JOURNAL OF CELLULAR PHYSIOLOGY i 4 m - 8 7 (1990)

Distribution of Vinculin in the Z-Disk of Striated Muscle: Analysis by Laser Scanning Confocal Microscopy LOUIS TERRACIO,* DAVID C. SIMPSON, LULA HILENSKI, WAYNE CARVER, ROBERT S. DECKER, NANCY VINSON, AND THOMAS K. BORG Departments of Anatomy, Cell Biology and Neurosciences (L.7., N.V.) and Pathology (L.H., W.C., T.K.B.), Departments of Medicine and Cell, Molecular and Structural Biology, Northwestern University, School of Medicine, Chicago, lllinois 606 1 1 (D.G.S., R.S.D.)

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib-like structure between the cell membrane and the Z-disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three-dimensional arrangement of vinculin in relation to the Z-disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X-Y and X-Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z-disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofibrillogenesis. Vinculin is a major cytoskeletal component that has been shown to be involved in a variety of basic cellular functions both in vivo and in vitro (Geiger et al., 1984; Burridge, 1986). This component exists in two forms: M R 150,000 protein, termed metavinculin (Siliciano and Craig, 1982,1987;Turner and Burridge, 19891, and M R 116,000 protein, called vinculin (Geiger, 1979; Feramisco and Burridge, 1980); however, the functional relationship between these two forms is not clear. Immunohistochemical localization in vitro has shown that vinculin is localized in close association with focal adhesions (Geiger et al., 1984; Woods and Couchman, 1988; Burridge et al., 1988). In addition, it appears that vinculin is closely associated with the cytoplasmic domain of integrins which are the receptors for extracellular matrix components (ECM) (Burridge et al., 1988; Terracio et al., 1989). This type of localization, along with biochemical investigations, has led to the theory that vinculin is an essential cytoskeleta1 component involved in the translation of information from the ECM to the internal milieu of the cell. The ECM: cytoskeletal interaction could be important in such functions as cell migration, adhesion, and alteration of phenotype (Hynes, 1987; Ruoslahti and Pierschbacher, 1987; Buck and Horwitz, 1987). The localization of vinculin in vitro has been confined to relatively few primary cells (Atherton et al., 1986; Aiherton and Behnke, 1988; Glukhova et al., 1986; Turner and Burridge, 1989; Borg et al., 1989; Hilenski et al., 1989). Most of these investigations have been in Q 1990 WILEY-LISS, INC.

striated muscle, where vinculin has been reported to form a rib-like structure, termed a costamere (Pardo et al., 1983a,b). The costamere was described as being between the cell membrane and the Z-disk of the contractile apparatus. The arrangement of vinculin in costameres in cardiac muscle was slightly different from skeletal muscle in that cardiac muscle showed a single rib whereas skeletal muscle tended to show a doublet (Pardo et al., 1983a,b). The function of the costameres has been proposed to provide alignment of the T-tubules as well as alignment of the Z-disks during contraction (Pardo et al., 198313). Immunohistochemical localization of integrin recognizing antisera in cardiac muscle in vivo and in vitro has also been shown to co-localize with vinculin (Borg et al., 1989; Terracio et al., 1989). These latter studies showing the colocalization of ECM components, their receptors and vinculin are indicative of the fact that the Z-line is a major site of mechanical tension in striated muscle (Friden, 1983; Wan and Ramirez-Mitchell, 1983). When increased mec anical stress is placed on myocytes, such as during myocardial ischemia, disruption of the vinculin costameres has been observed (Ganote and Vander Hide, 1987; Steenbergen et al., 1987). Because of the technical problems associated with

a

Received January 9, 1990; accepted June 11, 1990. *To whom reprint requestsicorrespondence should be addressed.

DISTRIBUTION OF VINCULIN IN STRIATED MUSCLE

immunolocalization in large cells such as striated muscle, the early investigations on the localization of vinculin were not clear as to whether the vinculin formed a rib-like costamere or was continuously distributed within the Z-disk. In this study we have used laser scanning confocal microscopy and optical sectioning through isolated neonate and adult-striated muscle cells from several different species and sections of embedded cardiac tissue to demonstrate that both polyclonal and monoclonal antibodies against vinculin were distributed within the Z-disk. The distribution of vinculin was similar to a known Z-disk component, alpha-actinin, but different from that of the B, integrins.

