Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.     

The Hazard of Acid Differentiation in Gomori's Method for Acid Phosphatase

In his original method for the histochemical demonstration of acid phosphatase, Gomori prescribed differentiation of incubated sections by rinsing them in 14% aqueous acetic acid, to remove the nonspecifically precipitated lead deposits. According to him, the enzymatically produced lead phosphate is not washed out by this procedure. As a result of recent improvements in tissue preparation and shorter incubation time, this staining reaction as it is used now is quite sensitive to an acetic acid wash. If this wash is used as recommended originally, it may completely abolish a truly positive reaction. To avoid falsely negative results, and to compare sections of normal and pathological tissue, omission of this differentiation by acetic acid is essential. The risk of mistaking nonspecific lead precipitates in the interpretation of a positive reaction is very small, and can be avoided by running a negative control slide in which no lead phosphate can be produced enzymatically.     

Multiple structures of thick filaments in resting cardiac muscle and their influence on cross-bridge interactions.

Based on two criteria, the tightness of packing of myosin rods within the backbone of the filament and the degree of order of the myosin heads, thick filaments isolated from a control group of rat hearts had three different structures. Two of the structures of thick filaments had ordered myosin heads and were distinguishable from each other by the difference in tightness of packing of the myosin rods. Depending on the packing, their structure has been called loose or tight. The third structure had narrow shafts and disordered myosin heads extending at different angles from the backbone. This structure has been called disordered. After phosphorylation of myosin-binding protein C (MyBP-C) with protein kinase A (PKA), almost all thick filaments exhibited the loose structure. Transitions from one structure to another in quiescent muscles were produced by changing the concentration of extracellular Ca. The probability of interaction between isolated thick and thin filaments in control, PKA-treated preparations, and preparations exposed to different Ca concentrations was estimated by electron microscopy. Interactions were more frequent with phosphorylated thick filaments having the loose structure than with either the tight or disordered structure. In view of the presence of MgATP and the absence of Ca, the interaction between the myosin heads and the thin filaments was most likely the weak attachment that precedes the force-generating steps in the cross-bridge cycle. These results suggest that phosphorylation of MyBP-C in cardiac thick filaments increases the probability of cross-bridges forming weak attachments to thin filaments in the absence of activation. This mechanism may modulate the number of cross-bridges generating force during activation.     

Loss of thick filaments from fast-twitch glucolytic muscle fibers of the pigeon pectoralis after chronic administration of dantrolene sodium.

Adult pigeons received dantrolene sodium, a skeletal muscle relaxant which blocks the release of calcium during excitation-contraction coupling, for 12 to 16 weeks. The pectoralis muscles of these birds were analyzed for changes occurring in the various fiber types of the muscle. Both histochemistry (ATPase and SDH activity) and electron microscopy (mitochondrial and lipid volume percentages) differentiated two fiber types. The two fiber-types consisted of fast-twitch glycolytic fibers (FG) and fast-twitch oxidative-glycolytic (FOG) fibers. After dantrolene treatment some FG fibers showed little or no ATPase activity. Dantrolene treatment also produced a disappearance of thick filaments in some FG fibers. We infer that the fibers without thick filaments are the ones lacking ATPase activity. The FOG fibers were nearly normal. Since drug-fed birds lose weight, a few birds were starved to determine whether the filament loss was related solely to the bird's loss in weight. No fibers in starved birds showed reduced ATPase activity or loss of thick filaments. In fibers that showed thick filament disappearance, the I-bands remained organized and intact, suggesting that the I-band maintains its integrity without interaction with the thick filaments. Changes in activity patterns may cause loss of thick filaments by inhibiting either their synthesis or assembly.     

