多头绒泡菌有丝分裂后细胞核重建过程的形态学研究

以进行自然同步核内有丝分裂的多头绒泡菌(Physarum polycephalum)原生质团为材料,应用常规制片和整体银染后制片的电镜技术研究了有丝分裂后细胞核的形态构建过程。形成新核仁的前体物质在有丝分裂中期时散在染色体区域的周围,末期时与染色体组一起到达两极。子细胞核刚形成时核仁物质与染色质混合,以后核仁物质相互汇合并同染色质逐渐分开,最后形成一个大核仁。染色质在有丝分裂后期开始解集缩,到两极后在新形成的子核中进一步松解。染色质在充分松解后又开始集缩活动,形成一些集缩比较紧密的染色质小块。随着细胞核的进一步发育在核膜和核仁之间形成许多大小不等,形状不规则的染色质团块。     

多头绒泡菌有丝分裂后细胞核重建过程的形态学研究

以进行自然同步核内有丝分裂的多头绒孢菌（Ｐｈｙｓａｒｕｍｐｏｌｙｃｅｐｈａｌｍ）原生质团为材料,应用常规制片和整体银染后制片的电镜技术研究了有丝分裂后细胞核的形态构建过程,形成新核仁的前体物质在有丝分裂中期时莠在染色体区域的周围,末期时与染色体组一起到达两极,子细胞核刚形成时核仁物质与染色质混合,以后核仁物质相互汇合并同染色质逐渐分开,最后形成一个大核仁,染色质在有丝分裂后期开始解集缩,到两极后在     

多头绒泡菌细胞核周期的电镜研究

多头绒泡菌PhysarumpolycophalumSchw的营养生长阶段为没有细胞壁的原生质团（合胞体）,内部众多的细胞核进行着同步的核内有丝分裂,本文电镜下研究了细胞核在有丝分裂周期中的结构变化。有丝分裂前期,染色质经松散改组和集缩形成染色体,核仁由中央移向边缘,并在近核膜处解体;中期核膜不消失,在核内形成纺锤体,核仁解体后的物质是不规则状散在于核内;有丝分裂后核膜的破裂处重新愈合,染色体解集缩成染色质,分散的核仁物质逐渐合并形成新的核仁。     

家蚕生存素基因在BmN4细胞有丝分裂中的功能分析

【目的】阐明生存素基因在家蚕Bombyx mori BmN4细胞有丝分裂中的功能。【方法】qRT PCR分析家蚕生存素基因BmSurvivin在家蚕5龄第3天幼虫不同组织(丝腺、中肠、马氏管、精巢、卵巢、脂肪体、皮细胞层和表皮)中的表达量;构建pIZT/V5-His-BmSurvivin-GFP (BG)融合载体并转染BmN4细胞,以免疫荧光标记检测BmSurvivin和第10位丝氨酸(Ser10)磷酸化的组蛋白H3(H3Ser10ph)在BmN4细胞有丝分裂分裂不同时期的定位。【结果】BmSurvivin在家蚕5龄第3天幼虫马氏管中表达量最高,其次是在丝腺和中肠中。成功构建pIZT/V5-His-BmSurvivin-GFP载体。免疫荧光结果显示,在BmN4细胞间期核中可以看到明显的GFP信号,涵盖了细胞核和细胞质区域,指示BmSurvivin的定位;随着细胞进入分裂期,GFP信号指示BmSurvivin与染色质共定位,待细胞形成明显的双取向时在纺锤体区域也可以看到GFP信号;后期随着姐妹染色单体分开,GFP信号指示BmSurvivin定位在染色质及胞质分裂区;末期随着两个新的子细胞形成GFP信号指示BmSurvivin仅定位在胞质分裂区。H3Ser10ph定位在BmN4细胞前中期染色质凝缩处,至中期信号最强,与整条染色体重合,后期信号消失。【结论】BmSurvivin与有丝分裂周期中染色质和纺锤体的动态变化相关。     

多头绒泡菌细胞核周期的电镜研究

多头绒泡菌Ｐｈｙｓａｒｕｍ ｐｏｌｙｃｅｐｈａｌｕｍ Ｓｃｈｗ的营养生长 没有细胞壁的原生质团（合胞体）,内部众多的细胞核进行着同步的核内有丝分裂,本文电镜下研究了细胞核在有丝分裂周期中的结构变化。有丝分裂前期,染色质经松散改组和集缩形成染色体,核仁由中央移向边缘,并在近核膜处解体;中期核膜不消失,在核内形成纺锤体,核仁解体后的物质呈不规则状散在于核内;有丝分裂后核膜的破裂处重新愈合,染色体解集缩     

