Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations

By taking advantage of a lethal phenotype characteristic of Caenorhabditis elegans embryos that fail to move, we have identified 13 genes required for muscle assembly and function and discovered a new lethal class of alleles for three previously known muscle-affecting genes. By staining mutant embryos for myosin and actin we have recognized five distinct classes of genes: mutations in four genes disrupt the assembly of thick and thin filaments into the myofilament lattice as well as the polarized location of these components to the sarcolemma. Mutations in another three genes also disrupt thick and thin filament assembly, but allow proper polarization of lattice components based on the myosin heavy chain isoform that we analyzed. Another two classes of genes are defined by mutations with principal effects on thick or thin filament assembly into the lattice, but not both. The final class includes three genes in which mutations cause relatively minor defects in lattice assembly. Failure of certain mutants to stain with antibodies to specific muscle cell antigens suggest that two genes associated with severe disruptions of myofilament lattice assembly may code for components of the basement membrane and the sarcolemma that are concentrated where dense bodies (Z- line analogs) and M-lines attach to the cell membrane. Similar evidence suggests that one of the genes associated with mild effects on lattice assembly may code for tropomyosin. Many of the newly identified genes are likely to play critical roles in muscle development and function.     

Suppression of active cross-bridge motions of isolated thick myofilaments in suspension by phenylmethylsulfonyl fluoride

Studies of photoelectron count autocorrelation function of light scattered from suspensions of thick filaments of Limulus telson muscle and scallop striated adductor muscle reveal that Ca2+ can activate cross-bridge motions of these isolated filaments. By treating suspensions of activated filaments with phenylmethylsulfonyl fluoride (PMSF), we can suppress active cross-bridge motions but not affect the Ca2+-dependent ATPase activity.     

Differentiation of metacoxal muscles in Periplaneta americana (L.) (Dictyoptera : Blattidae)

Flight muscles of the cockroach, Periplaneta americana (Dictyoptera : Blattidae) in different development stages (10 mm and 30 mm nymphs, and adult) are investigated for histochemical activity and by electron microscope. The 177 C muscle of 10 mm nymph shows low succinic dehydrogenase (SDH) and myosin-ATPase activities (+). Each myofibril is surrounded by an extensive network of sarcoplasmic reticulum. Regarding myofilament array, one thick filament is surrounded by 10–12 thin filaments. At the stage of 30 mm nymph, SDH and myosin-ATPase activities increase (+ +). Except for an increase in the number of mitochondria, electron microscopic features are similar to those in the 10 mm nymph. In the adult stage, both SDH and myosin-ATPase activities are highest. The distribution of sarcoplasmic reticulum and T-tubules is fundamentally unchanged, whereas the myofilament array is drastically changed, so that 6 thin filaments surround a thick one.     

Targeted disruption of nebulette protein expression alters cardiac myofibril assembly and function

To evaluate nebulette's role in cardiac myofibrils, cardiomyocytes expressing green fluorescent protein (GFP)-nebulette constructs were monitored for their ability to contract and myofilament protein distribution was analyzed. Cells expressing full-length GFP-nebulette appear unaffected and exhibit normal beating frequencies. Expression of the GFP linker and SH3 results in loss of the endogenous nebulette and tropomyosin; however, Z-line and thick filaments are undisturbed. Cells expressing either of these domains have dramatically reduced beating frequencies, consistent with the loss of thin filament proteins. This loss was inhibited by the addition of protease inhibitors during culturing. The GFP repeat domain disrupts both myofibrillogenesis and contraction in spreading cardiomyocytes, whereas introduction of this protein into well-spread cardiomyocytes results in localization at the Z-line and a 50% reduction in beating frequency. Ultimately, these cells form bundles containing the GFP repeat and many myofilament proteins. Interestingly, butanedione monoxime inhibition of contraction inhibited the formation of these bundles. These results show that the GFP-nebulette domains have a dominant-negative effect on the distribution and function of the sarcomeric proteins. Taken together with the observation that nebulette colocalizes with alpha-actinin in the pre-, nascent, and mature myofibrils, our data demonstrate the importance of this cardiac-specific nebulin isoform in myofibril organization and function.     

