Space Life Sciences, National Aeronautics and Space Administration Ames Research Center, Moffett Field, California 94035-1000
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ABSTRACT |
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Evidence is
accumulating to suggest that actin filament remodeling is critical for
smooth muscle contraction, which implicates actin filament ends as
important sites for regulation of contraction. Tropomodulin (Tmod) and
smooth muscle leiomodin (SM-Lmod) have been found in many tissues
containing smooth muscle by protein immunoblot and immunofluorescence
microscopy. Both proteins cofractionate with tropomyosin in the
Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are
solubilized by high salt. SM-Lmod binds muscle tropomyosin, a
biochemical activity characteristic of Tmod proteins. SM-Lmod staining
is present along the length of actin filaments in rat intestinal smooth
muscle, while Tmod stains in a punctate pattern distinct from that of
actin filaments or the dense body marker -actinin. After smooth
muscle is hypercontracted by treatment with 10 mM Ca2+,
both SM-Lmod and Tmod are found near
-actinin at the periphery of
actin-rich contraction bands. These data suggest that SM-Lmod is a
novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle
may be capped by Tmod in localized clusters.
actin cytoskeleton; tropomyosin binding protein; contraction bands; human 64-kDa autoantigen D1
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INTRODUCTION |
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SMOOTH MUSCLES are
found in many diverse organs throughout the body and perform numerous
essential functions, from peristalsis in the gastrointestinal tract to
maintenance of vascular tone (reviewed in Refs. 4,
26, 48, and 51 and references
therein). The subcellular morphology of smooth muscle is much
more uniform than skeletal muscle when viewed in the light microscope,
and the architecture and composition of the contractile apparatus in
smooth muscle is still not completely understood. Each smooth muscle
cell (SMC) contains intertwined "cytoskeletal" and
"contractile" domains that contain either nonmuscle actin and
intermediate filaments or muscle actin and myosin, respectively. The
cytoskeletal and contractile domains are anchored to each other at
dense bodies within the cytoplasm that contain -actinin and at dense
plaques on the cell surface that contain filamin and focal adhesion
proteins. The barbed ends of the actin filaments are anchored in these
dense bodies and dense plaques. This complex arrangement is thought to
be required for maintenance of subcellular organization during the
massive shape changes characteristic of smooth muscle contraction. Repeating structures analogous to striated muscle sarcomeres have not
been detected in smooth muscles despite considerable effort. However,
the presence of "contraction bands" or "zones" infrequently reported in histological tissue preparations (see e.g., Refs. 38 and 44) and isolated SMCs (see e.g., Refs.
6 and 17) suggests an underlying global organization for
the smooth muscle contractile apparatus.
In recent years, evidence has been accumulating to suggest that the
mechanism of contraction in smooth muscle may be more likely to
resemble motility in nonmuscle cells than contraction of skeletal or
cardiac muscle (see e.g., Refs. 30 and 55). Smooth muscle
contains a number of the same contractile proteins as striated muscle,
including actin, myosin, tropomyosin, CapZ/capping protein, and
-actinin. However, smooth muscle does not contain some contractile
proteins critical for the regulation of striated muscle contraction,
notably the troponins. Instead, smooth muscle expresses isoforms of
proteins, such as caldesmon (reviewed in Ref. 32) and
calponin (reviewed in Ref. 27), that are also required for
motility in nonmuscle cells and contain significant concentrations of
others, such as gelsolin (8) and profilin (7), that are expressed only at low levels in striated
muscle. In addition, some cytoskeletal proteins are expressed
predominantly or exclusively in smooth muscle, such as smoothelin
(reviewed in Ref. 57). These differing profiles of
tissue-specific expression in smooth muscle relative to other
contractile tissues suggest that, although smooth muscle contraction
may resemble nonmuscle contraction more than striated muscle
contraction, the mechanism of smooth muscle contraction is likely to be
in some ways unique.