从染色质到染色体的结构模型及今后研究展望

染色质是细胞形态学名词，指真核细胞有 丝分裂间期核内被碱性染料染色的物质。它相 当于生物化学上的核内DNA和蛋白质的复合 体。间期的染色质在有丝分裂时形成染色体。 染色体到有丝分裂间期又变为染色质。因此染 色质和染色体是有丝分裂周期中不同阶段的运 动形态。     

esiRNA分析人源驱动蛋白MKLP1在胞质分裂中的功能

为探讨人源驱动蛋白MKLP1在有丝分裂和胞质分裂中的作用,以E.coliRNaseⅢ制备MKLP1的3′UTResiRNA转染HeLa细胞,通过定量RTPCR、Western印迹检测MKLP1esiRNA对MKLP1基因的沉默效率.再利用FACS分析、免疫荧光染色和活细胞成像分析检测MKLP1表达缺失后在有丝分裂和胞质分裂不同时期的细胞形态学、细胞分裂指数、细胞百分数,动态观察有丝分裂和胞质分裂期间的表型改变,以系统分析MKLP1的功能.最后通过挽救实验验证MKLP1esiRNA的作用特异性.实验显示MKLP1esiRNA转染HeLa细胞能够有效地特异性消除MKLP1的表达,并被异位表达的MKLP1所挽救.MKLP1蛋白在有丝分裂后期和末期前期位于纺锤体中间带,在末期后期和胞质分裂的最后阶段集中于中间体的中心处.MKLP1表达缺失使中间体正确形成和胞质分裂的完成受到严重抑制,造成大量双多核细胞堆积.结果表明,MKLP1在胞质分裂中间体形成和有丝分裂末期前期向后期过渡过程中起关键作用,是纺锤体中间体中间带相关蛋白,为胞质分裂所必需.     

朱顶红生殖细胞胞质分裂的两种方式

大多数植物以形成细胞板方式完成胞质分裂过程,也有些植物以类似于动物和单细胞植物在赤道区形成收缩沟的方式而分成两部分。本工作应用电镜对朱顶红体外萌发9-18小时花粉管中的生殖细胞胞质分裂进行了研究。结果表明：70％的细胞表现的是第一种方式,30％却是第二种方式。即：朱硕红生殖细胞胞质分裂同时存在两种方式。前者最初以细胞板亚单位的形式出现于有丝分裂晚后期,它们聚集于成膜体的中央区域并于分裂末期融合成一个大的连续的单位(Fig．1-3)。大量新的微管形成于两组染色体之间(Fig．1)。分裂末期,细胞板形成并具胞质通道(Fig.2)。成膜体微管规则排列并穿过胞质通道向新形成的末期核伸展(Fig.2＆3)。这些微管与构成细胞板的质膜紧密联系(Fig．3)。后者则在有丝分裂后期开始(Fig．4),当两群染色体彼此分离时,生殖细胞质膜在中央区由两侧向内凹陷形成收缩沟。有时生殖细胞几乎被收缩沟分成两个部分(Fig．6)。发生缢缩的细胞中细胞器与具细胞板的无差异,但微管稀少并且排列紊乱(Fig．4＆5),染色体的状态使得难以准确区分细胞分裂时期。而且核膜的形成似乎始于有丝分裂后期、出现于染色体边缘(Fig.7)。有时尚有落后染色体出现(Fig.8)。据此认为：收缩沟的发生与核膜的重建、染色体的异常行为及微管无序有关。朱顶红生殖细胞同时存在两种方式的胞质分裂现象相当特殊,可能存在着两种胞质分裂机制。由于游离的生殖细胞在某种程度上类似于动物细胞,因而以缢缩方式完成胞质分裂是可能的。另一方面,生殖细胞对花粉管生长所处的环境极为敏感,体外培养造成生殖细胞不规剧分裂的可能性也应考虑。因此研究在柱头上萌发花粉管中的生殖细胞的胞质分裂是有意义的,此研究结果将有助于更好地理解生殖细胞胞质分裂的机制。     

朱顶红生殖细胞胞质分裂的两种方式(英文)

