【作者】 周瑶；

【导师】 顾福康；

【作者基本信息】 华东师范大学 ， 动物学， 2010， 博士

【摘要】 纤毛虫是原生动物中高度进化的类群,其皮层细胞骨架结构具有进一步的分化。纤毛虫微管胞器是皮层细胞骨架的主要组分。微管作为与细胞生命活动紧密联系的基本构架,成为原生动物学和细胞生物学领域研究的重要对象。目前,对进化程度较低的纤毛虫其皮层细胞骨架组成及纤毛器微管装配的了解较深入,而对于细胞结构高度特化的腹毛类纤毛虫,对其微管结构的研究尚处于初始阶段。探索腹毛类纤毛虫皮层微管胞器及其微管基本单元的组装机理对揭示真核细胞结构的复杂性、细胞结构的遗传及细胞调控等具有重要科学价值。除具有真核细胞中常见的细胞器外,腹毛类纤毛虫皮层细胞质中还存在着一类由膜性囊泡围着的特殊细胞器——射出胞器,该胞器可能在纤毛虫细胞生命活动中行使多种功能。目前对腹毛目纤毛虫的射出胞器,仅局限于对一二种纤毛虫其胞器的形态与形态发育的初步观察,对胞器的细微结构及其在细胞质中的发生和演化等方面尚待深入了解。探索腹毛类纤毛虫射出胞器的结构与功能等对进一步认识纤毛虫细胞中胞器结构的多样性及功能、胞器的发生、遗传及与细胞生命活动的关系等方面是有意义的。本文以腹毛目纤毛虫新伪尾柱虫(Pseudourostyla nova)为材料,应用荧光紫杉醇(FLUTAX)直接荧光标记、扫描电镜术、透射电镜术及电镜酶细胞化学等方法,以纤毛虫的皮层微管胞器和细胞质射出胞器为主对象进行了相关研究。所得结果分两部分报告如下：1新伪尾柱虫皮层微管胞器和其他胞质胞器的研究1.1皮层纤毛器微管的形态和形态发生应用荧光紫杉醇直接荧光标记法显示,腹毛目纤毛虫新伪尾柱虫腹皮层纤毛器微管胞器由口围带、波动膜、额腹横棘毛和左右缘棘毛等纤毛器微管、纤毛器基部附属微管组成。口围带基部含小膜托架及与托架相联系的肋壁微管；额腹横棘毛及缘棘毛基部含前纵微管束、后纵微管束、横微管束和周围微管束,其微管在不同棘毛基部的发达程度不一。纤毛器微管胞器的形态发生中,中腹棘毛瓦解时伴随着后仔虫口围带原基的发生及前仔虫口围带的更新,波动膜和中腹棘毛瓦解时伴随着波动膜和额腹横棘毛的发生,左、右缘棘毛的各1列缘棘毛前部和中部瓦解是伴随着前、后仔虫相应缘棘毛的发生；各类棘毛原基发生中,老结构瓦解产生的部分毛基体参与原基的形成,非原基区的老结构在新棘毛形成过程中逐渐退化瓦解,被新细胞吸收为胞质成分。结果表明,该纤毛虫的纤毛器基部微管与同属纤毛虫的同种微管胞器有差异,具有其种的特异性；在新纤毛器微管分化过程中,老纤毛器具有定位和物质贡献作用。并且,鉴于对新伪尾柱虫的皮层纤毛模式尚未正确描述的情况,本文将形态相似及位置相近的部分额棘毛和中腹棘毛等纤毛器区分开来,对该纤毛虫提供了进一步的形态学证据；纤毛虫细胞皮层中不同种纤毛器基部附属微管建构特征的差异对形态相似及位置相近的纤毛器的区分等也具有结构分类价值；所得结果也为揭示腹毛目纤毛虫皮层微管胞器的分化及建构的多样性提供了基础资料。1.2皮层微管胞器及其他胞质胞器的超微结构应用透射电镜术显示了腹毛目纤毛虫新伪尾柱虫的皮层表膜及表膜下细胞质、皮层微管胞器和其他细胞质胞器。与游仆虫类纤毛虫相比较,前者的腹、背皮层表膜下均含有规则排列的微管层,本种纤毛虫表膜下微管层厚度不一,且部分皮层表膜下无微管层,这也可能与尾柱虫类纤毛虫在运动状态下易弯曲及其细胞体柔软的形态学特征有关。并且,与进化程度较高、皮层纤毛器结构进一步分化的散毛亚目尖毛科、游仆亚目纤毛虫相比较,例如前两者的纤毛虫中,除皮层口纤毛器外,额腹横棘毛、左右缘棘毛较粗大,且棘毛是可数的,但新伪尾柱虫的额腹横棘毛、左右缘棘毛其棘毛较细小、棘毛数量多,且数目不稳定等,存在较明显的不同特征。据新伪尾柱虫纤毛器基部附属微管较为短小,加之细胞表膜下成束排列的微管结构与进化程度较高的纤毛虫的同种微管胞器表现出诸多差异的情况,作者认为,腹毛目纤毛虫棘毛基部微管的发达程度是与其各类群细胞的分化程度密切联系的,本文的结果不仅为说明不同类群纤毛器微管胞器的多样性提供了基础资料,也为纤毛虫的系统分类提供了相关的依据。2新伪尾柱虫射出胞器的研究2.1射出胞器的超微结构应用扫描电镜术和透射电镜术,显示了腹毛目纤毛虫新伪尾柱虫细胞质射出胞器的形态、发生及射出过程,分析了射出胞器的起源及功能。结果表明该纤毛虫的射出胞器不同于草履虫的刺丝泡射出胞器,其胞器分散分布在整个纤毛虫细胞质中,成熟的射出胞器在细胞内呈纺锤形,含体部、电子密度较高的轴杆部和多层膜的帽部；胞器射出时运动并接近细胞表膜,其前端与表膜融合,随即帽状结构连同轴杆样结构共同发射出细胞外,胞器射出物质位于细胞体纤毛区皮层表面,胞内的胞器残留物被细胞质吸收；由于胞器结构发育中伴随着高尔基体的发生,在高尔基体的周围存在大量的小囊泡,囊泡内包裹物质不断聚集,形成纤维状结构物,类似射出胞器中的低密度物质,因此推测该纤毛虫的射出胞器是由高尔基体附近膨大的小囊泡发育而来的；据在不同条件刺激下,细胞皮层表面显示出不同发射状态的胞器射出物形态,但胞器发射后在细胞腹皮层口纤毛器及其邻近位置则未见胞器射出物分布的迹象推断,新伪尾柱虫射出胞器可能具有一定的防御作用,但与细胞捕食无关。2.2射出胞器的电镜酶细胞化学为追踪新伪尾柱虫射出胞器发射后胞器残存物在纤毛虫细胞质内的命运,应用酶细胞化学技术结合透射电镜术,显示了处于不同阶段的射出胞器结构及其酶细胞化学反应特征。在未成熟射出胞器内的低电子密度区酸性磷酸酶反应颗粒均匀分布,推测酸性磷酸酶水解产生的能量可能与射出胞器的成熟及向表膜迁移运动有关；据射出胞器成熟时胞器中央的杆状体发射到胞外后,残存的部分低密度物质位于表膜下细胞质中,相应位置产生许多小囊泡,在小囊泡的膜上和膜内均有酸性磷酸酶反应颗粒的现象推测,射出胞器发射后在细胞内的残存物可能经历了类似于自噬泡消化的过程,其消化产物成为胞内物质利用、循环的一部分。更多还原

【Abstract】 The ciliates are the higher degree class of protozoan, and the cortical microtubular cytoskeletons of ciliates have some differentiation and specialization. The ciliature microtubular organelles are the important components of cortical microtubular cytoskeletons. The microtubules are the foundation which correlate to the ciliates activities closely, so it become the focus in the area of the protistology and cytobiology. To date, the related researches mainly focused on the cortical microtubular cytoskeletons of lower ciliates. However, the data are very limited about the extrusomes of the hypotrich ciliates. Study on the assembly mechanism of microtubular have important biological meaning for the further elucidation of heredity and regulation of ciliatures in ciliates.The cortical organelles in ciliates have some differentiation and specialization. Extrusomes are membrane-limited organelles and distributed widely