Projectin is an invertebrate connectin (titin): Isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle

Summary A filamentous protein was isolated from crayfish claw muscle. This protein had physiochemical properties very similar to vertebrate skeletal muscle connectin (titin), although its apparent molecular mass ( 1200 kDa) was considerably lower than that of connectin ( 3000 kDa). Polyclonal as well as monoclonal antibodies against chicken skeletal muscle connectin reacted with the 1200 kDa protein from crayfish claw muscle. Conversely, polyclonal antibodies against crayfish 1200 kDa protein crossreacted with chicken connectin. Circular dichroic spectra indicated the abundance of-sheet structure ( 60 %). Low-angle shadowed images showed filamentous structures (0.2 0.5m) by electron microscopy. Proteolysis of the 1200 kDa protein by -chymotrypsin or V8 protease rapidly resulted in formation of 1000 kDa or 1100 and 800 kDa peptides. The amino acid composition was very similar to those of vertebrate connectins and of honeybee flight muscle projectin. Based on the molecular weight and amino acid composition, the 1200 kDa protein is regarded to be crayfish projectin.Immunofluorescence and immunoelectron microscopy revealed that crayfish projectin was localized in the A/I junction area and A-band except for its centre region in crayfish claw muscles. Polyclonal antibodies against crayfish claw muscle projectin reacted with 1200 kDa projectin of honeybee and beetle flight muscle. A monoclonal antibody against chicken skeletal muscle connectin also reacted with honeybee and beetle projectin. Immunoelectron microscopic observations revealed that anti-crayfish projectin antibodies bound the connecting filaments linking the Z-line and the thick filaments up to the M-line of honeybee muscle sarcomere. Anti-crayfish projectin antibodies bound the I-band region near the Z-line of beetle flight muscle.It is concluded that the 1200 kDa projectin from crayfish claw muscle is an invertebrate connectin (titin). Recent work with locust flight muscle mini-titin (Nave & Weber, 1990) is in good agreement with the present study, except that the isolated minititin estimated as 600 kDa appears to be a proteolytic product ( 1100 kDa) of the parent molecule ( 1200 kDa).     

The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study

Summary Monospecific polyclonal antibodies against two exceptionally large proteins, titin (a-T) and nebulin (a-N) isolated from rabbit skeletal muscles, were raised in guinea pig. Using an immuno-pre-embedding method, we have localized at the ultrastructural level of resolution the reactivity sites in skinned muscle fibres. At resting length a-T and a-N antibodies recognize epitopes which only partially overlap. a-T antibodies decorate mostly the A band with at least four clearly distinguished lines of reaction and one line in the I band, all near the A/I limit; a-N antibodies bind to the same region, but with wider areas of reaction in both A and I bands. To study whether the localization of these reaction sites varies according to the sarcomere length, skinned rabbit psoas fibres were incubated at sarcomere lengths ranging from maximum shortening to overstretching. The results indicate that lines decorated by a-T move away from the Z disc when the sarcomere is lengthened. With respect to the M line, the behaviour was biphasic. When the sarcomere was stretched up to about 2.8 m, the decorated lines maintain almost the same distance from the M line. When the sarcomere is stretched beyond 2.8 m, all a-T epitopes move away from the M line and the molecule behaves elastically. At resting length the a-N decoration appears to be localized on three large adjacent bands at the I, A/I and A level. The a-N line of reaction at the edge of the A band moves away from the Z discs as the sarcomere lengthens, while a second line which seems to be localized at the tip of the thin filament moves away from M line when the sarcomere lengthens. In non-overlapping sarcomeres a-N antibodies decorate only the tip of the thin filaments. Our results indicate that titin forms a polar filament connecting the M line to the Z line. In short sarcomeres, the filament seems to have some connections with structures of the A band, since titin epitopes do not move during stretching. These connections are lost at longer sarcomere lengths. On the other hand, our results suggest that nebulin is probably not a constituent of the titin filament.     

