studies and those of others had
identified so far only one tropomodulin isoform, E-Tmod, which is
expressed in both skeletal and cardiac muscle (14-17). In this study,
we have identified and cloned a novel tropomodulin isoform from
vertebrates, Sk-Tmod, which is highly expressed in fast skeletal muscle
and is not present in slow skeletal or cardiac muscle. The high
conservation of Sk-Tmod sequences among vertebrates and its different
tissue and subcellular distribution as compared with E-Tmod suggest
that the Sk-Tmod isoform may be functionally distinct from the E-Tmod isoform.
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EXPERIMENTAL PROCEDURES |
Antibodies--
A panel of 19 monoclonal anti-tropomodulin
antibodies were generated using recombinant chicken E-Tmod as an
antigen (16) (Chris W. Grant, Custom Monoclonals, West Sacramento, CA).
The antibodies were purified from the hybridoma culture supernatants over a Protein G-Sepharose fast performance liquid chromatography column (Amersham Pharmacia Biotech, Uppsala, Sweden) and eluted with
0.2 M glycine-HCl, pH 2.8. The specificity of the
antibodies was characterized by two-dimensional gel electrophoresis,
followed by Western blotting (see below) and by immunofluorescence.
Monoclonal antibodies (mAbs) 17, 23, and 47 recognize a single
tropomodulin spot by Western blots in both heart and breast muscle
(pectoralis major, PM) (Fig. 1, left panel). In
contrast, mAbs 9 and 95 only recognize a tropomodulin spot in heart and
none in breast muscle (PM) (Fig. 1, right panel).
Mixing experiments demonstrate that tropomodulin spots from heart and
breast muscle (PM) do not comigrate (Fig. 1, bottom
left panel). By immunofluorescence, mAb 95 specifically recognizes E-Tmod, as demonstrated by staining of
embryonic breast muscle myofibrils, which contain E-Tmod (17), and its
inability to stain adult chicken breast muscle myofibrils, which
contain Sk-Tmod (data not shown and see below). In contrast, although mAb 17 recognizes both E- and Sk-Tmod isoforms on Western blots, it
preferentially recognizes Sk-Tmod by immunofluorescence since it does
not stain E-Tmod in the myofibrils of embryonic breast muscle but
stains the pointed ends of thin filaments in isolated myofibrils from
adult chicken breast muscle, which contain Sk-Tmod (data not shown and
see below).
Polyclonal antibodies to recombinant chicken Sk-Tmod (see below) were
generated in rabbits (R3577) and affinity-purified over a column of
recombinant chicken Sk-Tmod coupled to cyanogen bromide-activated Sepharose 4B (Sigma) by standard procedures. The monoclonal
anti-sarcomeric 
-actinin antibody (clone 9A2B8) (18) was generously
provided by Drs. S. and H. Holtzer (University of Pennsylvania,
Philadelphia, PA). The monoclonal anti-chicken fast C-protein antibody
(clone MF1) (19) developed by Dr. D. A. Fischman was obtained from the
Developmental Studies Hybridoma Bank developed under the auspices of
the NICHD and maintained by the University of Iowa, Department of
Biological Sciences, Iowa City, IA 52242. Polyclonal rabbit antibodies
generated against bovine brain -spectrin were prepared as described
previously (21). Rabbit anti-mouse IgG + IgM was purchased from Pierce.
Bodipy-labeled phallacidin, tetramethylrhodamine B
isothiocyanate-conjugated donkey anti-mouse IgG, and
rhodamine-conjugated goat anti-rabbit antibodies were purchased from
Molecular Probes, Inc. (Eugene, OR), Accurate Chemical and Scientific
Corp. (Westbury, NY), and Roche Molecular Biochemicals, respectively.
Comparison of Tropomodulin Isoforms by Two-dimensional
Electrophoresis--
Breast muscle (PM) from 12-day chicken embryos
and breast, heart biceps femoris (BF), and posterior latissimus dorsi
muscle from adult chickens (about 2 months old) were dissected and
washed with ice-cold phosphate-buffered saline plus protease inhibitors (17). Muscles were minced and then homogenized in a Polytron homogenizer with 10 volumes of 9.2 M urea and 1% (v/v)
2-
-mercaptoethanol, and their tropomodulin isoform composition was
characterized by two-dimensional gel electrophoresis followed by
Western blotting (17). The migration position of muscle -actin
(stained with Ponceau S) was used as a positional marker to compare the
mobility of tropomodulin isoforms electrophoresed on different gels.
For comparison of tropomodulins from adult chicken PM, lens, and
erythrocyte membranes, proteins were solubilized in 9.2 M urea, 1% 2--mercaptoethanol, and 2% Nonidet P-40 to improve the resolution of the first dimension. Membranes were prepared from chicken
erythrocytes according to Granger (22). Tropomodulin was
immunoprecipitated from adult chicken lens extracts using rabbit
polyclonal antibodies (R745) (20), as described previously for rat
lenses (23). Prior to two-dimensional gel electrophoresis, purified
rabbit skeletal muscle -actin was added to the immunoprecipitated lens tropomodulin and the erythrocyte membrane samples so that the
mobility of tropomodulin in the erythrocytes and lens could be compared
with that of the other muscle tissues.
Isolation of Chicken Sk-Tmod cDNAs--
Tropomodulin from
adult chicken breast muscle was immunoprecipitated as described (24)
using anti-tropomodulin mAb 17. Samples were electrophoresed on
7.5-15% acrylamide linear gradient SDS-polyacrylamide gels at pH 9.1 (20, 25), and polypeptides were transferred to polyvinylidene
difluoride membrane (0.45 µm; Millipore Corp., Bedford, MA) in CAPS
buffer (10 mM CAPS, pH 11, 10% methanol). The membrane was
stained briefly in 0.1% Ponceau S in 1% acetic acid and the region of
the membrane containing the tropomodulin band was excised and subjected
to tryptic digestion and microsequencing. The sequences of four
peptides were obtained (Dr. John Leszyk, University of Massachusetts
Medical School, Shrewsbury, MA) (underlined sequences in Fig. 2).
