Journal of Cell Science 112, 1111-1123 (1999) 1111 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0232

Tropomodulin assembles early in myofibrillogenesis in chick : evidence that thin filaments rearrange to form striated myofibrils

Angels Almenar-Queralt1, Carol C. Gregorio2 and Velia M. Fowler1,* 1Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 2Departments of Cell Biology and Anatomy, and Molecular and Cellular Biology, The University of Arizona, Tucson, AZ 85724, USA *Author for correspondence (e-mail: [email protected])

Accepted 10 February; published on WWW 23 March 1999

SUMMARY

Actin filament lengths in muscle and nonmuscle cells are dots often appear to be located between the closely spaced, believed to depend on the regulated activity of capping dot-like Z bodies that are stained for α-. Thus, in the at both the fast growing (barbed) and slow earliest premyofibrils, the pointed ends of the thin filaments growing (pointed) filament ends. In striated muscle, the are clustered and partially aligned with respect to the Z pointed end capping , tropomodulin, has been bodies (the location of the barbed filament ends). At later shown to maintain the lengths of thin filaments in mature stages of differentiation, the tropomodulin dots become myofibrils. To determine whether tropomodulin might also aligned into regular periodic striations concurrently with be involved in thin filament assembly, we investigated the the appearance of striated phalloidin staining for F- assembly of tropomodulin into myofibrils during and alignment of Z bodies into Z lines. Tropomodulin, differentiation of primary cultures of chick skeletal muscle together with the barbed end capping protein, CapZ, may cells. Our results show that tropomodulin is expressed early function from the earliest stages of myofibrillogenesis to in differentiation and is associated with the earliest restrict the lengths of newly assembled thin filaments by premyofibrils which contain overlapping and misaligned capping their ends; thus, transitions from nonstriated to actin filaments. In addition, tropomodulin can be found in striated myofibrils in skeletal muscle are likely due actin filament bundles at the distal tips of growing principally to filament rearrangements rather than to myotubes, where sarcomeric α-actinin is not always filament polymerization or depolymerization. detected, suggesting that tropomodulin caps actin filament Rearrangements of actin filaments capped at their pointed pointed ends even before the filaments are cross-linked into and barbed ends may be a general mechanism by which Z bodies by α-actinin. Tropomodulin staining exhibits an cells restructure their actin cytoskeletal networks during irregular punctate pattern along the length of cell growth and differentiation. premyofibrils that demonstrate a smooth phalloidin staining pattern for F-actin. Strikingly, the tropomodulin Key words: Tropomodulin, Actin, Myofibril, Muscle