Functional evidence for the similarity between smooth muscle contraction and nonmuscle motility comes from experiments using cytochalasin, a small molecule that inhibits elongation from the barbed ends of thin filaments, and latrunculin, a compound that sequesters actin monomers. Treatment with either cytochalasin or latrunculin inhibits motility of nonmuscle cells and also inhibits contraction and force generation in smooth muscle (5, 39, 46, 63). Actin filament dynamics thus appear to play an important role in smooth muscle contraction. Because filament growth occurs at the ends of actin filaments, these data make actin filament ends, and the proteins that regulate them in smooth muscle, of considerable interest.
Until now, relatively little attention has been paid to the potential role of actin filament capping proteins in smooth muscle contraction. Isoforms of two barbed-end capping proteins have been reported in smooth muscle: gelsolin, which both caps and severs filaments and is abundant in a variety of motile nonmuscle tissues (reviewed in Ref. 64), and CapZ/capping protein, which caps the barbed ends of actin filaments in striated and cardiac muscle as well as in nonmuscle tissues (31; reviewed in Ref. 13). The tropomodulin (Tmod) proteins are known to cap the pointed ends of actin filaments in striated and cardiac muscle as well as in the spectrin-actin membrane skeleton (reviewed in Refs. 21-23). My coworkers and I reported recently (12) that transcripts encoding several Tmod isoforms are expressed in tissues containing smooth muscle. Previously, we demonstrated that a protein called the 64-kDa human autoantigen D1 (16) was abundant in rat bladder (11) and that mRNA transcripts encoding this gene were present in all human tissues tested that contain smooth muscle (12). These observations led us to rename this protein smooth muscle leiomodin (SM-Lmod, gene LMOD1; see Ref. 12). SM-Lmod displays significant similarity to the Tmod family of pointed-end capping proteins in two functionally distinct regions of the Tmod molecule (10, 12, 53).
The Tmod family consists of four 40-kDa protein isoforms that are highly conserved among vertebrates but show no similarity to other known protein families (10, 12, 15). Erythrocyte (E)-Tmod (gene TMOD1) is present at the pointed "free" ends of actin filaments in striated and cardiac muscle and in the spectrin-actin membrane skeleton (21; reviewed in Refs. 22 and 23), and is expressed at low levels in a variety of mouse and human tissues (12, 15, 21, 33, 52). Neural (N)-Tmod (gene TMOD2) is expressed almost exclusively in embryonic and adult neural tissue (15, 58). Skeletal (Sk)-Tmod (gene TMOD4) is found at the pointed ends of fast skeletal muscle fibers (2) and is expressed exclusively in adult skeletal muscle (12, 15). Ubiquitous (U)-Tmod (gene TMOD3) is found in a wide variety of tissues (12, 15) and is the isoform most highly expressed in smooth muscle (12). Two biochemical functions are characteristic of Tmod family proteins: 1) binding of the protein tropomyosin (TM) (20) and 2) capping of the slow-growing ends of pure actin and TM-actin thin filaments (59, 60). The filament-capping function of E-Tmod is required in vivo for striated muscle function. In cardiomyocytes, both subcellular morphology and beating activity are disturbed after disruption of E-Tmod capping activity (29) or alteration of protein levels (54). With the recent suggestions that actin filament remodeling is required for smooth muscle contraction, it is of considerable importance to evaluate the presence of Tmod-like capping proteins in smooth muscle.
In this paper, I demonstrate that isoforms of the pointed-end capping
protein Tmod, and the larger, related protein SM-Lmod, are present in
many smooth muscle tissues by immunoblot and immunofluorescence analysis. Extraction of stomach smooth muscle by detergent and salt
indicates that SM-Lmod and Tmods as well as TM are found in the
Triton-insoluble cytoskeleton but can be extracted by high salt.