Degenerate primers to amplify Sk-Tmod cDNA were designed based on
the peptides TLQSLNIESNFITSAGMMSVIK and YKPVPDEPPNPTNVEETLR (see below,
Fig. 2). These primers were used to amplify a 300-bp cDNA fragment
by polymerase chain reaction using Taq polymerase (Promega,
Madison, WI) from a 
Zap adult chicken skeletal muscle cDNA
library (Stratagene, La Jolla, CA). This fragment was labeled with
[-32P]dCTP using a Random Primer kit (Stratagene) and
then used to screen the same library. Approximately 1 × 106 colonies were screened, and phage containing putative
Sk-Tmod cDNAs were isolated following the manufacturer's
instructions. Clones were analyzed by restriction mapping, and selected
clones were sequenced to identify overlapping fragments from the same cDNA sequence. Both strands from two of the longest clones (1.3 kb)
were sequenced.
Identification of Additional Sk-Tmod Sequences--
The
available tropomodulin sequences at GenBank together with the new
chicken Sk-Tmod cDNA sequence reported in this study, were used to
screen the expressed sequence tag data base (dbEST). A number of
overlapping ESTs from human, mouse, and zebrafish were identified that
showed higher levels of sequence similarity to the chicken Sk-Tmod than
to any other tropomodulin sequences. To determine the cDNA
sequences of clones corresponding to these ESTs, several cDNA
clones were obtained from the IMAGE Consortium clone collection (Genome
Systems, St. Louis, MO) and sequenced. The longest mouse cDNA
corresponded to amino acids 63-348 of chicken Sk-Tmod with a 95-bp
3'-UTR and the longest human cDNA corresponded to amino acids
20-348 together with a 122-bp 3'-UTR. The complete coding sequence of
the human Sk-Tmod was reconstructed from the sequences of five
additional human ESTs, which overlapped by 200-500 bp with the 1.1-kb
human Sk-Tmod cDNA sequence and extended ~120 bp upstream to
nucleotide 
66 before the start codon. A zebrafish cDNA clone was
also purchased from Genome Systems (St. Louis, MO), and it contained an
apparently full-length Sk-Tmod cDNA of 1.4 kb with 5'- and 3'-UTRs
of 62 and 328 bp, respectively, and encoded a protein of 343 amino acids.
Computational Analysis of Tropomodulin Sequences--
For
construction of the phylogenetic trees, the available ~40-kDa
tropomodulin sequences from GenBank, the chicken Sk-Tmod, and the
human, mouse, and zebrafish Sk-Tmods, were aligned with the program
CLUSTAL (26). A human EST encoding a putative N-Tmod with ~82%
identity to rat N-Tmod (27) was also included in the tree. A
tropomodulin homolog encoding a predicted protein of 324 aa present in
Caenorhabditis elegans was used to root the sequences of
vertebrate tropomodulins. Other larger, more distantly related proteins
such as the 64-kDa autoantigen D1 and a 684-amino acid protein in
C. elegans were not included in this analysis (28). To
optimize the alignment procedure, three different sets of gap opening
and extension penalties were used (10-0.05, 5-0.02, and 2-0.02).
Each alignment was used independently to calculate the phylogenetic
relationship between the aligned sequences. The distance method
implemented in the programs PROTDIST and NEIGHBOR (PHYLIP) (29) was
used to infer the phylogeny of Sk-Tmod sequences. The bootstrap
frequencies (100 cycles) were used to assess the reliability of the
different branching nodes of the final tree (30). Identical branching
arrangements were obtained with the three different alignment
conditions. The bootstrap frequencies shown in Fig. 4 correspond to the
alignment done with standard CLUSTAL parameters (10-0.05).
The nucleotide sequences used in these studies are available from
GenBank under the following accession numbers: Gallus
domesticus E-Tmod (L36678) (16), Mus musculus E-Tmod
(P49813) (14), Rattus norvegicus E-Tmod (U59241) (27),
Homo sapiens E-Tmod (A42336) (15), R. norvegicus
N-Tmod (U59240) (27), H. sapiens putative N-Tmod (AA203464),
C. elegans tropomodulin (AAB52440), Drosophila
melanogaster Sanpodo (AAC4506) (31), G. domesticus
Sk-Tmod (AF165215), Mus musculus Sk-Tmod (AA798934); H. sapiens Sk-Tmod (AF165217), and Danio rerio
Sk-Tmod (AI544990).
Northern Analysis--
Total mRNA from different adult
chicken tissues was extracted using Trizol reagent (Life Technologies,
Inc.) following the manufacturer's instructions. mRNAs were
resolved by electrophoresis on a 1.2% agarose, 1.2 M
formaldehyde gel and then transferred to a nylon Zeta-probe membrane
(Bio-Rad). Blots were hybridized with [-32P]dCTP
full-length Sk-Tmod cDNA in hybridization solution following standard protocols (32). Blots were stripped following the
manufacturer's instructions prior to incubating with other probes. For
detection of E-Tmod mRNA, a fragment containing the E-Tmod coding
sequence was obtained by the polymerase chain reaction and labeled with [-32P]dCTP using the random primer kit (Stratagene). A
60-bp [-32P]dCTP mouse 5.8 S ribosomal RNA fragment,
kindly provided by Dr. Vincent Mauro (Scripps Research Institute, La
Jolla, CA) was used as a probe to control for loading and integrity of
the mRNA. The migrations of the 28 S and 18 S ribosomal RNAs were
used to estimate the size of the tropomodulin mRNAs.
Indirect Immunofluorescence Microscopy--
Isolated myofibrils
from adult chicken breast muscle myofibrils were prepared as described
previously for rat psoas myofibrils (24). The immunostaining was
carried out basically as described (33). Briefly, coverslips were
washed with phosphate-buffered saline to remove excess fixative,
incubated with 3% bovine serum albumin in phosphate-buffered saline
for 30 min, and then incubated for 1 h with rabbit serum (R3577)
generated against recombinant Sk-Tmod (1:100 dilution) or mAb
anti--actinin antibody (1:400 dilution) followed by
rhodamine-conjugated goat anti-rabbit or rhodamine conjugated-donkey
anti-mouse secondary antibodies (1:200 dilution each) together with
bodipy-phallacidin (1:200 dilution).
To obtain muscle cryosections, small pieces of adult chicken biceps
femoris (BF) muscle were dissected, embedded in Tissue-Tek O.C.T.