INTRODUCTION Fowler, 1996, 1997; Littlefield and Fowler, 1998). In vitro, barbed and pointed end actin capping proteins can regulate Actin filaments are polymerized to strikingly uniform lengths filament elongation and depolymerization by nucleating new in a variety of elaborate supramolecular structures in filament assembly as well as by inhibiting monomer differentiated cells. Some dramatic examples include the ~1 association and dissociation from filament ends (Schafer and µm long thin filaments in striated muscle sarcomeres, the ~40 Cooper, 1995; Mullins et al., 1998; Weber et al., 1994). nm long actin filaments in the erythrocyte membrane skeleton, However, in vivo, the relative contributions of barbed and the ~2.5 µm long actin filament bundles in intestinal epithelial pointed end capping proteins to the initiation, regulation of cell microvilli, and the ~10 µm long actin filament bundles in rates and termination of filament growth are not well Drosophila cuticular bristles (for reviews, see Fowler, 1996; understood. Mooseker, 1985; Robinson and Cooley, 1997). Capping of the Striated muscle is an excellent system to address these fast growing (barbed) and slow growing (pointed) actin questions because both the barbed (CapZ) and pointed filament ends is believed to be important both for regulation of (tropomodulin) end capping proteins for the actin filaments filament assembly into these specialized architectural arrays, have been identified (Fowler, 1996), unlike other systems that and for stabilization of uniform filament lengths once the demonstrate uniform actin filament lengths. Furthermore, the filaments have been assembled (for recent discussions, see ~1 µm long thin filaments in muscle are organized into regular, 1112 A. Almenar-Queralt, C. C. Gregorio and V. M. Fowler repeating, contractile units called sarcomeres whose periodic over a period of several days (Cappers, 1960; Cooper and (striated) organization can be easily visualized by light Konigsberg, 1961; Stockdale and Holtzer, 1961). Here, we microscopy. In particular, thin filaments are aligned in an anti- have investigated the spatial and temporal distribution of parallel, polarized fashion in each sarcomere with the barbed tropomodulin during myofibril assembly with respect to other filament ends at the Z line and the pointed filament ends in the thin filament proteins. middle of the sarcomere (Huxley, 1960). Our results demonstrate that tropomodulin is associated with CapZ is believed to function early in myofibril assembly to the first nonstriated premyofibrils that appear during myofibril nucleate actin filament assembly, and to target and align the assembly, including actin filament bundles found at the barbed ends of the thin filaments at the Z line (i.e. to establish extreme distal tips of young growing myotubes. Furthermore, filament polarity). This idea was based initially on association tropomodulin staining along nonstriated premyofibrils appears of CapZ with nascent myofibrils in both skeletal and cardiac as an irregular pattern of dots which are often located between myocytes (Schafer et al., 1993, 1994). In a subsequent study, closely spaced dots of α-actinin, a marker for nascent Z bodies inhibition of CapZ’s actin capping activity in skeletal myotubes where the barbed ends of the thin filaments are located. The by microinjection of anti-CapZ antibodies or by dominant presence of both tropomodulin and CapZ along early negative transfection approaches resulted in disruption of nonstriated premyofibrils indicates that both the pointed and nonstriated premyofibrils or delays in formation of actin barbed filament ends of the thin filaments are capped from the striations, respectively (Schafer et al., 1995). initial stages of myofibril assembly in skeletal muscle cells. We In contrast, several experimental approaches indicate that conclude that transitions from nonstriated precursors to mature tropomodulin functions late in myofibril assembly to maintain striated myofibrils may be principally due to thin filament actin filament lengths and thin filament stability in mature rearrangements and alignments rather than to processes myofibrils in cardiac myocytes. Microinjection of an antibody involving extensive elongation or depolymerization of which inhibits tropomodulin’s actin capping activity into chick filaments. Restructuring the actin by filament cardiac myocytes leads to continuous (nonstriated) actin rearrangements may be a general mechanism for gradual filament staining along myofibrils, due to inappropriate remodelling of the cytoskeleton during cell growth and elongation of actin from the pointed ends of the thin filaments differentiation. (Gregorio et al., 1995). These cells are no longer capable of beating, demonstrating that maintenance of actin filament length by tropomodulin is required for contraction. MATERIALS AND METHODS Furthermore, recent experiments altering tropomodulin levels by infection with sense or antisense adenovirus vectors reveal Cell culture that reduction of tropomodulin levels in neonatal rat cardiac Skeletal muscle cultures were prepared from day 12 chick embryonic myocytes leads to disappearance of mature striated myofibrils pectoralis muscle basically as described (Nawrotzki et al., 1995; and accumulation of nonstriated actin filament bundles, Gilbert et al., 1996). For the biochemical studies, cells were plated at whereas increases in tropomodulin leads to a deficit of 2×105 cells/ml on collagen-coated 35 mm tissue culture dishes. For × 5 myofibrils together with myofibril disarray (Sussman et al., immunofluorescence analysis, cells were plated at 5 10 cells/ml on 1998a). In transgenic mice, tropomodulin overexpression in the collagen- (Sigma, St Louis, MO) or Matrigel- (Beckton Dickinson) coated glass coverslips. The same results were obtained with either myocardium after birth leads to development of dilated coating. Collagen was prepared as a 0.01% (w/v) solution in 0.1% cardiomyopathy (Sussman et al., 1998b). acetic acid. Autoclaved coverslips were coated with collagen for 2 The idea that tropomodulin functions to maintain thin hours at 37°C. Excess collagen solution was removed by several filament lengths once they have been assembled was originally washes in Dulbecco’s PBS (0.136 M NaCl, 2.6 mM KCl, 1.4 mM suggested by the absence of tropomodulin in nonstriated KH2PO4, 8 mM NaHPO4, pH 7.4). Alternatively, coverslips were myofibrils in primary cultures of embryonic chick cardiac coated with a drop of Matrigel for 2 hours at room temperature, excess myocytes (Gregorio and Fowler, 1995). Tropomodulin is one liquid was aspirated and the coverslips were dried before use. of the latest markers of myofibril assembly in this cell type, The cells were fed 24 hours after plating and then every other day and is only found assembled into myofibrils after the thereafter with Dulbecco’s modified Eagle’s medium (Gibco BRL, appearance of actin filament striations; i.e. after acquisition of Grand Island, NY), supplemented with 10% (v/v) ‘selected’ fetal bovine serum (Hyclone, Logan, Utah), 4% (v/v) chick embryo extract mature thin filament lengths. However, since primary cultures and 1% (v/v) antibiotics-antimycotics (Gibco BRL). On day three, the of cardiac myocytes isolated from hearts of day 6 chick medium was supplemented with 1 µg/ml cytosine β-D- embryos do not differentiate de novo in culture, the nature of arabinofuranoside (Sigma) to limit proliferation of contaminating myofibril assembly precursors and intermediates in this cell fibroblasts. Embryonic chick cardiac myocyte cultures were prepared model are somewhat controversial (e.g. see Dlugosz et al., from day 6 embryonic chick hearts (Gregorio and Fowler, 1995). 1984; Lin et al., 1989; Rhee et al., 1994). It thus remains an open question as to whether tropomodulin Antibodies might play a role in the assembly of actin filaments into muscle Monoclonal antibodies to tropomodulin were generated using sarcomeres, in addition to its role in maintaining filament recombinant chicken tropomodulin (E-Tmod) (Babcock and Fowler, length in the mature structures. To investigate this, we used 1994) as an antigen (Chris W. Grant, Custom Monoclonals, W. Sacramento, CA) and were purified over a Protein G-Sepharose primary cultures of chick skeletal myogenic cells, an column (Pharmacia, Upsala, Sweden). Monoclonal antibody 95 (mAb experimental system in which myoblasts differentiate de novo 95) was used for immunofluorescence staining; the specificity of this and fuse to form multinucleated myotubes in a relatively antibody is demonstrated in Fig. 1 using western blot analysis and synchronous manner. In these cells, contractile proteins are immunoprecipitation of [35S]methionine labelled proteins from day 5 synthesized coordinately and assembled into mature myofibrils chick skeletal myotubes. Monoclonal antibody 23 (mAb23) was used Tropomodulin assembly in skeletal muscle 1113 for the western blotting analysis of tropomodulin isoforms on 2-D gels the prints: ≥100 measurements were made from prints of the central since it recognizes both the E and Sk-tropomodulin isoforms present regions (shafts) of the myotubes. in chicken striated muscle (Almenar-Queralt et al., 1997). Polyclonal antibodies to recombinant chicken E-Tmod (Babcock and Fowler, Metabolic labelling, immunoprecipitation and 1994) were generated in rabbits and affinity purified by standard electrophoresis procedures procedures (Fowler and Adam, 1992) and characterized for specificity Myotubes were metabolically labelled for 3 hours with 200 µCi/ml of as for mAb 95; it recognized a single ~40 kDa polypeptide (data not Trans(35S)-label (ICN Pharmaceuticals, Inc., Irvine, CA) and proteins shown). The monoclonal anti-sarcomeric α-actinin antibody (clone were solublized and prepared for immunoprecipitation according to 9A2B8; Schultheiss et al., 1990) was a gift from Drs S. and H. Holtzer the method of Gregorio and Fowler (1995). 35S-labelled tropomodulin (University of Pennsylvania, Philadelphia, PA). Bodipy-labelled was immunoprecipitated from the extracts by addition of 2 µg of phallacidin, fluorescein conjugated-goat anti-rabbit IgG, rhodamine mAb95 that had been preadsorbed to Protein A Trisacryl beads (TRITC)-conjugated donkey anti-mouse IgG and Cy2-conjugated (Pierce) coated with rabbit anti-mouse IgG+IgM (H+L) (Pierce), goat anti-rabbit antibodies were purchased from Molecular Probes electrophoresed on 12% acrylamide mini-gels (Laemmli, 1970; Inc. (Eugene, OR), Boehringer Mannheim Biochemicals Fowler, 1990), and gels were dried down and exposed to film by (Indianapolis, IN), Accurate Chemical and Scientific Corporation standard procedures. For western blotting, gels were transferred to (Westbury, NY), and Pierce (Rockford, IL), respectively. mAb 95 was nitrocellullose membranes (Schleicher & Schuell, Keene, NH) conjugated with Alexa using an Alexa-594 labelling kit from (Towbin et al., 1979), and then incubated for 2 hours at room Molecular Probes, Inc. temperature with monoclonal anti-tropomodulin mAb 95 (10 µg/ml) (Fig. 1) followed by rabbit anti-mouse IgG+IgM (H+L) (1:1000) Immunofluorescence microscopy (Pierce, Rockford, IL) and 125I-Protein A (Fowler, 1990; Fowler et al., Skeletal muscle cells were fixed and stained as described (Gregorio 1993). and Fowler, 1995). To permeabilize the cells and minimize The tropomodulin isoform composition in skeletal and cardiac nonspecific binding of antibodies, the coverslips were pre-incubated myogenic cultures was characterized by 2-dimensional (2-D) gel in PBS containing 3% bovine serum albumin (BSA)(ICN, Aurora, electrophoresis followed by western blotting with mAb 23 (see OH) and 0.2% Triton X-100 for 30 minutes. For double staining of above). Briefly, chick skeletal and cardiac myogenic cells were tropomodulin with F-actin, cells were incubated with mAb 95 at 2 washed with ice-cold PBS plus protease inhibitors (Gregorio and µg/ml, followed by rhodamine-conjugated donkey anti-mouse IgG Fowler, 1995) and then extracted with 9.2 M urea plus 1% (v/v) β- (1:200) and bodipy-phallacidin (1:200). For double staining of mercaptoethanol for 30 minutes at room temperature on a rocking tropomodulin and α-actinin, cells were incubated with rabbit anti- platform. The total extract was collected and then centrifuged at chicken E-Tmod (2 µg/ml) and the monoclonal anti-α-actinin 15,000 g for 10 minutes. The resulting supernatants were antibody (1:400), followed by rhodamine-conjugated donkey anti- electrophoresed on two-dimensional gels (O’Farrell, 1975; as mouse IgG and fluorescein-conjugated goat anti-rabbit IgG (1:200). modified by Granger et al., 1982). The tube gels (worms) for the first Alternatively, cells were first incubated with Alexa-conjugated dimension contained 2% Ampholines, pH range 5-7, 1% Ampholines, mAb95 followed by incubation in normal mouse serum (1:50), then pH range 4-6 and 0.2% Pharmalytes, pH range 3-10 (Pharmacia). For incubated in the monoclonal anti-α-actinin antibody followed by Cy2- the second dimension, the worms were run on 7.5% to 15% linear conjugated goat anti-mouse antibodies (1:400). Stained coverslips gradient acrylamide gels, pH 8.6 (Laemmli, 1970; Fowler, 1990). were observed using a ×63 (1.4 NA) Plan-Apochromat or a ×100 (1.3 Nitrocellulose blots were probed with mAb 23 as described above. NA) Plan Neofluar objective lens on a Zeiss Axioskop microscope equipped with filters that exclude crossover between fluorochromes used. Digital images were acquired with a Princeton Instruments RESULTS 1317K CCD camera with a KAF 1400 chip (pixel size, 6.8 µm2) or a Princeton Instruments 1300Y CCD camera with a Sony Interline chip (pixel size, 6.7 µm2), using IP lab software. Images were Thick and thin filaments reorganize during myofibril processed for presentation using Adobe Photoshop. For some images, assembly in skeletal muscle cultures we applied a high pass filter to reduce the background. Separate To provide a framework for subsequent interpretation of our images of double labelled specimens were merged and aligned using biochemical and immunofluorescence analysis of Adobe Photoshop by superimposition of asymmetric identical tropomodulin assembly into myofibrils we, like others, used features (e.g. the edge of the cell, intersecting myofibrils); images thin section electron microscopy to analyze myofibril assembly captured the same day had a constant offset within a pixel. In some in our cultures of chick skeletal muscle myotubes (e.g. Antin experiments (indicated in the Fig. legends), images were acquired on et al., 1986; Shimada et al., 1967; for a recent review, see a Bio-Rad MRC 600 Confocal Microscope. Similar results were obtained by acquiring images with a CCD camera, confocal Franzini-Armstrong and Fischman, 1996). Examination of microscope or by recording images on film. myotubes from different days in culture shows that thin and thick filaments are present in bundles from the earliest days in Electron microscopy culture, but that a dramatic change in filament organization Myotubes grown on collagen-coated plastic tissue culture dishes were appears to take place during differentiation (Fig. 2). First, in fixed and processed for electron microscopy at different days in young myotubes in early cultures (~days 2-3), premyofibrils culture as previously described (Tilney and Tilney, 1994). Ultrathin which consist of loose bundles of thin (actin) filaments sections were cut parallel to the plate, mounted on grids, and stained interspersed between thick () filaments are observed all again with uranyl acetate and lead citrate (Reynolds, 1963). Sections along the length of the myotubes (Fig. 2A). The thin filaments were examined at 70 kV on a Hitachi HU600 electron microscope and insert into darkly stained dense bodies (Z bodies) which are photomicrographs taken at several different magnifications. Distances between Z bodies along premyofibrils or Z lines along myofibrils from irregularly distributed along the premyofibrils (Fig. 2A, long early and late days in culture, respectively, were analyzed from prints arrows); this, together with the misalignments of thin and thick of micrographs at ×20,000 to ×25,000 magnification. Straight lines filaments, accounts for the nonstriated appearance of were drawn between the centers of adjacent Z bodies (or Z lines), premyofibrils by immunofluorescence staining (see below, Fig. parallel to the long axis of the myofibrils, and measured directly on 3). Adjacent Z bodies are spaced at distances ranging from 0.6 1114 A. Almenar-Queralt, C. C. Gregorio and V. M. Fowler

