Structural organization of the Z-line protein, α-actinin, in developing skeletal muscle cells

DB (“‘Xl) DEVELOPMENTAI.

RIOLOGY

75, 231-238

(1980)

BRIEF Structural

Organization

NOTES

of the Z-Line Protein, a-Actinin,

in Developing

Skeletal Muscle Cells H. JOCKUSCH*.’ AND B. M. JOCKUSCH~~ *Institute of Neurobiology, ijEuropean Molecular

Uniuersity of Heidelberg, Im Neuenheimer Biology Laboratory, D-6900 Heidelberg

Received

March

15, 1979; accepted

Feld 347, D-6900 Heidelberg 1. and 1, Federal Republic of Germmy

in revised form June 18. 1979

The accumulation and spatial distribution of cu-actinin were studied in rat skeletal muscle cells developing in culture. Immunohistochemistry with an affinity column-purified antibody was employed. a-Actinin was found to accumulate in myoblasts prior to, and during, chain formation. In immature myotubes it displayed a longitudinal stress fiber-like pattern. During the period of spontaneous contractions, this pattern was gradually replaced by the assembly of cr-actinin in transversal Z-lines. This transformation could be prevented by paralyzing the culture with tetrodotoxin. a blocker of sodium channels.

tions using muscle cultures and an antibody directed against cu-actinin in conjunction The unit of the contractile apparatus of with indirect immunofluorescence. skeletal muscle, the sarcomere, is bordered by transverse disks, the Z-“lines,” to which MATERIALS AND METHODS the actin filaments are attached. In mature Chemicals and media. Collagenase Type cross-striated muscle fibers, the Z-lines of I (from Clostridium histolyticum), collagen the myofibrils are in register. A large pro(acid soluble from calf skin), Ficoll (MW portion, possibly more than half, of the approx 400,000), and tetrodotoxin were obprotein in the Z-line is a-actinin, a protein tained from Sigma Chemie, Munich. Mewhich can be purified as a soluble dimer dium A was 10% (v/v) horse-serum (heat with approx lOO,OOO-daltonsubunit molecinactivated) and 2% (v/v) chick embryo ular weight (Suzuki et al., 1976). In vitro extract in Dulbecco’s modified Eagle’s meexperiments indicate that a-actinin might dium containing 100 units of penicillin, 100 in fact be the constituent of the Z-line to pg of streptomycin, and 1 pg of amphoteriwhich the actin filaments are anchored tin per ml. Medium A-Hepes was medium (Go11 et al., 1972). Thus, cu-actinin may play A made 50 m&Y in N-2_hydroxyethylpipera key role in the molecular organization of azine-N-2-ethanesulfonic acid at pH 7.4. the myofibril. Muscle cultures. Limb muscle was colThree questions may be asked concernlected from I- to 3-day-old Sprague Dawley ing the role of a-actinin in muscle developrats, digested with collagenase, and dissoment: (1) Does cY-actinin accumulate signifciated according to Bekoff and Betz (1977). icantly prior to the appearance of a crossThe suspension was filtered through nylon striated pattern? (2) If so, what is the spagauze (with 20-pm square holes) and cential distribution of a-actinin in immature trifuged for 5 min at 250g. The cell sediment muscle fibers? (3) Is the assembly of (Y- was taken up in 2 ml medium A-Hepes and actinin into a Z-line pattern dependent on the suspension was underlayered with 2 ml muscle function? 7% Ficoll in medium A-Hepes. SedimentaThis work attempts to answer these question at unit gravity was allowed for 1 hr at 4°C. The muscle fibers (and fragments ’ To whom correspondence should be sent. INTRODUCTION

I1 0012-1606/80/030231-08$02.00/O Copyright 0 1980 h,v Academic Press. 11x All rights of reproduction in any form r~herved

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DEVELOPMENTAL BIOLOGY

thereof) preferentially settled into the Fico11 cushion. The upper layer containing mainly mononucleated cells was withdrawn. Yields were approx 2 x lo6 cells per rat. The cells were seeded into petri dishes containing collagenized glass coverslips of l&mm diameter. The standard seeding density was 8 x lo5 cells per 35mm dish; the plating efficiency was 35 to 50%. In some cases, cells were seeded at 2 X lo5 cells per 35-mm dish, and after 1 day the coverslips were transferred to dishes previously seeded at the standard density, to ensure better growth of the low-density culture on the coverslip. Antisera and immunofluorescence. The antibody employed, the staining procedure, and the photographic recording have been described (Jockusch et al., 1979). Briefly, an affinity column-purified rabbit antibody directed against porcine skeletal muscle (Yactinin was used in conjunction with fluorescein-labeled goat anti-rabbit antibody. Formaldehyde-fixed cells were made permeable to antibodies by a combined Triton X and acetone treatment. Since no yglobulin was bound to the affinity column when preimmune sera had been applied, the total preimmune serum at an appropriate dilution was used for control experiments. Observations were made, and photographs were taken, on the same day the staining had been performed. In some cases, cells fixed in formaldehyde were stored for several days at 4°C under 5 n&f sodium azide in calcium-magnesium-free phosphate-buffered saline. No differences in the results compared to those from cells delipidated and stained immediately after fixation were observed. RESULTS