(Sakura Finetek, Torrance, CA) in a cryomold (Miles, Elkhart, IN) and
immersed in liquid N2. Serial cross-sections of
approximately 10 µm thickness were obtained at 16 °C on a
Cryocut 1800 (Leica, Heidelberg, West Germany) and collected on a glass
coverslip. Sections were processed for immunofluorescence as described
above. Myofibrils and muscle sections were observed on a Zeiss Axioskop using a Zeiss 63× Plan-Apochromat objective lens (1.4 numeric aperture) and a Zeiss 25× multi-immersion objective (0.8 numeric aperture). Images were acquired with a cooled CCD-1300Y camera equipped
with a Sony interline chip (Roper Scientific, Trenton, NJ) using IP
laboratory software and then processed using Adobe Photoshop (Adobe
Systems Inc. San Jose, CA). In Fig. 9, images were recorded using TriX
(400 ASA) film.
Expression and Purification of Recombinant Sk-Tmod--
The
full-length Sk-Tmod cDNA was excised from the pBluescript plasmid
at EcoRI sites and subcloned in frame into the plasmid pGEX-KG (16) to express a glutathione S-transferase (GST)
fusion protein in E. coli. Proper orientation of all inserts
was confirmed by restriction mapping. For high yields, cells were grown
to an A605 of approximately 1 before inducing
with 1 mM
isopropyl-1-thio--D-galactopyranoside for 3 h, and
the GST fusion protein was purified as described for E-Tmod (16) with
some modifications. After affinity isolation of the GST fusion protein
on a glutathione column and release from the GST by thrombin cleavage,
Sk-Tmod was further purified by sequential anion exchange
chromatography on a Resource Q column (Amersham Pharmacia Biotech),
followed by a Mono Q column (Amersham Pharmacia Biotech). The purified
protein was concentrated and dialyzed by vacuum dialysis into 20 mM HEPES, pH 7.3, 80 mM KCl, 1 mM
dithiothreitol, 0.02% sodium azide, 20 µg/ml phenylmethylsulfonyl fluoride and stored frozen at 80 °C. The concentration of Sk-Tmod was determined by light absorption, using 
280 = 10.24 mM
1 cm1. The extinction
coefficient was calculated from the amino acid composition as described
(34) using the ProtParam tool available at the EXPASY web site. The
amino acid sequence of the GST linker that remains at the N-terminal
end of Sk-Tmod is GSPGISGGGGG, which is directly followed by the
Sk-Tmod sequence.
Actin Elongation Assays--
Measurements of actin elongation
were carried out as described previously (35,
36),2 using pyrenyl-labeled
rabbit skeletal muscle actin (10% pyrenyl-labeled) and short
gelsolin-capped actin filaments as nuclei for polymerization. Actin
polymerization was followed continuously for 5-7 h and measured again
the next morning. The fluorescence changes (excitation 366.5 and
emission 407 nm) were standardized against a Raman excitation peak and
measured in a photon counting fluorimeter (Photon Technology International, Princeton, NJ). All experiments were carried out at
20 °C with Mg2+-actin (converted from
Ca2+-actin as described previously (36) in a medium
containing 10 mM imidazole buffer, pH 7.0, 0.1 M KCl, 2 mM MgCl2, 1 mM
azide, 1 mM dithiothreitol, 0.5 mM ATP, and 0.1 mM CaCl2 (polymerization medium)).
Gelsolin-capped actin filaments used as nuclei for polymerization were
obtained by copolymerizing actin (usually 10 µM) with
gelsolin in the presence of calcium. Average sizes for filaments are
given in the figure legend as the ratios of actin:gelsolin. The
Kd of Sk-Tmod for capping the pointed ends of pure
actin filaments was estimated from the relative proportions of free and
capped pointed ends when F-actin was half-maximally polymerized as
described.2 For the preparation of gelsolin-capped,
tropomyosin-actin filaments as nuclei, rabbit skeletal muscle
tropomyosin (37) in excess over that necessary for filament saturation
(excess of about 1.0 µM) was mixed with G-actin before
copolymerization was started by the addition of salt. In addition, 1.0 µM tropomyosin was added directly to the assay medium to
ensure that elongating actin filaments were always saturated with
tropomyosin.2
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RESULTS |
Isolation of Sk-Tmod, a New Tropomodulin Isoform--
While
screening different muscles with a panel of mAbs generated against
E-Tmod, we observed a spot in chicken breast muscle that migrated
differently from E-Tmod on two-dimensional gels (Fig.
1). To determine whether this new
tropomodulin spot was a novel isoform of tropomodulin, sequences of
four peptides were obtained from tropomodulin immunoprecipitated from
adult breast muscle. Comparison of the peptide sequences to the chicken
E-Tmod amino acid sequence (16) demonstrated that all of the peptides were homologous but not identical to sequences in the C-terminal half
of E-Tmod (Figs. 2 and
3). Using degenerate primers from these
peptides, we isolated a polymerase chain reaction product, which was
then used to isolate a full-length Sk-Tmod cDNA from an adult
chicken skeletal muscle cDNA library. The complete nucleotide sequence of two of the longest clones (~1.3 kb) was determined and is
shown in Fig. 2. The sequence contains a single open reading frame of
1044 bp starting at nucleotide 56 (ATG) with a stop codon at position
1098 (TGA), followed by 131 bp of a 3'-untranslated region (3'-UTR),
containing a polyadenylation signal (Fig. 2, bold) (38) and
a short poly(A) tail. All four peptides obtained from the
immunoprecipitated protein are present in the predicted amino acid
sequence of the Sk-Tmod cDNA (Fig. 2, underlined).
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Fig. 1.
mAbs to recombinant chicken E-Tmod recognize
distinct tropomodulin spots on Western blots of two-dimensional gels of
adult chicken heart and breast muscle. Left panel, mAbs
23, 17 or 47 recognize a tropomodulin spot in heart and in adult breast
muscle (PM) samples. Right panel, mAbs 95 or 9 detect a tropomodulin spot only in heart muscle. Bottom
panels, coelectrophoresis of heart and breast muscle samples shows
that tropomodulin spots from these two muscles do not comigrate. The
positions of E- and Sk-Tmods are indicated.
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Fig. 2.
The nucleotide sequence of chicken Sk-Tmod
cDNA and its deduced amino acid sequence. The nucleotide
sequence of chicken Sk-Tmod is available from the GenBank data base
under accession number AF165215. The ~1.3-kb Sk-Tmod cDNA clone
starts 56 bp 5' of the initiation codon (lowercase letters at the 5' end). The sequence contains an open
reading frame of 1044 bp (capital letters),
followed by a 131 bp of a 3'-UTR (lowercase letters at the 3' end), which contains a putative
polyadenylation signal that is indicated in bold. The
predicted amino acid sequence for Sk-Tmod is presented under the
cDNA sequence. Peptide sequences obtained from microsequence of
immunoprecipitated Sk-Tmod are underlined.