long arrows) and the thick filaments become fairly well aligned between adjacent clusters of Z bodies (Fig. 2B, short arrows). At this stage, the M line is apparent in the center of the thick filament array in some sarcomeres (Fig. 2B, arrowheads). The lengths of the I bands are very variable in these intermediate cultures; some of the thick filament ends are almost juxtaposed to the Z bodies, while in other cases they are more distant (Fig. 2B, bracket). Finally, in later cultures (≥day 5), many myotubes start to contract spontaneously (twitch), myofibrils grow in length and width, and the thin and thick filaments become aligned into mature sarcomeres with well defined Z lines and regular I bands along the shaft of the long myotubes (Fig. 2C). High magnification micrographs reveal that the thin filaments from opposite Z lines overlap in the middle of the sarcomere (Fig. 2D, arrowheads), thus an H zone (gap in the middle of the sarcomere) is not present. While thin filament pointed ends cannot be discerned with certainty in these thin sections, the striated fluorescent staining patterns for tropomodulin and phalloidin indicate that thin filaments are likely to be between about 1 and 1.1 µm long in the mature sarcomeres (Fig. 3E,F), in agreement with previous estimates (Allen and Pepe, 1965; Fischman, 1967; Ohtsuki, 1979). It is not known if thin filament overlaps at the pointed ends in the sarcomeres are due to contraction during fixation or are the normal state of the mature striated myofibrils in cultured chick myotubes. These ultrastructural features of myofibril assembly are in agreement with previous studies of primary cultures of embryonic chick skeletal myotubes (Antin et al., 1986; Fig. 1. Monoclonal anti-tropomodulin antibody 95 (mAb95) Shimada et al., 1967; Shimada and Obinata, 1977), and closely specifically recognizes tropomodulin in chick skeletal muscle cells. resemble the ultrastructural features of myofibril assembly in SDS-extracts from day 5 embryonic chick skeletal myogenic cultures were electrophoresed on SDS-polyacrylamide gels and stained with developing vertebrate skeletal muscle in vivo (Allen and Pepe, Coomassie Blue (Coomassie, lane 1) or transferred to a 1965; Allen, 1973; Auber, 1969; Fischman, 1967; Kelly, 1969). nitrocellulose membrane and labelled with mAb95, followed by rabbit anti-mouse antibodies and [125I]-Protein A (Western, lane 2). Tropomodulin is associated with premyofibrils in a Cells from a day 5 culture were also metabolically labeled with punctate pattern and becomes striated concurrently [35S]methionine and tropomodulin was immunoprecipitated with with actin during myofibril assembly mAb95 (IP, lane 3). The positions of myosin and actin are indicated We used immunofluorescence staining to visualize when and on the left side of Lane 1, and the arrowhead in lane 3 indicates the where tropomodulin was assembled into myofibrils with position of tropomodulin (Tmod) (~40 kDa). respect to thin filament alignments during skeletal muscle differentiation. Phallacidin staining for F-actin was used to to 2.8 µm apart along the long axis of the premyofibrils; thus, reveal the location of thin filaments and whether they were many of them are considerably closer together than are Z lines organized into early nonstriated (premyofibril) or striated of mature myofibrils (2.2±0.15 µm). Occasionally, in early myofibrils (Epstein and Fischman, 1991; Franzini-Armstrong cultures, what appear to be nascent sarcomeres are observed in and Fischman, 1996; Gregorio and Fowler, 1996; Holtzer et al., which small groups of 1.6 µm long thick filaments are partially 1997). In young myotubes in early days of culture (e.g. day 2), aligned and positioned between adjacent Z bodies along the tropomodulin staining is already associated with nonstriated long axis of the premyofibril (Fig. 2A, short arrows). Thin premyofibrils running parallel to the long axis of the cells (Fig. filaments emerging from adjacent Z bodies appear to overlap 3A, arrows). At this early stage, the bodipy-phallacidin staining with one another; however, their lengths cannot be measured of premyofibrils is smooth and continuous (Fig. 3B, arrows), because the filaments in the loosely packed premyofibrils are consistent with the overlapping and misaligned actin filaments not perfectly straight and often leave the plane of section in the premyofibrils observed by thin section electron (Fischman, 1967, Allen and Pepe, 1965). microscopy (Fig. 2A). In contrast, tropomodulin staining is By days ~3-4 of culture, most cells have fused to form distributed in an irregular, finely punctate pattern along the myotubes and structures resembling sarcomeres appear more length of the identical premyofibrils (Fig. 3A, arrows). frequently along the central portion (the shaft) of the myotubes Later in culture, in slightly older myotubes (e.g. day 4), (Fig. 2B). However, it is common to find myofibrils at tropomodulin staining along the shafts of the myotubes now apparently earlier stages of assembly adjacent to the more appears as wider bands that are spaced ~1.8-2.0 µm apart in a striated myofibrils (Fig. 2B, asterisk), as well as at the tips of regular, periodic pattern along the myofibrils (Fig. 3C, myotubes (data not shown and see below). Z bodies are brackets). The bodipy-phallacidin staining of individual clustered in groups, often forming irregular Z lines (Fig. 2B, myofibrils is now somewhat uneven in appearance along their Tropomodulin assembly in skeletal muscle 1115 length (Fig. 3D, arrows) (also see McKenna et al., 1986; Sasaki myotubes now appear narrower and crisper and farther apart and Kuroda, 1994). It is not clear what the relationship of (~2.3 µm), taking on the typical striated appearance for uneven phallacidin staining is to the periodic bands of tropomodulin staining in mature myofibrils (Fig. 3E, arrows) tropomodulin staining. (Fowler et al., 1993). The inability to resolve tropomodulin In yet older myotubes later in culture (e.g. day 7), when the staining as two distinct stripes in each half sarcomere is due to myotubes have spontaneously begun to twitch, the bands of the overlapping of thin filament ends in the slightly contracted tropomodulin staining along myofibrils in the shaft of the sarcomeres typical of these myotubes (Fig. 2D) (also see