The entire cDNA sequences of projectin isoforms of crayfish claw closer and flexor muscles and their localization

Projectin is a giant protein related to twitchin and titin/connectin, that is found in arthropod striated muscle. The complete sequence of a 1 MDa projectin from Drosophila muscle was recently deduced from a thorough analysis of the genomic DNA (Southgate and Ayme-Southgate, 2001). Here we report the complete sequence for projectin from crayfish claw closer muscle (8625 residues; 962,634 Da). The N-terminal sequence contains 12 unique 19-residue repeats rich in glutamic acid (E) and lysine (K). This region, termed the EK region, is clearly distinguishable from the PEVK-like domain of Drosophila projectin. The sequence of crayfish flexor projectin differs from that of closer muscle projectin in that there is a 114-residue deletion and a 35-residue insertion in the N-terminal region. Immunofluorescence microscopy demonstrated that projectin is mainly localized within the sarcomeric A band in both closer and flexor muscles, although the N-terminal region was shown to extrude into the I band region. In the closer muscles, invertebrate connectin (D-titin) connects the Z line to the edge of the A band (Fukuzawa et al., 2001). We have shown that invertebrate connectin is also present in flexor muscle sarcomeres, although in very low abundance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments

Summary The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 m remained at about 0.8 m from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 m. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 m of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.     

Single-molecule measurement of elasticity of serine-, glutamate- and lysine-rich repeats of invertebrate connectin reveals that its elasticity is caused entropically by random coil structure

Invertebrate connectin (I-connectin) is a 1960 kDa elastic protein linking the Z line to the tip of the myosin filament in the giant sarcomere of crayfish claw closer muscle (Fukuzawa et al., 2001 EMBO J 20: 4826–4835). I-Connectin can be extended up to 3.5 μm upon stretch of giant sarcomeres. There are several extensible regions in I-connectin: two long PEVK regions, one unique sequence region and Ser-, Glu- and Lys-rich 68 residue-repeats called SEK repeats. In the present study, the force measurement of the single recombinant SEK polypeptide containing biotinylated BDTC and GST tags at the N and C termini, respectively, were performed by intermolecular force microscopy (IFM), a refined AFM system. The force vs. extension curves were well fit to the wormlike chain (WLC) model and the obtained persistence length of 0.37 ± 0.01 nm (n = 11) indicates that the SEK region is a random coil along its full length. This is the first observation of an entropic elasticity of a fully random coil region that contributes to the physiological function of I-connectin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of ultrastructural and contractile activation properties of crustacean (Cherax destructor) muscle fibres during claw regeneration and moulting

Summary Long-(SL>6m) and short-sarcomere (SL<4m) fibres were isolated from the claw muscle of the yabby (Cherax destructor) during limb regeneration and at different stages of the moult cycle. Long-sarcomere fibres were more susceptible to the changes resulting from the moult-induced atrophy compared with the short-sarcomere fibres. Signs of atrophy included fibre erosion, loss of myosin filaments, a reduction in the diameter of myosin filaments and changes associated with the Z line. The intracellular structure of the fibres, however, remained intact in both fibre types. Fibres taken immediately prior to ecdysis could not be fully activated with Ca²⁺ or Sr²⁺ without breaking. In contrast fibres taken within 4 h after ecdysis could develop and maintain full force when activated by Ca²⁺ or Sr²⁺. The results suggest that loss of myofibrillar proteins via the moult-induced atrophy and/or events associated with fibre elongation may occur in the period just prior to ecdysis and that these changes may be responsible for the fibres inability to function during the premoult stage. Results from this study showed that short-sarcomere fibres add sarcomeres by at least two different mechanisms (1) transverse sarcomere splitting and (2) Z line splitting. Long-sarcomere fibres appear to be elongated by mechanism (s) other than those used by short-sarcomere fibres which possibly involve large electron dense structures which are positioned between the myofibrils and within the A and I bands. Results from the regenerating chelae limb bud showed that sarcomeres form from separate units comprising myosin filaments and actin filaments anchored into Z lines respectively. These sub-sarcomeric units then join together to form sarcomeres. Myofibril formation is aided by electron dense regions which are closely associated with the membrane system. These fibres although short in length and still within the non-functional limb bud could be activated by Ca²⁺ and Sr²⁺ suggesting that full fibre function exists before the chelae become functional. Regenerating muscle fibres consisted predominately of fibres with short-sarcomeres.     

