Károly Trombitás

Elastic properties of the titin filament in the Z-line region of vertebrate striated muscle.

SummaryThe characteristics of the titin filament in the vicinity of the Z-line were investigated using immunoelectron microscopy. We used monoclonal titin antibodies T-11 and T-12 on single fibres of frog skeletal muscle, and on Z-line-extracted fibres. It is well established that the I-band region of titin is elastic. We find, however, that the elastic properties are not uniform. The T-12 epitope, which binds near the Z-line at the N1-line level, hardly changes position relative to the Z-line as the sarcomere is stretched. This demonstrates the functional inextensibility of the N1-Z-line region. After extreme stretch (above 6-μm sarcomere length), this zone finally does elongate; thus, the titin molecule in this region is intrisically elastic. The functional inextensibility seen at shorter sarcomere lengths may, therefore, be a result of binding of titin to the actin filament in the zone near the Z-line. When the Z-line was extracted, the T-12 epitope remained in the same position as in the unextracted fibres; it did not retract from the Z-line. Failure to retract implies that functional anchoring of titin is not exclusive to the Z-line, but includes some site closer to the A-band. Combined with the results of the above-mentioned stretch experiment, this result implies a likely binding of titin to the thin filament either focally at the N1 line or all along the entire N1-Z region. Thus, this region of titin is functionally stiff, but intrinsically elastic.

I-bands of striated muscle contain lateral struts

In electron micrographs of striated muscle, the I-band often shows a distinct cross-striation. The periodicity of this striation is near 40 nm and has been attributed to troponin, which is localized along the thin filament. However, the cross-striation is often so prominent as to be suggestive of physical structures running transversely across the I-band. We examined I-band ultrastructure using three independent methods: thin sections of chemically fixed specimens; freeze-fracture; and freeze-substitution. With all three methods we found transverse structures distributed throughout the I-band, many of which bridged the gap between neighboring filaments. Such structures were observed in each of the several species studied. In fish muscle in particular, which has a highly regular lattice, it was obvious that these structures gave rise to the observed periodicity.

Immunoelectron microscopic observations on tropomyosin localization in striated muscle

SummaryTropomyosin localization in striated muscle was studied by means of immunoelectron microscopy. Polyclonal and monoclonal antibodies to tropomyosin were allowed to diffuse into mechanically skinned single fibres dissected from frog semitendinosus muscle. Antibodies produced transverse I-band stripes with the expected periodicity of 38 nm. However, some differences were revealed among the various antibodies. While polyclonal antibodies generally showed 23 stripes, monoclonal antibodies showed an extra 24th stripe immediately adjacent to the Z-line, implying some structural/functional uniqueness of this terminal tropomyosin. Furthermore, the stripes did not always lie parallel to the Z-line. When the Z-line was straight or slightly skewed, the stripes generally were parallel to it. However, when Z-line skew was more severe, the stripes remained perpendicular to the fibre axis, indifferent to the Z-line skew. This may imply that the coupling of tropomyosin to the thin filament is not tight. Finally, the monoclonal antibodies themselves exerted an anomalous effect on the Z-line, apparently extracting or shifting some of its mass.

Contraction-induced movements of water in single fibres of frog skeletal muscle

SummaryAlthough X-ray diffraction measurements imply almost constant filament separation during isometric contraction, such constancy does not hold at the level of the isolated cell; cell cross-section increases substantially during isometric contraction. This expansion could arise from accumulation of water drawn from other fibre regions, or from water drawn into the cell from outside. To distinguish between these hypotheses, we froze single fibres of frog skeletal muscle that were jacketed by a thin layer of water. Frozen fibres were freeze-substituted, sectioned transversely, and examined in the electron microscope. In fibres frozen during contraction, we found large amounts of water just beneath the sarcolemma, less in deeper regions, and almost none in the fibre core. Such gradients were absent or diminished in fibres frozen in the relaxed state. The water was not confined to the myofibril space alone; we found large water spaces between myofibrils, particularly near mitochondria. Accumulation of water between myofibrils and around mitochondria implies that the driving force for water movement probably lies outside the filament lattice, and may therefore be osmotic. The fact that the distribution was nonuniform-highest near the sarcolemma and lowest in the core-implies that the water was likely drawn from the thin jacket surrounding the cell. Thus, the contractile cycle appears to be associated with water entry into and exit from the cell.

