蜜蜂飞翔肌收缩蛋白的电子显微镜鉴定

自五十年代肌丝滑行模型建立以来,关于脊椎动物骨胳肌的蛋白质成分,肌丝排列以及肌肉收缩时结构变化的研究取得了很大的进展。骨胳肌肌原纤维由粗、细肌丝有规律地排列所组成。对于肌肉收缩蛋白的选择性抽提,专一性抗体标记以及重组肌丝的研究,证实肌球蛋白存在于粗肌丝;肌动蛋白、原肌球蛋白和原宁蛋白存在于细肌丝(Huxley,A.F.,1957;Huxley,H.E;,1972)。昆虫间接飞翔肌的结构和生理特性有许多不同于脊椎动物骨胳肌的特点。蜜蜂飞翔肌肌原纤维虽然也包含有粗、细两     

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.     

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.     

螯虾腹屈肌的超微结构

用电子显微镜观察,发现螯虾(Procambarus clarkii)腹屈肌浅层(慢肌,tonic fiber)肌纤维和深层(快肌,twitch fiber)肌纤维的超微结构存在显著差异。浅层腹屈肌肌原纤维有相对长的肌节(5—10μm),肌原纤维直径较大,每根粗肌丝周围有9—12根细肌丝环绕,细肌丝与粗肌丝数量比约为6∶1;深层腹屈肌有相对短的肌节(3—4.5μm),肌原纤维直径较小,每根粗肌丝周围有6根细肌丝环绕,细肌丝与粗肌丝数量比为3∶1。以上结果提示,在决定螯虾腹屈肌收缩速度方面,可能肌原纤维直径大小比肌节长度更为重要。细、粗肌丝排列方式也可能与收缩速度有关。这与脊椎动物骨骼肌的情况是不同的。     

副肌球蛋白在意大利蜜蜂间接飞翔肌中的定位

肌肉收缩是由于肌原纤维中粗、细肌丝相互滑行的结果(Huxley,1988;Huxley,1983;Squire,1986)。许多无脊椎动物肌肉粗肌丝中除含有肌球蛋白外,还存在着含量不同的副肌球蛋白(陈明等,1984、1985)。我们曾经进行过一系列关于意大利蜜蜂(Apis mellifera ligustica Spin)间接飞翔肌原纤维排列及其粗肌丝亚丝结构的研究。间接飞翔肌的粗肌丝从Z-线延伸至另一Z-线(范世藩等,1966),分离的天然粗肌丝经变性剂(脲、胍)处理,可以散开成直径约为5nm的数根亚丝,在一些亚丝上     

鸣鸣蝉（Oncotympana maculaticollis Motsch）发声肌中肌原纤维的双阵列结构及莫发声原初过程的分析

给出了鸣鸣蝉发声肌肌原纤维的双阵列结构,其肌纤维中并存两种不同阵列的“快”和“慢”动肌原纤维（ＦＳＭ和ＳＳＭ）．ＦＳＭ和ＳＳＭ虽然由粗肌丝构成相同的阵列骨架,但细肌丝对粗肌丝的比例（ＲＴＩＦ）不同,分别为３：１和５：１．明显区别于单音调鸣声的蝉类发声肌肌原纤维的ＲＴＩＦ为３：１的单阵列结构,即与鸣鸣蝉变音调声产生的原初机制相适应．     

粘虫蛾飞行肌的发育：超微结构特征分析

应用电子显微镜对正常条件下饲养的0-16日龄粘虫Mythimna separata (Walker)雌蛾飞行肌超微结构的研究结果表明：肌原纤维直径。线粒体和横管的体积分量均随蛾龄的增加而增加,到7日龄达到最大值以后又随蛾龄的增加而下降;但是,肌节长度则随蛾龄的增加而缩短,到7日龄达到最短后又随蛾龄的增加而延长,从而使整条发育曲线呈“V”字形;肌原纤维和肌质网体积分量变化不大或无规律可寻;二位体在发育初期和未期的比例较高,而三位体在4和7口龄比例较高;肌丝排列从0～7日龄均是有序的,肌动蛋白丝(细丝)和肌球蛋白丝(粗丝)的数量比为3：1,粗丝的数量变化也不大,每根肌原纤维约有600根。但从10日龄开始肌丝排列出现紊乱,细丝全部消失,粗丝降解、数量减少了30%,从而使肌原纤维留下一片片的空白。根据这些结果,把7日龄前的飞行肌发育过程视为生长过程,而把10日龄后的视作降解过程。最后对粘虫蛾飞行肌与卵巢发育及其飞行能力变化的关系进行了讨论。     