MATERIALS AND METHODS Animals Isolated cells. Adult cardiac myocytes from rat, mouse, and cat were isolated by previously described procedures (Lundgren et al., 1984; Silver et al., 1983). Neonatal cardiac myocytes were isolated from 4-5day-old rats as previously described (Borg et al., 1984). The isolated rod-shaped adult myocytes were allowed to attach to laminin coated coverslips for 1hr, after which, the cells were fixed with 2% paraformaldehyde in 0.01 M phosphate buffer containing 50 mM KC1 (pH 7.4) and quenched with 0.1 M glycine in PBS. The isolated neonatal myocytes were panned to separate fibroblasts from myocytes. The myocytes were then allowed to attach to laminin coated coverslips at various times from 1hr to 4 days. Cells were fixed as described above for adult cardiac myocytes. Skeletal myocytes were isolated from young rats, 6 5 days post partum, by collagenase digestion (150 U/ml in Kreb's buffer) (Lundgren et al., 1984) for 60 rnin at 37°C in a reciprocating shaker. Partially separated myocytes were harvested by centrifugation and fixed as described above for adult cardiac myocytes. Tissue sections. Whole hearts were removed from anesthetized rats, rinsed in PBS, and fixed in either 2% paraformaldehyde in 0.01 M phosphate buffer or in cold Carnoy's fixative (Gabe, 1976) with methanol substituted for ethanol. The hearts were then processed for routine paraffin histology and the sections used for immunohistochemical staining. Antibodies Monoclonal antibodies a ainst vinculin were purchased from Sigma (Sigma hemicals, St. Louis, MO); however, these antibodies did not react with adult rat cardiac myocytes. Therefore, polyclonal antibodies were prepared against vinculin isolated from rat hearts (OHalloran et al., 1986). Vinculin fractions were identified by SDS-PAGE and immunoblots with antisera from Dr. Keith Burridge, Department of Anatomy and Cell Biology, University of North Carolina, Chapel Hill, North Carolina. The vinculin fractions were cut out of the gel, electroeluted, mixed with Freund's complete adjuvant and injected into rabbits. Subsequent boosts were done in a similar manner only using Freund's incomplete adjuvant. IgG's were purified from the antisera by Protein A affinity columns (Pharmacia,

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Uppsala, Sweden) by the manufacturer's instructions. The specificity of the IgG fractions was characterized by immunoblots of both one- and two-dimensional SDS-PAGE (OFarrell, 1975; Ben-Ze'ev, 1989) procedures using a crude cardiac cytoskeletal preparation and purified chicken smooth muscle vinculin. These techniques showed that the IgG fraction was monospecific for vinculin. Polyclonal antibodies against alphaactinin were purchased from Transformation Research (Framingham, MA). Anti-rabbit and anti-mouse IgGs and Fab's conjugated to FITC and Texas Red were purchased from Sigma and Cappel Laboratories (West Chester, PA). Polyclonal B, integrin antiserum (Gullberg et al., 1989) and B1 integrin antiserum specific for the cytoplasmic domain of the receptor (Marcantonio and Hynes, 1988) (a gift from Dr. Richard Hynes, MIT, Boston, MA) were used as controls.

