Nature and origin of gap filaments in striated muscle

K. TROMBITAS 0 P. H. W. W. BAATSEN 0 M. S. Z. KELLERMAYER 0 G. H. POLLACK 0 0 Bioengineering WD-12, University of Washington , Seattle, Washington 98195 , USA Summary Immunoelectron microscopy was used to study the nature and origin of 'gap' filaments in frog semitendinosus muscle. Gap filaments are fine longitudinal filaments observable only in sarcomeres stretched beyond thick/thin filament overlap: they occupy the gap between the tips of thick and thin filaments. To test whether the gap filaments are part of the titin-filament system, we employed monoclonal antibodies to titin (T-ll, Sigma) and observed the location of the epitope at a series of sarcomere lengths. At resting sarcomere length, the epitope was positioned in the I-band approximately 50 run beyond the apparent ends of the thick filament. The location did not change perceptibly with increasing sarcomere length up to 3.6 fan. Above 3.6 fan, the span between the epitope and the end of the A-band abruptly increased, and above 4 fan, the antibodies could be seen to decorate the gap filaments. Between - The discovery of titin (also called connectin), a giant structural protein, led eventually to the conclusion that the sarcomere contained, in addition to thick and thin filaments, a third longitudinal filament system composed of titin (Wang, 1985; Maruyama, 1986). On the basis of immunolocalization studies, it was suggested that titin formed a continuous filament connecting the M-line to the Logically, as the sarcomere is extended, the I-band domain of titin should be extended concomitantly. As the sarcomere is extended beyond thick and thin filament overlap, a segment of the titin filament should become visualizable in the gap. Although gap filaments have long been observable, there has been no direct evidence that they are made of titin. In earlier studies, the misalignment of titin epitopes that occurs at longer sarcomere length has made it impossible to obtain regular gap-filament labelling (Itoh et al. 1988; Ramirez-Mitchell et al. 1990; Wang, 1985). The present study was designed to define the nature and origin of gap filaments. To achieve this, we monitored the relative positions of a titin epitope that ordinarily lies Z-line (Wang et al. 19846; Fiirst et al. 1988; Itoh et al. 1988; Single fibres from frog semitendinosus muscle were mechanically Pierobon-Bormioli et al. 1990). These studies (see also Trombitas et al. 19906) further implied that at physiological sarcomere length only part of the titin strand was free and elastic: the I-band region could stretch with increases of sarcomere length, but the A-band domain appeared to be tethered to the thick filament. 5 and 6 fan, the epitope remained approximately in the middle of the gap. Even with this high degree of stretch, the label remained more or less aligned across the myofibril. The abrupt increase of span beyond 3.6 fan implies that the A-band domain of titin is pulled free of its anchor points along the thick filament, and moves toward the gap. Although this domain is functionally inextensible at physiological sarcomere length, the epitope movement in extremely stretched muscle shows that it is intrinsically elastic. Thus, the evidence confirms that gap filaments are clearly part of the titin-filament system. They are derived not only from the I-band domain of titin, but also from its A-band domain. close to the A-I junction, as the sarcomere was stretched. We confirm that the gap filament is indeed made of titin. However, the filament is derived not from titin's I-band domain exclusively: increasing tension apparently rips titin from its A-band tether, allowing a segment of the Aband domain to be translated into the gap. Materials and methods skinned by peeling off the sarcolemma in relaxing solution. The solution contained (HIM): calcium proprionate, 0.035; magnesium proprionate, 6; potassium proprionate 5; K2EGTA, 15; Mops, 117; Na2ATP, 4.4; Na2CP, 15.6; KOH, 57; pCa9.2, ionic strength 0.2, pH7.0). One end of each fibre was pinned down with a minutien pin (Fine Science Tools Inc., Cat. no. 26002-10) to a Sylgard elastomer dissection base on the bottom of a small Petri dish. The fibres were then stretched in small increments to the appropriate sarcomere length. Sarcomere length was set from 2.5 jjia to 6.0 fim. Then the fibres were fixed in a freshly prepared fomaldehyde/PBS fixative (3.7 % paraformaldehyde, 2.7 mM KC1, 1.5 mM KH2PO4,137 DIM NaCl, 8 mM Na2HPO4, pH 7.2) for 15 min at 4C, and washed three times in PBS for 30min per wash. The unspecific binding sites were blocked using PBS/1% BSA solution for 30 min. Conventionally available monoclonal titin antibody (Til, Sigma) was used for the immunolabelling experiments. The test fibres were then incubated for 24 h in the primary antibody solution (25^gml-1 mouse anti-titin IgG in PBS/BSA) at 4C. After washing the preparations three times with PBS/BSA for 30 min, the fibres were treated with secondary antibody solution (50 fig ml"1 rabbit anti-mouse IgG (Sigma) in PBS/BSA) for 24h, at 4C. Control fibres were incubated only in the secondary antibody solution under the same conditions as the test fibres. In addition, the overstretched fibres were labelled either with polyclonal tropomyosin antibody (Lemanski, 1979) as described earlier (Trombitas et al. 1990a), or with monoclonal troponin-T antibody (Sigma), in a manner similar to that used with antititin. The unbound secondary antibody was removed from the fibres by washing in PBS, for 30 min per wash, three times. Then the PBS was replaced with Mops buffer solution (20 mM K-Mops, 5mM MgCl2, pH6.8), and the fibres were fixed in 2.5% glutaraldehyde, 0.2% tannic acid, 10 mM MgCl2, 5mM EGTAand 20 mM K-Mops at pH6.8 for 30min at 4C, according to the method of Reedy and Reedy (1985). Subsequently, the fibres were washed in Mops buffer and in 100 mM potassium phosphate buffer. Then they were postfixed in 1% OsO4, 100 mM potassium phosphate buffer at pH 6.0 for 30 min at 0C, washed in the same buffer twice for 15 min each, and in water in the same way. Fibres were stained en bloc with 2 % aqueous uranyl acetate, dehydrated in a graded ethanol series, and embedded in Araldite. Ultrathin sections were cut with an LKB Ultratome III, stained with potassium permanganate and lead citrate, and observed and photographed with a Philips 420 electron microscope. In muscle stretched above a sarcomere length (SL) of 3.6 nm, a gap appears between the thick and thin filaments. In such a case, sarcomere continuity is maintained by the gap filaments (Fig. 1). Gap filaments associate with thick filaments in the A-band (arrows) and run across the gap into the I-band. They are obviously different from the thin filaments, having much smaller diameter. A fine periodicity of about 12-13 nm is occasionally observable along the gap filament both in the gap itself, and in the I-band (Fig. 1, arrowheads). Furthermore, immunolabelling experiments show that alth (...truncated)