in ciliated protozoa. Extrusomes play an important role in the life activity of ciliates. To date, the related researches mainly focused on the extrusomes of lower ciliates.On the contrary, the data are very limited about the extrusomes in hypotrich ciliates. The study on morphological characteristics, morphogenesis, and the location of extrusomes in hypotrich ciliates can supply the new data for further elucidation of the diversity and heredity of cell organelles, the relationship between function of organelles and cellular activity.The structure of ciliature microtubular organelles and extrusive organelles in Pseudourostyla nova were visualized using direct FLUTAX labeling, and the more delicate ultrastructure were observed by using scanning and transmission electron microscopy(SEM, TEM), and enzymo-cytochemistry. 1. The microtubular organelles and cytoplasm organelles in P. nova.1.1 The morphology and morphogenesis of microtubular organellesThe ciliature microtubular organelles in the ventral cortex of P. nova were visualized using direct FLUTAX. The organelles consist of adoral zone of membranelles (AZM), undulating membranes (UM), frontal-midventral-transverse cirri (FVTC), left and right marginal cirri (LMC and RMC) and the base-associated microtubules, etc. In the basal part of the AZM, there are membranelle brackets and the associated oral rib-wall microtubules. There are four types of microtubules at the base of FVTC:anterior longitudinal (ALM), posterior longitudinal (PLM), transverse (TM) and radiate microtubules (RM). These different types of microtubules differ from each other in developmental degree. During morphogenesis of the ciliature microtubular organelles, the disintegration of midventral cirri (MC) accompany with the generation of AZM primordia in opisthe and the regeneration of AZM primordia in proter; the disintegration of UM and MC accompany with the generation of UM and FVTC; the disintegration of LMC and RMC accompany with the generation of correspondence LMC and RMC. Among the generation of cirri primordia, part of basal bodies take part in formation of primordia, some old cirri (e.g. frontal, transverse and marginal cirri) could persist and remain unchanged for a long time and then they might absorbed by cell.As the result showing, The base-associated microtubules in the ventral cortex of P. nova possess some species-independent structural characters. During the process of the morphogenesis of the ciliatures, the old cirri play a role in positioning and material contribution. Moreover, in view of the cortex ciliature pattern of P. nova was not described correct yet, part of frontal cirri (FC) and MC which have similar morphology and location were separated in this article. The characteristics of base-associated microtubules among variation ciliatures have classificative value on separating the ciliatures which have similar morphology and location. The resule provide the foundation data for revealing the differentiation and diversity of cortical microtubular organelles in the hypotrich ciliates.1.2 The ultrustructure of microtubular and cytoplasm organelles in P.nova The cortical pellicle, cytoplasm beneath pellicle, cortical microtubular organelles and other organelles in hypotrich ciliates P. nova were observed by using transmission electron microscopy. Compared with Euplotes, the former has a tightly arranged microtubules layer