Fig. 2. Ultrastructural analysis of the organization of thick and thin filaments during myofibrillogenesis in primary cultures of embryonic chick skeletal myogenic cells. Electron micrographs of longitudinal sections of myofibrils in myotube shafts (i.e. the central portion of myotubes) from Early (A), Intermediate (Int., B) and Late (C,D) cultures (days, 2, 4, and 6, respectively). Long arrows, Z bodies or Z lines (A-C). Short arrows, thick filaments (A-C). Arrowheads, M lines (A-C); thin filaments (D). Brackets, I bands (B,C). Asterisk, a myofibril at an apparently earlier stage of assembly (B). Bars, 0.5 µm (A-C); 0.2 µm (D). 1116 A. Almenar-Queralt, C. C. Gregorio and V. M. Fowler

Fig. 3. The distribution patterns of tropomodulin change concurrently with actin filament reorganization during myofibril assembly. Fluorescence micrographs of mAb95 staining for tropomodulin (A,C,E) and phallacidin staining for F-actin (B,D,F) in chick skeletal myotube shafts from Early (A,B), Intermediate (Int., C,D) and Late (E,F) days of culture (days 2, 4, and 7, respectively). Arrows, premyofibrils (A,B,D). Brackets, tropomodulin bands (C). Arrows, mature tropomodulin striations (E); mature phallacidin striations (F) at the thin filament pointed ends. Arrowheads, phallacidin staining of thin filament barbed ends at the Z line (F). C and D are confocal micrographs. Bar, 10 µm.