Immunofluorescence Following fixation and quenching, the cells were rinsed in PBS and stained as previously described (Terracio et al., 1989).Briefly, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min and rinsed 3 times with PBS. Single staining. The cells or sections were incubated with the primary antibody for 30 min at 37"C, washed 3 times for 5 min with PBS, and incubated with either goat anti-rabbit FITC or goat anti-rabbit Texas Red for 30 min at 37°C. After being washed 3 times with PBS for 5 min, the coverslips were placed in PBS: glycerine (1:3) and mounted on hanging drop slides to prevent flattening which could distort the cells. Controls for the single staining were substitution of preimmune IgG or PBS for the primary antibody and bleaching of the reaction with exposure to UV light. The slides were examined on a BioRad MRC-600 laser scanning confocal microscope. Double staining. In order to better determine if vinculin co-localized with alpha-actinin and if the polyclonal and monoclonal anti-vinculin antisera co-distributed in myocytes, sequential double stainings were performed. The cells were incubated with primary antibody and washed as described above and then incubated with goat anti-mouse Fab-FITC for 60 min at 37°C. It is important to saturate the first antibody with the Fab-FITC so that the subsequent antibody and conjugate do not bind to the primary antibody. Saturating titers were determined by exposing anti-vinculin incubated cells to various concentrations of unconjugated Fab followed by goat anti-rabbit Fab-FITC. Lack of fluorescence indicated saturation of the primary antibody. Dilutions of 1/10 to 1/50 were tested. Saturation was achieved with a 1/20 dilution, and in all further experiments a 1/15 dilution was made to insure total saturation. The cells were washed and incubated with anti-vinculin or anti-alpha actinin as above. After washing 3 times with PBS, the cells were incubated with goat anti-rabbit IgG Texas Red for polyclonals or goat anti-mouse IgG Texas Red for monoclonals for 30 min at 37°C. The cells were washed with PBS and the coverslips were mounted as described above. Additional controls consisted of substitution of preimmune IgG and PBS for the primary antibodies. In addition, the

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order of staining for each antigen was reversed to assess the accuracy of the staining. Microscopy Laser scanning confocal microscopy was used to examine the cells and sections because it would allow W for analysis of the distribution of the antigens in a (3 three-dimensional configuration. Cells from each specimen were subjected to serial optical sectioning, usu- e I ally 1 km thick, in the X-Y plane. An X-Z section was then made through the midpoint of the cells. Double v) labelled cells were examined in the dual label config- n uration of the MRC-600, where both fluorochromes v) were excited at 514 nM and simultaneously recorded using separate and precisely matched photomultiplier tubes at 540 and 600 nM. The MRC-600 was gated to eliminate spectral overlap. Co-localization was determined by pseudocolor overlaying the digital images (Terracio et al., 1989; Gullberg et al., 1989) or transferring the images to a Universal Images (Ithaca, NY) Image IiAT digital image processing system for line scan analysis (Terracio et al., 1989; Gullberg et al., 1989).

A

IEF-

B

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RESULTS The specificity of the polyclonal rabbit anti-rat cardiac vinculin is demonstrated in Figure 1. The silver stain of a 2-D gel of a crude cytoskeletal extract of cardiac myocytes (Fig. la) reveals the presence of a number of proteins including vinculin and a-actinin. The immunoblot, however, demonstrates that the polyclonal anti-vinculin recognizes only the three common isoforms of vinculin and no other proteins (Fig. lb). The commercial monoclonal anti-vinculin yielded an essentially identical immunoblot (data not shown). Confocal imaging of isolated cardiac and skeletal muscle cells from a variety of species stained with anti-vinculin clearly demonstrated that vinculin was distributed within the Z-disk (Figs. 2, 4,5, 9, 10). Each optical serial section through either cardiac (Figs. 2,4) or skeletal muscle cells (Fig. 5 ) demonstrated a sarcomeric banding pattern similar to but not identical to that observed with the known Z-band component, alpha-actinin (Fig. 3, 6). The staining for alpha-actinin consistently appeared to be denser and thicker but identical in distribution to vinculin (Fig. 6). Both the polyclonal antibodies raised a ainst rat heart vinculin and the commercial monoc onal vinculin antisera showed a similar distribution in the same cell (data not shown). Both pseudocolor overlays (data not shown) and intensity line scans (Fig. 6) demonstrated that in all sections the staining for both vinculin and alphaactinin codistributed. Each peak of intensity for alphaactinin precisely coincided with the intensity peak for vinculin (Fig. 6). X-Z sections through cells stained for vinculin or alpha-actinin (Figs. 2-5) demonstrated that both proteins are distributed within the Z-disk and penetrate completely through the cell. The most intense staining is at the outer 1/3 of the rat cells (Figs. 2,5,6);however, staining is seen through the cell at the Z-disk. The only exception was at the nucleus and paranuclear regions which are known to be devoid of contractile elements. An X-Z cross-section through a Z-disk (Fig. 7) demon-