甲壳动物横纹肌的收缩蛋白质与调节机制

甲壳动物横纹肌肌原纤维的肌丝陈列,收缩蛋白质和收缩的Ca²⁺依赖性调节机制与脊椎动物横纹肌有不少差异.脊椎动物横纹肌、甲壳动物快肌与慢肌的粗丝与细丝的数量比依次为1:2,1:3和1:6,肌丝阵列各异.甲壳动物粗肌丝由肌球蛋白和副肌球蛋白组成,其分子装配与脊椎动物不同.细肌丝含有肌动蛋白、原肌球蛋白和肌钙蛋白,肌钙蛋白-T分子量较高,肌钙蛋白-C仅1个Ca²⁺结合位点.甲壳动物横纹肌兼有细肌丝调节与粗肌丝调节.     

横纹肌肌原纤维的第三肌丝──肌联蛋白

实验研究证明,在动物横纹肌肌原纤维中,除包含有粗肌丝、细肌丝外,还有纤肌丝的存在,肌联蛋白（肌巨蛋白）是具有挠性的线状蛋白质,分子量为3000 000,长度约为0.9μm,跨越肌原纤维的M－线和Z－线,形成纤肌丝.其生理功能是在粗肌丝装配中具有分子模板作用,并将粗肌丝稳定于肌原纤维肌小节中央以及可参与肌球蛋白活性的调节.     

Purification of native myosin filaments from muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.     

Analysis of the mechanisms of mitochondrial NADH regulation in cardiac trabeculae.

We have previously shown that increased cardiac work initially caused a rapid Ca(2+)-independent fall of mitochondrial [NADH] ([NADH](m)) to a minimum level, and this was followed by a slow Ca(2+)-dependent recovery toward control level (Brandes and Bers, Biophys. J. 71:1024-1035, 1996; Brandes and Bers, Circ. Res. 80:82-87, 1997). The purpose of this study is to improve our understanding of the factors that control [NADH](m) during increased work. [NADH](m) was monitored using fluorescence spectroscopy in intact rat trabeculae isolated from the right ventricular wall. Work was increased by increasing sarcomere length, pacing frequency, external [Ca(2+)], or by decreased temperature. The results were: 1) The initial fall of [NADH](m) during increased pacing frequency depends independently on increased myofilament work and on increased Ca(2+)-transport ATPase activity. 2) The [NADH](m) recovery process depends on average cytosolic [Ca(2+)] (Av[Ca(2+)](c)), but not on absolute work level. 3) The initial fall of [NADH](m) and the [NADH](m) recovery are similar whether increased work is associated with low frequency and high Ca(2+)-transient amplitude or vice versa (at the same myofilament work level and Av[Ca(2+)](c)). 4) The mechanisms associated with the smaller fall and recovery of [NADH](m) at 37 degrees C versus 27 degrees C, may be explained by lowered Av[Ca(2+)](c) and myofilament work. The NADH control mechanisms that operate at lower temperature are thus qualitatively similar at more physiological temperatures.     

蜜蜂间接飞翔肌粗肌丝的微丝结构

用能溶解肌球蛋白但不溶解副肌球蛋白的溶液(300 mM KCI,pH6.0)处理分离的蜜蜂间接飞翔肌粗肌丝,经数分钟后可以看到粗肌丝端头散开成为多根微丝,微丝数最多为7根。延长处理时间,可以见到粗肌丝中央部分只剩下直径约为5 nm的徽丝。实验结果支持我们以前提出的蜜蜂间接飞翔肌粗肌丝的结构模式,并指示贯穿肌小节、两端都和Z线相连的内芯至少部分由副肌球蛋白组成。只存在于A带的,由6根微丝形成的外套是由肌球蛋白分子组成。     

THE RELATIONSHIP BETWEEN MYOFILAMENT PACKING DENSITY AND SARCOMERE LENGTH IN FROG STRIATED MUSCLE

In cross-sections of single fibers from the frog semitendinosus muscle the number of thick myofilaments per unit area (packing density) is a direct function of the sarcomere length. Our data, derived from electron microscopic studies, fit well with other data derived from in vivo, low-angle X-ray diffraction studies of whole semitendinosus muscles. The data are consistent with the assumption that the sarcomere of a fibril maintains a constant volume during changes in sarcomere length. The myofilament lattice, therefore, expands as the sarcomere shortens. Since the distance between adjacent myofilaments is an inverse square root function of sarcomere length, the interaction of the thick and the thin myofilaments during sarcomere shortening may occur over distances which increase 70 A or more. The "expanding-sarcomere, sliding-filament" model of sarcomere shortening is discussed in terms of the current concepts of muscle architecture and contraction.     