SM-Lmod fulfills one of the functional requirements to be a Tmod family
member by binding muscle TM isoforms, as assayed by TM blot overlay of
bacterially expressed SM-Lmod protein and coimmunoprecipitation of
native SM-Lmod and TM from rabbit stomach smooth muscle.
Immunofluorescence microscopy demonstrates that Tmods and SM-Lmod are
present in tissues containing smooth muscle, although Tmods were not
expressed in all vascular smooth muscles examined. SM-Lmod and actin
filamentous structures are costained in smooth muscle in a pattern
distinct from that of Tmods. Confocal microscopy demonstrates that Tmod
staining is present in a punctate pattern that is distinct from
-actinin-stained dense bodies. In hypercontracted smooth muscle,
staining for both proteins is restricted to the periphery of
actin-containing contraction bands. These results demonstrate
for the first time that smooth muscle contains canonical Tmod proteins
as well as the larger SM-Lmod. Comparison of their subcellular
distribution in smooth muscle permits speculation concerning the roles
these proteins may play in smooth muscle contraction.
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MATERIALS AND METHODS |
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Recombinant protein expression and antibody production.
SM-Lmod protein and affinity-purified rabbit antibodies recognizing
SM-Lmod were prepared as described previously (11). Recombinant chicken E-Tmod protein was a gift from Dr. V. Fowler and
was prepared as described previously (3).
Affinity-purified rabbit antibodies recognizing human E-Tmod and TM
were obtained from Dr. V. Fowler and prepared as described previously
(21, 56). Monoclonal antibody A5044 specific for
-actinin was purchased from Sigma (St. Louis, MO). Secondary
antibodies were purchased from Kirkegaard and Perry (Gaithersburg, MD),
and phalloidin was obtained from Molecular Probes (Eugene, OR).
Protein immunoblots and tissue extraction. Whole tissue protein samples were prepared from freshly dissected rat tissues that had been plunge-frozen in liquid nitrogen. Tissues were ground under liquid nitrogen, and 10 volumes (wt/vol) of warm 2× SDS-PAGE sample buffer were added to the frozen powder. Samples were pipetted and vortexed rapidly to melt and mix the tissue powder, boiled for 2 min, sonicated, and centrifuged to pellet insoluble material. Samples of smooth muscle and epithelium fractions from rat small intestine were the kind gift of Dr. J. Black (Roswell Park Memorial Institute, Buffalo, NY). Approximately 20 mg of total protein from each tissue were separated on 10% polyacrylamide gels, pH 8.6, and blotted using standard procedures. Protein immunoblots were probed as described previously (62).
Rabbit stomachs were obtained from freshly killed adult rabbits, and wall muscle was dissected away from other stomach tissues, frozen in liquid nitrogen, and stored atTM blot overlays. Total protein (10 µg) from induced bacteria carrying an SM-Lmod expression plasmid (described in Ref. 11) and 0.2 mg of purified chicken E-Tmod and human Lmod protein were separated by SDS-PAGE on 10% gels, pH 8.6 (37), and blotted in Towbin transfer buffer containing 20% methanol and 0.1% SDS, according to standard procedures (see e.g., Refs. 21 and 56). Rabbit skeletal muscle TM and bovine brain TM were prepared as previously described (19, 50). Blot overlays were incubated with 2 mg/ml TM overnight at 4°C, and bound protein was visualized with anti-TM antibody as described previously (62).
Immunoprecipitation. Immunoprecipitation of SM-Lmod and Tmod from rabbit stomach wall muscle was performed essentially as described by Fowler et al. (24), with the exception that fresh rabbit stomach wall muscle was homogenized in Ringer buffer containing 2% Triton X-100, Ringer buffer containing 1 M KCl, or RIPA buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, and 0.01 M Tris, pH 7.2). Samples were sonicated five times for 2-s pulses and then centrifuged sequentially for 20 min at 2,000 g and 30 min at 50,000 g, and the 50,000 g supernatant was added to antibody-coated beads. After overnight incubation, beads were washed three times in the appropriate buffer solution and then mixed directly with 2× SDS-PAGE sample buffer. PAGE and protein immunoblots were performed as described above.
