The Z disc of skeletal muscle fibrils

Summary The structure of the Z disc has been studied in thin sections of striated muscle fibers from a wide variety of vertebrates. A common organization is found in all muscles examined. The disc shows a regular pattern made up of dense lines which seem to connect the actin filaments from adjacent sarcomeres. The lines are sometimes disposed to form a regular zigzag configuration; in other orientations with respect to the plane of the section the morphology is confused and, in still other images, the dense lines continuous with the actin filaments seem to go straight through the Z disc. In cross section this structure corresponds to a square pattern of considerable regularity. The intersections in the square pattern mark the location in the plane of the section of the actin filaments from adjacent sarcomeres. Dense lines form the edges of the squares and appear to represent condensations of Z-disc material, i.e., the lines in the zigzag. The possible origin of the structure as a product of the stretching of a membrane is discussed, together with functional interpretations of the Z disc.Postdoctoral fellow under USPHS Training Grant 2 G-707 to K. R. Porter.     

Properties of a clonal muscle cell line from rat heart

Two clonal cell lines have been derived from the thoracic aorta of embryonic rats. Both of these cell lines, at some stage of their development possess membranes capable of generating overshooting action potentials spontaneously. Contiguous cells of each of these lines are electrically coupled. Ultrastructural analysis consistently reveals the presence of rows of pinocytotic vesicles, a well-developed rough endoplasmic reticulum, massive tracts of thin filaments oriented parallel to the longitudinal axis of the cell and randomly dispersed intermediate sized filaments. The specific activities of the enzymes myokinase and creatine phosphokinase (CPK) increase 3- to 5-fold after growth has ceased. These two cell lines synthesize a muscle type CPK isoenzyme after the cessation of cell division. It is concluded that these cell lines proliferate as myoblasts and develop into cells which phenotypically resemble smooth muscle. A third clonal cell line, from fetal rat aorta, with properties of both smooth and skeletal muscle, is also described.     

Distribution of the Two Kinds of Myofilaments in Insect Muscles

Different insect muscles have been studied with the electronmicroscope and the distribution of the two kinds of myofilamentscompared. In muscles other than those of flight, each thickfilament is surrounded by 9–12 thin filaments, whereas,in the flight muscles, the contraction frequency of which ismuch higher, there are only 6 thin filaments surrounding eachthick one; nevertheless, in the flight muscles of some butterflies,the wing stroke frequency of which is particularly low, thereare 7–9 thin filaments. It seems then that there may bea relation between the ratio of the two kinds of myofilamentsand the frequency of muscular contraction. In the muscles which have more than 6 thin filaments surroundingeach thick one, the structure of the 7, line appears to be differentfrom that which was described in dipteran flight muscles. Apeculiar aspect of the M line is observed in lepidopteran flightmuscles.     

The organization and fine structure of the muscles of the scolex of the cysticercoid of Hymenolepis microstoma

The organization and fine structure of the muscles of the scolex of the cysticercoid of Hymenolepis microstoma are described. The contractile apparatus consists of thick (175–325 Å diameter × 1.4 μm) and thin (60–80 Å diameter × 1 μm) filaments. The thick filaments are occasionally attached to the thin filaments by cross bridges. The thin filaments are attached to the dense bodies or to a dense zone at the sarcolemma at muscle insertions. In contracted muscle the thick filaments appear as quasi-hexagonal arrays or in lines. Each thick filament is surrounded by an orbit of up to 12 thin filaments, which in turn may be shared by adjacent thick filaments. Thin filaments may be present in quasi-rectangular or hexagonal groupings indicating some low order degree of actin lattice. The fusiform dense bodies (1,500 Å × 900 Å), consisting of up to 25 discrete substructures, are distributed uniformly throughout the myofiber and/or attached to the sarcolemma at attachment plaques. The sarcoplasmic reticulum, consisting of a presumed anastomosing network of tubules is structurally connected to the sarcolemma by periodic deposits of electron opaque material. Sarcoplasmic extensions of the myofiber(s) contain the nucleus, Golgi complexes, rough endoplasmic reticulum, ribosomes, β-glycogen, mitochondria and membrane bound electron dense structures. Upon activation of the metacestode, groups of α-glycogen and enlargement of the rough endoplasmic reticulum were observed. Microtubules which were conspicuously absent from the sarcoplasm of the unactivated worms appeared adjacent to the myofibers in activated worms.     

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269-277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, "natural" F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca(2+)-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca(2+)-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca(2+). We suggest that twitchin inhibition of the actin-myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.     

Structural and functional reconstitution of thin filaments in the contractile apparatus of cardiac muscle.