Gregorio and Fowler, 1995). The tropomodulin striations also GFP-α-actinin (McKenna et al., 1986; Sanger et al., 1986; grow in length perpendicularly to the long axis of the Dabiri et al., 1997). myofibrils (Fig. 3E), reflecting the growth in myofibril width and increasing lateral organization of adjacent myofibrils that Clusters of thin filament pointed ends is also evident in thin section electron micrographs (Fig. 2C). (tropomodulin) appear to alternate with Z bodies (α- Interestingly, bodipy-phallacidin also stains mature actinin) along premyofibrils myofibrils in a pattern of narrow bright stripes (Fig. 3F, arrows) To investigate if the distribution of tropomodulin dots along which colocalize with the tropomodulin stripes in merged premyofibrils (clusters of thin filament pointed ends) might be images (not shown). In addition, a fainter stripe of phallacidin complementary to the distribution of Z bodies, (the presumed staining is present between the bright striations (Fig. 3F, location of the barbed filament ends) we used antibodies to α- arrowheads), corresponding to the location of the Z line, as actinin as a marker for Z bodies. α-Actinin cross-links actin demonstrated by colocalization with α-actinin (not shown). filaments at their barbed ends and is a classic marker for Preferential staining of thin filament pointed and barbed ends nascent Z bodies (Holtzer et al., 1997; Mckenna et al., 1986; by phalloidin (or phallacidin) has been observed previously in Sanger et al., 1986). For this experiment, myotubes from day mature myofibrils in cultured chick skeletal muscle cells (Antin 2 in culture were double stained with a rabbit polyclonal et al., 1986; Sasaki and Kuroda, 1994) as well as in isolated antibody to tropomodulin (Fig. 4A) and a monoclonal antibody myofibrils (Ao and Lehrer, 1995; Zhukarev et al., 1997 and to α-actinin (Fig. 4B). As described above for the references therein). This has been proposed to be due to the tropomodulin monoclonal antibody (Fig. 3A), tropomodulin presence of nebulin along thin filaments (Ao and Lehrer, 1995). staining is detected in a discontinous, punctate pattern along These immunofluorescence staining results demonstrate that the myofibrils. α-Actinin is also present in a distinct punctate tropomodulin is associated with early nonstriated premyofibrils pattern irregularly distributed along premyofibrils (Fig. 4B), as as well as with mature myofibrils, and implies that the thin expected from the irregular distribution of Z bodies along filament pointed ends are capped by tropomodulin at all stages premyofibrils at early stages in culture (Fig. 2A). Comparison of myofibril assembly during skeletal muscle differentiation. of red (α-actinin) and green (tropomodulin) staining in the Further, the irregular dot-like pattern of tropomodulin staining merged image (Fig. 4C) reveals that in general there is at early stages of differentiation suggests that tropomodulin- relatively little overlap (yellow) between the tropomodulin and capped pointed ends are clustered in regions along the α-actinin staining in early premyofibrils, except in regions premyofibrils. The changes in tropomodulin staining patterns where several myofibrils are superimposed or closely observed in myotubes on later days of culture (Fig. 3B,C) juxtaposed to one another (Fig. 4C, asterisks). In areas where suggest a temporal sequence of myofibril assembly in which single myofibrils can be clearly discerned, short stretches of the tropomodulin dots in premyofibrils coalesce and align to alternating red and green dots are frequently visible (Fig. 4C, form broader bands, followed by a transition to narrow stripes brackets). In some cases, gaps in between the individual in mature myofibrils. Such a rearrangement of pointed end tropomodulin or α-actinin dots are evident (Fig. 4C, left clusters would be similar to rearrangements of Z bodies bracket), whereas in other cases the red and green dots are observed by thin section electron microscopy (Fig. 2) (Kelly, immediately adjacent to one another (Fig. 4C, bottom right 1969), and by live cell microscopy of fluorescently-labelled or bracket). Interestingly, the distance between the α-actinin (or Tropomodulin assembly in skeletal muscle 1117 tropomodulin) dots varies from ~1.0 µm up to ~1.7 µm, in the 3), punctate staining for tropomodulin is detected along actin regions where separate red and green dots can be clearly filament bundles that extend all the way to the tips of the discerned (Fig. 4A-C, brackets). In comparison, the distances myotubes and stain continously with phallacidin (Fig. 5A,B). between α-actinin (or tropomodulin) striations in mature While not visible in Fig. 5, tropomodulin may also be striated myofibrils are considerably longer and more regular, associated with cortical actin filaments underlying the plasma at ~2.3 µm (Fig. 4D-F, brackets). membrane, based on our recent observation that tropomodulin We also attempted to directly compare the location of barbed is also localized on the sarcolemma in adult skeletal muscle and pointed ends by double staining myotubes for (A. Almenar-Queralt and V. M. Fowler, unpublished tropomodulin and CapZ. Unfortunately, we were unable to observations). reach a conclusion due to high background staining with the Double staining for tropomodulin and α-actinin available polyclonal and monoclonal antibodies to CapZ (data demonstrates that tropomodulin staining invariably extends not shown; also see Schafer et al., 1993). all the way to the distal ends of the premyofibrils at the tips Assuming that barbed ends are associated with Z bodies, the of the young myotubes (Fig. 6A,C, short arrows). However, alternating pattern of tropomodulin and α-actinin stained dots α-actinin staining extends to the tips of the cells only in some indicates that actin filament pointed and barbed ends are not (Fig. 6B) but not other (Fig. 6D) young myotubes; in this randomly distributed along the length of the premyofibrils. case, the α-actinin staining terminates well before the end of This in turn suggests that thin filaments whose barbed ends are the myotube (Fig. 6D; compare long arrow marking the end inserted into Z bodies are restricted to a relatively narrow range of α-actinin staining with short arrow marking the tip of cell). of lengths. By contrast, if the filaments were polymerized to a Myotube tips which are tropomodulin-positive but α-actinin- wide range of random and variable lengths, then the location negative represent about 30% of the population of myotube of the clusters of pointed ends would be unlikely to have had tips from these early days of culture. Double staining for α- any particular spatial relationship to the locations of Z bodies actinin and actin also demonstrates that α-actinin staining along the premyofibrils. does not always extend to the distal tips of phallacidin stained actin filament bundles in young myotubes (not shown). Tropomodulin is associated with nonstriated In contrast to the absence of α-actinin from the distal ends premyofibrils at the tips of growing myotubes, even of premyofibrils in some myotubes from early days of culture, in the absence of α-actinin α-actinin and tropomodulin are both detected all the way to the We also compared the distribution patterns of tropomodulin distal ends of the myofibrils in most myotubes (>90%) and α-actinin at myotube tips, which are believed to be sites obtained from late days of culture (e.g. days 5-7) (Fig. 6E, of new sarcomere addition during myofibril elongation tropomodulin; F, α-actinin). Note that the closely spaced dots (Holtzer et al., 1997; Peng et al., 1981; Sanger et al., 1986). In of α-actinin and tropomodulin in the distal region of a young myotubes obtained from early days in culture (days 2- myofibril at the tip of the myotube (Fig. 6E,F, short arrows) are