B

Fig. 1. a: A silver stain of a two-dimensional gel of a crude extract of cardiac myocytes. The arrow indicates the location of alpha-actinin and the arrow heads indicate the isoforms of vinculin. b An immunoblot of the proteins transferred from a companion gel of the one shown in A. The polyclonal anti-vinculin only recognizes the isoforms of vinculin. A = acidic; B = basic; IEF = isoelectric focusing; SDSPAGE = sodium dodecylsulfate-polyacrylamidegel electrophoresis.

strates that vinculin is distributed within the Z-disk, again with the most intense staining in the peripheral 113 of the cell. The distribution of vinculin staining in skeletal muscle cells isolated from neonatal rats did not always appear homogeneous. The majority of cells demonstrated the pattern described above where the vinculin was distributed within the Z-disk (Fig. 5); however, some cells demonstrated a pattern where the fluorescence was much more intense on the lateral margins than in the center of the Z disk (Fig. 5c). Thus in isolated cells, with conventional fluorescence, the dis-

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tribution appeared to be just as previously described for costameres; however, only when optical sections were closely examined was vinculin also seen to be distributed within the Z-disk (Fi . 5). This was also seen in isolated cardiac muscle cel s from the mouse (data not shown). As part of the controls, B1 integrin antiserum and an antibody directed at the cytoplasmic domains of the B1 integrin (Marcantonio and Hynes, 1988) were used to stain the myocytes (Fig. 8). Previous studies have shown that the B1integrin with specificity for collagen (Gullberg et al., 1989) colocalized at the Z-line with vinculin (Terracio et al., 1989). With confocal microscopy, it was apparent that the B1 integrin was localized in narrow stripes at the Z disk at the surface of the cells and not throughout the Z-disk (Fig. 8a,b). The localization of the B1 integrin specific for the cytoplasmic face was localized to the internal face of sarcolemma (Fig. 8c,d); however, the staining attern was wider than that of the B1 on the surface o the myocyte (Fig. 8a,b). Isolated neonatal myocytes reorganize their cytoskeleton and undergo myofibrillogenesis in vitro (Athertonet al., 1986; Hilenski et al., 1989).These cells were examined to determine whether vinculin was distributed in the Z-disk as observed in vivo. When optically sectioned, neonatal myocytes showed that vinculin was located in the focal adhesions with the substrate (Fig. 9a) and within the Z-disk of the contractile apparatus (Fig. 9b-d). While these data indicate that there may be two distinct patterns of vinculin staining (one in focal adhesions and one in Z-disks), there are occasional indications that these two regions may be connected (Fig. 9a,b). Further examination of these regions with high voltage electron microscopy should provide the increased resolution necessary to determine whether there are cytoskeletal connections between the focal adhesions and the Z-disk as well as the composition of these connections. To insure that the distribution of vinculin in the Z-disks was not strictly confined to isolated and cultured cells, sections of rat heart were also examined. Staining of paraformaldehyde fixed tissues resulted in high background autofluorescence and were not usable for immunofluorescence.The tissues fixed in Carnoy's exhibited reduced the autofluorescence. The distribution of vinculin in the in vivo heart was similar to that observed in the isolated cells (Fig. 10); however, the staining appeared more intense in the region of the intercalated disk than in isolated rat myocytes (Fig. 2). Optical sectioning again indicated that the vinculin was distributed within the entire Z-disk as observed in the isolated cells.

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DISCUSSION The data presented clearly show that vinculin is distributed within the Z-disk in isolated striated muscle, tissue sections of cardiac muscle, and in cultures of cardiac myocytes. This distribution is significant in understanding the potential physiological importance of vinculin in striated muscle. The pattern of vinculin distribution seen in this study is different from that described by a number of investigators using conventional fluorescence microscopy (Pardo et al., 1983a,b; Lin et al., 1989; Shear and Bloch, 1985; Geiger et al.,

Fig. 2. An isolated adult rat cardiac myocyte stained with antivinculin. Confocal optical sections through the cell in the X-Y plane ( a d ) indicate the presence of vinculin within the 2-disksalthough the intensity is brightest at the cell margins. "he distribution of vinculin within the Z-disk is confirmed in the X-2 section (e) taken through the same cell along the line drawn in d. Note that vinculin fluorescence is throughout the cell at the 2-disk except where the nucleus (N)is present. Bar = 10 kxn.

