gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres,...

Cell Motility 3:449-462 (1983)

Gamma Actin, Spectrin, and Intermediate Filament Proteins Colocalize With Vinculin at Costameres, Myof ibril-to-Sarcolemma Attachment Sites

Susan W. Craig and Jose V. Pardo

Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Localization of vinculin at the sarcolemma of striated muscle fibers defines an orthogonal lattice. The costameres of the lattice are the riblike bands of vinculin that run perpendicular to the long axis of the fiber, repeat in register with I bands of the subjacent myofibrils, and seem to couple the myofibril to the sarcolemma [Pardo et al 1982, 1983al. The colocalization studies presented in this paper show that gamma actin, spectrin, and intermediate filament antigens are additional components of this lattice of costameres. In addition, the results show that gamma actin and spectrin are also components of the internal network of collars, first visualized with antibody to desmin [Granger and Lazarides, 19781, that connects the myofibrils to each other at the level of the Z line.

Key words: myofibril to sarcolemma attachment, costamere, spectrin, actin, intermediate filaments, vinculin, fibronectin

INTRODUCTION

Researchers have long predicted the presence of an organized structure for attachment of myofibrils to the sarcolemma [eg, Bennett and Porter, 19531. Glimpses of this structure have been given in ultrastructural descriptions of fibrous connections or morphological specializations between Z lines, M lines, and the sarcolemma of both skeletal [Garamvolgyi, 1965; Pierobon-Bormioli, 19811 and cardiac muscle [Chiesi et al, 19811. However, until recently none of the molecular components of these myofibril to sarcolemma attachment sites nor the overall topographical arrangement of the sites at the sarcolemma were known.

Address reprint requests to Dr. Susan Craig, Department of Physiological Chemistry, The John Hopkins University School of Medicine, Baltimore, MD 21205.

0 1983 Alan R. Liss, Inc.

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Because there is a good probability that vinculin is a ubiquitous component of actin to membrane linkages in nonerythroid cells [Geiger et al, 19801, we used antibody to vinculin to search for the postulated sarcolemmal organization of myofibril-to-sarcolemma attachment sites. In the early work, we did find a two-dimensional lattice of vinculin associated with the sarcolemma of striated muscle cells [Pardo, et al, 1982, 1983a, 1983c, in press]. The transverse elements of the lattice, called costameres, are bands of vinculin that encircle the muscle fiber and repeat along its length with a periodicity corresponding to the subjacent sarcomeres. The costameres have a substructure consisting of densely clustered patches of vinculin; the patches are segregated into two rows which flank the Z line and overlie the I band of the underlying sarcomere. The membrane-Zssociated costameres appear to be coupled physically to the underlying sarcomeres because they widen and narrow in concert with the underlying I band in stretched and contracted muscle.

Verification of the hypothesis that costameres represent regions of attachment of the myofibrils to the sarcolemma will require molecular dissection of the structure and additional mechanical studies, but in the meantime colocalization studies can provide clues to the identity of those proteins that might be components of the costamere. Since the membrane to myofibril attachment sites are highly organized and are periodic with the structure of the underlying sarcomeres, it is easy to determine if a protein colocalizes with vinculin. In this paper we show, by the criterion of colocalization, that the membrane-associated lattice of alpha spectrin described by Repasky et a1 [1982] is part of the same lattice defined by the presence of vinculin. We also implicate gamma actin, intermediate filament proteins, and possibly fibronectin as presumptive components of this very same lattice.

MATERIALS AND METHODS Antibodies

The preparation and characterization of the affinity-purified rabbit antichicken gizzard vinculin has been described [Pardo et al, 1983al. Staining of cryostat sections of chicken muscles by this antivinculin is absorbed by preincubating the antibody with 130-kilodalton (kd) vinculin eluted from a preparative gel [Pardo et al, 1983a,c]. The production and characterization of anti-gamma actin has also been presented in detail [Pardo et al, 1983bl. This affinity-purified antibody recognizes gamma actin from avian gizzard and intestinal brush borders, but it does not recognize skeletal muscle alpha actin or beta actin from intestinal brush borders or human red cells. In addition, staining of cryostat sections by this antibody is absorbed by preincubating the antibody with homogeneous gamma actin prepared from chicken gizzard. [Pardo et al, 1983bl. The antispectrin IgG used in this study was a gift of Dr. E. Repasky and was found to recognize alpha spectrin specifically in chicken muscles [Repasky et al, 19821. Intermediate filament proteins were detected using a monoclonal prepared, characterized, and donated to us by Dr. R. Pruss [Pruss et al, 19811. This monoclonal antibody recognizes all known intermediate filament protein subunits and in addition, a ubiquitous 66K polypeptide thought to be a common subunit of all intermediate filament classes. Fibronectin was detected with a monoclonal antibody that is selective for avian fibronectin [Gardner and Fambrough, 19831. This monoclonal was given to us by Dr. D. Fambrough of the Carnegie Institute.

