SPECIAL TOPIC
Leiomodin and tropomodulin in smooth muscle

Catharine A. Conley

Space Life Sciences, National Aeronautics and Space Administration Ames Research Center, Moffett Field, California 94035-1000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca2+, both SM-Lmod and Tmod are found near alpha -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 alpha -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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 at -80°C. Frozen stomach wall muscle was ground under liquid nitrogen, and the frozen powder was added to 10 volumes of ice-cold Ringer buffer (80 mM NaCl, 2 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.1% glucose, and 6 mM KHPO4, pH 7.0) containing protease inhibitors (100 mg/ml phenylmethylsulfonyl fluoride, 5 mg/ml each of leupeptin and pepstatin A, and 1 mg/ml aprotinin) and 3 mM ATP to reduce actomyosin aggregation. The mixture was pipetted up and down until the tissue slurry was thawed, sonicated briefly to fragment DNA, and filtered through several layers of cheesecloth to remove large tissue particles. Aliquots of the filtered slurry were added to equal volumes of cold Ringer buffer either alone or containing 2 M KCl or 4% Triton X-100 and then incubated on ice with occasional vortexing for 15 min. Solutions were centrifuged sequentially at 2,000 and 50,000 g, with aliquots removed at each step. Gel samples were produced from supernatant fractions by adding 5× SDS-PAGE sample buffer to a final concentration of 1×, and pellets were resuspended to the equivalent volume of Ringer solution before addition of 5× sample buffer. PAGE and protein immunoblots were performed as described above.

TM 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 alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Protein expression of smooth muscle leiomodin (SM-Lmod) and tropomodulins (Tmods) in rat tissues. Equal amounts of total protein from the tissues or tissue fractions indicated were blotted and probed for either SM-Lmod or Tmods, as indicated. Protein sizes are indicated (right). Abd Mus, abdominal muscle; EOM, extraocular muscle; Sm Int, small intestine.

As a comparison, blots of the same tissues were probed with affinity-purified polyclonal antibodies that recognize at least E-Tmod, Sk-Tmod, and N-Tmod (V. Fowler and A. Lee, personal communication). Tmod-sized bands (40 kDa) were observed in all tissues examined, including both smooth and striated muscles as well as nonmuscle tissues (Fig. 1). These data greatly expand the number of rat tissues in which Tmod proteins have been identified. The poly-Tmod antibody recognizes a 40-kDa band rather strongly in lung and uterus, where U-Tmod transcripts are highly expressed but where E-Tmod transcripts are expressed at very low levels (12); thus it is likely that this antibody recognizes U-Tmod as well as the other Tmod isoforms. These data indicate that the anti-Tmod antibody appears to be polyspecific for multiple Tmod isoforms, although probably with differing affinities for the various proteins. In contrast, the antibody raised against human SM-Lmod is specific for SM-Lmod and does not recognize C-Lmod or Tmods on protein immunoblots.

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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Fig. 2.   Biochemical extraction of SM-Lmod, Tmod, and tropomyosin (TM) from rabbit stomach muscle. A Coomassie-stained gel is presented (top) to show the amount of protein that was blotted and probed with the antibodies indicated. The single lane (left) shows a sample of total stomach muscle homogenate (Tot) before fractionation. Each set of 3 lanes shows homogenate extracted with buffered physiological salt alone (P) or containing 2% Triton X-100 (T) or 1 M KCl (K). The sets of lanes correspond to the 50,000 g supernatant (50K sup) and 50,000 g pellet (50K pel) of this extraction. Prominent bands in the pellet lanes are actin (40 kDa), tropomyosin (38 kDa), and myosin heavy chain (200 kDa), indicating that this pellet fraction contains a crude myofibril preparation. The Coomassie-stained band at ~70 kDa is not SM-Lmod. Protein size markers are indicated (left).