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Fig. 3.
Amino acid sequence comparison of chicken Sk-
and E-Tmod. Alignment of the predicted amino acid sequence of
chicken Sk-Tmod with chicken E-Tmod (16). Identical amino acids are
boxed. Amino acid numbers are indicated on the
right.
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We have designated this new isoform Sk-Tmod since, as we describe
later, adult skeletal muscle is a major site of expression for this
isoform. Comparison of the cDNA sequences of chicken Sk- and
E-Tmods demonstrates that they are 64% identical along their entire
length, which indicates that these sequences are unlikely to be
alternatively spliced products from the same gene. In agreement with
this conclusion, the chicken E-Tmod and Sk-Tmod genes are located on
different chromosomes: E-Tmod is located on the chicken Z (sex)
chromosome and Sk-Tmod is located on a tiny
microchromosome.3 A striking
difference between the Sk-Tmod and E-Tmod cDNA sequences is the
difference in size of their 3'-UTRs. The chicken E-Tmod cDNA has a
long 3'-UTR of 2.5 kb (16), whereas the chicken Sk-Tmod cDNA has a
short 3'-UTR of 131 bp (Fig. 2).
Sk-Tmod Sequences Are Highly Conserved among Vertebrates--
The
chicken Sk-Tmod cDNA encodes a protein of 348 amino acids which is
62% identical and 75% similar to chicken E-Tmod (Fig. 3). The
calculated molecular mass and pI of Sk-Tmod are 39.2 kDa and 4.72, respectively, which is slightly smaller and more acidic than E-Tmod
(40.3 kDa and pI of 5), and is in agreement with the different
mobilities of the two isoforms observed on two-dimensional gels (Fig.
1). Interestingly, Sk-Tmod is 11 amino acids shorter than E-Tmod at the
C-terminal end (Fig. 3), a region proposed to be important for pointed
end capping activity (10). Notably, the recently described rat N-Tmod
is also shorter than E-Tmod at the C-terminal end (351 versus 359 aa, respectively) (27).
We have identified ESTs and obtained cDNA clones encoding putative
Sk-Tmods from several species, including mouse, human, and zebrafish.
Alignment of these sequences with other known tropomodulin sequences
allowed us to construct a phylogenetic tree using the 324-aa
tropomodulin homolog of C. elegans as a root. This revealed that Sk-Tmods form an independent conserved group, which is similar but
not identical to other vertebrate tropomodulins (Fig.
4) (14-16, 27, 28).
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Fig. 4.
Sk-Tmod sequences are highly conserved across
vertebrates and form an independent group in the tropomodulin
family. The amino acid sequences from the ~40-kDa tropomodulins
available in the data base were compared as described under
"Experimental Procedures" to infer their phylogenetic
relationships. The numbers at branch nodes correspond to
bootstrap frequencies for each particular branching relationship. The
324-aa tropomodulin sequence from C. elegans was used to
root the branches containing vertebrate sequences.
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Sk-Tmod Caps Thin Filament Pointed Ends--
E-Tmod is the capping
protein for the pointed ends of muscle thin filaments (5, 9, 28). To
determine the location of Sk-Tmod in chicken skeletal muscle, we used
polyclonal antibodies generated against Sk-Tmod to stain isolated
myofibrils from adult chicken breast muscle and compared the patterns
for Sk-Tmod staining and phalloidin staining for F-actin. In relaxed
myofibrils, phalloidin binds along the length of the thin filaments and
appears as a broad banding pattern with narrow gaps corresponding to
the H zones (gaps in the middle of the sarcomere with no actin
filaments) (Fig. 5A). Sk-Tmod
staining appears as a periodic doublet flanking the H zone in the
middle of the sarcomere, consistent with a localization at the thin
filament pointed ends (Fig. 5B; merged image in
C). In contrast, when myofibrils are stained for F-actin and
-actinin (Fig. 5, D and E) the merged image
clearly shows that -actinin staining is located at the Z line in the
middle of the thin filament I-Z-I array (Fig. 5F).
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Fig. 5.
Sk-Tmod is located at the pointed ends of
skeletal muscle thin filaments. Myofibrils were isolated from
relaxed chicken breast muscle and double-stained for F-actin
(A) and Sk-Tmod (B) or F-actin (D) and
-actinin (E) as described under "Experimental
Procedures." The merged images for the pairs are shown in
C-F. Sk-Tmod is localized at the pointed ends of thin
filaments in a doublet flanking the H zone (A-C,
arrowheads). In contrast, -actinin is localized at the Z
line of the sarcomere (D-F, arrows).
Bar, 5 µm.
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Among all actin capping proteins, E-Tmod is distinguished by its weak
capping activity for pointed ends of pure actin filaments (Kd ~ 0.1-0.4 µM) and its strong
capping activity for pointed ends of tropomyosin-actin filaments
(Kd < 1 nM) (36). This is a consequence
of the ability of E-Tmod to bind tropomyosin and actin (16, 36, 39). To
determine whether Sk-Tmod can cap actin filament pointed ends and
whether its actin capping activity is enhanced by tropomyosin, we
expressed chicken Sk-Tmod in E. coli and purified it to
homogeneity (see "Experimental Procedures"). The pointed end
capping activity of recombinant Sk-Tmod was evaluated from its ability
to inhibit elongation of actin filaments capped at their barbed ends by
gelsolin, using the fluorescence increase of pyrenyl actin to measure
actin polymerization (36). In the absence of tropomyosin, Sk-Tmod
inhibits the initial rate of elongation at the pointed filament end
with an estimated Kd of ~0.2 µM
(Fig. 6A). This indicates that
Sk-Tmod can bind directly to pure actin filaments at their pointed ends with about the same affinity as E-Tmod (~0.1-0.4 µM)
(36). Furthermore, Sk-Tmod also increases the critical concentration at
the pointed end as demonstrated by a decrease in the total F-actin at
steady state (i.e. lower end point of polymerization) (Fig.
6A), as previously observed for E-Tmod (36).
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Fig. 6.