Molecular Components of Costameres 451

lmmunofluorescence

Small strips of pectoralis, anterior latissimus dorsi (ALD), and posterior latissimus dorsi (PLD) from 4-10-week-old chicks were tied at resting length, dropped into freezing isopentane, and then mounted in O.C.T. compound (Miles Laboratories) on brass chucks. Cryostat sections, 4pm thick, were thaw-mounted onto gelatin- coated glass slides and fixed for 20-30 min in 3 % (w/v) formaldehyde in 0.123 M PO4 buffer, pH 7.3. The sections were rinsed in phosphate-buffered saline (PBS: 0.01 M PO4 buffer + 0.15 M NaC1, pH 7.5), incubated for 5 min in 0.5 mg/ml NaBH3 in PBS to quench unreacted aldehydes, and then rinsed twice in PBS. For indirect immunofluorescence, sections were overlaid with 15 pl of affinity purified antivinculin at 60 pg/ml; affinity-purified anti-gamma actin at 70 pg/ml; a 1:50 dilution of a 40% AmS04 fraction of rabbit anti-alpha spectrin; or 50 pg/ml of monoclonal antibody to intermediate filament protein. Staining time and washing protocols were as described [Pardo et al, 1983al. The rabbit antibodies were detected with affinity- purified fluorescein-coupled goat antirabbit (molar ratio fluroescein: protein = 6.75; Boerhinger-Mannheim) at 50 pglml in PBS + 0.4% bovine serum albumin (BSA); the mouse antibodies were visualized with affinity purified fluorescein-labeled goat antimouse IgG (F:P = 6.2; Boerhinger-Mannheim) at 10-20 pglml in PBS + 0.4% BSA. After the specimens were washed they were mounted in 50 mM Tris-HCL (pH 8.9), 0.1 M NaCl, 10% glycerol, 0.02% NaN3 containing 1 mg/ml of p-phenylene- diamine to retard fluorescence quenching [Johnson and Araujo, 19811. The samples were examined on an Ortholux I1 equipped for epifluorescence illumination with a 100-W Hg lamp, and Leitz 25 X 0.75 N.A. and Zeiss 63 X 1.4 N.A. oil immersion objectives. Leitz filters K455 or K480 were used in combination with the H2 cube. Photomicrographs were taken using a Vario-orthomat camera on Kodax Tri-X pan film and were developed in Acufine to an exposure index of 1,OOO. Fluorescence micrographs were printed on F5 Kodabromide paper.

RESULTS AND DISCUSSION

Observations were made on cryostat sections of pectoralis, ALD, and PLD muscles from eleven chickens. Transverse and longitudinal cryostat sections were used to determine the localization of alpha spectrin, gamma actin, intermediate filament antigen, and fibronectin with respect to vinculin. Simultaneous localization of two proteins was unnecessary because the location of vinculin is well defined in relation to the banding pattern of the underlying myofibril. With respect to any one of the five proteins examined, ALD, PLD, and pectoralis showed qualitatively similar patterns. Therefore, examples of localization patterns have been chosen randomly from the results in the different muscles.

Appearance of Vinculin, Spectrin, Gamma Actin, and Intermediate Filament Antigen in Transverse Sections of Chicken Skeletal Muscle

Vinculin. In transverse sections, vinculin is detectable only at the sarcolemma [Pardo et al, 1982, 1983aI (Fig. 1A). It is evident from Figure 1A that the organization of this membrane-associated vinculin can be appreciated only in oblique and longitudinal sections that graze the sarcolemma of the fiber. In such sections, vinculin is

452 Craig and Pardo


found in an extensive two-dimensional lattice at the cortex of the fiber [Pardo et al, 1982, 1983al (Fig. 1B). The lattice looks like a sheath or stocking on the fiber. The elements of the lattice that run perpendicular to the long axis of the fiber are the costameres (arrows Fig. IB); the costameres are periodic, in register with the I band of the subjacent myofibril, and seem to be coupled physically to the myofibrils [Pardo et al, 1982, 1983al.