Four aspects of creep phenomena in striated muscle

Summary Four aspects of the slow creep of tension and sarcomere lengths observed during fixed-end tetani are studied with computer simulations, using the instantaneous steady-state (adiabatic) approximation. (1) Most aspects of fixed-end creep phenomena can be simulated in the presence of the passive forces which correctly produce initially shortened end sarcomeres. However, the very large maximum tensions observed with fibres of low resting force for sarcomere lengths > 3.0m cannot be simulated within the adiabatic approximation. (2) Random variations in the passive tension-length curve between different sarcomeres can predict the reported incidence of contracting sarcomeres in the middle of the fibre, while avoiding significant tension creep when a central segment is length-clamped. They can also reverse the velocity of these sarcomeres during creep in fibres with high resting tension, as observed by Altringham and Bottinelli (1985). At sarcomere lengths of 3.4m we find that spatial variations in passive tension strength also contribute to tension creep. (3) Crossbridge fluctuations in active tension have been estimated from the sliding-filament model, and do not contribute significantly to tension creep. (4) The need for inter-sarcomere stiffness or other mechanisms to produce an additional slow rise in tension at long times, and to smooth the sarcomere length distribution, is assessed.Mathematical symbols used for muscle variables and parameters, including standard parameter values _– slope of active tension-velocity curve in contraction per unit isometric tension - ₊ slope (as above) in extension per unit isometric tension - _i equals _± for sarcomere number i, (i = 1, ...,n) - l_i random sarcomere length shift for passive tension, sarc. no. i - l r.m.s. value of the passive length shifts - l_i random sarcomere length fluctuation from active tension - l r.m.s. value of the active length fluctuations - decay exponent per sarcomere for extra end tension strength - i sarcomere number [i = l(middle), ...,n(end)] - l_i(t) length of sarcomere i at time t - l_end average sarcomere length of a fibre segment at one end - l_* slack length per sarcomere - L total length of fibre - coefficient of viscous passive tension n number of sarcomeres in half-fibre - exponent for length dependence of extra end tension - t time - _C correlation time for active tension fluctuations - r overall rate of the contraction cycle - R₀ isometric ATP-ase rate per half-sarcomere - T(t) total tension per filament at time t - T(l, ) steady-state tension function (per filament) - T₀(l) isometric active tension for sarc. length l - T_Pp(l) homogeneous passive tension function of sarc. length l - T_{P i}(l_i) inhomogeneous passive tension for sarc. no. i, length l_i - T_P,o strength of passive tension function T_P(l) (at 3.6 m) - T_{i E}(l_i) extra end tension function for sarc. no. i - T_{E i} strength of extra end tension function for sarc. no. i (at 3.6 m) - T_E,o strength of extra end tension (T _E ⁿ ) at end of fibre (i = n) - extension velocity per sarcomer - exponent for length dependence of passive tension     

A Titan but not Necessarily a Ruler: Assessing the Role of Titin During Thick Filament Patterning and Assembly

The sarcomeres of striated muscle are among the most elaborate and dynamic eukaryotic cellular protein machinery, and the mechanisms by which these semicrystalline filament networks are initially patterned and assembled remain contentious. In addition to the acto‐myosin filaments that provide motor function, the sarcomere contains titin filaments, comprised of individual molecules of the giant Ig‐ and fibronectin domain‐rich protein titin. Titin is the largest known protein, containing many structurally distinct domains with a variety of proposed functions, including sarcomere stabilization, the prevention of over‐stretching, and returning to resting length after contraction. One molecule of titin, which binds to both the Z‐disk and the M‐line, spans a half‐sarcomere, and is proposed to serve as a “molecular ruler” that dictates the spacing of sarcomeres. The semirigid rod‐like A‐band region of titin has also been proposed to act as a scaffold for thick filament formation during muscle development, but despite decades of research, this hypothesis has not been rigorously tested. Recent studies in zebrafish have brought into question the necessity for the A‐band region of titin during the early stages of sarcomere patterning. In this review, we give an overview of the many different roles of titin in the development and function of striated muscle, and address the validity of the “molecular ruler” model of myofibrillogenesis in light of the current literature. Anat Rec, 297:1604–1614, 2014. © 2014 Wiley Periodicals, Inc.     