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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Fig. 3.   Binding of TM isoforms to SM-Lmod and E-Tmod by blot overlay. The Coomassie-stained gel (left) shows total protein from bacteria transformed with a control plasmid (pET16), a plasmid containing an expression construct of SM-Lmod (pET64), and purified SM-Lmod protein. Two duplicate blots of these samples and purified E-Tmod protein, probed with either brain (middle) or muscle TM (right), demonstrate that SM-Lmod binds muscle TM but not brain TM, while E-Tmod binds both muscle and brain TM.

To assess further whether native SM-Lmod binds to smooth muscle TM, I performed immunoprecipitation experiments using both the SM-Lmod and poly-Tmod antibodies on a rabbit stomach wall muscle preparation extracted under several buffer conditions (Fig. 4). The affinity-purified SM-Lmod antibody immunoprecipitated Coomassie-staining amounts of protein with the predicted size of SM-Lmod from muscle extracted with 2% Triton X-100 or RIPA (Fig. 4A, asterisks), which was recognized by affinity-purified SM-Lmod antibodies obtained from a different rabbit (Fig. 4B). The poly-Tmod antibody immunoprecipitated a single protein band at 40 kDa under all three buffer conditions, although Coomassie-staining amounts were apparently not obtained (Fig. 4, A and C). It appears that binding of the SM-Lmod antibody to SM-Lmod is inhibited by 1 M KCl, although binding of the poly-Tmod antibody was not reduced (Fig. 4, B and C).


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Fig. 4.   Immunoprecipitation of proteins from rabbit stomach smooth muscle by SM-Lmod and poly-Tmod antibodies. Rabbit stomach muscle extracted in physiological buffer containing 2% Triton X-100 (T) or 1 M KCl (K) or in RIPA buffer (R) was used for immunoprecipitation with SM-Lmod (SM-Lmod IP) or poly-Tmod (Tmod IP) antibodies. Samples of the immunoprecipitate supernatant (s) and bead pellet (b) were separated by SDS-PAGE and stained with Coomassie (A), which identifies a faint band with the expected size of SM-Lmod in the T and R lanes of the SM-Lmod IP (asterisks). Additional bead samples were blotted and probed with antibodies recognizing the proteins indicated (B and C). Protein size markers are indicated in A (left).

When blots of each immunoprecipitate were probed with an anti-TM antibody, TM was present in the SM-Lmod immunoprecipitate only in the 2% Triton extract (Fig. 4B), suggesting that native SM-Lmod does bind native TM in rabbit stomach smooth muscle but that this binding is disrupted by RIPA buffer. In contrast, TM was present in the Tmod immunoprecipitate only in the RIPA buffer extract (Fig. 4C). One possible explanation for this observation is as follows: in the 2% Triton extract, the only Tmod immunoprecipitated was soluble to begin with and not bound to TM in the pellet; in the 1 M KCl extract, both Tmod and TM were extracted from the pellet but did not bind to each other, while in the RIPA extract, the Tmod and TM were both extracted from the pellet and bound to each other either in vivo or subsequently. Alternatively, it is possible that the amount of TM coimmunoprecipitated with Tmod from the 2% Triton extract was simply below the level of detection of this blot and that the larger amount of Tmod obtained from the RIPA extract coimmunoprecipitated sufficient TM to be detected.

To test cross-reactivity of the SM-Lmod and poly-Tmod antibodies, blots of each immunoprecipitate were probed with the other antibody. When a blot of the SM-Lmod immunoprecipitate was probed with the poly-Tmod antibody, no 40-kDa Tmod was detected, indicating that the SM-Lmod immunoprecipitate did not contain detectable amounts of Tmod, and the poly-Tmod antibody did not immunoprecipitate SM-Lmod, indicating that these antibodies do not cross-react under native conditions (Fig. 4, B and C). These data also indicate that SM-Lmod and Tmod do not directly bind to each other at detectable levels under any of these conditions.

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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Fig. 5.   Immunofluorescence localization of SM-Lmod in rat tissues. Asterisks indicate small blood vessels. SM-Lmod staining is similarly intense in these blood vessels as in other smooth muscles in the same field of view. Tissues are adjacent serial sections to the corresponding panels in Fig. 6. Bars, 20 µm.



