洋葱花粉母细胞细胞内、细胞间微梁骨架的超微结构观察(英文)

以洋葱(AlliumcepaL.)花粉母细胞为材料,采用DGD包埋去包埋原位技术,对花粉母细胞不同发育时期的细胞内、细胞间微梁骨架的超微结构进行了电镜观察。结果发现,花粉母细胞核内存在粗细不等的微梁骨架,与核仁和染色体紧密相连,随着发育的推移,其均一性发生改变。在核周有核纤层样的结构存在,与细胞核和胞质中的微梁骨架紧密相连,到前期结束时解体。洋葱花粉母细胞内具有发达的胞质微梁骨架,这种结构在减数分裂前期Ⅰ变化不明显。在胞间连接(胞间连丝和胞质通道)内,也有精细的微梁骨架分布,并且与两端细胞中的骨架相连。在凝线期的花粉母细胞中观察到细胞融合现象,有胞质或核内微梁骨架与穿壁转移的胞质小球和核小球内骨架相连。此时细胞核偏向一边,但细胞的其余部位仍充满了胞质微梁骨架。初步探讨了核微梁骨架与核仁和染色体之间的关系,核纤层与细胞核之间的关系,以及细胞内、细胞间微梁骨架与细胞融合之间的关系     

细胞的骨架系统

细胞骨架是一类复杂的蛋白质纤维结构,广泛地存在于动物细胞、植物细胞甚至一些原生动物与酵母中。细胞骨架按分布区域可分为胞质骨架和细胞核骨架,胞质骨架又具有三种类型:微管、微丝和中等纤维.胞质骨架和核骨架以及三种胞质骨架之间的结构、性质和功能上是有所区别的,但另一方面它们又协调地参予细胞的一系列生理活动,共同组成了细胞的骨架系统。六十年代初,波特(K·Porter)等第一次用电镜证明了细胞质中骨架结构的多样性,他们发现几乎每一个真核细胞的胞质中都存在三种类型的骨架结构,即微管、微丝和中等纤维。之后,对它们的结构、性质和功能进行了深入的研究。七十年代以来,在细胞核中又发现了一个形态类似于胞质骨架、蛋白质性质的网架结构——细胞核骨架(简称核骨架)对它可能的作用也有了初步的认识,这些发现丰富了骨架系统的内容。现在,已经证实胞质骨架和核骨架在结构与功能上是密切联系的,两者构成了统一的细胞骨架体系,对细胞生长、运动及细胞分化等过程起着重要的作用。     

烟草花粉母细胞细胞融合过程中细胞骨架的超微结构观察

应用普通电镜和DGD去包埋技术 ,研究了烟草花粉母细胞中的细胞融合现象及细胞融合过程中细胞骨架的变化。观察发现 ,处于凝线期的花粉母细胞 ,其内含物 ,包括细胞器和染色质 ,主要通过胞质通道向相邻细胞发生转移。DGD去包埋观察发现 ,花粉母细胞中核骨架与细胞质内及胞间连丝和胞质通道内胞质骨架连接成一个整体。在整个细胞融合过程中 ,均有核骨架纤维与染色质相连。本文讨论了细胞骨架在细胞融合过程中的作用     

洋葱花粉母细胞细胞内、细胞间微染骨架的超微结构观察

以洋葱（Allium cepa L.）花粉母细胞为材料,采用DGD包埋去包埋原位技术,对花粉母细胞不同发育时期的细胞内、细胞间微染骨架的超微结构进行了电镜观察。结果发现,花粉母细胞核内存在的粗细不等的微染骨架,与核仁和染色体紧密相连,随着发育的推移,其均一性发生改变。在核周有核纤层样的结构存在,与细胞核和胞质中的微染骨架紧密相连,到前期结束时解体。洋葱花粉母细胞内具有发达的胞质微染骨架,这种结构在减数分裂前期Ⅰ变化不明显。在胞间连接（胞间连丝和胞质通道）内,也有精细的微染骨架分布,并且与两端细胞中的骨架相连。在凝线期的花粉母细胞中观察到细胞融合现象,有胞质或核内微梁骨架与穿壁转移的胞质小球和核小球内骨架相连。此时细胞核偏向一边,但细胞的基余部位仍充满了胞质微染骨架,初步探讨了核微染骨架与核仁和染色体之间的关系,核纤层与细胞核之间的关系。以及细胞内、细胞间微染骨架与细胞融合之间的关系。     

小麦胚乳细胞内,细胞间胞质骨架分布格局的超微结构观察

以小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）幼嫩胚乳为材料,经ＴｒｉｔｏｎＸ１００抽提、ＤＧＤ（ｄｉｅｔｈｙｌｅｎｅｇｌｙｃｏｌｄｉｓｔｅａｒａｔｅ）渗透、包埋,制备去包埋剂超薄切片,对细胞内、细胞间胞质骨架的分布格局与特征进行了电镜观察。由所获图像可见,胞质骨架呈主要由微管、微丝组成的三维网络结构;特别值得注意的是,有不少５～７ｎｍ的微丝在多处从网络表层向胞壁界面方向突出,并时而可见其横贯分界壁连接相邻骨架网络而将相邻细胞骨架联成一体。胚乳组织中微丝的跨胞分布以两种形式存在,直径达１００～２００ｎｍ微丝束的跨越和单个微丝的分散贯穿,看来这与该组织中开放态胞间通道与正常胞间连丝同时并存相吻合。初步讨论了微丝参与正常胞间连丝结构的可能性。     