THE ORGANIZATION OF CONTRACTILE FILAMENTS IN A MAMMALIAN SMOOTH MUSCLE

Ordered arrays of thin filaments (65 A diameter) along with other apparently random arrangements of thin and thick filaments (100–200 A diameter) are observed in contracted guinea pig taenia coli rapidly fixed in glutaraldehyde. The thin-filament arrays vary from a few to more than 100 filaments in each array. The arrays are scattered among isolated thin and thick filaments. Some arrays are regular such as hexagonal; other arrays tend to be circular. However, few examples of rosettes with regular arrangements of thin filaments surrounding thick filaments are seen. Optical transforms of electron micrographs of thin-filament arrays give a nearest-neighbor spacing of the thin filaments in agreement with the "actin" filament spacing from x-ray diffraction experiments. Many thick filaments are closely associated with thin-filament arrays. Some thick filaments are hollow circles, although triangular shapes are also found. Thin-filament arrays and thick filaments extend into the cell for distances of at least a micron. Partially relaxed taenia coli shows thin-filament arrays but few thick filaments. The suggestion that thick filaments aggregate prior to contraction and disaggregate during relaxation is promoted by these observations. The results suggest that a sliding filament mechanism operates in smooth muscle as well as in striated muscle.     

Localized deposition of lead phosphate precipitate in glycerinated rabbit psoas muscle during ATP-induced contraction

The location of ATP-ase sites within sarcomeres was studied by allowing glycerinated rabbit psoas muscle to shorten under load in a solution containing ATP and lead nitrate. The lead phosphate precipitate formed during the contraction is located mainly within the A-band of shortened sarcomeres. In fibers glycerinated at rest length, the heaviest precipitate deposits are located at the center of the A-band, roughly corresponding to the double overlap zone, with lesser precipitate concentrations at the A-l boundary contraction bands. In fibers glycerinated at stretched length, shortened sarcomeres with heavy contraction bands at the A-l boundary and patent H-bands have heavy precipitate deposits over the contraction bands and no localization to the center of the A-band. The precipitate is thus seen to be most concentrated at those regions where one would expect to find the best contact between thick and thin filaments. Physiological experiments were performed. Evidence is presented that lead ions can substitute for calcium in the contraction process, altering the time course of contraction.     

Control of filament length by the regulatory light chains in skeletal and cardiac myosins

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.     

Specific activity of cell-surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake

Activity of extracellular acid phosphatases was measured at single-cell level in bacterioplankton groups defined by their morphology and size, in acidified mountain Lake Certovo, during the 2003 season, with a method based on use of the substrate ELF97 phosphate which provides fluorescent precipitates upon hydrolysis by phosphatases. The bacterial cell-associated precipitates were quantified by image analysis. A specific, conspicuous, apparently homogeneous morphotype of curved cells of approximately 5 microm average length, despite its low total biomass (average of 4%), contributed significantly (in average by 31%) to the total bacterioplankton phosphatase activity in Lake Certovo (ranging from 1.0 to 12.7 micromol l(-1) h(-1), using ELF97 phosphate as a substrate). Bacterial filaments (> 10 microm), although comprising in average 85% of bacterioplankton biomass, contributed to the total bacterioplankton activity only by 45%. Biomass-specific activity of extracellular (cell-surface) phosphatases of the main bacterioplankton morphotypes increased in the order filaments < cocci and rods < curved cells. The biomass-specific activity of bacterioplankton extracellular phosphatases (0-300 nmol microgC(-1) h(-1)) was generally highest in the spring and decreased gradually during summer. These changes could result from seasonal changes in the phosphorus status of the lake and from subsequent regulation of enzyme expression by bacteria.     

Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.     