Fig. 4. The pointed and the barbed ends of actin filaments are not randomly distributed along nonstriated premyofibrils. Fluorescence micrographs of chick skeletal myotube shafts on early (A-C) and late (D-F) days of culture, fixed and double-stained for tropomodulin (A,D) and α- actinin (B,E). The merged images of tropomodulin (green) and α- actinin (red) are shown in C and F. Brackets, positions of α-actinin dots (A-C); positions of α-actinin stripes at the Z line (D-F). Asterisks, overlaps of 2 or more adjacent premyofibrils (C). Staining for tropomodulin was with a polyclonal rabbit antibody. D-F are confocal micrographs. Bar, 5 µm. 1118 A. Almenar-Queralt, C. C. Gregorio and V. M. Fowler

organization at the tips of myotubes containing mature striated myofibrils may not be the same as in immature myotubes containing nonstriated premyofibrils. One possibility is that the termini of mature striated myofibrils are adhesion plaques required for stable sites of contact with the substratum, rather than lamellipodial-like extensions responsible for myotube elongation in young myotubes. Exploration of this idea will require further study. Embryonic chick skeletal and cardiac muscle cells express the same isoform of tropomodulin (E-Tmod) Our previous study with embryonic chick cardiac myocytes demonstrated that tropomodulin is not detected along nonstriated actin filament bundles in these cells and is Fig. 5. Tropmodulin is associated with actin filaments at the distal assembled late during myofibril assembly, after myofibrils ends of premyofibrils at myotube tips. Fluorescent micrographs of have become striated (Gregorio and Fowler, 1995). To chick skeletal myotube tips from early cultures (day 3) fixed and investigate whether the expression of a different tropomodulin double stained for tropomodulin with mAb95 (A) and for F-actin with isoform during early skeletal myofibrillogenesis could account phallacidin (B). Tropomodulin extends all the way along the actin for these differences, we examined the isoform composition of filament bundles to the distal tip of the cell (arrows). Bar, 10 µm. tropomodulin by 2-D gel electrophoresis followed by western blotting with a monoclonal antibody that recognizes the two tropomodulin isoforms that have been identified in chicken continuous with mature striated myofibrils containing periodic striated muscles (E-Tmod and Sk-Tmod) (Almenar-Queralt et α-actinin and tropomodulin striations (Fig. 6E,F, arrowheads). al., 1997). The results from this experiment demonstrate that a Interestingly, the transition between striated and punctate single isoform of tropomodulin is present in early (day 2) and staining patterns is fairly abrupt, as reported previously late (day 7) skeletal cultures, as well as in cardiac myocytes (Holtzer et al., 1997; Peng et al., 1981). In higher magnification (Fig. 7a-c). Co-electrophoresis of either day 2 skeletal muscle merged images of tips in myotubes from early and late days in cells and cardiac myocytes (Fig. 7d), or day 7 skeletal muscle culture (not shown), tropomodulin dots are detected in between cells and cardiac myocytes (Fig. 7e) demonstrates that the α-actinin dots, as described above for nonstriated myofibrils tropomodulin polypeptides expressed in both cell types along the shafts of young myotubes (Fig. 4A-C). comigrate identically on 2-D gels, regardless of the day in These observations indicate that actin filament pointed ends culture for the skeletal cells. These results suggest that a single are capped by tropomodulin and partially aligned in the earliest tropomodulin isoform is present in embryonic chick skeletal appearing actin filament bundles that have assembled at the tips muscle cells and in cardiac myocytes. This isoform is E-Tmod of the young growing myotubes. Interestingly, the presence of based on co-migration with E-Tmod from adult chicken heart α-actinin at myotube tips in most myotubes late in culture, but (data not shown) (Almenar-Queralt et al., 1997). Therefore, the absence of α-actinin at myotube tips in some young isoform differences are unlikely to account for the different myotubes early in culture, suggests that thin filament distribution patterns of tropomodulin during myofibril

Fig. 6. Tropomodulin is found at the very tips of premyofibrils where sarcomeric α-actinin is often absent. Fluorescence micrographs of chick skeletal myotube tips from early (A- D) and late (E,F) days of culture fixed and double stained for tropomodulin (A,C,E) and α-actinin (B,D,F). Short arrows, tips of myotubes (A-F). Long arrows, termination of α-actinin staining (C,D). Arrowheads, transition between punctate (nonstriated) and striated staining regions in tips from late cultures (E,F). Tropomodulin was stained with a rabbit polyclonal antibody in (A) and Alexa- conjugated mAb95 in C and E. Bars: 5 µm (A,B); 10 µm (C-F). Tropomodulin assembly in skeletal muscle 1119

