THE ANATOMICAL RECORD 244:148-154 (1996)

lmmunocytochemical Electron Microscopic Study and Western Blot Analysis of Troponin in Striated Muscle of the Fruit Fly Drosophila melanogaster and in Several Muscle Cell Types of the Earthworm

Eisenia foetida MAR ROYUELA, ROSA GARCIA-ANCHUELO, M. PAZ DE MIGUEL, M. ISABEL ARENAS, BENITO FRAILE, AND RICARDO PANIAGUA

Department of Cell Biology and Genetics, University of Alcala de Henares, Madrid, Spain

ABSTRACT Background: There is little information about troponin in invertebrate muscles, and no previous references to this protein in annelid muscles have been found. The aim of this paper was to study the presence and distribution of troponin in different muscle cell types from the earth- worm Eiseniu foetida (the muscular body wall, and the inner and outer muscular layer of the pseudoheart). These results were compared with those obtained in the transversely striated muscle of Drosophila melum- guster and in skeletal and smooth muscles of the mouse.

Methods: Immunocytochemical electron microscopic study and Western blot analysis using anti-TnT antibodies were employed in this study.

Results: Troponin immunoreaction was detected in the mouse skeletal muscle, the fly flight muscle, and earthworm obliquely striated muscles (body wall musculature and inner muscular layer of the pseudoheart). Im- munolabeling for TnT in all these muscle cells appeared in moderate amounts at any point along the sarcomere length, except for the central zone of the A band (H band). This suggests that troponin molecules were located along the thin filaments. The density of immunogold particles was similar in the three muscles, and thus the amount of troponin in each mus- cle type was proportional to the number and length of actin filaments in each. Troponin was found in neither the mouse smooth muscle nor the outer muscular layer of the earthworm pseudoheart. The latter muscle showed an ultrastructural pattern that was intermediate between ob- liquely striated and smooth muscle. The estimated molecular weight for TnT in the earthworm was 55 kDa; this is higher than the weight of this protein in the mouse skeletal muscle (40 kDa) but similar to that of the D. melumguster muscle (52 kDa).

Conclusions: Troponin is present in both types of striated muscle (trans- versely striated and obliquely striated) of the earthworm with a distribu- tion that is very similar to that observed in the mammalian striated muscle. As in vertebrates, troponin is absent in the smooth muscle of the earth- worm. Discrepancies in the classification of some invertebrate muscles are common in the literature, and the use of distinctive markers, such as tro- ponin, may improve our understanding of the nature and properties of many invertebrate muscles showing an ultrastructural pattern that does not resemble any of the classic muscle types.

Key words: Troponin, Obliquely striated muscle, Striated muscle, Dro-

o 1996 Wiley-Liss, Inc.

sophila melumguster muscle, Oligochaete annelid muscle

Troponin is a regulatory protein of muscular contrac- tion in striated muscle cells. In vertebrates, this pro- tein is localized on the thin filament, with a periodicity of approximate]y 40 nm, and consists of three subunits Received June 1 9 7 19g5; accepted September 25, 1995.

Address reprint requests to R. Paniagua, Department of Cell Biol- ogy and Genetics, University of Alcald de Henares, E-28871 Alcald de (TnT, TnI, and TnC) in equimolar proportions (Ohtsuki

et al., 1986). Troponin is activated by the increase in Henares, Madrid, Spain

0 1996 WILEY-LISS. INC

TROPONIN EISENIA FOETIDA MUSCLES 149

cytosolic levels of Ca++ ions, which bind to TnC and permit myosin ATPase activity and actin-myosin in- teraction (Collins et al., 1991). Troponin is absent in vertebrate smooth muscle cells, where another protein, calmodulin, is involved in muscular contraction. When cytosolic Ca' + levels increase, the calmodulin-Ca+ +

complex binds to the myosin light chain kinase, thereby phosphorylating the myosin light chain and permitting actin-myosin interaction (Trybus, 1989).