Recent X-ray Diffraction and Electron Microscope Studies of Striated Muscle

The sliding filament model for muscular contraction supposes that an appropriately directed force is developed between the actin and myosin filaments by some process in which the cross-bridges are involved. The cross-bridges between the filaments are believed to represent the parts of the myosin molecules which possess the active sites for ATPase activity and actin-binding ability, and project out sidewise from the backbone of the thick filaments. The arrangement of the cross-bridges is now being studied by improved low-angle X-ray diffraction techniques, which show that in a resting muscle, they are arranged approximately but not exactly in a helical pattern, and that there are other structural features of the thick filaments which give rise to additional long periodicities shown up by the X-ray diagram. The actin filaments also contain helically arranged subunits, and both the subunit repeat and the helical repeat are different from those in the myosin filaments. Diffraction diagrams can be obtained from muscles in rigor (when permanent attachment of the cross-bridges to the actin subunits takes place) and now, taking advantage of the great increase in the speed of recording, from actively contracting muscles. These show that changes in the arrangement of the cross-bridges are produced under both these conditions and are no doubt associated in contraction with the development of force. Thus configurational changes of the myosin component in muscle have been demonstrated: these take place without any significant over-all change in the length of the filaments.     

Helical order in tarantula thick filaments requires the "closed" conformation of the myosin head

Myosin heads are helically ordered on the thick filament surface in relaxed muscle. In mammalian and avian filaments this helical arrangement is dependent on temperature and it has been suggested that helical order is related to ATP hydrolysis by the heads. To test this hypothesis, we have used electron microscopy and image analysis to study the ability and temperature dependence of analogs of ATP and ADP.Pi to induce helical order in tarantula thick filaments. ATP or analogs were added to rigor myofibrils or purified thick filaments at 22 degrees C and 4 degrees C and the samples negatively stained. The ADP.Pi analogs ADP.AlF4 and ADP.Vi, and the ATP analogs ADP.BeFx, AMPPNP and ATPgammaNH2, all induced helical order in tarantula thick filaments, independent of temperature. In the absence of nucleotide, or in the presence of ADP or the ATP analog, ATPgammaS, there was no helical ordering. According to crystallographic and tryptophan fluorescence studies, all of these analogs, except ATPgammaS and ADP, induce the "closed" conformation of the myosin head (in which the gamma phosphate pocket is closed). We suggest that helical order requires the closed conformation of the myosin head but is not dependent on the hydrolysis of ATP.     

Purification and biochemical properties of complex flagella isolated from Rhizobium lupini H13-3

1. The complex flagella of Rhizobium lupini H13-3 differ from plain bacterial flagella in the fine structure of their filaments dominated by conspicuous helical bands, in their fragility and their resistance against heat decomposition. To elucidate the basis of these differences, the composition of complex filaments and their subunits was analysed. 2. Isolated complex flagella containing the filament and hook protions were purified by differential centrifugation. Hooks were separated by ultracentrifugation after acid degradation of filaments at pH 2. The complex filaments consist of 43 000 dalton monomers (cx-flagellin), the hooks are composed of 41 000 dalton subunits. 3. Amino acid analysis of cx-flagellin indicated the presence of approx. 417 amino acid residues. These comprise 47% hydrophobic residues and 21% Asp and Glu (or amides), but no Cys, His, Pro and Trp. No carbohydrate, phosphate or lipid moieties have been detected. Fingerprint analysis after tryptic digestion yields approx. 36 peptides, about half of them clustered in the neutral region. A comparison with the composition of varous known flagellins from plain flagella indicates a 7% higher content of hydrophobic amino acid residues in complex filaments; this is largely compensated for by the higher content of Glu and Asp (presumably as Gln and Asn) in plain filaments. 4. Immunodiffusion and immunoelectrophoresis of cx-flagellin yield single precipitin bands indicating homogeneity. In contrast, isoelectric focusing lead to three close-running bands around pH4.7. When isolated, the two major bands again produced an "isoelectric spectrum" suggesting that it reflects an allomorphism of cx-flagellin. 5. Self-assembly experiments with cx-flagellin lead to coiled fibres including helical regions, but not to intact filaments. The products resemble heat-denatured complex filaments and may represent intermediates between monomers and complete polymers.     