Immunofluorescence microscopy.
For multiple tissue screening, adult rats were anesthetized with
halothane and perfused through the heart first with phosphate-buffered saline (PBS) solution until organs bleached, followed by freshly prepared 2% paraformaldehyde in PBS. Tissues were excised and fixed in
2% paraformaldehyde in PBS for 1 h on ice, followed by three
15-min incubations in ice-cold 50 mM NH4Cl in PBS to quench unreacted aldehydes. Tissues were infiltrated with 30% sucrose in PBS
overnight at 4°C and frozen in Tissue-Tek OTC (optimum cutting
temperature compound) on a liquid nitrogen-cooled metal block. To
obtain rat intestinal circularis tissue, rats were euthanized with
halothane, and then 6-mm rounds of small intestine were cut and placed
in fixative, or in hypercontraction solution (PBS with 10 mM
CaCl2) for 10 min before fixation, and subsequently treated as described above. Cryosections (8 µm) were mounted on coverslips, permeabilized in 0.2% Triton X-100 in PBS for 20 min, and blocked in
1% BSA in PBS for 1 h at room temperature. Sections were
incubated in affinity-purified antibodies that recognize erythrocyte TM and multiple Tmod isoforms at a final concentration of 10 mg/ml, affinity-purified antibodies to SM-Lmod at 2.5-5 mg/ml, or a fresh 1:200 dilution of -actinin monoclonal antibody A5044 (Sigma) in PBS
with 2% gelatin for 2 h at room temperature. Sections were washed
in four changes of PBS and then incubated in PBS containing 2%
gelatin, fluorescein-conjugated secondary antibody, and
rhodamine-phalloidin for 1 h, washed again, and mounted in
Aqua-Polymount. For costaining, both antibodies were diluted
into the same primary incubation buffer, and after sections were
washed, they were coincubated in fluorescein-conjugated and Texas
red-conjugated secondary antibodies. Slides were examined by
conventional epifluorescence microscopy, and images were collected with
a Pentamax charge-coupled device camera (Princeton Instruments, CA) or
by confocal microscopy using a Bio-Rad (Hercules, CA) MRC 1024 confocal microscope.
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RESULTS |
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Tissue-specific expression of SM-Lmod and Tmod proteins.
Previously, it was demonstrated that SM-Lmod transcripts were present
in all tissues that contain smooth muscle present on a human multiple
tissue RNA dot blot (12). In addition, an
affinity-purified polyclonal antibody that I raised against human
SM-Lmod recognized a single ~70-kDa band on protein immunoblots of
rat bladder and extraocular muscle (11). To develop a more
complete picture of the tissue-level expression of proteins recognized
by the SM-Lmod antibodies, I probed immunoblots containing a variety of
rat tissues. These antibodies strongly recognized one band at ~70 kDa
in many tissues that contain smooth muscle and also recognized more
weakly a band of the same size in some striated muscles (Fig.
1). The faint higher molecular weight
band present in a few of the rat tissues is not observed in immunoblots
of the corresponding tissues from rabbit (data not shown) and, thus, is
likely to result from cross-reactivity with an unrelated rat protein.
In smooth muscle and epithelial cells purified from rat small
intestine, the SM-Lmod band was present exclusively in the smooth
muscle fraction (Fig. 1), suggesting that it is predominantly a smooth
muscle-specific protein. Interestingly, the affinity-purified
antibodies to SM-Lmod did not recognize any bands in heart, where a
second Lmod isoform, cardiac (C)-Lmod (gene LMOD2), is most
highly expressed (12).
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Extraction of Lmod, TM, and Tmod from rabbit stomach muscle.