myofibrils in skeletal muscle may be due in large part to filament rearrangements and alignments, rather than to extensive filament polymerization or depolymerization processes. Thin and thick filaments rearrange to form mature striated myofibrils The concept that filament rearrangements are a major operative process in vertebrate skeletal muscle myofibrillogenesis has been widely accepted for assembly of thick filaments into mature myofibrils (Epstein and Fischman, 1991; Fischman, 1970; Franzini-Armstrong and Fischman, 1996). Myosin µ Fig. 7. A single tropomodulin isoform is expressed during myofibril appears to polymerize directly into 1.6 m long thick filaments assembly in chick skeletal and cardiac myogenic cells. Extracts of (a) and shorter thick filament precursors are not observed in day 6 cardiac myocytes, (b) day 2 skeletal myogenic cells, (c) day 7 differentiating skeletal muscle myotubes in vertebrates (Allen skeletal myogenic cells, (d) a mixture of day 2 skeletal myogenic and Pepe, 1965; Auber, 1969; Fischman, 1967). Our cells and cardiac myocytes and (e) a mixture of day 7 skeletal observations in this study suggest that similar mechanisms may myogenic cells and cardiac myocytes were prepared and analyzed by also be important for thin filament assembly; namely, that thin 2-D gel electrophoresis. Gels were transferred to nitrocellulose filaments may be preassembled and capped at both ends first, membranes and labelled with mAb23 followed by rabbit anti-mouse 125 and then subsequently be integrated into mature myofibrils by IgG and [ I]Protein A. Only the region of the membrane containing a gradual process of filament rearrangments and alignments. tropomodulin is shown. Identical results were obtained using several While our data do not allow us to determine directly the other monoclonal and polyclonal anti-tropomodulin antibodies that recognize epitopes located in different regions of the molecule (data lengths of thin filaments in nascent or immature sarcomeres, it not shown). is likely that thin filament lengths are restricted to a relatively narrow range based on the central location of tropomodulin dots (i.e. clusters of thin filament pointed ends) between assembly in chick skeletal muscle cells and in cardiac closely-spaced sets of α-actinin dots (i.e. clusters of barbed myocytes. ends) (Fig. 4). This conclusion assumes that thin filament barbed ends are associated with Z bodies along early premyofibrils; this will clearly require direct testing with DISCUSSION improved antibodies to CapZ. In Fig. 8, we have depicted two possibilities for thin filament assembly into mature myofibrils, The polymerization and perfect alignment of thin and thick a Variable Length model (A1) in which the nascent thin filaments into myofibrils in striated muscle is a dramatic filaments are somewhat shorter or longer than their mature example of supramolecular assembly in eukaryotic cells; the length of ~1 µm, and a Fixed Length model (A2) in which thin mechanisms by which this occurs are still incompletely filaments have been polymerized initially to their final ~1 µm understood. In this study, we sought to contribute to what is lengths in early nonstriated premyofibrils. A previous study known about the roles of actin filament capping proteins in thin demonstrating that heavy meromyosin-decorated thin filaments filament assembly and length regulation in skeletal muscle by in nonstriated premyofibrils have mixed polarities (Shimada focusing on tropomodulin, the capping protein for the pointed and Obinata, 1977) is consistent with either model. We propose ends of the thin filaments (Fowler, 1996). We found that that misalignments of thin filament ends at early stages (Fig. tropomodulin is associated with nonstriated premyofibrils early 8A), rather than large variations in thin filament lengths, can in skeletal muscle differentiation, indicating that thin filament account for the irregular sizes, variable spacings and occasional pointed ends are capped well before the precise alignments of overlaps of tropomodulin and α-actinin stained dots in early thin filaments into organized, mature sarcomeres. The premyofibrils (Fig. 4). Next, we propose that the preassembled discontinuous, punctate appearance of tropomodulin staining thin filaments are organized into striated myofibrils by a along premyofibrils indicates that thin filament pointed ends process of filament sliding to align their barbed and pointed are clustered into discrete regions and implies a greater degree ends at the Z line and at the central bare zone of the thick of thin filament organization than had been heretofore filaments, respectively (Fig. 8B,C). During this process, suspected. Our observation that some tropomodulin-stained sarcomere lengthening occurs simply as a consequence of dots alternate periodically with α-actinin-stained dots (Z rearrangements and alignments of the preassembled thin bodies) further indicates that clusters of pointed ends are filaments (Fig. 8A-C). positioned between the nascent Z bodies; i.e. that thin filaments Sanger and colleagues have also proposed a model of originating from adjacent Z bodies are oppositely polarized myofibril assembly in which nascent myofibrils are composed with their barbed ends at the Z bodies and their pointed ends of ‘mini-sarcomeres’ that increase in length during later stages in the middle of the nascent sarcomeres. The regular of myofibril assembly (Sanger et al., 1986; Rhee et al., 1994). distribution of pointed and barbed ends along nonstriated This model was based principally on elegant time-lapse premyofibrils implies that newly assembled thin filaments are observations of the movements of α-actinin dots in living polymerized to a relatively restricted range of lengths from the skeletal or cardiac cells using rhodamine-labelled α-actinin or earliest stages of myofibrillogenesis in skeletal muscle. Thus, GFP-α-actinin, which directly demonstrate separations and transitions from nonstriated premyofibrils to mature striated realignments of adjacent dots to form the regular, striated Z 1120 A. Almenar-Queralt, C. C. Gregorio and V. M. Fowler