beneath the pellicle, but the arrangement of microtubules in P. nova are random and discontinuous, it is speculated that have relationship with the morphology characteristic of Urostyla. Compared with higher development ciliates, such as Sporadotrichina and Euplotina, the FVTC, LMC and RMC are stronger, and the number of cirris is limited. But the FVTC, LMC and RMC are relatively smaller in P. nova. According to the significant difference among the different kinds of ciliates, this study further demonstrated that the development of cirri base-associated microtubules have relationship with the differentiation and specialization of cell. The aim of the present study is to futher elucidate the diversity of the ciliature microtubular organelles, and also provide the new data for classification in hypotrich ciliates.2. Study on extrusive organelles in hypotrichous ciliate P. nova2.1 The ultrastructure of extrusome in P. novaThe distribution, morphological characteristics, morphogenesis process, and extrusive process of the extrusive organelles in hypotrichous ciliate P. nova was observed by using scanning and transmission electron microscopy. The results show that the extrusomes in P. nova are different from the trichocysts in Paramecium. Extrusomes in P. nova are distributed irregularly throughout cytoplasm and the mature organelles are situated in cytoplasm beneath the pellicle; The anterior end of P. nova extrusomes possesses a cap-like structure consisted of multilayered membranes, It seems that during extrusion, cap and shaft of the extrusive organelles can be discharged outside the pellicle and the remaining non-discharged substance of the organelles is electron-lucent. The pellicular structure seems to be able to become intact again through membrane-fusion after extrusion. Vesicles irregular in size and shape, the vesicles are located densely in cytoplasm area around Golgi apparatus. They contain fibrous substance showing a low electron density similar to that of the remaining substance of the extrusionSo we can speculate that the extrusomes of P. nova might originate from the Golgi apparatus and become matured in cytoplasm.The observation that different fixative could cause various status of extrusomes suggests that these extrusomes play some defensive roles. The phenomenon that no extrusomes are distributed in cytoplasm regions surrounding AZM indicates that the extrusomes bear no relations with the predatory process of P. nova ciliate.2.2 The expression of acid phosphatase in extrusomeIn order to elucidate the destiny of remained substances after extrusion of the extrusomes, The ultrastructure of extrusomes of hypotrichous ciliate P. nova was observed by using transmission electron microscopy and enzymo-cytochemistry. In the extrusomes, acid phosphatase was found distributed only in the area with low electron density, so we speculate that the energy which generate through hydrolyzation of acid phosphatase maybe have relationship on the migration of mature extrusomes. A large quantity of acid phosphatase were demonstrated in the region with the residual structural substance of extruded extrusomes by enzymo-cytochemical electron microscopy in the present study. It could be speculated that the residual substance of extrusomes in P. nova might be reabsorbed and reused by ciliate cell via food vacuoles and take part in material recycling of the cell.更多还原