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Fig. 6.   Immunofluorescence localization of Tmods in rat tissues. Asterisks indicate small blood vessels. Tmod staining is less intense in some blood vessels than for other smooth or striated muscle in the same field of view. Tissues are adjacent serial sections to the corresponding panels in Fig. 5. Bars, 20 µm.

In contrast, staining by the poly-Tmod antibody was observed in some but not all of the smooth muscle tissues examined, while appearing in all striated muscles (Fig. 6). Poly-Tmod staining was observed in all visceral smooth muscles examined, as well as airway smooth muscle, extraocular smooth muscle, and a blood vessel in stomach wall muscle (Fig. 6). However, poly-Tmod antibodies labeled some blood vessels in bladder and uterus rather weakly and labeled cardiac vessels very weakly or not at all (Fig. 6).

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 alpha -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 alpha -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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Fig. 7.   Costaining of SM-Lmod, Tmods, or TM (as indicated) with alpha -actinin in longitudinal rat circularis smooth muscle. Color panels show merged images. Tmod staining is punctate and does not colocalize with alpha -actinin-staining dense bodies. SM-Lmod and TM staining is more uniform along the cells. Bar, 5 µm.

As an additional evaluation of the distribution of these proteins within the smooth muscle contractile apparatus, I stained sections of hypercontracted small intestine with phalloidin for actin filaments and antibodies recognizing alpha -actinin, SM-Lmod, poly-Tmod, and TM (Fig. 8). The phalloidin-stained actin filaments show distinct contraction bands, intense stripes of phalloidin staining that run perpendicularly through many SMCs (Fig. 8, left). Interestingly, alpha -actinin, SM-Lmod, and poly-Tmod antibodies all show a somewhat banded staining pattern as well (Fig. 8, right), but this staining is not overlaid with the actin contraction bands in the merged images (Fig. 8, middle). All three of these antigens are present at the periphery of the contraction bands, where the phalloidin staining is less intense. In contrast, the TM staining appears uniform along the hypercontracted SMCs (Fig. 8, bottom). These data indicate that alpha -actinin, Tmods, and SM-Lmod are concentrated at the edges of the contraction bands in hypercontracted smooth muscle, while TM is distributed more uniformly. Because SM-Lmod is present along the length of the filaments in normally fixed smooth muscle, these data suggest that SM-Lmod may be changing its subcellular distribution during hypercontraction.


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Fig. 8.   Costaining of alpha -actinin, SM-Lmod, Tmods, or TM (as indicated) with phalloidin-stained actin filaments in hypercontracted longitudinal rat circularis smooth muscle. Color panels show merged images. Actin contraction bands are visible as dense phalloidin-stained regions. alpha -Actinin, SM-Lmod, and Tmod staining is present only at the periphery of the contraction bands, while TM staining is uniform throughout. Bar, 10 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -actinin (Fig. 7, top). Because Tmods bind to the pointed ends of actin filaments in both striated muscle (2, 24) and nonmuscle tissues (56), this observation is strongly suggestive that the pointed ends of actin filaments are localized to discrete subcellular regions in smooth muscle, as well. Although this hypothesis has never before been suggested, additional support can be found in Small et al. (49) in their figures showing supercontracted smooth muscle fragments called "stars." In that report, supercontracted smooth muscle fragments appear as starlike structures, with alpha -actinin and myosin localized to the center of the structure and actin filament bundles protruding outward in all directions. Because dense bodies containing alpha -actinin are the anchor sites for the barbed ends of actin filaments, the pointed ends must be at the points of the star. Suggestively, the star actin filament bundles usually become narrow at the tips in fluorescent images (see Ref. 49, Figs. 1 and 3), and in electron micrographs of single stars, the tips of the actin filament bundles appear to contain multiple filaments with ends in close proximity (see Ref. 49, Figs. 5 and 6). The recognition that the pointed ends of actin filaments, as well as the barbed ends, could be localized to discrete subcellular regions may have significant implications concerning the structure and function of the smooth muscle contractile apparatus.