The muscle contractile apparatus has a highly ordered liquid crystalline structure. The molecular mechanism underlying the formation of this apparatus remains, however, to be elucidated. Selective removal and reconstitution of the components are useful means of examining this mechanism. In addition, this approach is a powerful technique for examining the structure and function of a specific component of the contractile system. In this study we have achieved the structural and functional reconstitution of thin filaments in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with gelsolin. Under these conditions no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached 135 +/- 64% of the original level. The augmentation of tension was attributable to the elongation of reconstituted filaments. As another possibility for augmented tension generation, we suggest the presence of an inhibitory system that was not reconstituted. In any case, the thin filaments of the cardiac contractile apparatus are considered to be assembled so as not to develop the highest degree of tension. Incorporation of the tropomyosin-troponin complex fully restored Ca2+ sensitivity without affecting maximum tension. The present results indicate that a muscle contractile apparatus with a higher order structure and function can be constructed by the self-assembly of constituent proteins.     

Ca2+- and S1-induced conformational changes of reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy: structural evidence for three States of thin filament

Rabbit skeletal muscle alpha-tropomyosin (Tm) and the deletion mutant (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056] were used to investigate the interaction between actin and tropomyosin or actin and troponin (Tn) by means of fluorescence resonance energy transfer (FRET). FRET between Cys-190 of D234Tm and Gln-41 or Cys-374 of actin did not cause any significant Ca2+-induced movement of D234Tm, as reported previously for native Tm [Miki et al. (1998) J. Biochem. 123, 1104-1111]. FRET did not show any significant S1-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of TnI, and Gln-41 and Cys-374 of actin on thin filaments reconstituted with D234Tm (mutant thin filaments) were almost the same as those on thin filaments with native Tm (wild-type thin filaments) in the absence of Ca2+. Upon binding of Ca2+ to TnC, these distances on mutant thin filaments increased by approximately 10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+-induced conformational change of thin filaments [Miki et al. (1998) J. Biochem. 123, 324-331]. The rigor binding of myosin subfragment 1 (S1) further increased these distances by approximately 7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with S1. This indicates that a further conformational change on thin filaments was induced by S1 rigor-binding (S1-induced or open state). Plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed that the curve for wild-type thin filaments is hyperbolic, whereas that for mutant thin filaments is sigmoidal. This suggests that the transition to the S1-induced state on mutant thin filaments is depressed with a low population of rigor S1. In the absence of Ca2+, the distance also increased on both wild-type and mutant thin filaments close to the level in the presence of Ca2+ as the molar ratio of S1 to actin increased up to 1. The curves are sigmoidal for both wild-type and mutant thin filaments. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding. For mutant thin filaments, the transition from the closed state to the open state in the presence of ATP is strongly depressed, which results in the inhibition of acto-myosin ATPase even in the presence of Ca2+. The present FRET measurements provide structural evidence for three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states) for the regulation mechanism of skeletal muscle contraction.     

Fine structure of the myotendinous junction of lathyritic rat muscle with special reference to connectin, a muscle elastic protein

The fine structure of the myotendinous junction of the skeletal muscle of lathyritic rats caused by β-aminopropionitrile was investigated. In the junction there are finger-like processes of muscle fibers, in which thin filaments were extended from the last Z lines of myofibrils and attached to the sarcolemma of the processes. By the heavy meromyosin decoration technique, these thin filaments were identified as actin filaments. In the lathyritic muscle, the thin filaments were markedly fewer in number and distributed sparsely in the sarcoplasm.The content of connectin, an elastic protein, which is localized in myofibrils and also in sarcolemma was significantly decreased in the lathyritic muscle. A possible relationship between the changes in the fine structure of the myotendinous junction and in the connectin contents is discussed.     

Ca(2+)- and s1-induced movement of troponin T on mutant thin filaments reconstituted with functionally deficient mutant tropomyosin

The deletion mutant (D234Tm) of rabbit skeletal muscle alpha-tropomyosin, in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing, inhibits the thin filament activated myosin-ATPase activity whether Ca(2+) ion is present or not [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056]. Fluorescence resonance energy transfer (FRET) showed substantial changes in distances between Cys-60 or 250 of troponin T (TnT) and Gln-41 or Cys-374 of actin on wild-type thin filaments corresponding to three states of thin filaments [Kimura et al. (2002) J. Biochem. 132, 93-102]. Troponin T movement on mutant thin filaments reconstituted with D234Tm was compared with that on wild-type thin filaments to understand from which the functional deficiency of mutant thin filaments derives. The Ca(2+)-induced changes in distances between Cys-250 of TnT and Gln-41 or Cys-374 of F-actin were smaller on mutant thin filaments than on wild-type thin filaments. On the other hand, the distances between Cys-60 of TnT and Gln-41 or Cys-374 of F-actin on mutant thin filaments did not change at all regardless of whether Ca(2+) was present. Thus, FRET showed that the Ca(2+)-induced movement of TnT was severely impaired on mutant thin filaments. The rigor binding of myosin subfragment 1 (S1) increased the distances when the thin filaments were fully decorated with S1 in the presence and absence of Ca(2+). However, plots of the extent of S1-incuced movement of TnT against molar ratio of S1 to actin in the presence and absence of Ca(2+) showed that the S1-induced movement of TnT was also impaired on mutant thin filaments. The deficiency of TnT movement on mutant thin filaments causes the altered S1-induced movement of TnI, and mutant thin filaments consequently fail to activate the myosin-ATPase activity even in the presence of Ca(2+).     