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Fig. 3. Confocal optical sections through an adult rat cardiac myocyte stained for alpha-actinin. Alpha-actinin is present in all sections of the X-Y plane (4). An X-Z section (e) verifies this distribution. Bar = 10 pm.

Fig. 4. A series of confocal optical sections through an adult feline cardiac myocyte stained with ICN-monoclonalanti-vinculin. Vinculin is distributed within the Z-disk in all the sections. ( a 4 in a similar pattern to the rat (Fig. 1) and mouse (data not shown). In addition, there is an intense reaction a t the region of the intercalated disk of the cell (arrows). The X-Z section through the same cell confirms the distribution pattern of the vinculin within the Z-disk. Bar = 10 pm.

DISTRIBUTION OF VINCULIN IN STRIATED MUSCLE

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a-actinir vinculin

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i

1

Fig. 6. An optical section near the center of an adult rat cardiac myocyte double stained for alpha-actinin (a) and vinculin (b).The distribution patterns are nearly identical as demonstrated by the intensity scan across the line indicated in the figures and shown in c. The intensity of alpha-actinin staining was always greater, but the distribution was very similar but not identical to vinculin. Bar = 10 pm.

Fig. 5 . Freshly isolated skeletal muscle from a 5-day-old neonatal rat. Micrographs (a-d) are confocal optical sections in the X-Yplane. Micrograph e is an X-Z section through the same cell along the line indicated in d. Vinculin staining is present in the Z-disk in each section through the cell indicating that the distribution is within the Z-disk and not strictly in a costamere pattern. This distribution is verified by the X-Z section (e). Bar = 10 pm.

1984; Tokuyasu, 1989). This difference in staining could be accounted for if the antiserum used in this study cross reacted with alpha-actinin or some other Z-disk protein. The 2-D immunoblot clearly shows that the antibodies used recognize only the isoforms of vinculin. This antiserum was raised against rat heart vinculin, whereas most other studies used chicken gizzard vinculin. It is possible that the antiserum used in this study recognized unique epitopes on mammalian vinculin. This is supported by the fact that the monoclonal anti-vinculin does not stain adult rat cardiac myocytes while the polyclonal anti-vinculin does. Also, the monoclonal anti-vinculin yields primarily a focal adhesion staining pattern in cultured myocytes with an occasional cell showing Z-banding while the polyclonal recognizes both the focal adhesions and Z-disk. Others, using the chicken gizzard anti-vinculin monoclonal antibody (Lin et al., 19891, report only a focal adhesion staining pattern in cultured chick myo-

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Fig. 7. An isolated cardiac myocyte sectioned in the X-Y plane (a) and an X-2 plane through a Z-disk as indicated by the line on the cell in a (b).The X-Z section (b) demonstrates that vinculin fluorescence is most intense at the periphery of the cell but is present through the Z-disk. Bar = 25 pm.

cytes. However, the same group in a later publication (Schultheiss et al., 1990) have demonstrated occasional costamere staining in the cultured cardiac myocytes. Another explanation for the fact that we have localized vinculin not only at the periphery of the cell but also within the Z-disk is the increased resolution, sensitivity, and optical sectioning properties of the confocal microscope. When we previously used conventional fluorescence microscopy to study vinculin stained myocytes (Terracio et al., 1989), the staining pattern appeared to be as a costamere similar to that previously reported by others (Pardo et al., 1983a,b). The same was true for the cells used in this study; however, optical sectioning demonstrated that the distribution of vinculin was within the Z-disk. Both X-Y and X-Z sections show that the immunofluorescence is most intense at the peripheral of the cell; however, the fluorescence is present at a reduced intensity through the center of the Z-disk (Figs. 2, 7). Since vinculin has been described as being associated with the T-tubules (Pardo et al. 1983a), it is possible that the distribution seen in the optical sections is due to the inability of the confocal microscope to resolve T-tubule staining on each side of the Z-disk as parallel stri es. We do not think that is the case for two reasons: 1)t e resolution of the confocal microscope is superior to conventional fluorescence microscopes. Our particular instrument can resolve about 0.15 km. 2) We can resolve the staining of another antigen, titin, which is known to be distributed immediately adjacent to both sides of the Z-disk (data not shown). Therefore, we feel that confocal microscopy has enabled us to better describe the distribution of vinculin in mammalian striated muscle. Vinculin in striated muscle appears to be in a complex associated with other proteins that are hypothesized to function in signal transduction from the ECM via connections with integrins (Horwitz et al., 1986; Buck and Horwitz, 1987) and in force transmission as