Ultrastructural investigations on the anterior adductor muscle of a brachiopoda, Lingula unguis

The brachiopoda, Lingula unguis, has a pair of anterior adductors located in the center of the shell. Each muscle consists of an opaque and a translucent portion which is constructed of smooth and obliquely-striated muscle respectively. According to our ultrastructural observations, the opaque portion seems to have two types of cells. They differ only in the diameters of their thick myofilaments. The fine structure of their cell organelles resembles each other. We measured the diameters of the thick myofilaments in each type of cell to distinguish between the two cell types. About 500 measurements of myofilament diameters were made for each type of cell and statistically analyzed. For one type of cell, the distribution of diameters of the thick myofilaments fit a normal distribution curve with a peak at 37-60 nm. The distribution of diameters of the thick myofilaments for the other type fit a curve in which two normal distribution curves having peaks at 37-60 and 75-97 nm respectively partially overlapped. According to these results, we suggest that the opaque portion contains two types of cells, each having a different distribution of thick myofilament sizes.     

螫虾腹屈肌的肌球蛋白-副肌球蛋白丝

利用甘油梯度离心方法分离和纯化螯虾腹屈肌粗肌丝,电子显微镜照片显示粗肌丝上有数条纵行条纹,指示其可能由数根亚丝所组成。粗肌丝的 SDS-聚丙烯酰胺凝胶电泳表明其含有肌球蛋白和副肌球蛋白,肌球蛋白仅包含有二种轻链。副肌球蛋白类晶体呈针状,具有14.5nm 和72.5nm 的横纹周期。实验结果表明,螯虾腹屈肌粗肌丝是肌球蛋白-副肌球蛋白丝。     

Molecular and Subcellular-Scale Modeling of Nucleotide Diffusion in the Cardiac Myofilament Lattice

Contractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions.     

Molecular and Subcellular-Scale Modeling of Nucleotide Diffusion in the Cardiac Myofilament Lattice

Contractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions.     

螯虾横纹肌粗肌丝超微结构的计算机图象分析

本文在微机ＡＳＴ／３８６和真彩色图形采集卡ＣＡ－５４０构成的趁科象处理系统上开发了一个软件,包括用于对动物横纹肌粗肌丝的超微结构进行分析的专用模块,一般图象处理系统的通用模块和文件管理模块三大部分。本系统对动物横纹肌粗肌丝的亚结构从旋转功率谱的角度分析了其对称性的存在,并运用图象的迭加,旋转平衡和旋转滤波等手段来处理,显示其对称性;同时,对存在的不对称结构也作了相应的分析处理。利用本系统,对螯虾的     

Frequency-dependent acceleration of relaxation involves decreased myofilament calcium sensitivity

The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxation. Force of contraction increases with frequency, while simultaneously a frequency-dependent acceleration of relaxation occurs. While frequency dependency of calcium handling and sarcoplasmic reticulum calcium load have been well described, it remains unknown whether frequency-dependent changes in myofilament calcium sensitivity occur. We hypothesized that an increase in heart rate that results in acceleration of relaxation is accompanied by a proportional decrease in myofilament calcium sensitivity. To test our hypothesis, ultrathin right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophorically loaded with the calcium indicator bis-fura 2. Twitch and intracellular calcium handling parameters were measured and showed a robust increase in twitch force, acceleration of relaxation, and rise in both diastolic and systolic intracellular calcium concentration with increased frequency. Steady-state force-intracellular calcium concentration relationships were measured at frequencies 1, 2, 3, and 4 Hz at 37 degrees C using potassium-induced contractures. EC(50) significantly and gradually increased with frequency, from 475 +/- 64 nM at 1 Hz to 1,004 +/- 142 nM at 4 Hz (P < 0.05) and correlated with the corresponding changes in half relaxation time. No significant changes in maximal active force development or in the myofilament cooperativity coefficient were found. Myofilament protein phosphorylation was assessed using Pro-Q Diamond staining on protein gels of trabeculae frozen at either 1 or 4 Hz, revealing troponin I and myosin light chain-2 phosphorylation associated with the myofilament desensitization. We conclude that myofilament calcium sensitivity is substantially and significantly decreased at higher frequencies, playing a prominent role in frequency-dependent acceleration of relaxation.     