The protein sequence similarity of SM-Lmod to the Tmods suggests that
SM-Lmod may associate with smooth muscle actin filaments biochemically,
as do Tmods. To address this question, I examined the distribution of
SM-Lmod, Tmod isoforms, and TM in a crude myofibril preparation from
rabbit stomach wall muscle that was extracted in physiological buffer
with or without 2% Triton X-100 or in high-salt conditions known to
dissociate TM and Tmod from thin filaments (28). In the
presence of physiological buffer with or without detergent, SM-Lmod,
Tmod, and TM proteins were found in the pellet fraction (Fig.
2, lanes P and T,
50K pel), demonstrating directly that SM-Lmod is a component of the
Triton-insoluble cytoskeleton. In the presence of 1 M KCl, all three
proteins dissociated from the pelletable material (Fig. 2, lane
K, 50K pel). These data suggest that SM-Lmod, as well as Tmod and
TM, associate with the actin-containing Triton-insoluble cytoskeleton
under physiological conditions.
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TM-binding activity of SM-Lmod.
The SM-Lmod protein sequence displays a truncated amino terminus
relative to the 40-kDa Tmod sequences (12). The amino
terminus of E-Tmod is known to be essential for function: the
TM-binding activity of E-Tmod is localized within its 180 amino-terminal amino acids (3). Binding of E-Tmod to
skeletal muscle TM was greatly reduced by removal of the 30 amino-terminal amino acids of E-Tmod, and the erythrocyte TM-binding
site was localized to a region within amino acids 90-194 of
E-Tmod. Because SM-Lmod is missing the 30 amino-terminal amino-acids of
the 40-kDa Tmods as well as significant homology to Tmod sequences from
amino acids 85 to 170, a question is raised as to whether SM-Lmod can
in fact bind any TM isoforms. I investigated the TM-binding activity of bacterially expressed SM-Lmod using the same blot-overlay assay that
was employed to demonstrate the TM-binding specificity of E-Tmod
fragments (3, 52). Figure 3
shows that SM-Lmod does bind to rabbit skeletal muscle TM on blot
overlays, although apparently with lower affinity than does E-Tmod, and
does not bind bovine brain TM. Preliminary evidence suggests that
SM-Lmod also binds pig stomach smooth muscle TM (data not shown),
although the relative affinities and TM binding site(s) on the SM-Lmod
molecule remain to be determined. These results indicate that SM-Lmod
displays at least the TM-binding ability of the Tmod family.
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Immunofluorescence localization of SM-Lmod and Tmods in rat
tissues.
To localize SM-Lmod and Tmods within various tissues that contain
smooth muscle, I examined sections of rat tissues by immunofluorescence microscopy (Figs. 5 and 6). In all rat
tissues examined, the anti-SM-Lmod antibody bound to cells that were
morphologically smooth muscle (Fig. 5) and contained large
quantities of filamentous actin by phalloidin staining (data not
shown). Even in tissues that did not contain large quantities of Lmod
by immunoblot, immunofluorescence staining for SM-Lmod could be found
in small blood vessels (e.g., heart; Fig. 5). Additionally, a small
subset of striated muscle fibers from mixed fiber-type muscles,
including laryngeal and extraocular muscles (Fig. 5) and abdominal wall
muscle (data not shown), were observed to stain with the anti-SM-Lmod
antibodies. Interestingly, the SM-Lmod staining in extraocular muscle
displays a broad doublet pattern (Fig. 5), rather than the narrow
staining bands observed for Tmod in an adjacent serial section (Fig.
6). These data suggest that Tmod and
SM-Lmod may be localized to different parts of the contractile
apparatus in striated muscle.
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Subcellular distribution of SM-Lmod, Tmods, and other contractile
proteins in smooth muscle.
To evaluate the subcellular distribution of SM-Lmod and Tmods in
relation to other contractile proteins, I stained longitudinal circularis muscle sections with SM-Lmod, poly-Tmod, and TM polyclonal antibodies, as well as a monoclonal antibody specific for -actinin, and examined the sections in a confocal microscope (Fig.