Vertebrate muscle cell classification of striated and smooth muscle is not quite adequate for invertebrate muscles. Transversely striated muscle cells are present in arthropods (Smith, 1961; Bonilla et al., 1992). Ob- liquely striated muscle is found in nematodes (Rosen- bluth, 1965), annelids (Hanson, 1957; de Eguileor et al., 1987; Royuela et al., 19951, and molluscs (Morrison and Odense, 1974; Matsuno and Kuga, 1989). Smooth muscle is reported in molluscs (Matsuno, 1987; Morri- son and Odense, 1974) and echinoderms (Matsuno, 1987). Nevertheless, there is no complete agreement on the classification of many invertebrate muscles. Inter- mediate cell types between transversely striated mus- cle and obliquely striated muscle have been reported in annelids (Bouligand, 1966), and intermediate types be- tween obliquely striated muscle and smooth muscle have been observed in annelids (Naitoh and Matsuno, 1985) and molluscs (Matsuno, 1987).

Biochemical methods have demonstrated troponin in some muscles of nematodes (Kimura et al., 1987), mol- luscs (Ojima and Nishita, 1986, 1992; Ojima et al., 1990), and arthropods (Bullard et al., 1988; Garone et al., 1991; Lehman et al., 1976; Nishita and Ojima, 1990; Mieguel et al., 1992). Morphological demonstra- tion of troponin has recently been carried out in the nematode muscle using immunofluorescence micros- copy (Nakae and Obinata, 1993). All these troponin containing muscles are presumably transversely or ob- liquely striated, but troponin has also been reported in the smooth muscle of the bivalve Chlamys nipponensis (Ojima and Nishita, 1986). However, other authors failed to find troponin in the smooth muscle of the bi- valve Pseudocardium sachalinensis (Chiba et al., 1992). We have not found references to troponin in an- nelid muscles.

The aim of the present report was to investigate the distribution of troponin in several types of earthworm muscle, classified according to ultrastructural pattern, using immunoelectron microscopy and Western blot analysis. The results were compared with those ob- tained in transversely striated muscle of the fruit fly and skeletal and smooth muscles of the mouse.

MATERIAL AND METHODS

The tissues studied were psoas (striated muscle) and intestinal wall muscle (smooth muscle) from the mouse (Mus musculus), thoracic flight muscle (transversely striated muscle) from the fruit fly (Drosophila melano- gaster), and muscular body wall (obliquely striated muscle) and pseudoheart muscle (consisting of both ob- liquely striated muscle and a muscle of doubtful clas- sification which seems to be intermediate between smooth muscle and obliquely striated muscle) from the earthworm (Eisenia foetida, Oligochaeta). The animals were anaesthetized with ether and killed, and the mus- cles mentioned above were removed for the following

studies: conventional electron microscopy (two animals of each species), immunoelectron microscopy (five ani- mals of each species), and Western blotting analysis (the number of animals that was necessary in each case to obtain 100 mg of muscular tissue). The primary an- tibody used was mouse antirabbit TnT troponin anti- body, which was supplied by Amersham (Buckingham- shire, UK).

The specificity of the primary antibody was tested by Western blotting analysis as described by Towbin et al. (1979). Tissues were homogenized in 0.5 M Tris-HC1 buffer (pH 7.4) containing 1 mM EDTA, 12 mM 2-mer- capto-ethanol, and 1 mM phenylmethylsulphonyl flu- oride (PMSF). The homogenates were centrifuged at 10,OOOg for 30 min. After boiling for 2 min at 98"C, aliquots of 20 p1 homogenate were separated in SDS- polyacrylamide slab minigels (12% Wh), according to the procedure of Laemmli (1970). Separated proteins were transferred for 4 h to 0.25A nitrocellulose paper, and thereafter the nitrocellulose sheets were stained with Ponceau red, soaked in blocking solution (1 M glucose, 1% BSA, 0.5% Tween-20,10% glycerol in PBS, pH 7.3) overnight at 37°C and then incubated with the primary antibody at 1:400 dilution in blocking solution for 3 h. After extensive washing with PBS-Tween-20, the sheets were incubated with a peroxidase-labeled second antibody (goat antimouse biotinylated immuno- globulin) (Biocell, Cardiff, UK), at 1/1,000 dilution in blocking solution. The filters were developed with en- hanced chemiluminescence (ECL) Western blotting analysis, following the procedure described by the manufacturer (Amersham).