Ultrastructure of the bulla seminalis in Cadra cautella walker (Pyralidae : Lepidoptera)

The bulla seminalis of Cadra cautella (Lepidoptera : Pyralidae), which serves as a pump in the translocation of sperm from the bursa copulatrix to the spermatheca and as an egg crusher for oosorption, is covered by a layer of branching and intertwining visceral muscles. The sarcomeres have perforated Z-disks allowing the penetration of myosin filaments during contraction, and the myofilament array consists of 10–12 actin filaments surrounding a thick myofilament. The ultrastructure suggests that the muscles are supercontracting muscles. The fragmentation of the Z-disks and the length changes over which the muscles operate indicate they may also be superextending muscles. Combined with an internal pleated cuticular lining, the musculature permits the bulla seminalis to be expanded to 4 times its normal size to accommodate a number of ova simultaneously, and to contract with sufficient force to rupture the ova during oosorption. Continuous accumulation of large numbers of empty shells in the bulla of 7-day-old females, stretches the wall of the bulla. This change, which appears to be irreversible, may reduce the efficiency of the muscles in aging females.     

New Turbidimetric Device for Measuring Cell Concentrations in Thick Microbial Suspensions

A photoelectric turbidimeter is described for use in measuring cell concentrations in thick suspensions. Its sample cuvette provides a path length continuously adjustable between 0.01 and 20.00 mm. By reducing the path length, it is possible to measure the optical density in thick cell suspensions without dilution. Moreover, the method described is rapid and simple, and only small amounts (less than 1 ml) of cell suspensions are required. The method is applicable to cell concentrations ranging up to 10⁹/ml for yeast.     

Population structure in Ascaris suum (Nematoda) among domestic swine in Denmark as measured by whole genome DNA fingerprinting

We here analyze the population structure in the pig roundworm, Ascaris suum, among domestic pigs in Denmark using a whole-genome DNA fingerprinting technique, "amplified fragment length polymorphism" (AFLP) analysis. With these data, we can extract absolute gene frequency variance components and G-statistics for 135 independent nucleotide polymorphisms. The average proportion of total variance partitioned between Jutland and Zealand is less than 3% of the total variance, implying no restriction in gene flow between worms from different regions in Denmark. The average gene frequency difference between two farms widely separated in Jutland represents 5% of the total genetic variance of these two farms combined. Conversely, worms from different hosts within these two farms are more subdivided, with an average of 12% of the total variance in gene frequencies within farms being distributed between hosts. This result implies substantial single generation inbreeding due to founder effects in the establishment of adult worms in single hosts. Absolute variance components extracted from the gene diversities also showed significant differences, with the among-host variance being greater that the between-farm and between-region values. This little geographical variation is discussed in relation to the hierarchic structure of the Danish swine production system. Comparison of our results with other studies on parasitic roundworms, suggests that patterns of host dispersal effectively control patterns of worm gene flow. Furthermore, the potential spread of anthelminth resistance among A. suum may thus be rapid, due to the flow of infected hosts within the domestic swine stocks in Denmark.     

Effects of cellular morphology on the viscoelastic behavior of high hematocrit RBC suspensions

Using a constant-amplitude (+/- 1 degree) oscillatory Couette viscometer (f = 0.01-1.0 Hz), we have measured the viscous (eta') and elastic (eta") components of the complex viscosity at 25 degrees C for shape-transformed human RBC suspended in isotonic buffer at 80% hematocrit. Morphology-altering drugs employed were: ECHINOCYTIC AGENT 2,4-dinitrophenol (DNP, 0.1-5 mM); STOMATOCYTIC AGENT chlorpromazine hydrochloride (CPZ, 0.01-0.1 mM). All suspensions exhibited decreasing eta' and eta" with increasing frequency. Compared to biconcave, control RBC suspensions, salient effects of shape transformation included: 1) for DNP, a dose-related elevation of both eta' and eta", with a 850% increase in eta' and a 2500% increase in eta" at 5 mM and the lowest frequency; 2) for CPZ, a dose-related elevation of both eta' and eta", with a 170% increase in eta' and a 280% increase in eta" at 0.1 mM and the lowest frequency; 3) for both DNP and CPZ, the elevations of eta' and eta" were inversely related to frequency. Using 2 mM DNP and various concentrations of CPZ, both eta' and eta" could be returned to control with 0.08 mM CPZ; further increases of CPZ at constant DNP led to elevations of both components. Comparisons of eta' and eta" to steady shear viscometric data indicated that neither a nominal shear rate approach nor a RMS complex viscosity technique was able to completely reconcile these data; a modified Kelvin-Voigt model proved useful in evaluating cellular versus membrane contributions to eta". These results indicate that RBC morphology is an important determinant of the oscillatory behavior of RBC suspensions and suggest the usefulness of the technique for studies of drug-membrane interactions.