Sk-Tmod inhibits elongation from the pointed
ends of actin and tropomyosin-actin filaments. A,
effect of Sk-Tmod on the elongation rate of gelsolin-capped actin
filaments. Elongation was initiated by the simultaneous addition of
gelsolin-capped actin filaments (final concentration 10 nM;
gelsolin:actin, 1:10) and polymerizing salts to a 2.5 µM
solution of rabbit skeletal muscle G-actin (10% pyrenyl-actin),
containing 0 ( ,  ), 0.3 ( ), 0.6 ( ), and 1.5 ( )
µM recombinant Sk-Tmod. End points of polymerization were
determined after 24 h and were 1.91 µM F-actin in
the absence of Sk-Tmod (i.e. 0.69 µM G-actin,
which is the pointed end critical concentration), and 1.45, 1.21, and
1.23 µM F-actin for 0.3, 0.6, and 1.5 µM
Sk-Tmod, respectively. This corresponds to a maximal increase in the
G-actin concentration to ~1.38 µM at steady state
(i.e. about a 2-fold increase in the pointed end critical
concentration), as described previously for E-Tmod (Ref. 36 and
Footnote 2). B, effect of Sk-Tmod on the elongation rate at
the pointed ends of tropomyosin-actin filaments. The elongation assay
was carried out as for A, except that the assay medium
contained 1.5 µM G-actin, 1.18 µM rabbit
skeletal muscle tropomyosin, and 6 nM gelsolin:actin seeds
(0.9 µM F-actin) (gelsolin:actin, 1:150) (, ), with
the addition of 7 (×), 15 (), and 30 () nM Sk-Tmod.
The end points of F-actin polymerization were 1.74 µM for
the control in the absence of tropomodulin (corresponding to ~0.66
µM G-actin; the pointed end critical concentration), and
1.62, 1.75, and 1.65 µM F-actin for 7, 15, and 30 nM Sk-Tmod. These are not significantly different from the
end point in the absence of tropomodulin, as described previously for
E-Tmod (Ref. 36 and Footnote 2).
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In the presence of skeletal muscle tropomyosin, nanomolar
concentrations of Sk-Tmod completely block elongation from the pointed ends of the actin filaments and do not increase the critical
concentration (Fig. 6B), similarly to E-Tmod
(36).2 The Kd for Sk-Tmod capping of
tropomyosin-actin filaments is estimated to be less than ~7
nM, from this experiment (Fig. 6B). The much
higher capping activity of Sk-Tmod for a tropomyosin-actin filament
than for pure actin is similar to E-Tmod and suggests that Sk-Tmod also
binds tropomyosin in addition to actin at the pointed filament end. In
fact, a tropomyosin blot overlay assay (16, 24) demonstrates that
skeletal muscle tropomyosin binds directly to Sk-Tmod (data not shown).
Tissue Expression Pattern of Sk-Tmod--
A Sk-Tmod probe detects
a single band of approximately 1.3 kb in adult breast muscle (PM) but
not in heart, brain, cerebellum, spleen, lung, gizzard, liver,
intestine, ovary, or thymus (Fig. 7,
A and B). In contrast, an E-Tmod probe detects
three bands of approximately 1.4, 2.1, and 2.6 kb in heart but not in
breast muscle (PM) (Fig. 7A, right
panel, asterisks) or in the other tissues (data
not shown). Similar sizes for E-Tmod messages have been reported
previously and are most likely due to utilization of different
polyadenylation signals (15, 16). Although Sk-Tmod is the predominant
tropomodulin isoform in adult chicken breast muscle, E-Tmod is also
present in breast muscle, albeit in low amounts, and can be detected by
Northern blots of poly (A)+ RNA (16) or by grossly
overexposing the Western blots of two-dimensional gels (data not
shown).
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Fig. 7.
Tissue expression pattern of Sk- and E-Tmod
in adult chickens. A, Northern blot of total RNA from
heart and breast (PM) muscle probed with the full-length
Sk-Tmod cDNA (A, left) or the fragment
containing the coding region of chicken E-Tmod (A,
right). The Sk-Tmod probe detected a ~1.3-kb mRNA as
indicated by the arrowhead. The E-Tmod probe detected three
mRNA bands of approximately 2.8, 2.0, and 1.4 kb
(asterisks on the right). The positions of the 28 S and 18 S ribosomal RNAs are indicated on the left. B,
Northern blot of total RNA from adult chicken breast muscle and various
non-muscle tissues probed with the full-length Sk-Tmod cDNA
(B, top) or a fragment complementary to the 5.8 S
ribosomal RNA (B, bottom) as a loading and
mRNA integrity control. The positions of the 28 S and 18 S
ribosomal RNAs are indicated on the left. C,
adult chicken lens, erythrocyte membranes (RBC), or breast
(PM) muscle samples were electrophoresed on two-dimensional
gels separately (C, left) or mixed in pairs
(C, right) and followed by Western blotting with
the polyclonal antibody R745, which recognizes both E- and Sk-Tmod
isoforms.
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E-Tmod was originally identified in human erythrocytes and later shown
to be in the rat lens (data not shown, and Refs. 23 and 39). The
tropomodulin isoform composition of chicken lens fiber cells and
erythrocytes was characterized by two-dimensional gel electrophoresis
followed by Western blotting, because the very low amounts of mRNA
contained in these biosynthetically inactive tissues make Northern blot
analysis difficult. Fig. 7C shows that a single tropomodulin
spot is detected in both adult chicken lens and erythrocytes
(RBC) (Fig. 7C, left
panel). Mixing experiments of lens or erythrocytes
(RBC) with adult breast (PM) muscle (Fig. 7C, right panel) demonstrate that the
tropomodulin spots in these tissues comigrate, indicating that Sk-Tmod
rather than E-Tmod predominates in chicken lens and erythrocytes.
Sk-Tmod Replaces E-Tmod during Development of Chicken Skeletal
Breast Muscle--
Muscle growth and an increase in muscle activity
(as seen in flapping movement) are remarkable during the postnatal
development of chicken breast muscle. The changes in the physiological
properties of the muscle during development are paralleled by shifts in
the expression of distinct isoforms for each sarcomeric component (13).
To determine whether tropomodulin isoform expression changes during
breast muscle development in vivo, we first compared message levels of E- and Sk-Tmod by Northern blotting. Sk-Tmod messages are
detected exclusively in adult breast muscle and not in either day 12 or
day 18 embryonic breast muscle (Fig.