Elastic Properties of Connecting Filaments Along the Sarcomere

The elasticity of the connecting filament--the filament that anchors the thick filament to the Z-line--has been investigated using rigor release, freeze-break and immunolabelling techniques. When relaxed insect flight muscle was stretched and then allowed to go into rigor, then released, the recoil forces of the connecting filaments caused sarcomeres to shorten. Thin filaments, prevented from sliding by rigor links, were found crumpled against the Z-line. Thus, rigor release experiments demonstrate the spring-like nature of the connecting filaments in insect flight muscle. In vertebrate skeletal muscle, however, the same protocol did not result in sarcomere shortening. Absence of shortening was due to either smaller stiffness of connecting filaments and/or higher stiffness of the thin filaments relative to insect flight muscle. The spring-like nature of the connecting filament was confirmed with the freeze break technique. When the frozen sarcomeres were broken along the A-I junction, the broken connecting filaments retracted to the N1-line level, independently of the thin filaments, demonstrating the basic elastic nature of these filaments. To study the elastic properties of the connecting filaments along the sarcomere, the muscle was labelled with monoclonal antibodies against a titin epitope near the N1-line, and another very near the A-I junction in the I-band. Before labelling, fibers were pre-stretched to varying extents.(ABSTRACT TRUNCATED AT 250 WORDS)

Thick filaments of striated muscle are laterally interconnected

Earlier reports from this and other laboratories indicated that thick filaments may be interconnected along their length by rung-like structures. This study was carried out to test whether these interconnections are genuine structures; whether they appear in different muscle types; and whether they arise from myosin cross-bridges. We studied insect flight muscles because of their well-known ultrastructure, and frog heart and rabbit psoas muscles secondarily. Ultrastructure was examined with freeze-fracture; with conventionally prepared thin sections; and with negative stain. All three methods showed rung-like interconnections between thick filaments. The interconnections spanned the length of the cross-bridge zone, i.e., along all but the central bare zone of the thick filament. They were observed consistently in relaxed, activated, and rigor states. We considered potential artifacts that might cause apparent interconnections where none existed in vivo, but were unable to identify a source of artifact common to all methods. Several features of the interconnections imply that for the most part they may be composed of S-1 heads from adjacent thick filaments binding to one another at their tips.

Rigor bridge angle: Effects of applied stress and preparative procedure

The effect of applied force on the rigor cross-bridge angle was studied in insect flight muscles. Both conventionally prepared thin sections and freeze-fracture methods were used. We exploited a technique in which thin filaments could be dislodged from their anchor points on the Z-lines, so that they could no longer transmit tension to the bridges. The rigor bridge angle in such sarcomeres was no different from the rigor bridge angle in sarcomeres with intact thin filaments, even with tension applied to the specimen. Thus, bridge tension does not change the rigor angle. In addition, we found that the bridge angle did not always assume the expected 45° value. In the freeze-fracture specimens the angle was generally closer to 90°, and in conventionally prepared specimens we found angles ranging from 45° to 135° depending on the experimental condition. Thus, the rigor angle does not appear to be fixed at 45°.

Filament lengths in frog semitendinosus and tibialis anterior muscle fibres

SummaryIn frog semitendinosus muscle the descending limb of the length-tension curve is shifted rightward relative to that of tibialis anterior. Both the plateau right corner and the zero-force intercept are equally shifted. To investigate the reason for this shift, we compared filament lengths in the two muscles. Single fibres were mechanically skinned, stretched to reveal filaments clearly, incubated in a solution containing one of several antibodies to enhance filament visualization, and examined by electron microscopy. We found no differences of filament length. Thick filament lengths were 1.62 and 1.61 μm, respectively. I-segment lengths were measured by two methods. With the first, filament length was the same for both muscles, 1.95 or 1.98 μm, depending on the value taken for the troponin repeat; with the second it was 1.92 and 1.94 μm, respectively, for the two muscles. These differences are insignificant. Thus, the reported differences of shape of the length-tension curve are not explainable in terms of differences of filament length.