荧光标记法检测不同毒物对细胞骨架的影响

细胞骨架(Cytoskeleton)主要由微管(Microtubule,MT)、微丝(Microfilament,MF)以及中间丝(Intermediate filament,IF)这三种类型组成。它们在细胞的形态维持、物质运输、信号转导、能量转换及细胞的运动和分裂等多个过程中发挥着重要的作用。其中,由肌动蛋白组成的微丝是真核细胞中含量最丰富的一种蛋白复合体,以解聚时的球状肌动蛋白G-actin(Globular actin)或聚合时的纤丝状肌动蛋白F-actin(Filamen-tous actin)形式存在。正常细胞中肌动蛋白两种形态的转换处于动态平衡,共同行使细胞的变形运动、胞质分裂、基质附着和胞间连接等多…     

BHK_(21)细胞的中间纤维-lamina-核骨架体系

以系列选择性抽提技术与显示细胞骨架的整装电镜技术为基础,应用免疫胶体金标记与蛋白质成份的双向电泳分析技术,研究了BHK_(21)细胞的中间纤维-lamina与核骨架(核基质)结构体系及其主要的蛋白成份。BHK_(21)细胞的中间纤维-lamina与核骨架是在结构上相互联系,贯穿于核与质的网络体系。中间纤维单丝直径为10nm,能很好地被抗波形蛋白抗体-金颗粒所标记,生化分析同样说明BHK_(21)细胞中间纤维的主要成份是波形蛋白(vimentin),其分子量为55KD,等电点为5.6。中间纤维网在胞质内呈极性分布,与lamina密切联结。BHK_(21)细胞的lamina能被抗lamin A与C的单克隆抗体-金颗粒标记。双向电泳分析证明,lamina含有三种蛋白成份,即lamin A,B,C,其分子最分别为68KD,70KD与62KD,lamin A,C等电点均为6.9—7.2,而lamin B偏酸,其等电点为5.8。BHK_(21)细胞核骨架纤维网也可以被清晰的显示,其蛋白成份较为复杂,在双向电泳谱上经常出现多个清晰的斑点,很可能含有肌动蛋白(actin)。298KD核基质蛋白的单克隆抗体-金颗粒能准确的标记核骨架纤维。     

皮动蛋白及微丝相关蛋白2/3复合物介导的细胞运动的分子机制

皮动蛋白(cortactin)是一种含有特殊重复序列结构域的微丝肌动蛋白结合蛋白,它直接参与了细胞皮层(cortex)微丝细胞骨架的组建。它又是细胞内Src类酪氨酸蛋白激酶的主要底物之一,代表了一类高度保守的胞内皮层信号蛋白质家族。近几年来,对于细胞运动分子机制的研究取得很大进展,利用组织培养细胞进行的体外实验证明。皮动蛋白能够活化微丝相关蛋白2／3复合物(actin related protein 2／3 complex,Arp2／3 complex),调控皮层微丝细胞骨架的组装,在细胞运动过程中具有重要作用。     

Rho GTPases和细胞凋亡

细胞凋亡涉及细胞骨架的形态学改变,Rho GTPases在细胞骨架改变中起着至关重要的作用。近年来的研究揭示了Rho蛋白家族在肌动蛋白(actin)聚合、解聚及actin-myosin的分子调节机制。同时越来越多的研究表明,Rho GTPases在巨噬细胞吞噬凋亡小体中也发挥了关键作用。本综述就Rho GTPases信号途径在细胞凋亡中细胞骨架的结构改变及凋亡小体被吞噬过程中的作用进行具体讨论。     

微管微丝交联因子1的结构与功能

微管微丝交联因子1(microtubule actin cross-linking factor 1,MACF1)是一种新的细胞骨架交联蛋白,属于血影斑蛋白(spectraplakin)家族成员之一,包含3个基本结构域即N末端结构域、杆状结构域及C末端结构域.其主要功能是交联微丝微管细胞骨架,参与细胞信号转导、蛋白质运输、胚胎发育以及疾病发生等过程.近年来,MACF1在细胞骨架动力学过程中的作用备受关注.现就该分子的结构与功能的最新研究进展进行综述.     