Tension and stiffness of frog muscle fibres at full filament overlap

Summary Stiffness measurements in activated skeletal muscle fibres are often used as one means of estimating the number of attached crossbridges on the assumption that myofilament compliances do not contribute significantly to the fibre compliance. This assumption was tested by studying the effects of sarcomere length on fibre stiffness in the plateau region of the length-tension diagram (from 1.96 to 2.16m sarcomere length in the tibialis anterior muscle of the frog). Lengthening of the sarcomere across this region in fact, produces only an increase in the proportion of actin filament free from cross-bridges without altering the amount of effective overlap; no change in fibre stiffness is therefore expected if actin filaments are perfectly rigid. The results show that while tetanic tension remained constant within 1.5%, as the sarcomere length was increased from 1.96 to 2.16m fibre stiffness decreased by about 4%, indicating that a significant proportion of sarcomere compliance is localized in the actin filaments. A simple model based on the sliding filament theory was used in order to calculate the relative contribution of actin filaments to fibre compliance. In the model it was assumed that fibre compliance resulted from the combination of crossbridge compliance (distributed over the overlap zone) in series with thin filament and tendon compliances. The calculations show that the experimental data could be adequately predicted only assuming that about 19% of sarcomere compliance is due to actin filament compliance.     

Influences of sarcomere length and selective elimination of myosin filaments on the localization and orientation of triads in rat muscle fibres

Summary Ultrastructural features of internal membrane systems directly concerned with the excitation-contraction coupling were observed in chemically skinned muscle bundles prepared from Wistar rat extensor digitorum longus muscle to clarify two questions: (1) whether triads localization and orientation are influenced by the sarcomere length and (2) whether triads localization and orientation are influenced by the selective elimination of myosin filaments. The distance between triads and Z-lines depends on the sarcomere length: it increase with sarcomere length. There is a highly significant (p<0.01) positive correlation between sarcomere length and the distance between triads and Z-line. The distance between Z-line and triads is dependent on sarcomere length, but the width of junctional gap remains constant when the sarcomere length was changed. Incubation in a concentration of KCl, which dissolves the myosin filaments. The localization and orientation of triads was not altered by the elimination of myosin filaments, however, the distance between the Z-line and triads becomes shorter when the myosin filaments was completely eliminated. There were significant differences (p<0.01) between control and myosin filament eliminated fibres in the distances between Z-lines and triads (over 2 m). These results indicate that the distance between triads and Z-lines depend on the sarcomere length and that there may be some connection(s) between triads and the myofibrils. There is that the elastic component responsible for tethering the triads in their normal position is interrupted either because it is normally attached to the myosin filaments, or because it is extracted by the conditions that dissociate myosin filaments.     

Active tension generation in isolated skeletal myofibrils

Summary Single or double myofibrils isolated from rabbit psoas muscle were suspended between a fine needle and an optical force transducer. By using a photodiode array, the length of every sarcomere along the specimen could be measured. Relaxed specimens exhibited uniform sarcomere lengths and their passive length-tension curve was comparable to that of larger specimens. Most specimens could be activated and relaxed four to five times before active force levels began to decline; some specimens lasted for 10–15 activation cycles. Active tension (20–22°C) was reproducible from contraction to contraction. The contractile response was dependent on initial sarcomere length. If initially activated at sarcomere lengths of 2.7 m, one group of sarcomeres usually shortened to sarcomere lengths of 1.8–2.0 m, while the remaining sarcomeres were stretched to longer lengths. Myofibrils that were carefully activated at shorter initial sarcomere lengths usually contracted homogeneously. Both homogeneous and inhomogeneous contractions produced high levels of active tension. Calcium sensitivity was found to be comparable to that in larger preparations; myofibrils immersed in pCa 6.0 solution generated 30% of maximal tension, while pCa 5.5–4.5 resulted in full activation. Active tension at full overlap of thick and thin filaments ranged from 0.34 to 0.94 N mm^-2 (mean of 0.59 N mm^-2±0.13 sd. n=65). Even allowing for a maximum of 20% nonmyofibrillar space in skinned or intact muscle fibres, the mean tension generated by isolated myofibrils per cross-sectional area is higher than by fibre preparations.     