For electron microscopy immunohistochemistry, prior to fixation, extended muscles were incubated for 30 min in a solution of 0.6 M KI, 0.02 M Na2S203, 1 mM EGTA, and 0.02 M Tris-HC1 (pH 7.5) according to de Eguileor et al. (1988) to maintain muscular relaxation. Thereafter, tissues were fixed for 90 min in 0.1 M phos- phate-buffered mixture of 2.5% paraformaldehyde and 0.5% glutaraldehyde (in equal proportions), a t pH 7.4. Afterwards, the material was washed, dehydrated, and embedded in Lowicryl K4M. Ultrathin sections were placed on drops of 0.2 M Tris buffer containing 0.1% glycine and 1% BSA. Then they were incubated for 2 h a t room temperature with the primary antibody at 1:20 dilution. After washing with Tris buffer, the sections were incubated with 15 nm gold-labeled IgG goat an- timouse immunoglobulin (Biocell), a t M O O dilution in 20% goat serum-PBS buffer (pH 7.6) for 2 h at room temperature. After incubation with the second anti- body, the sections were washed with Tris buffer and distilled water and counterstained with uranyl acetate for 20 m at room temperature.

The specificity of the immunohistochemical proce- dure was checked by incubation of sections with 1) non- immune serum instead of the primary antibody, 2) pri- mary antibody that had previously been absorbed with the purified antigen, and 3) primary antibody that had previously been absorbed with a nonrelated antigen (substance P, 10 nmol/ml, 48 h, at 4°C). As positive controls, sections from the mouse striated and smooth muscles mentioned above were immunolabeled as the same time as the invertebrate muscles.

For conventional electron microscopy tissues were incubated in the above-mentioned solution and fixed

150 M. ROYUELA ET AL.

K Da

97.4 - 66.2 -

42.7-

31.5-

21.5-

14.4 -

MW M-Sk Y a m E-BW E-Ps D-FI

Fig. 1. Western blotting analysis of the TnT troponin subunit in different muscle types after 12% SDS-polyacrylamide gel electro- phoresis. MW: molecular weight. M-Sk: mouse skeletal muscle. M-Sm: mouse smooth muscle. E-BW Eisenia foetida body wall mus- cle. E-Ps: Eisenia foetida pseudoheart muscle. D-FI: Drosophila flight muscle.

for 6 h in 3% glutaraldehyde in 0.1 M cacodylate, buff- ered at pH 7.2 with 0.1 M sodium cacodylate, rinsed in the cacodylate buffer, postfixed for 4 h in 1% osmium tetroxide in the cacodylate buffer, rinsed again in cac- odylate, dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections were double-stained with ura- nyl acetate and lead citrate.

A semiquantitative study of the immunoreaction density in the different bands of the sarcomere was carried out in the muscle types studied. Calculations were made on electron micrographs taken at a magni- fication of x 20,000. For each muscle type, five electron microscopy sections (grids) containing abundant longi- tudinally sectioned myofilaments were selected. Each section was obtained from a different animal. In each of these sections, 20 areas (256 pm2) showing only longi- tudinally sectioned myofilaments were selected at ran- dom. In each of these areas, the surfaces occupied by the I bands, the central zone of the A bands (H bands), and the remaining surface of A bands were measured using an image analyzer and the number of immu- nogold particles in each band was counted. Since a dis- tinct H band was rarely observed in the invertebrate muscles, although a solution to maintain muscular re- laxation was used, the central zone of the A band in these muscles (1/4 of the A band including the M line) was assumed to be the H band for quantitations. The results were expressed as number of immunogold par- ticles per square micrometer. From the average values obtained for each of the five sections, the means and SD for each muscle type were calculated.