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Fig. 8. Adult rat cardiac myocytes stained with an antibody against B, Inkgrin (a,b)and the cytoplasmic domain of the B,-integrin (c,d). These confocal optical sections near the midpoint of the cell in the X-Y plane demonstrate the expected appearance of an antigen distributed in a costamere-like pattern. Bar = 10 pm.

well as myofibrillar alignment with the sarcolemma (Shear and Bloch, 1985; Borg et al., 1989; Hilenski et al., 1989). In striated muscle, vinculin appears to play an analogous role by its arrangement in the Z-disk. The Z-disk region represents a specialized region where ECM components, their transmembrane rece tors, and certain cytoskeletal components are locaEzed. While this region appears to be reinforced to resist mechanical deformation, the Z-disk also repre-

DISTRIBUTION OF VINCULIN IN STRIATED MUSCLE

of confocal optical sections at ~ L steps ~ i9, ~A series , m through a neonatal cardiac mvocvte after 24 hours in vitro. In addition to vinculin being locatkd in the focal adhesions (a,b),vinculin is also located within the Z-disk of the newly formed sarcomeres (bd). Bar = 10 pm.

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Fig. 10. A confocal optical section of rat papillary muscle demonstrating that vinculin is located within the Z-disk as well as at the intercalated disk (arrow). Optical serial sections (a-e) through the section indicated that the vinculin was present throughout the Z-disk as in the Bar = 25 pm.

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sents the principal point of mechanical transmission of force and, therefore, may be prone to injury (Friden, 1983; Wang and Ramirez-Mitchell, 1983; Steenbergen et al., 1987). If vinculin is located within the Z-disk rather than being restricted to rib-like costameres, then its role in resistance of mechanical deformation and transmission of contractile force would be enhanced. Vinculin may also play a role in myofibril alignment as well as coordination with the sarcolemma (Borg et al., 1989) and its specializations such as the T-tubules (Pardo et al., 1983a,b). Being distributed at restricted sites on the cytoplasmic side of the sarcolemma and within the Z-disk is more compatible with this hypothesis than with costamere distribution alone. In skeletal muscle there is more torque and deformation due to the length of the fiber than in the shorter cardiac myocytes. This may explain why the costameric patterns in skeletal muscle are slightly different in these two striated muscles. The strengthening of the Z-disk region may also be important in normal development, where mechanical stimulation such as increased pressure or volume is thought to play an im ortant role in the formation of the heart (Borg and 1perracio, 1989). During development, the vinculin is believed to be involved in insuring proper alignment of the developing myofibrils. Localization in developing cardiac muscle by Tokuyasu (1989) has shown that vinculin is first localized near the surface of the sarcolemma and is colocalized with the myofibrillar protein, titin. This is a similar pattern to that seen in vitro with assembly of myofibrils in neonatal myocytes (Borg et al., 1989; Hilenski et al., 1989). In vitro investigations have shown that vinculin is associated with the focal adhesion sites and closely associated with receptors for ECM components (Damsky et al., 1985; Buck and Horwitz, 1987;Terracio et al., 1989; Borg et al., 1989). The function of this particular anatomical arrangement has been proposed to be that of providing mechanical strength as well as a system for the translation of mechanical information from the ECM to the internal components of the cells. In addition, the latter studies have shown that ECM and ECM receptors (integrins) may influence the pattern of the myofibrillar development both in vivo and in vitro. The sequence of events appears to be the initial recognition of the substrate or ECM component by integrins followed by the a pearance of vinculin and the subsequent formation o the myofibril (Borg et al., 1989; Hilenski et al., 1989). While the timing and regulation of these events have yet to be determined, it appears that there is a coordination involving the cytoskeleton that is important to the assembly of myofibrils. The structural role of vinculin in the Z-disk may also be important in the adult where increases in mechanical stimulation are important in muscle hypertrophy (Borg and Terracio, 1989) and in certain diseases such as myocardial infarction (Steenbergen et al., 1987). During hypertrophy, as a result of mechanical stimulation, there is an increase in ECM components, especially collagens (Borg and Terracio, 1989). Some of the collagens are attached to the surface of the myocytes at a region just lateral to the Z-disk. The attachment of