8A, left
panel). When the same blot is reprobed with the E-Tmod
specific probe, the three characteristic E-Tmod messages of
approximately 1.4, 2.1, and 2.6 kb are detected in both day 12 or 18 day embryonic breast muscle but not in adult breast muscle (Fig.
8A, right panel).
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Fig. 8.
The expression of Sk- and E-Tmod is
developmentally regulated during skeletal muscle differentiation.
A, Northern blot of total RNA from days 12 and 18 embryonic
(Emb) or adult breast (PM) muscle probed with
full-length chicken Sk-Tmod cDNA (left) or a fragment
containing the coding region of chicken E-Tmod (right). A
single band corresponding to a Sk-Tmod message of approximately 1.3 kb
is detected in samples from adult but not embryonic breast
(PM) muscle. Three E-Tmod message sizes were detected in
embryonic but not adult breast muscle. The position of the 28 S and 18 S ribosomal RNAs are indicated on the right. B,
samples from breast (PM) muscle at different stages of
development were homogenized with urea and subjected to two-dimensional
gel electrophoresis followed by Western blotting with mAb 23 that
recognizes both E and Sk-Tmod. The locations of E- and Sk-Tmod are
indicated. A minor spot more acidic than E-Tmod is also detected with
this antibody (arrow, top panel). This may indicate the
existence of yet another tropomodulin isoform or a post-translational
modification present at earlier stages of breast muscle
development.
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In agreement with the Northern analysis, Western blots of
two-dimensional gels show that E-Tmod is the most abundant tropomodulin isoform detected in breast muscle from day 12 embryos (Fig.
8B) and from day 20 embryos (just before hatching) (data not
shown). Soon after hatching (2 days), the more acidic Sk-Tmod isoform starts to be detected (Fig. 8B), and, as development
proceeds, the E-Tmod spot disappears and is replaced by the Sk-Tmod
isoform. Thus, in breast muscle from chickens 1 week old or older, the Sk-Tmod isoform is the predominant tropomodulin isoform detected (Fig.
8B).
Sk-Tmod Is Enriched in Adult Fast Skeletal Muscle Fibers, Whereas
E-Tmod Is Enriched in Slow Skeletal Muscle Fibers--
Adult breast
muscle consists mainly of fast muscle fiber types, while embryonic
breast muscle resembles adult slow fiber types in its physiological
properties (40). Studies of isoform expression for other sarcomeric
proteins have shown that the embryonic isoform often predominates in
adult chicken slow fiber types as well as in cardiac muscle, while a
different isoform is expressed in adult fast fibers (e.g.
myosin light chain or C-protein) (13). To further characterize the
expression of Sk-Tmod and E-Tmod in different fiber types, we
immunolocalized tropomodulins in serial cross sections of frozen adult
chicken BF muscle which contains both fast and slow muscle fiber types.
A monoclonal antibody specific for fast twitch C-protein (MF1) was used
as a marker for fast muscle fiber types (19) and its staining pattern
was compared with that of mAb 95, which specifically recognizes E-Tmod,
and mAb 17, which preferentially recognizes Sk-Tmod by
immunofluorescence (see "Experimental Procedures").
The staining pattern obtained with the fast C-protein antibody reveals
the heterogeneity in the muscle fibers in BF (Fig. 9B). Most fibers stain
brightly for fast C-protein (Fig. 9B, double arrowhead), a subset of fibers appear moderately stained
(Fig. 9B, short arrow), while in a few
fibers fast C-protein is hardly or not at all detected (Fig.
9B, long arrow). Overall, the
intensity of the fiber staining for E-Tmod and fast C-protein is
complementary to each other. For instance, the minority of the fibers
that appear brightly stained for E-Tmod are not stained for fast
C-protein (Fig. 9, A and B, long
arrow), indicating that E-Tmod is enriched in slow muscle
fiber types. Conversely, most fibers that do not stain for E-Tmod stain
brightly for fast C-protein (compare Fig. 9, A and
B, double arrowhead). On the other
hand, fibers stained for Sk-Tmod also contain fast C-protein (Fig. 9,
B and C, short arrow,
double arrowhead) and fibers with hardly any
detectable Sk-Tmod are brightly stained for E-Tmod but not fast
C-protein (Fig. 9, A-C, long arrow).
Additionally, we found that by Western blot of two-dimensional gels,
Sk-Tmod and E-Tmod are both present in BF, whereas Sk-Tmod is the only
tropomodulin isoform detected in posterior latissimus dorsi muscle,
which consists of fast muscle fiber types (data not shown).
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Fig. 9.
Sk-Tmod predominates in skeletal fast muscle
fiber types while E-Tmod predominates in slow muscle fiber types.
Serial cross-sections of frozen adult chicken BF muscle were stained
for E-Tmod (A), fast twitch C-protein (B), or
Sk-Tmod (C). E-Tmod is most abundant in slow muscle fiber
types (A, long arrow), where fast twitch
C-proteins (B, long arrow) and Sk-Tmod
(C, long arrow) are hardly detected. Some fibers
contain a moderate amount of E-Tmod (A, short
arrow), fast twitch C-protein (B, short
arrow), and Sk-Tmod (C, short arrow).
Sk-Tmod is most abundant in the fast muscle fiber types that are
brightly stained for fast C-protein (B and C,
double arrowheads) and do not contain E-Tmod (A,
double arrowhead). E-Tmod staining is also detected at the
sarcolemma of muscle fibers (A, arrowhead). The
brighter staining obtained for fast C-protein on the right edge of the
tissue section is most likely due to a fold or a difference in
thickness of the section. Bar, 50 µm.
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Together, these results suggest that Sk-Tmod predominates in fast
twitch fiber types. However, fibers that are moderately stained for
fast C-protein are also stained for E- and Sk-Tmod (Fig. 9,
A-C, short arrow) indicating that
some fibers contain both tropomodulin isoforms. Hybrid fibers
containing both fast and slow isoforms of different myofibrillar
protein isoforms are reported to be quite frequent and may indeed
represent the rule rather than the exception (13).
E-Tmod Colocalizes with -Spectrin in Distinct Subsarcolemmal
Domains--
E-Tmod was first identified as a component of the
spectrin-actin membrane skeleton in human erythrocytes (15, 39).
