Yasunori Takezawa

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.

Extensibility of the actin and myosin filaments in various states of skeletal muscle as studied by X-ray diffraction.

The effects of length changes applied to resting, contracting and rigor muscles on the reflection spacings of the X-ray diffraction patterns were summarized. The spacing changes of the actin- and myosin-based meridional reflections as a function of tension relative to an isometric tension of active muscle (P0) were linear and almost identical in the active and rigor states, showing that the extension of both filaments is Hookenian and does not depend upon the states of muscle. In addition to their length changes caused by tension generation, there are small but significant length changes of both filaments due purely to activation of muscle. The actin and myosin filaments are elongated by approximately 0.36% and approximately 0.43%, respectively under the maximum active tension. The results indicate that a large part of the sarcomere compliance of an active muscle is caused by the extensibility of the myofilaments. Inspection of the behavior of the meridional and layer-line reflection spacings reveals that there is a close relationship between the extensibility and helical twisting of the actin filaments under active and passive forces. The extension caused by tension is associated with an unwinding of right-handed helices following the actin monomers in the filament. At the pointed end of the filament could rotate anticlockwise through one fifth the complete turn during contraction.

Backward Movements of Cross-Bridges by Application of Stretch and by Binding of MgADP to Skeletal Muscle Fibers in the Rigor State as Studied by X-Ray Diffraction

The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by approximately 23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by approximately 33 and 17%, respectively, when rigor frog muscle was stretched by approximately 4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10-20 degrees) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state.

Morphological evaluation of chondrogenic potency in passaged cell populations.

The present study describes the morphological assessment of chondrogenic potency during a cell expanding process through serial subculturing of rabbit chondrocytes at different levels of population doublings (PD) in a T-flask with a conventional polystyrene surface. The passaged populations were seeded on a high-density collagen surface (CL surface) and in a collagen gel (CL gel) scaffold to evaluate the planar and spatial morphologies of the chondrocytes, respectively, as well as the gene expressions of mRNA for collagen types I and II. The planar morphological estimation was based on roundness (R(c)) of chondrocyte cells at different PD values after 1 day incubation on the CL surface. The frequency of round-shaped cells with R(c)>0.9 (f(R)) decreased with increasing PD values, accompanied by an increase in collagen type I mRNA level. At PD=17.8, the frequency reached f(R)=0.12, which was less than one-sixth of that at PD=0. A similar trend was found with respect to the passaged chondrocytes embedded in the CL gels by estimating the spatial morphology in terms of sphericity (S(c)) determined 4 days after seeding. With an increase in PD value, the frequency in spherical-shaped cells with S(c)>0.9 (f(S)) decreased and the mRNA expression of collagen type I increased, giving f(S)=0.28 at PD=17.8 which was less than a quarter of that at PD=0. From these results, the cell morphologies on the CL surface and in the CL gel were proposed as indicators for understanding chondrogenic potentials concerning the phenotypes and differentiated states in the population during cell expansion, ultimately leading to quality control of tissue-engineered cartilage.

Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 × 105 cells/cm3 underwent a transition to spindle‐shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 × 106 cells/cm3 exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology‐relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 × 105 cells/cm3 gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 × 106 cells/cm3. In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 × 105 cells/cm3 showed appreciable down‐regulation in collagen type II relating to differentiation and up‐regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 × 106 cells/cm3. These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.

Evaluation of vertical cell fluidity in a multilayered sheet of skeletal myoblasts

The procedure for fabricating a multilayered cell sheet has been developed by combining multiple sheets using a thermo-responsive surface and stamp system. Confocal laser scanning microscopy revealed that the fluidity of a multilayered sheet of skeletal myoblasts could be estimated as vertical diffusivity and changed upon addition of dermal fibroblasts.

Growth and differentiation potentials in confluent state of culture of human skeletal muscle myoblasts

The transitional behaviors of myoblasts toward differentiation were investigated in the cultures at the low and high seeding densities (respectively, X(0)=1.0x10(3) and 2.0x10(5) cells/cm(2)). In the culture at the low seeding density, an increase in confluence degree accompanied a decrease in growth potential (R(p)), being R(p)=0.85 and 0.11 at t=48 and 672 h, respectively. Myoblasts seeded at the high density resulted in the immediate cessation of growth with keeping the low range of R(p)=0.02-0.09 throughout the culture. The reduction of R(p) led to the generation of three subpopulations of cells in proliferative, quiescent and differentiated states. Close cell contacts in the confluent state of high seeding culture induced cell quiescence to a higher extent with suppressing differentiation.

Synergic stimulation of laminin and epidermal growth factor facilitates the myoblast growth through promoting migration.