7). In the optical sections obtained, the
poly-Tmod staining is strikingly punctate and does not colocalize with
-actinin (Fig. 7, top), although it does appear that the
poly-Tmod puncta frequently occur near dense bodies. In contrast, the
staining patterns for SM-Lmod and TM are relatively uniform along the
length of the actin filaments (Fig. 7, middle and
bottom). These data indicate that SM-Lmod and Tmods may be
associated with different parts of the contractile apparatus in smooth
muscle. Furthermore, the punctate staining pattern for Tmod suggests
that the slow-growing pointed ends of actin filaments could be
localized to small discrete regions within SMCs.
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DISCUSSION |
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Smooth muscle is capable of exerting force over a considerable range of lengths (5, 39), and in some cases even single SMCs have been shown to contract to less than one-third their resting length (34, 47). Although several models have been proposed for the structure of the smooth muscle contractile apparatus that might accommodate this broad length-tension relationship (reviewed in Refs. 4, 26, 48, and 51 and references therein), a consensus has not yet been reached. Recently, evidence has accumulated to suggest that remodeling of actin filaments is required during smooth muscle contraction (5, 30, 39, 46, 55, 63), which begs the question of what proteins in smooth muscle might regulate filament remodeling. In this report, I have demonstrated the presence of the pointed-end capping protein Tmod in smooth muscle, as well as the related protein SM-Lmod (Fig. 1). Previously, I had shown that transcripts of the U-Tmod isoform are most abundant in smooth muscle (12), suggesting that the predominant Tmod protein in smooth muscle is U-Tmod. Extraction of stomach smooth muscle with detergent or high salt demonstrates that SM-Lmod and Tmod cofractionate with the actin-binding protein TM, in that they are present in a Triton-insoluble crude myofibril preparation but are extracted by high salt (Fig. 2). These data do not conclusively demonstrate that SM-Lmod and Tmod are bound directly to actin filaments in smooth muscle, but since Tmod has been shown to bind the pointed ends of actin filaments in both muscle and nonmuscle tissues (reviewed in Refs. 21-23), it is likely that Tmod is also bound to the pointed ends of actin filaments in smooth muscle.
SM-Lmod as a functional member of the Tmod family. SM-Lmod can be considered a member of the Tmod family on the basis of sequence similarity alone (12); however, the final determination of whether the Lmods should be considered bona fide members of the Tmod gene family lies in the evaluation of their biochemical function. The TM-binding activity of E-Tmod has been localized to two separate regions in its 180 amino-terminal amino acids, which bind either muscle or nonmuscle TM (3). SM-Lmod is divergent from the canonical 40-kDa Tmod sequences in this region (12) and is missing the entire nonmuscle TM binding sequence. Consistent with this observation, SM-Lmod does not bind bovine brain TM on blot overlays (Fig. 3). However, SM-Lmod does in fact bind skeletal muscle TM on blot overlay, although apparently to a lesser extent than E-Tmod (Fig. 3), and coimmunoprecipitates smooth muscle TM from rabbit stomach wall muscle (Fig. 4). This result is somewhat surprising, given the previous demonstration that truncation of the 34 amino-terminal amino acids of E-Tmod abolished specific binding to skeletal TM (3). However, when the SM-Lmod and E-Tmod sequences are aligned, the SM-Lmod sequence begins at amino acid 27 of E-Tmod (12), and it is conceivable that these six additional amino acids are enough to restore the skeletal muscle TM binding site. It is possible that a more precise dissection of the skeletal muscle TM-binding domain of the 40-kDa Tmods will demonstrate that only the first 20-24 amino acids are not essential.