鸣鸣蝉发声肌的结构观察

鸣鸣蝉Onvotympana maculaticollit Motsch的发声肌平均含193个初级肌束,多数初级肌束含9-10条肌纤维,其顶、底瑞的附着结构仅由柱状粘和细胞层组成。每条肌纤维约含1 900根肌原纤维,多数肌原纤维的长,宽和截面分别约0.77μm、0.68μm和0.53μm².井约含200根粗肌丝,其粗细肌丝的比值一般为3∶1。肌小节的长度和z线的宽度分别约3μm 和0.2μm.三联管分别位于距两端z线约0.75μm处。肌原纤维、线粒体和微气管-肌质网的面积系数分别约31.3%、46.O%和11.9%。肌小节中粗肌丝纵贯两端z线,中间无1带;细肌丝由z线相向延伸到肌小节中央,其空区约0.15-0.25μm,并无M线。这些结构特征不仅使发声肌能够利用有限的几何空间产生最大的张力,并可适应高速串的收缩运动。     

穴居狼蛛腿肌的显微和亚显微结构

用光学显微镜、电子显微镜和小角 X 射线衍射技术研究了穴居狼蛛(Lycosa singriensis)腿肌的构造。腿肌的肌原纤维显示 I 带和 A 带构成的典型的横纹,肌节长度比脊椎动物骨胳肌长得多,约为5.3μm。A 带中间存在 H 带和 M 线区。在肌原纤维中,长4.3μm,直径200(?)的粗肌丝排成中心间距为约600(?)的六角阵列,每根粗肌丝周围环绕着8—12根直径60(?)的纽肌丝。粗肌丝中心的显著浅染提示粗肌丝可能由肌球蛋白围绕着副肌球蛋白核心组成。X射线衍射实验结果指示,静息状态活肌肉中,肌球蛋白横桥形成435(?)重复螺旋,横桥水平的轴向间距为145(?)。静息状态在位固定的腿肌的电镜照片还显示与肌动蛋白细肌丝接触的肌球蛋白横桥,文中讨论了这一颇有兴趣的结果。     

饥饿和交配对小地老虎飞行肌发育的影响

小地老虎Agrotis ypsilon(Rottemburg)成虫飞行肌的发育常受一些因素影响而发生变化,为探讨饥饿和交配行为对飞行肌发育的影响,通过电子显微镜对雌虫飞行肌(背纵肌)的肌原纤维、线粒体结构进行观察,结果显示:4日龄饥饿雌虫,肌原纤维直径、肌节长度、肌原纤维体积均显著(P<0.05)小于取食的。7日龄饥饿雌虫肌原纤维直径、肌节长度、肌原纤维体积分数较4日龄的差异均不显著(P≥0.05),而7日龄饥饿的肌原纤维直径显著(P<0.05)大于7日龄取食的;羽化10 d后,饥饿雌虫肌节长度显著(P<0.05)大于取食雌虫的,而肌纤维体积分数和线粒体体积分数均却小于后者。7、10、13日龄交配雌虫肌原纤维横切直径分别显著(P<0.05)小于同日龄非交配的;7、10、13日龄交配雌虫肌原纤维体积分数显著(P<0.05)小于非交配的,线粒体体积分数虽然无差异(P≥0.05),但是交配雌虫的早在4日龄便已明显(P<0.05)减小。上述结果表明:正常取食的小地老虎飞行肌4日龄后会发生降解现象;饥饿抑制飞行肌前期发育和中期的降解,而促进成虫末期肌原纤维的分解;交配能促进飞行肌的降解。     

Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms

We have investigated the molecular bases of muscle abnormalities in four Drosophila melanogaster heldup mutants. We find that the heldup gene encodes troponin-I, one of the principal regulatory proteins associated with skeletal muscle thin filaments. heldup3, heldup4, and heldup5 mutants, all of which have grossly abnormal flight muscle myofibrils, lack mRNAs encoding one or more troponin-I isoforms. In contrast, heldup2, an especially interesting mutant wherein flight muscles are atrophic, synthesizes the complete mRNA complement. By sequencing mutant troponin-I cDNAs we demonstrate that the molecular basis for muscle degeneration in heldup2 is conversion of an invariant alanine residue to valine. We finally show that degeneration of heldup2 thin filament/Z-disc networks can be prevented by eliminating thick filaments from flight muscles using a null allele of the sarcomeric myosin heavy chain gene. This latter observation suggests that actomyosin interactions exacerbate the structural or functional defect resulting from the troponin-I mutation.     

THE FILAMENT LATTICE OF COCKROACH THORACIC MUSCLE

The fine structure of the tergo-coxal muscle of the cockroach, Leucophaea maderae, has been studied with the electron microscope. This muscle differs from some other types of insect flight muscles inasmuch as the ratio of thin to thick filaments is 4 instead of the characteristic 3. The cockroach flight muscle also differs from the cockroach femoral muscle in thin to thick filament ratios and diameters and in lengths of thick filaments. A comparison of these latter three parameters in a number of vertebrate and invertebrate muscles suggests in general that the diameters and lengths of the thick filaments and thin to thick filament ratios are related.     