Spectrin. As reported by Repasky et al [1982] spectrin is highly concentrated at the muscle fiber cortex (Fig. lC,D). In addition, as also noted by Nelson and Lazarides [1983], there is an internal reticular pattern of fluorescence that is much dimmer than the fluorescence associated with the cortex. This fine reticulum is punctuated by brighter foci of fluorescence (Fig. 1C). Because of the difference in fluorescence intensity between the cortex and the internal reticulum, it is hard to reproduce both patterns accurately in the same picture. A very short exposure time was used for the print in Figure 1C so that the reticulum would be seen, but as a consequence the cortex-associated fluorescence is unnaturally diffuse. Figure 1 C also shows that not all the fibers have the same degree of internal staining. Fiber heterogeneity with respect to the spectrin reticulum was observed in the three types of muscle examined; the basis for it is not known but could reflect the heterogeneity of fiber types in these muscles.

Gamma actin. The localization pattern of gamma actin is similar to that of spectrin. The cortex of the fiber is strikingly fluorescent, and inside the fiber there is a fainter reticulum that is devoid of foci (Fig. 2A,B). The internal reticulum resembles the pattern shown when isolated Z-disc sheets are stained with antidesmin [Granger and Lazarides, 19781, suggesting that this pattern reflects the presence of gamma actin located at the periphery of the myofibrils. Evidently, the pattern does not represent staining of thin filaments in the myofibrils because transverse sections stained with an antibody that recognizes both alpha and gamma actin [Pardo et al, 1983bI show the expected fluorescent cross-sections of myofibrils (Fig. 2C,D). Staining of the myofibrils in Figure 2C is so intense that the comparatively dimmer staining of the internal reticulum and fiber cortex does not stand out.

Intermediate filament antigen (IFA). Transverse sections stained for IFA with the monoclonal antibody provided by Pruss et a1 [1981] show an internal reticulum that is brighter than the cortex associated stain (Fig. 3A,B). The reticulum is more prominent in some fibers, usually the less well preserved fibers, than in others, as can be seen by comparing the fluorescence and phase micrographs of the two fibers in Figure 3A,B. Perhaps ice damage unmasks antigenic determinants or causes intermediate filaments to collapse together forming larger, hence more visible, masses. The IFA reticulum is the pattern expected from the known locations of intermediate

Fig. 1. Immunofluorescence localization of vinculin and spectrin in chicken skeletal muscle. A. Transverse section of ALD stained for vinculin, showing restriction of vinculin to the fiber cortex. B. Longitudinal section grazing the surface of a pectoralis fiber stained with antivinculin. Vinculin is organized at the sarcolemma in a two dimensional network. Arrows mark the transverse bands or costameres that are periodic with I bands of the underlying myofibrils. C . Transverse section stained with antispectrin. ,Note intense fluorescence at cell margin and the fainter internal reticulum punctuated by bright foci. Fibers in ALD, PLD, and pectoralis show heterogeneity with respect to the presence of detectable internal spectrin stain. D. Phase micrograph of C .

454 Craig and Pardo

Fig. 2. Immunofluorescence of gamma actin and alpha actin plus gamma actin in transverse sections of chicken skeletal muscle. A. Section of ALD stained for gamma actin. The cortex of the fibers is extremely bright and there is a dimmer internal reticulum that outlines the periphery of the myofibrils. B. Phase micrograph of A. C. Trans- verse section stained with antibody that recognizes both alpha and gamma actin. Cross-sections of the myofibril bundles are brightly stained in all the fibers. D. Phase micrograph of C.


Fig. 3. Transverse sections of chicken skeletal muscles stained for intermediate filament antigen or fibronectin. A. Pattern of intermediate filament antigen in ALD. The internal reticulum is more brightly stained than the cortex of the fiber. B. Phase micrograph of A. C . A section of PLD stained with antifibronectin; only the cortex of the muscle fibers is fluorescent. D. Phase micrographs of C.