Limits of titin extension in single cardiac myofibrils

Summary Passive force and dynamic stiffness were measured in relaxed, single myofibrils from rabbit ventricle over a wide range of sarcomere lengths, from 2–5m. Myofibril stretch up to sarcomere lengths of 3 m resulted in a steady increase in both force and stiffness. The shape of the length-force and the length-stiffness curves remained fully reproducible for repeated extensions to a sarcomere length of 2.7 m. Above this length, myofibrillar viscoelastic properties were apparently changed irreversibly, likely due to structural alterations within the titin (connectin) filaments. Stretch beyond 3 m sarcomere length resulted in a markedly reduced slope of the passive force curve, while the stiffness curve became flat. Thus, cardiac sarcomeres apparently reach a strain limit near a length of 3 m. Above the strain limit, both curve types frequently showed a series of inflections, which we assumed to result from the disruption of titin-thick filament bonds and consequent addition of previously bound A-band titin segments to the elastic I-band titin portion. Indeed, we confirmed in immunofluorescence microscopic studies, using a monoclonal antibody against titin near the A/I junction, that upon sarcomere stretch beyond the strain limit length, the previously stationary antibody epitopes suddenly moved into the I-band, indicating A-band titin release. Altogether, the passive force/stiffness-length relation of cardiac myofibrils was qualitatively similar to, but quantitatively different from, that reported for skeletal myofibrils. From these results, we inferred that cardiac myofibrils have an approximately two times greater relative I-band titin extensibility than skeletal myofibrils. This could hint at differences in the maximum passive force-bearing capacity of titin filaments in the two muscle types.     

Positioning of actin filaments and tension generation in skinned muscle fibres released after stretch beyond overlap of the actin and myosin filaments

Summary Skinned fibres from frog semitendinosus muscle were stretched in relaxing solution from a sarcomere length of 2.5 m to greater sarcomere lengths, and then shortened back to the original length. Fibres could be stretched up to sarcomere lengths of 3.3 m, and reshortened fully. If the original stretch was to a sarcomere length greater than 3.3 m, the extent of recovery was dependent on the magnitude of the stretch and the number of times the stretch/shorten cycle was repeated. When the original stretch was to sarcomere lengths beyond overlap of the thick and thin filaments, the thin filaments did not re-enter the thick filament array but buckled at the A-I junction. If these fibres were subsequently activated and contracted, the thin filaments re-entered the thick filament array, taking up approximately their former positions, and allowing reduced development of isometric tension.Finally, the present observations support the view that calcium-induced interactions of actin and myosin filaments in the presence of ATP play an important role in the organization of myofibrillar structure during differentiation (compare Hayashiet al. 1977; Shimada & Obinata, 1977).     

Nebulin is a full-length template of actin filaments in the skeletal muscle sarcomere: an immunoelectron microscopic study of its orientation and span with site-specific monoclonal antibodies

Summary Nebulin, a giant myofibrillar protein with size variants from 700 to 900 kDa in various skeletal muscles, has been proposed to constitute a set of inextensible filaments anchored at the Z-line and coextensive with actin filaments. To elucidate the architectural organization of this fourth set of myofilaments in the skeletal muscle sarcomere, we performed immunoelectron microscopic localization of epitope profiles of a number of site-specific monoclonal antibodies against cloned human nebulin fragments of known sequence loci. Monoclonal antibody N113, which is directed to fragment ND8 at approximately 300 residues away from the C-terminus, labelled the edges of Z-lines in both human quadriceps muscle and rabbit psoas muscle. Monoclonal antibody N101, which is directed to fragment NB5 near the N-terminal side, is localized to a single locus at 0.89 m from the Z-line in human quadriceps muscle and 0.80m from the Z-line in rabbit psoas muscle. Additionally, monoclonal antibody N109, which is directed to fragment NA3 on the carboxy side of the adjacent fragment NB5, is localized at 0.76 m away from the Z-line in rabbit psoas muscle. This one-to-one correspondence between epitope loci and sequence loci demonstrates that a single nebulin polypeptide spans the length of the thin filament with its C-terminus anchored at the Z-line. The epitope spacings of site-specific antibodies are consistent with the notion that the nebulin filament is uniform in mass density along its length.We conclude that the thin filament, as defined morphologically by electron microscopy, is a composite filament of the conventional actin thin filament (actin/tropomyosin/troponin) and coextensible nebulin polypeptides which act as full-length molecular templates that regulate or stabilize colaterally the actin filament in the skeletal muscle sarcomere.     

