粘虫蛾飞行肌超微结构的研究

应用电子显微镜对粘虫雌蛾Mythimna separata(Walker)飞行(背纵)肌的研究结果表明,其肌原纤维由500-700根肌球蛋白丝(粗丝)组成,每根粗丝由6根肌动蛋白丝(细丝)环绕排列成六角形,每根细丝精确地位于两根粗丝间1/2处,从而使粗丝和细丝的比为1：3。肌节较短,长度约2.2-2.6μm。肌原纤维之间充满着线粒体和横管。每个肌节约有线粒体三个,横管二根。线粒体约占肌纤维体积的40%,而横管为7%。每根横管准确地位于肌节的1/4、3/4处,或Z线和中膈的中央,并与肌质网交接形成二位体(dyads)或三位体(triads)。肌质网相当不发达,约占肌纤维体积的2.5%。但其分布很有特色,即除了紧贴于肌原纤维周围的由单层液泡组成的肌质网以外,在中膈处还有一层横穿于肌原纤维的肌质网。和其它同步飞行肌的结构和功能分析比较的结果还表明,粘虫蛾飞行肌具有较善于飞行的结构。     

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。     

群居型和散居型东亚飞蝗雌成虫飞行肌的超微结构

应用电子显微镜对群居型和散居型东亚飞蝗Locusta migratoria manilensis（Meyen）雌成虫背纵肌进行了比较观察。结果表明：群居型和散居型成虫背纵肌具有类似的亚细胞结构,飞行肌的肌原纤维具有1∶3粗细丝比例,每根粗丝由6根细丝环绕排列成六角形结构。飞行肌的发育和线粒体的形成均是渐进的过程,在不同日龄成虫间存在差异。肌节长度为2.1～3.4 μm;7和10日龄时群居型成虫肌节长度小于散居型;7日龄群居型肌原纤维直径显著大于散居型。背纵肌内线粒体含量约占肌纤维的20%～43%,两型飞蝗之间存在着显著的差异,7日龄时群居型线粒体占肌原纤维的比例高达42.96%,而散居型的只有22.45%;10日龄群居型线粒体含量为41.32%,散居型线粒体29.98%。上述差异可能是东亚飞蝗群居型成虫飞行能力显著强于散居型成虫的重要原因之一。     

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。     

昆虫飞行肌蛋白质

昆虫飞行肌的肌原纤维不仅含有粗肌丝、细肌丝、纤肌丝,还含有很多其它蛋白质参与肌原纤维的组装和调节,文章介绍了10余种蛋白质的结构、功能及其在肌原纤维中的位置和功能,对于了解昆虫飞行肌的发育和探索昆虫飞行能力差异的原因具有重要意义。     

棉铃虫飞翔肌的超微结构

利用电子显微镜观测表明,棉铃虫Helicoverpa rmigera (Hubner)飞翔肌的肌原纤维由400～800根肌球蛋白丝组成,每根肌球蛋白由6根肌动蛋白丝环绕排列成六角形,肌节长度2.0～3.5μm,线粒体占飞翔肌的体积达42.38%～48.57%,微气管组织较为发达。初羽化棉铃虫肌原纤维和线粒体的发育基本完成,横管系统的发育相对较慢,羽化3日后趋于成熟,至5日龄占飞翔肌的体积达3.31%～3.54%。表明棉铃虫具有适宜飞行的飞翔肌结构。采自渤海海面距海岸线80km的迁飞蛾子飞翔肌基本结构和实验种群无明显的区别,但迁飞过程中的能量代谢导致线粒体内脊疏松而出现大量空洞。     

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.     

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     

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     

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。     

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。     

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.     