The effect of calcium activation of skinned fiber bundles on the structure of Limulus thick filaments

Here we present evidence that strongly suggests that the well-documented phenomenon of A-band shortening in Limulus telson muscle is activation dependent and reflects fragmentation of thick filaments at their ends. Calcium activation of detergent-skinned fiber bundles of Limulus telson muscle results in large decreases in A-band (from 5.1 to 3.3 microns) and thick filament (from 4.1 to 3.3 microns) lengths and the release of filament end fragments. In activated fibers, maintained stretched beyond overlap of thick and thin filaments, these end fragments are translocated to varying depths within the I-bands. Here they are closely associated with fine filamentous structures that also span the gap between A- and I-bands and attach to the distal one-third of the thick filaments. End-fragments are rarely, if ever, present in similarly stretched and skinned, but unstimulated fibers, although fine "gap filaments" persist. Negatively stained thick filaments, separated from skinned, calcium-activated, fiber bundles, allowed to shorten freely, are significantly shorter than those obtained from unstimulated fibers, but are identical to the latter with respect to both the surface helical array of myosin heads and diameters. Many end-fragments are present on grids containing thick filaments from activated fibers; few, if any, on those from unstimulated fibers. SDS-PAGE shows no evidence of proteolysis due to activation and demonstrates the presence of polypeptides with very high molecular weights in the preparations. We suggest that thick filament shortening is a direct result of activation in Limulus telson muscle and that it occurs largely by breakage within a defined distal region of each polar half of the filament. It is possible that at least some of the fine "gap filaments" are composed of a titin-like protein. They may move the activation-produced, fragmented ends of thick filaments to which they attach, into the I-bands by elastic recoil, in highly stretched fibers.     

Electron microscopic and optical diffraction analysis of the structure of scorpion muscle thick filaments

We rapidly and gently isolated thick filaments from scorpion tail muscle by a modification of the technique previously described for isolating Limulus thick filaments. Images of negatively stained filaments appeared to be highly periodic, with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrograph images were detailed and similar to optical diffraction patterns from Limulus and tarantula thick filaments. Analysis of the optical diffraction patterns and computed Fourier transforms, together with the appearance of the filaments in the micrographs, suggested a model for the filaments in which the myosin cross-bridges were arranged on four helical strands with 12 cross-bridges per turn of each strand, thus giving the observed repeat every third cross-bridge level. Comparison of the scorpion thick filaments with those isolated from the closely related chelicerate arthropods, Limulus and tarantula, revealed that they were remarkably similar in appearance and helical symmetry but different in diameter.     

Mechanism of chalaza formation in quail eggs

The present study was conducted to determine the mechanism of chalaza formation in eggs of the Japanese quail Coturnix japonica and to determine the production site of chalaza materials in the oviduct. Electrophoretic profiles of the chalaza materials showed six bands of 480, 320, 210, 180, 96, and 58 kDa following Coomassie Blue staining and one band of 600 kDa after immunoblotting. An antiserum was produced against the 180-kDa band. This antiserum and an antiserum generated against the 600-kDa protein were used as probes to detect chalaza materials. Immunofluorescent and immunoelectron-microscopic observations revealed that chalazae and chalaziferous layers overlaid to approximately 40 μm upon the vitelline membrane of the ovum were composed of the same materials as those produced by both types of secretory cells in the luminal and glandular epithelia at the infundibulum. We propose that the mechanism of chalaza formation is as follows: (1) chalazae first appear as fine filaments at the presumptive sharp and blunt ends of the ovum at the infundibulum; (2) these filaments are twisted into a lead fiber while the ovum is rotating and descending in the magnum; (3) at the posterior end of the magnum, the lead fiber is anchored to the thick egg white and lifted outward with the chalaziferous sublayers when the inner egg white is liquefied by absorbing water; (4) the lead fiber and chalaziferous sublayers are twisted further into the chalaza in the uterus.     