Immunocytochemical electron microscopic study and Western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit flyDrosophila melanogaster, the earthwormEisenia foetida, and the snailHelix aspersa

Summary The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. the muscles studied were: transversely striated muscle with continuous Z lines (flight muscle fromDrosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snailHelix aspersa), obliquely striated body wall muscle from the earthwormEisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.     

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.     

Fuel mobilization for energy metabolism in the silkmoth, Bombyx mori, during reproductive processes

Flight muscles of male moth, B. mori seem to utilize carbohydrate preferentially as a source of energy for all its acrobatic movements during the search for female moth. Depletion of triacylglycerol from flight muscles without affecting its level from fat body suggests that this lipid fraction serves as a source of energy in flight muscles during insemination processes. Significant depletion of triacylglycerol and glycogen from flight muscles of female moth after egg laying indicates that they are used to meet the energy requirement of female during oviposition activity. Depletion of proteins from flight muscles of male and female insects suggest that these proteins are transported to the accessory reproductive glands to meet their protein demand.     

Localization of connectin-like proteins in leg and flight muscles of insects

In leg muscle sarcomeres of a beetle, approximately 6 mum sarcomere length at rest, projectin ( approximately 1200 kDa) was located on the myosin filament up to 2 mum from the both ends of the filament, using immunofluorescence and immunoelectron microscopy. On the other hand, projectin linked the Z line to the myosin filament and bound on the myosin filament in beetle flight muscle, approximately 3-4 mum sarcomere length at rest. Connectin-like protein ( approximately 3000 kDa) was detected by immunoblot tests in beetle, bumblebee and waterbug leg muscles. Immunofluorescence and immunoelectron microscopic observations revealed that the connectin-like protein linked the myosin filament to the Z line in beetle leg muscle.     

Passive tension and stiffness of vertebrate skeletal and insect flight muscles: the contribution of weak cross-bridges and elastic filaments.

Tension and dynamic stiffness of passive rabbit psoas, rabbit semitendinosus, and waterbug indirect flight muscles were investigated to study the contribution of weak-binding cross-bridges and elastic filaments (titin and minititin) to the passive mechanical behavior of these muscles. Experimentally, a functional dissection of the relative contribution of actomyosin cross-bridges and titin and minititin was achieved by 1) comparing mechanically skinned muscle fibers before and after selective removal of actin filaments with a noncalcium-requiring gelsolin fragment (FX-45), and 2) studying passive tension and stiffness as a function of sarcomere length, ionic strength, temperature, and the inhibitory effect of a carboxyl-terminal fragment of smooth muscle caldesmon. Our data show that weak bridges exist in both rabbit skeletal muscle and insect flight muscle at physiological ionic strength and room temperature. In rabbit psoas fibers, weak bridge stiffness appears to vary with both thin-thick filament overlap and with the magnitude of passive tension. Plots of passive tension versus passive stiffness are multiphasic and strikingly similar for these three muscles of distinct sarcomere proportions and elastic proteins. The tension-stiffness plot appears to be a powerful tool in discerning changes in the mechanical behavior of the elastic filaments. The stress-strain and stiffness-strain curves of all three muscles can be merged into one, by normalizing strain rate and strain amplitude of the extensible segment of titin and minititin, further supporting the segmental extension model of resting tension development.     

The developmental profile of paramyosin and other myofibrillar proteins of larval and imaginal thoracic muscles of Lepidoptera

The synthesis of paramyosin and other myofibrillar proteins of the thoracic muscles of the tobacco hornworm Manduca sexta was studied by immunological and electrophoretical methods during the histolysis of the larval thoracic muscles and the differentiation of the indirect flight muscles. Antigens of the myofibrillar proteins in the thoracic muscles of the last-larval stage cross reacted with those in the flight muscles of the adults against polyspecific antibodies from actomyosin and monospecific antibodies from paramyosin. After the breakdown of the larval thoracic muscles (2 days from larval-pupal ecdysis) these antigens can no longer be detected in the thorax. The results indicate an almost complete removal of the larval thoracic muscles. Paramyosin could be identified again in a homogenate of the thoracic muscles of animals on the 13th day from larval-pupal ecdysis. Paramyosin is the first protein found during the differentiation of the flight muscles. The other myofibrillar proteins could be identified in thoracic homogenates of pharate adults of Manduca sexta on the 14th and 15th day from larval-pupal ecdysis. On the 14th day from larval-pupal ecdysis the dorso-longitudinal muscle and the tergosternal muscles show cross-striation, and the appearance of most of the electrophoretical results are in accordance with immunological and morphological findings. The myofibrillar proteins of the indirect flight muscles of Manduca sexta are synthesized de novo during metamorphosis.