Interestingly, we noticed E-Tmod staining associated with the
sarcolemma of many muscle fibers in BF muscle (Fig. 9A,
arrowhead). This staining is specific for E-Tmod because it
is eliminated by pre-incubating mAb 95 with a 100-fold molar excess of
purified E-Tmod, but not Sk-Tmod, prior to incubation with the muscle
sections (data not shown). Furthermore, we obtained the same
sarcolemmal staining using another E-Tmod specific monoclonal antibody
(mAb 9) (Fig. 10A), which
recognizes an epitope of E-Tmod located in a different region of the
molecule than the epitope recognized by mAb 95 (data not shown). These
observations suggested that E-Tmod might be a component of the
subsarcolemmal spectrin-actin membrane skeleton in skeletal muscle.
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Fig. 10.
E-Tmod colocalizes with -spectrin at discrete subsarcolemmal domains in
skeletal muscle fiber cells. Serial cross-sections of frozen adult
chicken BF muscle were double stained for E-Tmod (A), or
-spectrin (B). E-Tmod together with -spectrin
localizes at the sarcolemma of all the muscle fiber types (A
and B, arrows). Higher magnification images show
that E-Tmod is localized in distinct puncta along the sarcolemma
(C, arrowhead), which colocalize with
-spectrin (E, arrowhead). The merged image is
shown in D (merged). The asterisk in
D and E indicates a capillary brightly stained
for -spectrin. Bar in A and B, 25 µm. Bar in C-E, 2 µm.
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-Spectrin has been described to be concentrated at subdomains along
the sarcolemma that overlay the I bands in skeletal muscle and are
termed costameres (41). To explore the association of E-Tmod with the
subsarcolemmal spectrin lattice, we double-stained cross-sections of BF
muscle for E-Tmod and -spectrin. Fig. 10 shows that E-Tmod as well
as -spectrin are associated with the sarcolemma of all muscle fiber
types contained in this muscle (Fig. 10, A and B,
arrows). A higher magnification view of an oblique BF
section demonstrates that E-Tmod staining is not uniformly distributed
along the sarcolemma, and instead appears to be concentrated in
distinct and periodic puncta ~2 µm apart, which colocalize with the
-spectrin puncta (Fig. 10, compare C with D,
arrowheads).
This result suggests that E-Tmod is associated with the
-spectrin-containing costameres overlying the I bands in chicken skeletal muscles.
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DISCUSSION |
Tropomodulins are a family of actin filament pointed end capping
proteins. The first tropomodulin isoform identified in vertebrates was
E-Tmod, a ~40-kDa protein whose expression in mammals is restricted to terminally differentiated cells including erythrocytes, lens fibers,
neurons and striated muscle (9, 28, 42). Subsequently, another
~40-kDa tropomodulin isoform, N-Tmod, was identified in embryonic and
adult neurons (27). ~40-kDa tropomodulin homologs have also been
identified in flies (Sanpodo) (31) and in C. elegans (28,
31). Larger, more distantly related proteins with limited regions of
similarity to portions of the ~40-kDa tropomodulins have also been
identified, including a ~64-kDa protein in smooth muscle and
extraocular muscle (43, 44) as well as a longer ~60-kDa (predicted)
protein from C. elegans (28, 31). In this study, we report
the identification of a third ~40-kDa tropomodulin isoform from
vertebrates, which we have named Sk-Tmod, based on its high level of
expression in skeletal muscle.
The three known ~40-kDa vertebrate tropomodulin isoforms are products
of different genes and are all about 60-65% identical (75-90%
similar) to each other at the amino acid level. The high sequence
homology between tropomodulin isoforms implies that they most likely
share functional properties. Indeed, here we show that Sk-Tmod shares
all the known functional properties described for E-Tmod (9); it is
associated with the pointed ends of muscle thin filaments in
vivo and completely blocks actin elongation from the pointed ends
of tropomyosin-actin filaments in vitro (Kd < 7 nM). Like E-Tmod, Sk-Tmod also
inhibits elongation from the pointed ends of pure actin filaments but
with considerably lower affinity (Kd ~ 0.2 µM). Thus, we propose that a defining feature of this
family of proteins is the ability to block the elongation of
tropomyosin-actin filaments from their pointed ends, mediated by their
binding to both actin and tropomyosin (9). N-Tmod has been shown
previously to interact with tropomyosin similarly to E-Tmod, but its
actin capping activity has not yet been investigated (27). Thus,
verification of this hypothesis will require determination of the
tropomyosin-actin filament capping activities for N-Tmod and the other
recently identified tropomodulin homologs from invertebrates.
On the other hand, the sequences of tropomodulins in each independent
group in the tropomodulin family (E-, N-, and Sk-Tmod groups) are
highly conserved across vertebrates (28 and Fig. 4 of this study). This
might indicate that different tropomodulin isoforms have acquired minor
but functionally significant differences in their sequences that have
been preserved through evolution. This study and those of others have
described several observations supporting this idea. First,
tropomodulin isoforms exhibit strikingly different tissue expression
patterns (28, 42). For example, in adult chickens, Sk-Tmod is found in
fast skeletal muscle, erythrocytes, and lens. In contrast, E-Tmod is
found in heart and slow skeletal muscle while N-Tmod is expressed in
brain.4 A second observation,
and probably the one that most strongly supports the idea of functional
specificity for each tropomodulin isoform, is the sorting of Sk- and
E-Tmod isoforms to different cytoskeletal structures within the same
skeletal muscle cell (e.g. myofibrils and costameres,
respectively). This may indicate that other regions of the molecule,
not involved in tropomyosin or actin binding, might be responsible for
interactions with other sarcomeric or cytoskeletal components and
determine unique functions, which are as yet undescribed.
On the other hand, the tropomodulin isoform composition in the same
tissue appears to differ between species. For instance, chicken lens
and erythrocytes contain Sk-Tmod (Fig. 7) while mouse lens and
erythrocytes contain E-Tmod.4 This may indicate that
tropomodulins could have redundant functions and that their
differential expression in tissues and between species may reflect the
regulatory constraints of different developmental programs. Future
studies on the genomic organization and the intron/exon structure of
tropomodulins may also provide an understanding of the role that
regulatory or structural regions play in generating and maintaining
this tropomodulin isoform sequence diversity.