Sarcomere structures in the rabbit psoas muscle as revealed by fluorescent analogs of phalloidin

Summary The fluorescent analogs of phalloidin (rhodamine-and fluorescein-phalloidin) bind tightly to the skinned fibres of rabbit psoas muscle at essentially the same sites as phalloidin and mainly stain the known regions of actin localization in the sarcomere: the thin filaments and Z bands. On both sides of the Z bands, unstained zones were observed, suggesting the presence of proteins tightly bound to the thin filaments. In myofibrils which are stretched to such an extent that the actin and myosin filaments do not overlap, stained bands could also be seen at the myosin-band border, which suggests the localization of actin at these sites.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : IV. Microfilament Distribution and Cell Shape in Untransformed, Transformed, and Revertant Balb/c 3T3 Cells

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells.     

Re-Exposure of Tapes at High Temperature and Pressure in the Lycopodium Clavatum Spore Exine

Lamellations are visualizable through the staining commonly used in transmission electron microscopy during exine formation on Lycopodium and other spores, and the nexine of pollen grains. The lamellations so exposed consist-of dark tapes at either side of an unstained (white) line. Neither tapes nor white lines are visualizable in the exine of mature spores of Lycopodium. The continued presence of lamellations having tape-white line spacing has been demonstrated with inorganic tracers in the nexine of pollen in which lamellations otherwise appeared to be absent. Through high contrast staining methods for TEM we have observed lamellations in the residual exine following heat treatment (350[ddot]C) of mature spores of Lycopodium clavatum. The surface of these residual exines was etched by treatment with hot 2-aminoethanol and filaments were observed to protrude from the etched surfaces. The residual exine stained darkly. Lycopodium spores heated to 300[ddot]C at 1 kb pressure had long filaments exposed at the surface of the residual exine (sporopollenin). Sections of the pellet remaining after heat and pressure treatment also included bundles of closely parallel filaments and masses of isolated filaments. The filaments were stained while the exine residue, assumed to include sporopollenin, was not. Isolated filaments produce a stable metachromasia with toluidine blue indicating the presence of many closely spaced sites of negative charge. The staining of intact exines with basic dyes may result from anionic sites on filaments embedded within the exine rather than being due to sporopollenin. The results of our experiments indicate that the filaments are more resistant to heat and pressure and 2-aminoethanol than is sporopollenin. We propose that the trilamellar elements commonly called tapes and white lines might be composed of two filaments bridged by polybasic molecules.     

The variable twist of actin and its modulation by actin-binding proteins

Previous studies demonstrated that actin filaments have variable twist in which the intersubunit angles vary by approximately +/- 10 degrees within a filament. In this work we show that this variability was unchanged when different methods were used to prepare filaments for electron microscopy. We also show that actin-binding proteins can modulate the variability in twist. Three preparations of actin filaments were photographed in the electron microscope: negatively stained filaments, replicas of rapidly frozen, etched filaments, and frozen hydrated filaments. In addition, micrographs of actin + tropomyosin + troponin (thin filaments), of actin + myosin S1 (decorated filaments), and of filaments frayed from the acrosomal process of Limulus sperm (Limulus filaments) were obtained. We used two independent methods to measure variable twist based on Fourier transforms of single filaments. The first involved measuring layer line intensity versus filament length and the second involved measuring layer line position. We measured a variability in the intersubunit angle of actin filaments of approximately 12 degrees independent of the method of preparation or of measurement. Thin filaments have 15 degrees of variability, but the increase over pure actin is not statistically significant. Decorated filaments and Limulus filaments, however, have significantly less variability (approximately 2 and 1 degree, respectively), indicating a torsional stiffening relative to actin. The results from actin alone using different preparative methods are evidence that variable twist is a property of actin in solution. The results from actin filaments in the presence of actin-binding proteins suggest that the angular variability can be modulated, depending on the biological function.     

Cytological differentiation of human fetal skeletal muscle.