Filament overlap affects TnC extraction from skinned muscle fibres

Summary Recent studies on calcium regulation of muscle contraction selectively extract troponin C (TnC) from skinned skeletal muscle fibres with a low ionic strength rigor solution containing a Ca²⁺/Mg²⁺ chelator. As previous results from this laboratory and others demonstrate a crossbridge effect, especially rigor, on many of the properties of TnC, the effects of filament overlap on TnC extraction from skinned rabbit psoas muscle fibres were investigated. Tension-pCa relationships at a sarcomere length of 2.7 m were determined before and after a 5 min TnC extraction at sarcomere lengths of 2.3, 2.5, 2.7, 3.1, 3.3 or 3.5 m with 20 mm Tris, pH 7.8, 5 mm EDTA. The decrease in the post-extraction maximum Ca²⁺ activated tension, an indicator of the amount of TnC extracted, was linearly related to the overlap of the thick and thin filaments with decreases in tension being associated with a decrease in filament overlap. The smaller fibre diameter at the longer sarcomere length could facilitate diffusion of TnC from fibre segments. However, the wide range of measured diameters, 40–120 m, accounted for only 14% of the observed tension decrement and shrinking the fibre with polyvinylpyrrolidone did not increase the tension decrement. Increasing the sarcomere length before extraction was also found to decrease the TnC content of fibre segments along with the post-extraction maximum tension. Thus, TnC appears to be preferentially extracted from non-overlap than overlap regions of the sarcomere. These results further indicate that rigor crossbridges affect TnC other than through increased Ca²⁺ binding and that under the conditions used here, they retard its extraction.     

Sarcomeric visco-elasticity of chemically skinned skeletal muscle fibres of the rabbit at rest

The giant muscle protein titin (connectin), contained in the gap filament that connect a thick filament to the Z-line in a sarcomere, is generally considered to be responsible for the passive force (tension) and visco-elasticity in resting striated muscle. However, whether it can account for all the features of the resting tension response remains unclear. In this paper, we examine the basic features of the ‘sarcomeric visco-elasticity’ in a single resting mammalian muscle fibre and attempt to account for various tension components on the basis of known structural features of a sarcomere. At sarcomere length of ∼2.6 μm, the force response to a ramp stretch of 2–5% is complex but can be resolved into four functionally different components. The behaviour displayed by the components ranges from pure viscous type (directly proportional to stretch velocity, ranging from 0.1 to 30 lengths s⁻¹) to predominantly elastic type (insensitive to stretch velocity at 1–2 s time scale); simulations show two components of visco-elasticity with characteristically different relaxation times. The velocity-sensitive components (only) are enhanced by filament lattice compression (dextran – 500 kD) and by increased medium viscosity (dextran – 12 kD); also, the relaxation time of visco-elasticity is longer with increased medium viscosity. Amplitude of all the components and the relaxation time of visco-elasticity are increased at longer sarcomere length (range ∼2.5 – 3.0 μm). The study, and quantitative analyses, extend our previous work on intact muscle fibres and suggest that the velocity-sensitive tension components in intact sarcomere arise from interactions between sarcomeric filaments, filament segments and inter-filamentary medium; the two components of visco-elasticity arise from distinct regions of titin (connectin) molecules. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sarcomere length behaviour along single frog muscle fibres at different lengths during isometric tetani