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filaments in the Z-disc scaffold surrounding each myofibril at the level of the Z line [Lazarides and Hubbard, 1976; Granger and Lazarides, 19781. Since gamma actin and spectrin are organized in a similar reticulum, it is likely that they are also localized at the periphery of the myofibrils.

Fibronectin (FN). Staining for fibronectin is extremely dim and confined to the fiber cortex (Fig. 3C,D). The small quantity of fibronectin associated with the sarcolemma is evident by comparison with the brilliant fluorescence of nearby con- nective tissue cells.

Appearance of Vinculin, Spectrin , Gamma Actin, and Intermediate Filament Antigen in Longitudinal Sections of Chicken Skeletal Muscles

In contrast to vinculin, the proteins spectrin, gamma actin, and IFA are detected easily throughout the chicken muscle fibers. This situation could have made it difficult to distinguish the membrane-associated fluorescence from the internal fluorescence in longitudinal sections. Fortunately, however, both spectrin and gamma actin are highly enriched at the sarcolemma compared to elsewhere in the fiber (Figs. 1C,D,2A,B). This difference in fluorescence intensity enables us to identify and separate the sarcolemma-associated fluorescence in longitudinal sections.

Spectrin (Fig. 4A,C) and gamma actin (Fig. 5A,C) are organized in a two- dimensional lattice at the sarcolemma. As in the case of vinculin (Fig. lB), there are fluorescent foci at the intersections of the longitudinal and transverse components of the lattice. These foci occur at the ends of Z lines (arrows Fig. 4C). The fluorescent bands of spectrin and gamma actin that are transverse to the long axis of the fiber repeat in register with the I bands of the subjacent layer of myofibrils.

The difference in fluorescence intensity between sarcolemma-associated and myofibril-associated gamma actin (Fig. 2A) is also readily apparent in longitudinal sections (Fig. 5E); here, the edge of the fiber is brilliantly fluorescent while the Z line-associated fluorescence of the myofibrils is just barely discernible. Obviously, it is easy to identify the sarcolemma-associated gamma actin.

Consistent with our previous findings in mouse diaphragm [Pardo et al, 1983b], gamma actin is also associated with mitochondria in these chicken muscles (Fig. 5F,G arrows). However, in mouse diaphragm, the sarcolemma-associated gamma actin was not as intense as it is in the chicken muscles, and we could not detect myofibril- associated stain at all. The reason for this variation is not known.

For both spectrin and gamma actin, the width of the stained bands in the myofibrils is consistently less than the width of the I band and closely approximates the Z line (Fig. 6A-D). These data together with the reticular patterns observed in transverse sections (Figs. lC, 2A) demonstrate that the network of spectrin and gamma actin encircles each myofibril in the plane of the Z disc. The similarity between the location of spectrin and gamma actin and the organization of desmin reported by Lazarides and coworkers [1982] is obvious.

Intermediate filament antigen. Our results from localization of the intermediate filament antigen confirm those from previous studies on localization of desmin at the periphery of Z discs and in filamentous structures that run along the long axis of the fiber in close association with the plasma membrane [Lazarides and Hubbard, 1976; Lazarides et al, 19821. In addition, we observe a distinct two-dimensional lattice, which seems to be located at the cortex of the fiber (Fig. 7A,C). At the level of resolution of light microscopy, this IFA lattice is identical with the sarcolemma- associated lattice visualized with antibodies to vinculin, spectrin, and gamma actin.


Fig. 4. Longitudinal sections of chicken skeletal muscles stained with antispectrin. The cortex-associated fluorescence (A, PLD; C, ALD) is organized in a bright two-dimensional lattice. Fluorescent foci (arrows, C) occur at intersections of the longitudinal and transverse lattice components. The transverse fluorescent bands are in register with the I band of underlying myofibrils. B. Phase micrograph of A, showing that fluorescence of A is in the sarcolemmal plane. D. Phase micrograph of C, showing the myofibril banding pattern.

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Fig. 6. Myofibril-associated gamma actin and spectrin overlie the Z line, and not the entire I band of the myofibrils. Fluorescence (A) and phase (B) micrographs of an ALD stained with antigamma actin. The fluorescent bands of gamma actin in A (arrows) are narrower than the corresponding I bands (arrows) shown in the phase micrograph (B). C. Fluorescence micrograph of an ALD fiber stained with antispectrin. D. Phase micrograph of C showing that I bands are wider than the corresponding fluorescent bands (arrows) of the myofibrils in C.