RESULTS Western Blotting Analysis

The results of Western blot analysis showed a single band for all muscles studied except for the mouse smooth muscle which did not immunoreact to TnT an- tibodies. The bands were at the following molecular weights: 40 kDa in the mouse, 52 kDa in D. melano- gaster, and 55 kDa in E. foetida (Fig. 1).

lmmunoelectron Microscopy The thoracic flight muscle of D. melanogaster

showed the characteristic pattern of transversely stri- ated muscle, although the sarcomeres of this muscle differed from those of the vertebrate striated muscle in several aspects, including sarcomere length (4 pm), thin myofilament length (2.6 pm), the length (2.6 pm) and diameter (16 nm) of thick myofilaments, the third thick filament ratio (3/1), and other minor ultrastruc- tural features (Shafiq, 1963; Smith, 1961). In both mouse skeletal muscle (Fig. 2) and insect flight muscle (Fig. 3), immunolabeling for TnT appeared in moderate amounts at any point along the sarcomere length, ex- cept for the central zone of the A band (a clear H band in the mouse muscle and a central zone containing the M line in the insect muscle). This suggests that tropo- nin molecules were located along the thin filaments. The density of immunogold particles, expressed as number of immunogold particles per square microme- ter, was similar in the mouse (3.4 2 0.3 in the I band, 3.2 k 0.3 in the A band, and 0.2 * 0.01 in the H band) and the insect (3.3 * 0.3 in the I band, 3.4 ? 0.3 in the A band, and 0.1 * 0.01 in the central zone of the A band).

The muscle cells in the body wall of the earthworm had a single myofibril which was arranged in a spiral course around the longitudinal axis of the cell (Fig. 4). The myofibril showed the ultrastructure characteristic of obliquely striated muscle. There were sarcomeres consisting of both thick and thin myofilaments ar- ranged in parallel arrays. On one plane of view, the myofilaments appeared to be oriented obliquely to the Z bands rather than perpendicularly, and, on another plane of view (perpendicular to the former), the Z and I bands were seen together. Immunoreaction to TnT in this muscle was observed in the three planes of view only in the A bands and I bands (Figs. 5-7), and the immunolabeling density (immunogold particles per square micrometer) was similar to that found in the mouse and insect muscle (3.6 * 0.4 in the I band and 3.5 k 0.4 in the A band). Although a distinct H band could not be delimited, the central zone of the A band (1/4 of the A band) usually appeared devoid of immunolabeling (0.2 k 0.02).

The earthworm pseudoheart muscle consisted of two layers: an inner layer (Figs. 8, 9) displaying the ultra- structure of the obliquely striated muscle and an outer layer (Figs. 10-12) showing an ultrastructural pattern that was more similar to that of vertebrate smooth muscle than to that of obliquely striated muscle. Thick myofilaments in this outer layer appeared arranged regularly, but no sarcomeric organization (A and I bands with Z discs) was evident. TnT immunoreaction in the cells of the inner layer (Figs. 8,9) was like in the cells of the muscular body wall, whereas muscle cells in the outer layer (Figs. 11, 12), like the mouse smooth muscle, did not immunolabel (Fig. 13).

DISCUSSION In the present study troponin was detected using

mouse-derived antibodies against the TnT subunit of this protein. Western blot analysis in mouse skeletal muscle confirmed the specificity of the antibody used, showing a single band at the corresponding molecular

Fig. 2, 3. Immunolabeling to troponin in two types of transversely striated muscles: mouse skeletal muscle (Fig. 2) and Drosophilu flight muscle (Fig. 3). Immunoreaction is located along the thin filaments. H. H band M. M lines: Z. Z lines. Fie. 2: X 50.000. Fie. 3: X 20.000.

planes (XY, YZ, and ZX). A bands (A) and I bands (I) are visible. Tissue was fixed for conventional electron microscopy with glutaral- dehyde and osmium tetroxide. x 9,000.