Flight muscle myofibrillogenesis in the pupal stage of Drosophila as examined by X-ray microdiffraction and conventional diffraction

In the asynchronous flight muscles of higher insects, the lattice planes of contractile filaments are strictly preserved along the length of each myofibril, making the myofibril a millimetre-long giant single multiprotein crystal. To examine how such highly ordered structures are formed, we recorded X-ray diffraction patterns of the developing flight muscles of Drosophila pupae at various developmental stages. To evaluate the extent of long-range myofilament lattice order, end-on myofibrillar microdiffraction patterns were recorded from isolated quick-frozen dorsal longitudinal flight muscle fibres. In addition, conventional whole-thorax diffraction patterns were recorded from live pupae to assess the extent of development of flight musculature. Weak hexagonal fluctuations of scattering intensity were observed in the end-on patterns as early as approximately 15 h after myoblast fusion, and in the following 30 h, clear hexagonally arranged reflection spots became a common feature. The result suggests that the framework of the giant single-crystal structure is established in an early phase of myofibrillogenesis. Combined with published electron microscopy results, a myofibril in fused asynchronous flight muscle fibres is likely to start as a framework with fixed lattice plane orientations and fixed sarcomere numbers, to which constituent proteins are added afterwards without altering this basic configuration.     

Caenorhabditis elegans UNC-96 is a new component of M-lines that interacts with UNC-98 and paramyosin and is required in adult muscle for assembly and/or maintenance of thick filaments

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent "needles." By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel approximately 47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.     

Ultrastructure of Developing Flight Muscle in Drosophila. I. Assembly of Myofibrils

In order to evaluate the effects of specific mutations on sarcomere assembly and function in vivo, we describe the course of normal development of Drosophila indirect flight muscle (IFM) in staged pupae using electron microscopy. We find that no contractile assemblies remain in larval muscle remnants invaded by imaginal myoblasts, establishing that myofibrils in IFM assemble de novo. Stress-fiber-like structures or other template structures are not prominent before or during sarcomere assembly. By 42 hr pupation (eclosion 112 hr), thick and thin filaments have appeared simultaneously in slender, interdigitated arrays between regularly spaced Z-bodies. Each tiny, uniformly striated myofibril forms within a "sleeve" of microtubules, and both microtubules and myofibrils are attached to the cell membrane at each end of the fiber from the initial stages of assembly. Later in pupation, the microtubule "sleeves" disassemble. Sarcomere number appears to remain constant. We saw no evidence that terminal sarcomeres are sites for addition of new sarcomeres or that Z-lines split transversely, producing new, very short sarcomeres. Rather, initial thick and thin filaments and sarcomeres are much shorter than adult length. Sarcomere length increases smoothly and coordinately from 1.7 to 3.2 μm, reflecting increase in filament lengths and indicating that myosin and actin molecules must be incorporated into filaments after sarcomere formation. Myofilaments are not seen scattered in the cytoplasm at any time, nor do we detect filaments that could be in the process of being "trolleyed" along myofibrils into positions of lateral register. Myofibril diameter increases uniformly from 4-thick filaments to 36-thick filaments across, by peripheral addition of myofilaments. At each successive stage, all sarcomeres in a fiber attained similar length and diameter. Initial thick filaments are solid but within several hours these and all subsequently assembled thick filaments appear hollow. Initial Z-bodies do not show any internal lattice and are more irregularly shaped than adult Z-discs.     

The site of paramyosin in insect flight muscle and the presence of an unidentified protein between myosin filaments and Z-line.

The position of paramyosin in insect flight muscle was determined by labelling myofibrils with antibody to paramyosin and examining them by fluorescent and electron microscopy.Antiserum to dung beetle paramyosin had antibodies to another protein as well as to paramyosin. Specific anti-paramyosin bound to the H-zone of Lethocerus myofibrils showing paramyosin was exposed only in that region. Antibodies to the other protein bound at the ends of the A-band.The exposure of antigenic sites in the two regions of the myofibril depended on the extent of contraction in the myofibril: the sites at the end of the A-band were most exposed in rest-length myofibrils and those at the H-zone in shortened ones.Antibody-labelling in stretched bee muscle showed that the protein at the ends of the sarcomere extended from myosin filaments to Z-line.The high resting elasticity of insect flight muscle and hence its capacity for oscillatory contraction may be due to the protein between myosin filaments and Z-line.