Histochemical demonstration of hyaluronic acid molecules by Alcian Blue

Summary Specimens of vitreous humour (monkey eye), Wharton jelly (human umbilical cord) and commercial hyaluronates were immersed in buffered fixative solutions containing either aldehydes and Alcian Blue, or aldehydes and Alcian Blue with MgCl₂ as electrolyte. Two MgCl₂ concentrations were used, 0.025m and 0.3m. Immersion in both solutions induced formation of precipitates which were postfixed in OsO₄, dehydrated and embedded for thin section electron microscopy. The use of the same fixative solution produced morphologically comparable precipitates from all three materials. The precipitates, especially after fixation in the presence of electrolyte, were composed of linear, unbranched filaments, frequently aggregated into bundles. The filaments were considered to be molecules of hyaluronic acid.Part of this work was presented at the 10th Meeting of the European Club for Ophthalmic Fine Structure, Copenhagen, September 3–4, 1982.     

Diffraction rings obtained from a suspension of skeletal myofibrils by laser light illumination. Study of internal structure of sarcomeres.

Diffraction rings corresponding to the first, second, and third order were obtained by laser light illumination from a suspension of rabbit glycerinated psoas myofibrils (diameter, 1-2 microns; average length of the straight region, 44 microns; average sarcomere length, 2.2-2.6 microns) of which the optical thickness was appropriately chosen. Dispersed myofibrils were nearly randomly oriented in two dimensions, so that the effects of muscle volume were minimized; these effects usually interfere significantly with a quantitative analysis of laser optical diffraction in the fiber system. The diameters of diffraction rings represented the average sarcomere length. By using this system, we confirmed the ability of the unit cell (sarcomere) structure model to explain the intensity change of diffraction lines accompanying the dissociation from both ends of thick filaments in a high salt solution. The length of an A-band estimated from the relative intensity of diffraction rings and that directly measured on phase-contrast micrographs coincided well with each other. Also, we found that myofibrils with a long sarcomere length shorten to a slack length accompanying the decrease in overlap between thick and thin filaments produced by the dissociation of thick filaments.     

Effects of DMSO, pH, stretch and calcium on the thick filaments in an amphibian smooth muscle.

Conventional fixation with glutaraldehyde fixatives at pH 7.4 did not preserve/promote thick filaments in Bufo smooth muscle. However, if pH was lowered to 6.0, thick filaments were present and addition of dimethyl sulfoxide (DMSO) led to a greater number of thick filaments. Stretch alone had little effect on the presence of thiber seen. Calcium had little effect on the numbers of thick filaments, but it affected the appearance of the thick filaments. Low calcium (10(-7) M) caused a higher proportion of rod-shaped filaments, while in high calcium (10(-3) M) most thick filaments were ribbons. The great lability of the thick filaments in amphibian smooth muscles makes them ideal for studying factors which affect the appearance of thick filaments in smooth muscle, but it also raises the question of the degree of aggregation of myosin in the living cell.     

Hydrostatic pressure studies of native and synthetic thick filaments: II. Native thick filaments from rabbit skeletal muscle

Native thick filaments isolated from freshly prepared rabbit psoas muscle were found to be resistant to pressure-induced dissociation. With increasing pressure application and release, a bimodal distribution of filament lengths was observed. The shorter filament length is associated with filament breakage at the center of the bare zone, while the longer length is associated with relatively intact filaments. Intact filaments and filament halves decrease in length by no more than 20% after exposure to and release of 14,000 psi. Bimodal distributions were not observed in equivalent experiments performed on filaments isolated from muscle glycerinated and stored at -20 degrees C for 6 months. Instead, filament dissociation proceeds linearly as a function of increasing pressure. Filaments prepared from muscle glycerinated and stored for 2 and 4 months exhibited pressure-induced behavior intermediate between the filaments prepared from fresh muscle and filaments prepared from muscle stored for 6 months. Since there appears to be no difference in the protein profiles of the various muscle samples, it is possible that stabilization of the native thick filament against hydrostatic pressure arises from trapped ions that are leached out over time.