The dynamic behaviors of human skeletal muscle myoblasts were investigated in the culture on a laminin-coated surface in the presence of 100 ng/ml epidermal growth factor (EGF) in medium. The coexistence of laminin and EGF caused the enhancement of myoblast migration, giving an average migration rate of 62.0 microm/h, which was 2.7 times that on a plain surface. This encouraged migration could be a driving force to separate the dividing cells from each other, accompanied by shortened disjunction time of daughter cells to complete cytokinesis. In addition, the synergic effect of laminin and EGF led to the promotion of myoblast growth with keeping a relatively high fraction of proliferative cells during the culture for 150 h, which is considered to arise from the reduced frequency of cell-cell contacts during cytokinesis and thereby suppressing the process towards myotube formation after cell division.

Small-Angle X-ray Diffraction of Muscle Using Undulator Radiation from the Tristan Main Ring at KEK

Time-resolved X-ray diffraction of muscle has demanded ever-increasing flux into small sample volumes with low beam divergence. Results are reported of static and time-resolved small-angle X-ray diffraction studies on muscle fibers using a hard X-ray undulator installed in the Tristan main ring at KEK, Tsukuba, Japan, as an innovative source of synchrotron radiation more intense and better collimated than that available with the Photon Factory bending-magnet beamline. Static studies used the low divergence of the source to obtain detailed high-quality diffraction patterns of stable muscle states. The diffraction patterns from live skeletal muscles showed the numerous (over 100) meridional reflections. The well collimated beam from the undulator made it possible to clearly resolve, with an angular resolution of ca 700 nm, the closely spaced diffraction peaks arising from the two halves of the thick filaments centred on the M lines in a sarcomere, in addition, the diffraction peaks from the thin filaments on opposite sides of the Z bands could be resolved with an angular resolution of ca 1000 nm. The detailed structure of the meridional pattern defines the nature of the molecular packing in the thick and thin filaments. Time-resolved experiments using a focusing mirror aimed to prove cross-bridge states in striated muscle fibers by collecting X-ray diffraction data at a 0.185 ms time resolution from sinusoidally oscillating chemically skinned rabbit muscle fibers during active contraction and in rigor. When sinusoidal length changes at 500 Hz with a peak-to-peak amplitude of 0.6% of the muscle length were applied to a small fiber bundle, the tension showed a simple elastic response during the length oscillation. In the active muscle the intensity of the 14.5 nm myosin-based meridional reflection changed out of phase with the tension change during the oscillating length change. In contrast, in the rigor muscle it occurred in phase with the tension change. The high time-resolved experiments provide an insight into the coupling between conformational changes and force generation of the actomyosin cross-bridges. These studies provide a preview of the expected gains for muscle studies from the more widespread use of undulator radiation at third-generation synchrotron sources.

X-ray diffraction studies on the structural changes of rigor muscles induced by binding of phosphate analogs in the presence of MgADP.

To clarify the structure of the ATP hydrolysis intermediates (ADP.Pi bound state) formed by actomyosin crossbridges, the effects of various phosphate analogs in the presence of MgADP on the structures of the thin and thick filaments in glycerinated rabbit psoas muscle fibers in the rigor state have been investigated by X-ray diffraction with a short exposure time using synchrotron radiation. When MgADP and phosphate analogs such as metallofluorides (BeFx = 3,4 and AlF4) and vanadate (VO4(Vi)) were added to rigor fibers in the presence of the ATP-depletion backup system, the intensities of the actin-based layer lines were markedly weakened. The greatest effect (approximately 50% decrease in intensity) was observed in the presence of BeFx among the analogs examined. The intensity distribution of the 5.9 nm actin-based layer line shifted towards that observed in the Ca(2+)-activated fibers, while the first actin layer line at approximately 1/36.7 nm-1 retained a rigor-like profile with an intensity weakened by approximately 50%. The intensity of the equatorial 10 reflection increased while that of the 11 reflection changed little, resulting in only a small increase (approximately 1.7 fold) in the intensity ratio of the 10 to the 11 reflection. No resting-like pattern appeared upon the addition of MgADP and BeFx. These results indicate that a substantial fraction (approximately 40%) of the myosin heads dissociate from actin but the detached heads remain in the vicinity of the actin filaments when MgADP and BeFx bind. The states produced by binding phosphate analogs to a rigor muscle differ from the resting-like state produced by adding them to a contracting muscle (Takemori et al., J. Biochem. (Tokyo) 117 (1995) 603-608). Our conclusion put forward to explain the data is that one of the two heads of a crossbridge is detached and the other retains a rigor-like attachment.