Fig. 8. Models for thin filament Models for Thin Filament Rearrangements during rearrangements during myofibril Assembly in Skeletal Muscle assembly in skeletal muscle. In early nonstriated premyofibrils (A, A1. Early-Variable Length A2. Early-Fixed Length Early), thin filaments are partially aligned with their barbed ends overlapping in the Z bodies (green OR ovals) and their pointed ends (red arrowheads) clustered (pink ovals) in the middle of nascent sarcomeres. In the Variable Length model (A1), newly assembled thin filaments are somewhat shorter or B. Intermediate longer than their mature length and in the Fixed Length model (A2), thin filaments have polymerized to their mature length of ~1.0 µm. At intermediate stages of myofibril assembly (B, Intermediate), thin filament pointed ends become increasingly well aligned in the C. Mature ZZ middle of the sarcomere (red ovals), as do barbed ends and Z bodies (green ovals), and sarcomere lengths increase. In mature striated myofbrils (C, Mature), precise alignments of the pointed (red bars) and barbed (green bars) ends of the ~1.0 µm long thin filaments leads to a further increase in sarcomere lengths. The distances between the sets of Z bodies (green ovals) in A and B and the lengths of the thin filaments (black lines) are drawn roughly to scale so that in the mature myofibril (C), the thin filaments are ~1.0 µm long and the Z lines (green bars) are about 2.3 µm apart, as observed. Note that thick filaments are not included, for simplicity. Additionally, thin filament lengths in mature myofirils may not all be exactly 1.0 µm long, based on a report of variability in the number of striations for thin filaments isolated from embryonic pectoralis muscle (Ohtsuki, 1979). lines of mature sarcomeres (Dabiri et al., 1997; McKenna et undetected by our antibody, electron dense Z bodies are also al., 1986; Sanger et al., 1986). Increases in sarcomere lengths not observed in the distal regions of premyofibrils at the have also been observed previously by thin section electron extreme tips of Xenopus myotubes by thin section electron microscopy in differentiating skeletal muscle in vivo in microscopy (Peng et al., 1981). This suggests that α-actinin is chickens (Allen, 1973), and in cultured Xenopus muscle cells not required for clustering and aligning filament ends during (Peng et al., 1981). However, the Sanger group’s idea that early stages of myofibril assembly. In nonmuscle cells, bipolar sarcomere lengthening results from filament elongation differs myosin filaments have been suggested to play a role in cross- from our model that sarcomere lengthening results principally linking and gathering actin filaments into oppositely polarized from thin filament rearrangements. In contrast, in invertebrates, bundles in the leading lamellipodium of migrating cells sarcomere lengthening during maturation does result from (Verkhovsky et al., 1995). A similar mechanism could also elongation of both thin and thick filaments (i.e. polymerization) operate in skeletal muscle, based on a report that nonmuscle rather than from filament rearrangements (Reedy and Beall, (but not sarcomeric) myosin is enriched near the tips of young 1993, and references therein). myotubes in primary cultures of rat skeletal myogenic cells (Fallon and Nachmias, 1980; for a discussion of this question, What is responsible for early thin filament see Franzini-Armstrong and Fischman, 1996). alignments and subsequent rearrangements during Another candidate which could orchestrate initial thin myofibrillogenesis? filament alignments as well as subsequent reorganizations of Z Initial alignments of pointed and barbed ends of thin filaments bodies into striated myofibrils is , a giant sarcomeric along nonstriated actin filament bundles are likely to require protein which extends from the Z line to the M line. Titin is an actin-binding protein(s) which can recognize thin filament proposed to function as a template to organize thin and thick polarity and ends. Clues to what might be involved come from filaments into mature sarcomeres based on its molecular size, examination of components at the distal ends of nonstriated layout in the sarcomere, ability to interact directly with actin filament bundles near myotube tips; sites of new multiple sarcomeric components and presence of different sarcomere assembly (Holtzer et al., 1997). Surprisingly, isoforms (i.e. different templates) in different striated muscle discontinuous dot-like patterns of tropomodulin (i.e. clusters of types (for recent reviews, see Gregorio et al., 1999; Trinick, pointed ends) that are observed in distal portions of nonstriated 1994). Association of the N-terminal portion of titin with α- actin filament bundles at myotube tips are not always actinin and other Z band and I band components, together with accompanied by α-actinin dots in many young, growing association of the C-terminal portion of titin with thick myotubes (Fig. 6). While we cannot exclude the possibility that filaments, could establish the central position of the thick nonsarcomeric α-actinin isoforms could be present and be filaments with respect to the flanking location of thin filaments Tropomodulin assembly in skeletal muscle 1121 in the sarcomere. Interactions of myosin heads with oppositely and Wang, 1996) could be explained by thin filament polarized thin filaments could then contribute directly to rearrangements and increasing alignments during myofibril aligning thin filaments into their final positions. Thus, the final assembly. In cardiac muscle, restriction of thin filament lengths stages of thin filament alignment would likely depend on the and thus capping by tropomodulin may not occur until after the precision of myosin thick filament alignment in the A band, thin filaments have been aligned into polarized sarcomeres by which is predicted to be determined by the late-assembling a titin-myosin scaffold (Littlefield and Fowler, 1998). myosin-binding proteins: C protein and M protein (Lin et al., 1994; van der Ven and Furst, 1997). Interestingly, thin filament Actin filament rearrangements in nonmuscle cells lengths as well as alignments have also been proposed to be Actin filament rearrangements are emerging as an important directly dependent on correct assembly of a myosin thick mechanism for restructuring the actin cytoskeleton in a variety filament-titin scaffold (Littlefield and Fowler, 1998). of nonmuscle cell types. For example, in cytokinesis in animal cells, the contractile ring appears to be formed by recruitment Thin filament assembly is different in cardiac as of preexisting actin filaments into the cleavage furrow from compared to skeletal muscle polar regions of the cell cortex (Cao and Wang, 1990). Actin The observations reported here are strikingly different from filaments in stress fibers also undergo continuous movements what we had earlier observed in cultured embryonic chick and reorganization in living cells (Wang, 1987). It has also been cardiac myocytes, where tropomodulin is not associated with proposed that in budding yeast, cell cycle-dependent nonstriated myofibrils (Gregorio and Fowler, 1995). Indeed, movements of actin patches and cables may take place largely tropomodulin is one of the latest markers of myofibril assembly by rearrangements of preexisting actin filaments rather than by and is found assembled into myofibrils only after the actin polymerization and depolymerization processes (Karpova appearance of actin filament striations. The differences in the et al., 1998). All of these processes are distinct from the rapid assembly properties of tropomodulin in cultured skeletal and and extensive bursts of actin polymerization that take place cardiac muscle cells cannot be explained by differences in their during activation of platelets or chemotaxis of amoeboid cells tropomodulin isoform, which is E-Tmod in both cell types. (Cano et al., 1991; Eddy et al., 1997; Hartwig, 1992), or from Furthermore, preliminary characterization of de novo myofibril the transient assembly of actin filaments in membrane- assembly in explants from precardiac regions of avian embryos associated foci in the lamellipodia of cultured cells (Schafer et indicates that tropomodulin is assembled late, after the al., 1998). The relative contributions of regulated actin appearance of actin striations (D. Rudy, P. B. Antin and C. C. polymerization or filament rearrangements to assembling actin Gregorio, unpublished observations). This indicates that late filaments with defined lengths (e.g. microvilli, erythrocyte assembly of tropomodulin in cultured cardiac myocytes is not actin filaments, Drosophila bristles) remain to be elucidated. an artifact of isolated cells cultured in vitro. Therefore, differences in the temporal and spatial pattern of incorporation The authors thank Rénald Gilbert and Takashi Mikawa (Cornell of tropomodulin in cardiac as compared to skeletal myofibrils University Medical College, New York, NY) and Dorothy Schafer may reflect intrinsic differences in thin filament and myofibril (Washington University, St Louis, MO) for valuable advice on assembly mechanisms (Gregorio, 1997). establishing chick skeletal myogenic cells, Elizabeth Repasky (Roswell Park Cancer Institute, Buffalo, NY) for advice on 2-D gel What accounts for the ability of tropomodulin to cap thin electrophoresis, Klaus Hahn for the use of his microscope, Ryan filament pointed ends in nonstriated myofibrils in skeletal but Littlefield for help with the image acquisition and processing, George not cardiac muscle? We had proposed earlier that newly Klier and Dan Peterson for assistance with the confocal microscopy, polymerized actin filaments in nonstriated myofibrils in cardiac and Lewis Tilney (University of Pennsylvania, Philadelphia, PA) and myocytes are of indeterminate and variable lengths (Gregorio Malcolm Wood for assistance with the electron microscopy. This and Fowler, 1996; Gregorio, 1997). Differences in nascent thin work was supported by grants from the National Institutes of Health filament lengths and tropomodulin assembly in skeletal and (GM34225) to V. M. Fowler and to C. C. Gregorio (HL 57461), and cardiac muscle could be at least partly a consequence of the by a grant from the Human Frontiers Science Program to V.M.F and interaction (either directly or indirectly) of tropomodulin with C.C.G. the skeletal muscle-specific protein, nebulin (Gregorio, 1997; Littlefield and Fowler, 1998). 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