I , I , - - Figs. 5-7. Troponin immunoreaction in the body wall of Ezseniu

foetidu. Myofilaments are sectioned in the planes ZX (Fig. 51, YZ (Fig Fig. 4. Muscle cells in the body wall of Eiseniu foetidu showing the characteristic ultrastructural features of obliquely striated muscle. The helicoidally arranged myofilaments have been sectioned in three

6), and XY (Fig. 7). Immunolabeling is observed in the A (A) and I 0) bands. M, M line. Fig. 5: x 23,000. Fig. 6: x 26,000. Fig. 7: x 57,000.

Figs. 8,9. Troponin immunolabeling in the inner muscular layer of the pseudoheart from Eisenia foetida. The ultrastructure and immu- noreaction are similar to those observed in the body wall muscle (Figs. 6, 7). A, A bands; I, I bands. Fig. 8: x 70,000. Fig. 9: x 46,000.

Figs. 11, 12. Troponin immunolabeling in the inner muscular layer of the pseudoheart from Eisenia foetida. No troponin immunolabeling is observed in either longitudinally (Fig. 11) or cross-sectioned (Fig. 12) myofilaments. Fig. 11: x 54,500. Fig. 12: x 72,500.

Fig. 13. Absence of troponin immunoreaction in mouse intestinal Fig. 10. Muscle cell in the outer muscular layer of the pseudoheart from Eisenia foetida. The cell shows abundant, parallelly arranged myofilaments, but no sarcomeric organization is apparent. Tissue was fixed for conventional electron microscopy with glutaraldehyde and osmium tetroxide. x 15.000.

smooth muscle. x 27,500.

TROPONIN EISENIA FOETIDA MUSCLES 153

weight (MW), approximately 40 kDa (Ebashi et al., 1972). When Western blotting analyses were carried out in invertebrate muscles using the same antibody, a single band also appeared for each species, but the MW of the protein detected was different (52 kDa in Dro- sophila and 55 kDa in the earthworm). This agrees with previous biochemical data on the variability in the MW of TnT in invertebrates: 55 kDa in the nema- tode Ascaris (Kimura et al., 1987), 40 kDa in the bi- valve Chlamys (Ojima et al., 1990), 55 kDa in the bi- valve Aequipecten (Lehman et al., 1980), 40 kDa in the merostomata Limulus (Lehman et al., 1976), 40 kDa in the crustacean Homarus (Nishita and Ojima, 1990); 50-55 kDa in the insect Lethocerus (Bullard et al., 19881, and 50-55 kDa in Drosophila (Bernstein et al., 1993). The MW value found for the TnT of Drosophila in the present study is within these values (52 kDa). Although we have found no references to troponin in annelids, the present findings show this TnT also has a MW of about 55 kDa.

Troponin is absent in the vertebrate smooth muscle (Collins, 1976). Smooth muscle contraction is regulated by the calmodulin-Ca++ complex which binds to the myosin light chain kinase and causes the phosphory- lation of the myosin light chains. This phosphorylation permits actin-myosin interaction and the subsequent muscular contraction. We failed to find troponin in the outer muscular layer of the earthworm pseudoheart. On the basis of its ultrastructural pattern, this muscle was difficult to classify; the abundance and arrange- ment of thick myofilaments suggested a striated mus- cle, whereas the absence of Z bands suggested a smooth muscle. The absence of troponin in this muscle con- firmed the latter opinion. Troponin has been reported in some bivalve muscles that were considered of the smooth type (Ojima and Nishita, 19861, whereas other authors failed to find this protein in similar muscles of other bivalve species (Chiba et al., 1992). These differ- ent results might be attributed to interspecific differ- ences; however, it is more likely that they are due to a mistaken classification of the troponin-positive mus- cles, which would actually be obliquely striated and not of the smooth type. Discrepancies in the classification of some invertebrate muscles are common in the liter- ature (Bouligand, 1966; Naitoh and Matsuno, 1985). The use of distinctive markers, such as troponin, may improve our understanding of the nature and proper- ties of many invertebrate muscles showing an ultra- structural pattern that does not resemble any of the classic muscle types.