Fig. 5 . Localization of gamma actin in longitudinal sections of skeletal muscle. A. Section of pectoralis showing organization of gamma actin in a two-dimensional lattice. B. Phase micrograph of B. C. Another example of the gamma actin lattice in a pectoralis fiber showing the costameres more clearly. D. Phase micrograph of C illustrating that the fluorescent costameres in C overlie the I bands of subjacent myofibrils. E. Longitudinal section below the sarcolemma of a pectoralis fiber, illustrating the difference in fluorescence intensity betwen cortex-associated gamma actin and gamma actin at the 2 lines of internal myofibrils. F. Longitudinal section of PLD showing association of gamma actin with mitochondria (arrow). G. Phase micrograph of F; mitochondria are labeled with arrow. Note also that the fluorescent bands in F are skinnier than the I bands in G.

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Because the myofibrillar IFA stain is more intense than the cortex-associated fluorescence (Fig. 3A), identification of the lattice as a sarcolemma-associated structure depends on observing the sarcolemmal plane by phase microscopy (eg, Fig. 7C,D). The other identifying characteristic of sarcolemma-associated IFA is the prominent presence of the longitudinal components. Despite evidence from electron microscopy that intermediate filaments run longitudinally between Z discs of the sarcomeres [Price and Sanger, 1979; Wang and Ramirez-Mitchell, 19831 we and others [Bennett et al, 1979; Lazarides and Hubbard, 1976; Granger and Lazarides, 19781 have found that in immunofluorescence stains of myofibrils and myofibers the transverse banding pattern in register with the Z line is the most prominent and often the only pattern visible. Possibly the myofibril-associated, longitudinal arrays of 100- A filaments observed by electron microscopy are masked by other proteins and are not consistently available to react with antibody in our sample preparation.

Fibronectin. In longitudinal sections, fibronectrin sometimes shows indication of a periodic arrangement (Fig. 7E), but this is not clear as it is for the other proteins we have localized. Also, the muscle-associated fibronectin is so dim that it has not been possible to record on film the faint banding patterns that we have occasionally seen. Thus, it is uncertain whether fibronectin, a cell surface-associated glycoprotein, is organized in the costamere pattern, as defined by proteins on the cytoplasmic surface of the membrane. Such co-localization would be important to establish potential transmembrane linkages between the muscle cytoskeleton and the extracel- lular matrix.

In stretched chicken ALD, the vinculin-containing costamere is seen to be composed of two closely spaced rows of densely clustered patches which flank the Z line [Pardo et al, 1983al. In the present study, we found that the organization of IFA and spectrin, but not that of gamma actin, in costameres also shows a change in stretched ALD, seeming to form a ladderlike structure over the Z line (data not shown). Because of the low resolution of the light microscope, these observations are only tentative, and immunoultrastructural localization studies are needed to determine the localization of these proteins with respect to the substructure of the costamere.

CONCLUSION

Based on these co-localization studies, we conclude that gamma actin, spectrin, and intermediate filament antigens could be structural components of the lattice of sarcolemma-to-myofibril attachment sites defined earlier by the localization of vinculin [Pardo et al, 1982, 1983al. In addition, spectrin [Nelson and Lazarides, 1983; this paper] and gamma actin are components of the internal Z-disc scaffold, originally

Fig. 7. Immunofluorescence localization of intermediate filament antigen (IFA) and fibronectin (FN) in longitudinal sections of skeletal muscle. A. Pectoralis fibers stained with anti-IFA show a two-dimensional lattice associated with the sarcolemma. This lattice is identical with that seen with antibodies to gamma actin, spectrin, and vinculin. B. Phase micrograph of A. C., Sarcolemmal lattice in PLD stained with anti-IFA. Both longitudinal and transverse components of the lattice are seen. D. Phase micrograph of C showing that the lattice is present in the same focal plane as the sarcolemma. E. Immunofluorescence localization of fibronectin in PLD. Occasionally periodicity of the fibronectin tufts is observed. F. Phase micrograph of E.

462 Craig and Pardo

defined by the localization of desmin [Granger and Lazarides, 19781 that connects the myofibrils to each other at the level of the Z line.

ACKNOWLEDGMENTS

This work was supported by NIH grant AI-13700 and the Muscular Dystrophy Association. We thank Lucy Robinson for printing the micrographs.

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gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres,...

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