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the collagen is mediated by collagen specific integrins which colocalize with vinculin in the Z-disk (Terracio et al., 1989). This arrangement is thought to provide increased strength to the myocytes, especially at the Z-disk, during the increased mechanical tension associated with hypertrophy. In infarcted regions of cardiac muscle, the noncontracting myocytes are thought to be subjected to a high shear force from the surrounding healthy, contracting myocytes. These shear forces could possibly cause the rupture of the collagen that is attached to the cell surface near the Z-disk. This rupture of the collagen connection via the transmembrane integrins might also lead to damage of the cytoskeleton and eventually cause the disruption of the myofibrils. Staining patterns for vinculin on ischemic myocytes have shown a diffuse pattern near the sarcolemma indicating a disruption of the arrangement of vinculin at the cell membrane and myofibrillar disarray (Ganote and Vander Heide, 1987; Steenbergen et al., 1987). Determining the precise chronology and interaction of vinculin with other components of the Z-disk will be essential to our understanding of the structure and function of vinculin in striated muscle. Confocal microscopy clearly provides an excellent analytical tool for the analysis of the three dimensional distribution of antigens such as vinculin; however, high resolution investigations with electron microscopy will also be necessary to provide essential data on the localization of vinculin in association with other cytoplasmic components in striated muscle.

ACKNOWLEDGMENTS The authors wish to thank Dr. Kristofer Rubin and Donald Gullberg for able assistance with the B1 antisera to hepatocytes; Dr. Keith Burridge for the antivinculin and vinculin from avian smooth muscle; Dr. Richard Hynes for antiserum to the cytoplasmic domain of the B1 integrin; and Dr. Ron Jyring for supplying the isolated mouse and rat cardiac myocytes. These investigations were supported in part by grants from the NIH HL-42249, HL-40424, HL-24935, and HL-37669 to T.K.B. and L.T. In addition, support from the South Carolina and Georgia Affiliates of the Heart Association to L.L.H. is gratefully acknowledged. The support of NIH HL-33616 and the American Heart Association of the Chicago Metropolitan Heart Association to R.S.D. is gratefully acknowledged. LITERATURE CITED Atherton, B. T., and Behnke, M. M. (1988) Structure of myofibrils at extra-junctional membrane attachment sites in cultured cardiac muscle cells. J. Cell Sci., 89:97-106. Atherton, B.T., Meyer, D.M., and Simpson, D.G. (1986) Assembly and remodeling of myofibrils and intercaiated discs in cultured neonatal rat heart cells. J. Cell Sci., 86:233-248. Ben-Ze’ev, A. (1989) Cell shape and cell contacts. In: Cell Shape: Determinants, Regulation and Regulatory Role. W.D. Stein and F. Bronner, eds. Academic Press, Inc., San Diego, pp 9&119. Borg, T.K., and Terracio, L. (1989) Interaction of the extracellular matrix with cardiac myocytes during development and disease. In: Cardiac Myocyte-Connective Tissue Interactions. T. Robinson, ed. S. Karger A.G., Basal, Switzerland, pp 113-129. Borg, T.K., Rubin, K., Lundgren, E., Borg, K., and Obrink, B. (1984) Recognition of extracellular matrix components by neonatal and adult cardiac myocytes. Dev. Biol., 104:8696.

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Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy.

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib-like structure between the cell membrane and t...
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