The results of the present immunocytochemical study revealed that troponin is present in the trans- versely and obliquely striated muscles of the inverte- brate species studied. In vertebrate striated muscle, troponin is located along the thin myofilaments (Oht- suki et al., 1986). In the invertebrate striated muscles examined here, the H band was barely observed despite the use of a solution to maintain muscular relaxation. This agrees with previous accurate ultrastructural studies of insect (Shafiq, 1963) and oligochaete (de Eguileor et al., 1988) muscles. Thus, it should not be concluded that troponin molecules are specifically as- sociated with the thin filaments because thin myofila- ments might be overlapped in the center of the sarcom- ere. However, the absence of immunogold particles in

the center of the A band in D. melanogaster and E. foetida might be interpreted in two ways: the thin fil- aments are not overlapped or the thin filament tips are devoid of troponin. A less convincing explanation would be that troponin is associated with both thick and thin filaments but it is lacking on the central seg- ment of thick filaments. Therefore, present results agree with the notion that troponin distribution in the invertebrate striated muscle studied here is similar to that found in vertebrate striated muscle. The immuno- labeling density is similar in all the striated muscles studied, and thus the amount of troponin in each mus- cle type is proportional to the number and length of actin filaments in each.

Differences in physiological behavior between verte- brate and invertebrate striated muscles seem to be re- lated with the affinity of troponin for Ca++ rather than with the amount of troponin. The TnC subunit of troponin has four binding sites for Ca+ + andlor Mg+ +. Sites I and I1 are directly involved in muscular contrac- tion, whereas the principal role of sites I11 and IV is the integration of the troponin complex (Ohtsuki et al., 1986; Morimoto and Ohtsuki, 1987). Collins et al. (1991) have classified striated muscles into four cate- gories according to the number of TnC sites that bind Ca+ + . Vertebrate skeletal muscle, with four Ca+ +

binding sites, forms group A. Vertebrate cardiac mus- cle, with three binding sites (11, 111, and IV) forms group B. The transversely striated muscle of D. mela- nogaster belongs to group C, which only binds Ca+ + in sites I1 and IV. This group also includes the striated muscles of other arthropods, such as the lobster (Ga- rone et al., 1991), the crayfish (Kobayashi et al., 1989a1, and the horseshoe crab (Kobayashi et al., 1989b). The obliquely striated muscle of some bivalves, such as Chlamys nipponensis and Patinopecten yessoen- sis (Ojima and Nishita, 1992), are classified as group D which only binds Ca+ + at site IV. The low Cat + af- finity in these type D muscles is complemented by the additional use of the same mechanism that acts in the smooth muscle contraction: the calmodulin-Ca + + com- plex (Kendrick-Jones et al., 1970; Takahashi et al., 1990). The obliquely striated muscle of E. foetida stud- ied here might belong to this group, although studies of Ca+ +-binding sites in its TnC have not been reported.

The efficiency of muscular contraction is also related with the development of the sarcoplasmic reticulum and T tubules (sarcotubular system), which account for the release of the Ca++ that binds to TnC and pro- motes muscular contraction. The sarcotubular system is very developed in transversely striated muscles of vertebrates and invertebrates and permits rapid and continuous contractions. The sarcotubular system is less developed in obliquely striated muscles, and their contraction is not so rapid, immediate and continuous, although, due to their myofilament distribution, it is more resistant to tension. In the smooth muscle, the sarcoplasmic reticulum is even less abundant, and con- tractions are slower but more prolonged than in the striated muscles (Morrison and Odense, 1974).

ACKNOWLEDGMENTS The authors thank Mr. Antonio Priego for technical

assistance and Ms. Carol F. Warren from the I.C.E. of

154 M. ROYUELA ET AL.

the University of Alcala de Henares for linguistic as- sistance.

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