The ultrastructural differentiation of several different muscles was investigated in human fetuses ranging in age from 13 weeks to neonatal. At approximately 16 weeks of gestation cell cluster containing both myotubes and satellite cells lie enclosed by a newly formed basal lamina and show evidence of fusion. The development of organelles is evident in myoblasts, proceeds as the cells transform into myofibers, and continues in the neonate. Filament synthesis occurs primarily in the cell periphery where thin filaments appear to align themselves in relations to parallel arrays of ribosome-studded thick filaments: Z line formation follows the appearance of thin filaments. Intermediate filaments, approximately 10-12 nm thick, were also consistently observed in perinuclear regions and distal to filament assembly. Although sarcoplasmic reticulum (SR) development is closely related to fibril formation, connections between Z lines and SR are not consistent, thus supporting the conclusion that SR does not evoke the formation of the Z line. Bristlecoated vesicles appear to be the precursors of elements of the SR, possibly the lateral sacs. Development of the transverse tubules, as invaginations of the sarcolemma, is closely associated with the formation of lateral sacs since the latter occur along the sarcolemma as soon as transverse tubules appear. Cytological differentiation is similar, though not identical, in several different muscles. During the last trimester muscle fibers show some evidence of diversity mainly of variation in Z line width. In gerneral the results suggest that the sequence and stages of human myogenesis are similar to those of other species.     

Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.     

Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin

Muscle needs an elastic framework to maintain its mechanical stability. Removal of thin filaments in rabbit skeletal muscle with plasma gelsolin has revealed the essential features of elastic filaments. The selective removal of thin filaments was confirmed by staining with phalloidin-rhodamine for fluorescence microscopy, examination of arrowhead formation with myosin subfragment 1 by electron microscopy, and analysis by SDS-PAGE. Thin section electron microscopy revealed the elastic fine filaments (approximately 4 nm in diameter) connecting thick filaments and the Z line. After removal of thin filaments, both rigor stiffness and active tension generation were lost, but the resting tension remained. These observations indicate that the thin filament-free fibers maintain a framework composed of the serial connections of thick filaments, the elastic filaments, and the Z line, which gives passive elasticity to the contractile system of skeletal muscle. The resting tension that remained in the thin filament-free fibers was decreased by mild trypsin treatment. The only protein component that was digested in parallel with the decrease in the resting tension and the disappearance of the elastic filaments was alpha-connectin (also called titin 1), which was transformed from the alpha to the beta form (from titin 1 to 2, respectively). Thus, we conclude that the main protein component of the elastic filaments is alpha-connectin (titin 1).     

Differences in I band structure,sarcomere extensibility,and electrophoresis of titin between two muscle fiber types of the perch (Perca fluviatilis L.)

I bands of perch muscle contain crosslinks that interconnect the thin filaments as well as the connecting filaments and link them to the membrane systems and to other intermyofibrillar material. In shortened sarcomeres cross connections seem to pile up in the I band and at the A/I band junction. Sarcomeres of red fibers, which in another study are reported to have steep passive stress-strain curves, were pulled apart when they were stretched by about 40% of their slack length. Sarcomeres of white fibers could be stretched by almost 100% and still display filament alignment. Sarcomeres of the red fibers also differed from those of white fibers in the distance over which connecting filaments could be discerned, the presence of an extra transverse filament in their NI lines, the electrophoretic mobility of titin, and the titin/actin ratio.     

The contractile nature of the tubular-like cells of the parietal layer of the Bowman's capsule in male mice. An immunochemical and ultrastructural study.

The tubule-like cells (TLC) of the parietal layer of the Bowman's capsule of the renal corpuscle in normal male mice are provided with bundles of thin microfilaments (50-70 A). Moreover, the ultrastructural observations demonstrated that similar thin filaments (50-70 A) are located in the flattened cells of the parietal layer of the Bowman's capsule and in the tubular cells. All the above cells bind antimyosin-like antibodies. Considering the correspondence between the immunochemical and ultrastructural findings, it is suggested that the microfilaments located in TLC contain a 'myosin-like' protein.     

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.     

Alterations in flightin phosphorylation inDrosophila flight muscles are associated with myofibrillar defects engendered by actin and myosin heavy-chain mutant alleles

Flightin is a 20-kD myofibrillar protein found in the stretch-activated flight muscles ofDrosophila melanogaster. Nine of the eleven isoelectric variants of flightin are generatedin vivo by multiple phosphorylations. The accumulation of these isoelectric variants is affected differently by mutations that eliminate thick filaments or thin filaments. Mutations in the myosin heavy-chain gene that prevent thick filament assembly block accumulation of all flightin variants except N1, the unphosphorylated precursor, which is present at much reduced levels. Mutations in the flight muscle-specific actin gene that block actin synthesis and prevent thin filament assembly disrupt the temporal regulation of flightin phosphorylation, resulting in premature phosphorylation and premature accumulation of flightin phosphovariants. Cellular fractionation of fibers that are devoid of thin filaments show that flightin remains associated with the thick filamentrich cytomatrix. These results suggest that flightin is a structural component of the thick filaments whose regulated phosphorylation is dependent upon the presence of thin filaments.This work was supported by National Science Foundation Grant IBN-9253045.