Summary A detailed investigation of sarcomere lengthening and shortening during fixed-end tetani has been made along frog muscle fibres stretched over a large range of sarcomere lengths. A variety of sources of error common in such measurements are quantitated and give an uncertainty in sarcomere length of about 53–62 nm. The difference in sarcomere length calculated from the left and right first orders at rest was 21 nm ±16 nm and this is suggested to be a measure of Bragg artefact. The laser diffraction measurements showed that the shortening end regions decrease in size during contraction and that the magnitude of shortening is increased at greater fibre extensions. The average length change and sarcomere length of the central and end regions was 0.10 m (2.85 m) and –0.37 m (2.66 m), respectively. The sarcomere length of the end regions at the end of creep was regularly observed to be <2.1 m. An unexpected finding was the occasional observation of striations in the transition zone between lengthening and shortening regions which remained nearly isometric during a period of tension rise during creep. Measurements of diffraction order linewidth do not suggest increased sarcomere length dispersion in these areas. A smooth transition from shortening to lengthening was always observed. Although our data are in general agreement with the models proposed by Morgan, Mochon and Julian (Biophys. J. 39 (1982) 189–96) and Edman and Reggiani(J. Physiol. (Lond.) 351 (1984) 169–98), specific differences which do exist are discussed.     

Structurally differentDrosophila striated muscles utilize distinct variants of Z-band-associated proteins

Summary Monoclonal antibodies raised against four proteins from insect asynchronous flight muscle have been used to characterize the cross-reacting proteins in synchronous muscle ofDrosophila melanogaster. Two proteins,-actinin and Z(400/600), are found at the Z-band of every muscle examined. A larger variant of-actinin is specific for the perforated Z-bands of supercontractile muscle. A third Z-band protein, Z(210), has a very limited distribution. It is found only in the asynchronous muscle and in the large cells of the jump muscle (tergal depressor of the trochanter). The absence of Z(210) from the anterior four small cells of the jump muscle demonstrates that cells within the same muscle do not have identical Z-band composition. The fourth protein, projectin, > 600 kDa polypeptide component of the connecting filaments in asynchronous muscle, is also detected in all synchronous muscles studied. Surprisingly, projectin is detected in the region of the thick filaments in synchronous muscles, rather than between the thick filaments and the Z-band, as in asynchronous muscles. Despite their different locations, the projectins of synchronous and asynchronous muscles are very similar, but not identical, as judged by SDS-PAGE and by peptide mapping. Projectin shows immunological cross-reactivity with twitchin, a nematode giant protein that is a component of the body wall A-band and shares similarities with vertebrate titin.     

Myosin heads contact with thin filaments in compressed relaxed skinned fibres of frog skeletal muscle

Summary When skinned skeletal muscle fibres with rest sarcomere length (L=2.5 m) are compressed by the addition of various concentrations ([PVP]) of polyvinylpyrrolidone, the relation between the 1,0 spacing (d) of thick filament lattice and [PVP] has been known to break at d of around 35 nm, resulting in a steeper slope of the relationship at d > 35 nm. To clarify the cause of this, X-ray diffraction and crosslinking experiments were carried out. Thed versus [PVP] relationship of stretched fibres (L=3.5m) breaks at a d of around 29 nm. The difference between these characteristic d values, 35-29=6 nm, is close to the diameter of thin filaments (8 nm). The crosslinking efficiency of formaldehyde between myosin heads and thin filament surface, measured by radial stiffness increase, was found to begin to markedly increase when the relaxed fibre with rest L was compressed to a d of nearly 35 nm. In addition to these results, the dversus [PVP] relationship obtained in rigor and in high [Mg²⁺] (30mM) relaxing solutions, and the crosslinking efficiency seen in high [Mg²⁺] solutions supported our previous hypothesis that in normal relaxing solution (containing 1mM Mg²⁺) the probability of myosin heads coming into contact with the thin filament surface abruptly increases at d near 35 nm in fibres with rest L.     

Changes in diffraction patterns with length in single muscle fibres at rest

The addition of a simple X-Y sampling circuit to a closed circuit television system (CCTV) permits measurements of a narrow profile of the laser diffraction patterns from single frog muscle fibres at rest. Results confirm that maximum intensity occurs at 2.95–3.00 m, but a positive linear relationship between the dispersions of sarcomeres and sarcomere lengths from 2.7–3.8 m is obtained in four isolated single fibres.