AND

DISCUSSION

Specificity of the anti-a-actinin antibody. The affinity column-purified anti-cuactinin antibody was used at concentrations of around 50 pg/ml. Preimmune serum from the same rabbit, diluted 1:50 to give a pro-

VOLUME 75,198O

tein concentration of -1.5 mg/ml, gave diffuse and very weak background fluorescence (Figs. la, b). Preabsorption of the antibody with purified a-actinin abolished subsequent staining of Z-lines in isolated myofibrils (not shown). The exact correspondence of fluorescence and Z-line location in myofibrils has been documented (Masaki et al., 1967; Lazarides and Burridge, 1975; Jockusch et al., 1979). From these data we conclude that our antibody is specific and that its binding indicates the presence of cu-actinin or immunologically related molecules, such as precursors and degradation products of skeletal muscle (Yactinin, a-actinin differing in some posttranslational modification from skeletal muscle a-actinin, or some iso-a-actinin, i.e., product of a gene different from, but related to, the skeletal muscle a-actinin gene. This ambiguity could only be resolved by biochemical techniques. In this paper, “a-actinin” is used as a simplified term to mean “a-actinin and/or immunologically related proteins.” a-Actinin in the early development of primary muscle cultures. In mixed fibroblast/myoblast cultures, myoblasts could often be identified by a typical spindle or fish-like shape. Staining with anti-cu-actinin showed that mononucleated myogenic cells at that stage may already have significantly more ar-actinin than fibroblasts (Figs. lc, d). The fluorescence appeared homogeneously distributed in most cells (Figs. 2a, b, c), yet an indication of a longitudinal fiber structure could be seen in some cases (Fig. Id, arrow). When myoblasts associated in chains, members of the same chain sometimes had very different contents of a-actinin (Figs. 2d, e). This indicated that the intensity of antibody staining was not simply a function of cell mass per area but rather reflected the stage of biochemical differentiation. Subsequently, mononucleated cells fused to form myotubes. Even the smallest fusion products, i.e., myotubes with 2-3 nuclei, were distinguished by a

FIG. 1. Staining of cultured rat myotubes and fibroblasts with preimmune serum (negative control) (a. b) and of a myoblast with affinity column-purified anti-a-actinin (c, d). (a. b) Myotubes (arrows) were from a 4day-old culture seeded at standard density in which spontaneous contractions were seen. (c. d) Myoblast (arrows) and fibroblast-like cells were from a culture seeded at low density 2 days prior to fixation. (a. (‘) Phase contrast; (b. d) IJV fluorescence. x 480. 233

FIG. 2. Single (a, b, c) and chain-forming (d, e) myoblasts from low-density cultures. Cultures were fixed 1 day (a, b, c) and 2 days (d, e) after seeding and stained with anti-o-actinin. (d, e) Note the two adjacent myoblasts, one of which (arrows) fluoresces more strongly than the other. (a, d) Phase contrast; (b, c, e) fluorescence (phase-contrast picture of c not shown). x 480. 234

BRIEF NOTES

235

FIG. 3. Immature myotubes stained with anti-a-actinin. Four-day-old cultures seeded at low (a, bl and standard (c, d) densities. Spontaneous contractions were seen in the culture seeded at standard density. Note the intense staining of the myotube in comparison to the fibroblast-like cells (a, arrow) and the longitudinal distribution of cu-actinin (d, arrow). (a, c) Phase contrast; (b. d) fluorescence. x 480.

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DEVELOPMENTAL BIOLOGY

VOLUME 75,198O

BRIEF NOTES

high content of a-actinin which showed a longitudinal organization (Figs. 3a, b). Longitudinal a-actinin-containing filaments also characterized immature myotubes of larger size (Figs. 3c, d). In a permanent myogenic cell line, L6 (Yaffe, 1968), mononucleated cells had a low cY-actinin content comparable to that of 3T3 fibroblasts, while fusion products stained intensely with anti-a-actinin (unpublished results). Irrespective of the uncertainty with respect to the nature of the antibody binding protein, anti-a-actinin may be used to study the progress of muscle development. In early stages it is mainly the accumulation of Lu-actinin which distinguishes more advanced myogenic cells from less differentiated ones and nonmyogenic cells (cf. Allen et al., 1979). Fiber maturation and conditions preventing the formation of Z-lines. From day 4 of culture onward, myotubes contracted spontaneously. During the following 3 days they assumed the cylindrical shape of a muscle fiber with the nuclei in peripheral bulgings of the cytoplasm. At that stage the cross-striation pattern became clearly visible in phase-contrast optics (cf. Jockusch et al., 1979). As expected, cy-actinin in these fibers was localized in a Z-line pattern (Fig. 4a). However, in the flat “feet” by which myotubes were attached to the substratum, the longitudinal, “stress fiber-like” pattern of cY-actinin distribution persisted (Fig. 4b). In some cases, when the feet were very much stretched out on the substratum, a periodic substructure of these fibers could