The actin capping activity of Tmod has been shown to require a region to its carboxy-terminal half (29), which is conserved with a central portion of SM-Lmod (12). Unfortunately, attempts to assess the actin capping activity of SM-Lmod were inconclusive because of precipitation of protein aggregates during the capping assay (C.A. Conley, R. Kuhl, A.-M. Weber, and V. M. Fowler, unpublished data). These data, in conjunction with the observations that SM-Lmod displays broader bands of staining in striated muscle than Tmod (Figs. 5 and 6), may be indicative of an actin-bundling or side-binding activity for SM-Lmod. Future studies using electron microscopy should resolve this issue.SM-Lmod and Tmod in tissues containing smooth muscle.
Immunofluorescence staining of a variety of tissues that contain
smooth muscle with antibodies specific for SM-Lmod and Tmod isoforms
(Fig. 4) indicates that SM-Lmod is expressed at high levels in all
identifiable SMCs (Fig. 5). However, Tmod expression appears more
variable: although Tmod staining is found in both vascular and visceral
smooth muscle (Fig. 6), some vascular tissue does not stain brightly
(Fig. 6; e.g., bladder, uterus, heart, extraocular muscle).
Interestingly, a number of cytoskeletal protein isoforms have been
shown to display variable expression in vascular compared with visceral
SMCs (see e.g., Refs. 18, 45, 57, and 61). For example, dramatically
different isoforms of the actin-associated protein smoothelin are
expressed in chicken gizzard relative to blood vessels
(61). Smooth muscle -actin is highly expressed in adult
vascular smooth muscle but has recently been shown to be expressed at
significantly lower levels in adult visceral muscle (45).
Smooth muscle myosin heavy chain is widely expressed in smooth muscle
tissues (41); however, a 2.3-kb fragment of the promoter
region drives expression of a transgene predominantly in vascular
tissue (25). Although the immunostaining data are suggestive, it is not known whether the poly-Tmod antibody recognizes all 40-kDa Tmod isoforms with equal affinity. Thus the reduced staining
by this antibody in some vascular smooth muscles relative to visceral
smooth muscle may indicate either that Tmods are expressed in lower
levels in some vascular smooth muscles or that different Tmod isoforms
are expressed in specific parts of the vascular tree, or both.
Subcellular distribution of SM-Lmod, Tmod, and other contractile proteins in intestinal circularis smooth muscle. During smooth muscle contraction, the contractile actin and myosin become concentrated near the periphery of each SMC (see reviews, Refs. 4 and 48). The observation that SM-Lmod is found near actin filament bundles in the periphery of normally fixed intestinal circularis SMCs (Figs. 5 and 7) suggests that SM-Lmod is likely to be associated predominantly with contractile actin filaments. The more uniform distribution of Tmods in the same tissue may have several alternate explanations. First, Tmods may bind to the cytoskeletal as well as the contractile actin filaments of smooth muscle. Alternatively, the diffuse staining for Tmods may reflect the distribution of a soluble pool, because immunoblotting data indicate the presence of soluble Tmod in rabbit stomach smooth muscle (Fig. 3). Further experiments using extracted SMCs will be required to distinguish between these possibilities.
Visualization of poly-Tmod staining in a confocal microscope, rather than a standard fluorescence microscope, demonstrates that Tmod proteins are localized to discrete subcellular structures (Fig. 7, top right). These structures are not dense bodies, although they frequently occur near dense bodies stained for ![]() |
ACKNOWLEDGEMENTS |
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I thank Dr. Velia M. Fowler for generosity and support and Drs. Marian Ludgate and Jennifer Black for generous sharing of tissues and reagents.
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FOOTNOTES |
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C. A. Conley was the recipient of a National Institutes of Health National Research Service Award postdoctoral fellowship and is currently supported by the National Aeronautics and Space Administration Fundamental Biology program.
Address for reprint requests and other correspondence: C. A. Conley, Space Life Sciences, MS 239-11, NASA Ames Research Center, Moffett Field, CA 94035-1000 (E-mail: cconley{at}mail.arc.nasa.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 September 2000; accepted in final form 22 December 2000.
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