Expression of Different Tropomodulin Isoforms in Muscle Correlates
with Changes in Thin Filament Lengths--
Our results show an
interesting correlation between the tropomodulin isoform expressed and
the changes in thin filament lengths that take place during the
development of chicken breast muscle. Decoration of thin filaments from
adult breast muscle with antibodies to troponin invariably revealed 24 striations at regular intervals of 38 nm, whereas in embryonic muscle
the number of striations varied between 25 and 29 (45). This suggests
that the thin filaments are all ~0.9 µm long in adult breast
muscle, whereas they vary from ~0.95 to 1.1 µm in embryonic breast
muscle. Our results show that E-Tmod is present in embryonic breast
muscle and thus appears to be associated with sarcomeres in which the
thin filaments are longer and have a wider range of lengths. In
contrast, Sk-Tmod is present in adult breast muscle and thus appears to
be associated with sarcomeres in which the thin filaments are shorter
and have a more precise length distribution. To further explore this
idea, it will be necessary to compare directly the expression of
tropomodulin isoforms in other muscles in which thin filament lengths
have been measured.
How might different tropomodulin isoforms influence the relative
precision or variability in thin filament length regulation? One
possibility is that tropomodulin isoforms could have different effects
on the dynamics of actin monomer exchange at the thin filament pointed
ends. For instance, in vivo, Sk-Tmod might provide, directly
or indirectly, a tighter capping activity that would more precisely
stabilize thin filament lengths in fast skeletal muscles. In contrast,
E-Tmod might cap the thin filaments less tightly in embryonic breast
muscle, heart, or adult slow skeletal muscle leading to a more
extensive exchange of monomeric actin at their pointed ends, and
consequently a wider variation in lengths.
Although Sk-Tmod has a similar affinity as E-Tmod for tropomyosin-actin
filament pointed ends in vitro, differences in the affinity
of tropomodulin isoforms for thin filament pointed ends in
vivo could be influenced by other sarcomeric components. Potential candidates are tropomyosin isoforms and nebulin. Skeletal muscle cells
have two tropomyosin isoforms, and , that are developmentally regulated at the transcriptional level such that the -tropomyosin gene is repressed at hatching (46). Thus, in adult breast muscle, / is the principal isoform found (47-50). Furthermore, the ratio of /, /, and / dimers differs between mature muscle
fiber types (51, 52).
Nebulin is a long actin-binding protein that extends from the Z line to
the thin filament pointed end and has been proposed to function as a
molecular template to specify thin filament lengths in skeletal muscle
(53-55). The existence of different nebulin isoforms which differ in
their molecular size has been correlated with the length of thin
filaments in different skeletal muscle types and during differentiation
of chicken breast muscle (56-58). One can speculate that distinct
nebulin isoforms might interact with specific tropomodulin isoforms to
regulate tropomodulin affinity for the pointed ends and thereby produce
thin filaments of different lengths for physiologically distinct types
of muscle (5). However, cardiac muscle does not contain nebulin (55),
yet it contains E-Tmod, which might suggest that nebulin is not
necessarily required by E-Tmod to regulate the length of thin filaments.
Sorting of Tropomodulin Isoforms to Different Cytoskeletal
Structures--
Interestingly, in fast skeletal muscle fibers, which
coexpress both E and Sk-Tmod, E-Tmod appears to be associated with the -spectrin-containing costameric network overlying the I bands at the
sarcolemma while Sk-Tmod is associated with the myofibrils within the
same cell. What can account for restriction of E-Tmod to the sarcolemma
in hybrid muscle fibers which express both E and Sk-Tmod? One
possibility is that Sk-Tmod binds thin filament pointed ends more
tightly and thus competes for E-Tmod binding to this site. Similarly,
E-Tmod may bind more tightly to costameric actin filaments than
Sk-Tmod. In this case, we might expect the existence of specific
sequences in tropomodulin isoforms to target the tropomodulins to their
appropriate binding partners in each structure. However, it is
important to point out that the basis for sorting of different
tropomodulin isoforms to different cytoskeletal structures is likely to
be complex since in some muscle fibers (i.e. slow and mixed)
E-Tmod is detected associated with myofibrils and at the sarcolemma.
Costameres were first described as vinculin-rich areas forming rib-like
elements flanking the Z lines of underlying myofibrils in skeletal
muscle (59, 60). Subsequently, spectrin was found to be enriched in
costameres overlying the I bands, in transverse elements over the M
line and in fine longitudinal strands connecting the costameres (41,
61-65). To date, a variety of other proteins including desmin,
integrins, dystrophin, and components of the spectrin-based membrane
skeleton have also been localized to elements of the costameric network
(64, 66), but very little is known about their molecular organization.
Our observation that E-Tmod is a component of the costameric network
suggests that similar to erythrocytes, the spectrin-based membrane
skeleton underlying the costameric network is built by short actin
filaments capped by E-Tmod at their pointed ends and cross-linked by
spectrin (9).
It has been proposed that costameres could play a mechanical role in
anchoring the sarcomeric cytoskeleton of the most peripheral myofibrils
to the membrane via intermediate filaments and also in linking the
sarcolemma to the extracellular matrix via integral membrane proteins
(60, 63, 64, 67, 68). In addition to a mechanical role, costameres have
also been proposed to be involved in signal transduction across the
membrane through focal adhesion proteins such as integrins, talin, and
vinculin, or through the dystrophin-based membrane skeleton, which
appears to interact with enzymatic or regulatory proteins (64). Thus,
E-Tmod in costameres may function in a structural capacity to stabilize and limit the lengths of actin filaments in a spectrin-actin network, thereby influencing the clustering of focal adhesion components and/or
dystrophin-associated proteins at costameres. This could regulate, in
turn, the signaling pathways controlling skeletal muscle fiber growth
or atrophy.
Prospects--
While studies of isolated proteins have been useful
in identifying protein function, they may be less helpful in
distinguishing properties of closely related isoforms. The correlation
of tropomodulin isoform expression with differences in the contractile
properties of different muscle types suggests the possibility that
tropomodulins might also be involved in thin filament linked regulation
of contraction together with tropomyosins and troponins (for a
discussion, see Ref. 5). In vivo, it is likely that the
functional advantages produced by each molecular phenotype have
contributed to preserve certain combinations of sarcomeric isoforms in
each muscle. Recently, several functional studies have been published
reporting that closely related sarcomeric isoforms of tropomyosin and
troponin are not functionally redundant in vivo (Refs.
69-71; for a review, see Ref. 13). Thus, genetic approaches to gene
inactivation and gene replacement of tropomodulin in animal models may
provide new insights into the physiological consequences of altering
tropomodulin isoform expression during development and differentiation.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Cell Biology, MB24, Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8277; Fax: 858-784-8753; E-mail: velia@scripps.edu.