237

be distinguished which was similar to the discontinuous distribution of a-actinin in stress fibers of fibroblasts (Lazarides and Burridge, 1975). When the spontaneous contractions of myotubes were arrested by 1 fl tetrodotoxin (TTX), a blocker of voltage-sensitive sodium channels (cf. von Hahn and Honegger, 1974), the myotubes did not assume a cylindrical shape. Furthermore, the cu-actinin was not organized into a Z-line pattern but assumed a distribution in irregular chains of well-separated small specks (Fig. 4c), or, when TTX was given later, in parallel longitudinal arrays (Fig. 4d). When TTX was given after the onset of spontaneous contractions, the Zline pattern of cu-actinin persisted for at least five days. In spontaneously detached myotubes, a speckled distribution of a-actinin reminiscent of TTX-blocked myotubes was often observed (Fig. 4a, arrow). Thus repeated contractions along a constant axis might be a critical condition for the assembly of (Yactinin in a Z-line pattern in which myofibrils are in register. Analogously to the transition from /3- and y- to a-actin in muscle development (cf. Merlie et al., 1977) the a-actinin molecules assembled in Z-line could be of a different molecular species from those in the longitudinal arrangement. Z-line a-actinin would then have to be synthesized de novo. Alternatively, Lu-actinin preexisting in the cytoplasm, possibly in submembrane regions (Hagopian and Spiro, 1970), could be activated to assemble in Z-lines in the course of function-dependent maturation.

FIG. 4. Maturation of myotubes and effects of detachment and tetrodotoxin. Primary rat muscle cultures were fixed and stained with anti-ol-actinin after 7 days in culture. (a, b) No treatment; (c) 1 fl tetrodotoxin from day 1 onward; (d) 1 +I4 tetrodotoxin from day 2 onward. Spontaneous contractions were seen in the control culture from day 4 onward. No contractions were seen in the tetrodotoxin-treated cultures. Note: Absence of cross striation in detached, rounded myotube (a, arrow); transition between cross striation in cylindrical part of myotubes and longitudinal striation in “foot” of myotube (b, arrow); and speckled (c, arrow) and partially longitudinal (d, arrow) distribution of cu-actinin in tetrodotoxin-treated cultures. No indication of cross striation was found in culture Cc), but a few incompletely cross-striated myotube segments were observed in culture (d). In order to show internal cell structure, prints were exposed longer than for the foregoing figures. Fluorescence, x 480.

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DEVELOPMENTAL BIOLOGY

We thank Mrs. Beate Segnitz and Miss Kristin Kelley for excellent assistance, and Dr. M. K. Reedy for critical reading of the manuscript. This work was supported by grants Jo 84/5 and Jo 55/8 from Deutsche Forschungsgemeinschaft. REFERENCES ALLEN, R. E., STROMER,M. H., GOLL, D. E., and ROBSON,R. M. (1979). Accumulation of myosin, actin, tropomyosin, and oc-actinin in cultured muscle cells. Develop. Biol. 69, 655-660. BEKOFF, A., and BETZ, W. J. (1977). Physiological properties of dissociated muscle fibres obtained from innervated and denervated adult rat muscle. J. Physiol. 271, 25-40. GOLL, D. E., SUZUKI, A., TEMPLE, J., and HOLMES,G. R. (1972). Studies on purified a-actinin. I. Effect of temperature and tropomyosin on the cr-actinin/Factin interaction. J. Mol. Biol. 67, 469-488. HAGOPIAN, M., and SPIRO, D. (1970). Derivation of the Z-line in the embryonic chick heart. J. Cell Biol. 44.683-687.

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HAHN, H. P. VON, and HONEGGER,C. G. (1974). Anima1 neurotoxins in neurobiological research. Experientia 30, 2-7. JOCKUSCH,H., JOCKUSCH,B. M., and BURGER,M. M. (1979). Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence. J. Cell Biol. 80. LAZARIDES,E., and BURRIDGE, K. (1975). a-Actinin: Immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell 6, 289-298. MASAKI, T., ENDO, M., and EBASHI, S. (1967). Localization of 6 S component of a-actinin at Z-band. J. Biochem. 62.630-632.

MERLIE, J. P., BUCKINGHAM,M. E., and WHALEN, R. G. (1977). Molecular aspects of myogenesis. Cur. Top. Develop. Biol. 11,61-114. SUZUKI, A., GOLL, D. E., SINGH, I., ALLEN, R. E., ROBSON,R. M., and STROMER,M. H. (1976). Some properties of purified skeletal muscle o-actinin. J. Biol. Chem. 251,6860-6870. YAFFE, D. (1968). Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Nat. Acad. Sci. USA 61, 477-483.