Contraction bands have been reported in the literature since the early 1900s, on the basis of both histological staining in the light microscope (see e.g., Refs. 38 and 44) and fluorescence labeling of specific contractile proteins (6, 17). Draeger et al. (17) demonstrated that contraction bands appear in isolated SMCs upon contraction and further showed that alpha -actinin staining and dense bodies are localized to the periphery of the actin-containing contraction bands in isolated cells. These data agree with the staining of alpha -actinin at the periphery of contraction bands in hypercontracted smooth muscle tissue (Fig. 8, top) and, together, they indicate that barbed ends of actin filaments are concentrated at the edges of the contraction bands. The demonstration that Tmod is also found at the periphery of the contraction bands in hypercontracted smooth muscle suggests that the pointed ends of the actin filaments could also be concentrated at the edges of the contraction bands. This may indicate that individual actin filaments span the contraction bands in individual SMCs. If so, by measuring the width of contraction bands it should be possible to obtain an estimate of the average length of actin filaments in hypercontracted smooth muscle. In preparations shown here (Fig. 8), contraction bands range from a few micrometers up to 10 µm in width, although it is possible that the wider bands include parts of more than one cell in a single contraction band. These dimensions are not inconsistent with the reported length for actin filaments in contracted cell fragments of 4.5 µm (49). It is important to note, however, that the length of actin filaments in hypercontracted cells may not at all reflect the length of actin filaments in relaxed cells, since actin filament polymerization has been shown to be required for contraction. Measurement of the distance between clusters of alpha -actinin and Tmod spots in the confocal images of normal smooth muscle (Fig. 7) gives a number closer to 2 µm. Electron microscope studies on relaxed and contracted smooth muscle fragments labeled for both ends of the actin filaments will undoubtedly be required to resolve this issue.

The observation that SM-Lmod costains along the length of contractile actin filaments in normally fixed smooth muscle (Figs. 5 and 7) but that it is present at the periphery of contraction bands in hypercontracted smooth muscle (Fig. 8) raises a number of possibilities concerning the role of SM-Lmod in smooth muscle and muscle contraction. One hypothesis is that SM-Lmod redistributes during muscle contraction such that it associates along actin filaments in relaxed muscle but is only associated with the ends of actin filaments, or is excluded from the contraction bands, in hypercontracted smooth muscle. Redistribution from the cytosol to the plasma membrane in contracting smooth muscle has been reported for calponin (42) and protein kinase C (36, 40). Analysis of the distribution of soluble vs. bound SM-Lmod in relaxed and hypercontracted cells would address this question.

An alternative but nonexclusive hypothesis is that SM-Lmod does not only associate with actin filaments but also binds to other components of the contractile apparatus that are redistributed during contraction. Several interesting possibilities are raised on consideration of the polyproline motif that is located in the carboxy terminus of SM-Lmod. In Drosophila, one of the tropomyosin genes displays muscle-specific alternative splicing to create a larger protein, producing a tropomyosin with a carboxy-terminal extension containing a polyproline motif (35). This heavy tropomyosin protein has been proposed to act as a link between actin thin filaments and myosin thick filaments, possibly transmitting tension directly to the regulatory troponin complex (43). Recently, it was shown that this carboxy-terminal extension is responsible for localizing glutathione-S-transferase to myofibrils in the indirect flight muscles of Drosophila (9). Although Lmod homologues are not present in Drosophila, an alternatively spliced form of the Drosophila Tmod homologue sanpodo was recently identified that contains an amino-terminal proline-rich extension (1; GenBank accession no. AAF57067). It could be that, in vertebrates, SM-Lmod acts as a link between smooth muscle thin filaments and other components of the contractile apparatus. Biochemical analyses to identify binding partners for the Lmods are a promising area for future research.


    ACKNOWLEDGEMENTS

I thank Dr. Velia M. Fowler for generosity and support and Drs. Marian Ludgate and Jennifer Black for generous sharing of tissues and reagents.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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