Ultrastructural Observation on the Intra- and Intercellular Microtrabecular Network of the Pollen Mother Cells in Onion (Allium cepa)

Using DGD embedment-free electron microscopy, ultrastructural observation on the intra- and intercellular microtrabecular network (MN) of the pollen mother cells (PMC) of the whole meiotic prophase Ⅰ in onion ( Allium cepa L.) was performed. Complex nuclear MN was observed in the nucleus of PMCs, spreading throughout the nuclear region. The nucleolus and chromosomes were connected with the MN filament network. The uniformity of nuclear MN changed with the development of the PMCs. A lamina-like structure surrounded the nucleus and joined the MN in nucleus and in cytoplasm, but it disassembled at the end of prophase Ⅰ. There was also a complex cytoplasmic MN in PMCs, without obvious variation during the prophase Ⅰ. Furthermore, MN in cytoplasmic connections (plasmodesmata and cytoplasmic channels) was noticed to link the frameworks in two neighboring PMCs into one entity. Cytomixis was observed at synizesis of prophase Ⅰ in this experiment, and MN in cytoplasm and in nucleus was noticed to distribute in these granules which migrated from one PMC into its neighboring cell. At this time the nucleus moved aside from center of the PMC, but the rest of the cell was still fulfilled with MN filaments. The relationships of nuclear MN with nucleolus and chromosomes, lamina with nucleus, as well as intra- and intercellular MN with cytomixis are discussed in this paper.     

The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections

High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.     

Getting connected: actin-based cell-to-cell channels in plants and animals

It has been known for more than one hundred years that plant cells are interconnected by cytoplasmic channels called plasmodesmata. This supracellularity was generally considered to be an exotic feature of walled plants containing immobile cells that are firmly enclosed within robust walls. Unexpectedly, intercellular channels in mobile animal cells have been discovered recently. These are extremely dynamic and sensitive to mechanical stress, which causes their rapid breakage and retraction. Both plasmodesmata and nanotubular cell-to-cell channels are supported by the actin cytoskeleton and exclude microtubules. In this article, we discuss the relevance of cell-to-cell channels not only for intercellular communication but also for the development and morphogenesis of multicellular organisms. We also suggest possible parallels between the cell-to-cell transport of endosomes and intracellular pathogens.     

Arp2/3 complex-mediated actin polymerisation occurs on specific pre-existing networks in cells and requires spatial restriction to sustain functional lamellipod extension

The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3-mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex-mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3-mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3-mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3-mediated networks are intrinsically three-dimensional and multilayered by nature and, as such, cannot sustain significant polarized extension. Furthermore, such networks polymerize only at preferred locations in extracted cells, corresponding to pre-existing Arp2/3 networks, suggesting that the specific molecular organization of the actin cytoskeleton, in terms of structure and/or biochemical composition, dictates the location of Arp2/3 complex-mediated actin polymerization. We propose that successful lamellipod extension depends not only on localized actin polymerization mediated through local signalling, but also on spatial restriction of the Arp2/3 complex-mediated nucleation of actin polymerization, both in terms of location within the cell and ultrastructural organization of the resulting network.     

Nuclear–cytoskeletal linkages facilitate cross talk between the nucleus and intercellular adhesions

The linker of nucleoskeleton and cytoskeleton (LINC) complex allows cells to actively control nuclear position by coupling the nucleus to the cytoplasmic cytoskeleton. Nuclear position responds to the formation of intercellular adhesions through coordination with the cytoskeleton, but it is not known whether this response impacts adhesion function. In this paper, we demonstrate that the LINC complex component SUN2 contributes to the mechanical integrity of intercellular adhesions between mammalian epidermal keratinocytes. Mice deficient for Sun2 exhibited irregular hair follicle intercellular adhesions, defective follicle structure, and alopecia. Primary mouse keratinocytes lacking Sun2 displayed aberrant nuclear position in response to adhesion formation, altered desmosome distribution, and mechanically defective adhesions. This dysfunction appeared rooted in a failure of Sun2-null cells to reorganize their microtubule network to support coordinated intercellular adhesion. Together, these results suggest that cross talk between the nucleus, cytoskeleton, and intercellular adhesions is important for epidermal tissue integrity.     

A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells

The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.     

Live imaging reveals that the Drosophila actin-binding ERM protein, moesin, co-localizes with the mitotic spindle

Members of the vertebrate ezrin-radixin-moesin (ERM) protein family crosslink the actin cytoskeleton and the cell membrane and are, therefore, considered cytoplasmic regulators of cell adhesion, cell movement and membrane trafficking. Here we demonstrate that besides its cytoplasmic functions Drosophila moesin, the only ERM protein in Drosophila melanogaster, exhibits a dynamic cell cycle-dependent nuclear localization. In a small fraction of cells and at a low level, moesin can be detected in interphase nuclei in regions complementary to the chromatin; its level rapidly increases during prophase and it co-localizes with the actin network surrounding the mitotic spindles throughout mitosis. We also found that the predicted single nuclear localization signal in moesin is not necessary for the nuclear accumulation of the protein. FRAP experiments confirmed this finding and further revealed that the mitotic localization of moesin is highly dynamic. Immuno-histochemical staining for moesin demonstrated the existence of spindle association in wild-type embryos. The biological relevance of this phenomenon is indicated by the mitotic phenotypes detected in S2 cells treated with moesin RNAi, and awaits future exploration.     

Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites

Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite's shape and the gliding motility.