大多数植物以形成细胞权方式完成胞质分裂过程,也有些植物以类似于动物和单细胞植物在赤道区形成收缩沟的方式而分成两部分。本工作应用电镜对朱顶红体外萌发９～１８小时花粉管中的生殖细胞胞质分裂进行了研究。结果表明：７０％的细胞表现的是第一种方式、３０％却是第二种方式。即：朱顶红生殖细胞胞质分裂同时存在两种方式。前者最初以细胞板亚单位的形式出现于有丝分裂晚后期,它们聚集于成膜体的中央区域并于分裂末期融合成一个大的连续的单位（Ｆｉｇ．１～３）。大量新的微管形成于两组染色体之间（Ｆｉｇ．１）。分裂末期,细胞板形成并具胞质通道（Ｆｉｇ．２）。成膜体微管规则排列并穿过胞质通道向新形成的末期核伸展（Ｆｉｇ．２＆３）。这些微管与构成细胞板的质膜紧密联系（Ｆｉｇ．３）。后者则在有丝分裂后期开始（Ｆｉｇ．４）,当两群染色体彼此分离时,生殖细胞质膜在中央区由两侧向内凹陷形成收缩沟。有时生殖细胞几乎被收缩沟分成两个部分（Ｆｉｇ．６）。发生缢缩的细胞中细胞器与具细胞板的无差异,但微管稀少并且排列紊乱（Ｆｉｇ．４＆５）,染色体的状态使得难以准确区分细胞分裂时期。而且核膜的形成似乎始于有丝分裂后期、出现于染色体边缘（Ｆｉｇ．７）。有时尚有落后     

胡杨小孢子发生及微管骨架变化与异常研究

利用压片法和间接免疫荧光结合DAPI(4′,6-diamidino-2-phenylindole)染色法,对胡杨小孢子母细胞减数分裂过程中微管骨架变化和染色体行为进行观察研究。结果表明:（1）胡杨小胞子母细胞减数分裂进程中染色体行为正常,其中:偶线期可观察到单价体,中期Ⅰ会出现落后染色体,末期Ⅰ和末期Ⅱ的核仁呈现动态变化。（2）胡杨小孢子发生过程中细胞内微管骨架呈动态变化过程,其中:中期Ⅱ形成平行纺锤体以及三极纺锤体;末期Ⅱ未观察到典型的成膜体结构,同时型胞质分裂受子核间辐射微管系统调节,通过胞质向心收缩而发生,胞质分裂后形成四边形和四面体型四分体。（3）胡杨小孢子母细胞减数分裂过程中还存在各种异常细胞学现象,其中:中期Ⅱ平行纺锤体发生融合;中期Ⅱ 和后期Ⅱ孢母细胞两个纺锤体间的胞质会出现裂沟;四分体时期存在三分体和二分体,并产生天然2n花粉和连体花粉。     

Ultrastructure of Mitosis and Cytokinesis In the Colorless Flagellate Katablepharis Ovalis Skuja

ABSTRACT. Mitosis and cytokinesis in Katablepharis ovalis , a colorless flagellate, was investigated. Two new flagella are produced prior to prophase, resulting in a motile quadriflagellate cell during mitosis. the inner array of microtubules of the feeding apparatus disappears before prophase begins. the nuclear envelope disperses during prophase, apparently being converted into rough endoplasmic reticulum. the chromatin condenses and the nucleolus disperses with spindle microtubules appearing oriented perpendicular to the longitudinal axis of the cell. At metaphase, the chromatin is condensed as a single disc-shaped mass and rough endoplasmic reticulum flanks the chromatin mass on each side. Groups of spindle microtubules pass through tunnels in the rough endoplasmic reticulum and through electron-translucent areas of the chromatin. the spindle microtubules end at a number of minipoles in the cytoplasm. Vesicles, ribosomes, mitochondria and endoplasmic reticulum migrate among the spindle microtubules. There is no polar body or any electrondense area associated with the spindle poles. the basal bodies of the flagella remain attached to the axonemes and do not participate in mitosis. In anaphase, the chromatin separates and migrates to the poles. During telophase, the nuclear envelope reforms from the rough endoplasmic reticulum and the nucleoli reappear. the spindle microtubules are persistent during telophase. Cytokinesis occurs by longitudinal fission, starting at the anterior end and progressing posteriorly. Cytokinesis may be driven by elongation of the spindle microtubules since there is no visible structure associated with the furrowing.     

Dissociation of cytokinesis initiation from mitotic control in a eukaryote

Cytokinesis is initiated only after mitotic exit in eukaryotes. However, in the insect (procyclic) form of an ancient protist, Trypanosoma brucei, a blockade at the G2/M checkpoint results in an enrichment of anucleate cells (zoids), suggesting separated regulations between mitosis and cytokinesis (X. Tu and C. C. Wang, J. Biol. Chem. 279:20519-20528, 2004). Polo-like kinases (Plks) are known to play critical roles in controlling both mitosis and cytokinesis. A single Plk homologue in T. brucei, TbPLK, was found to be capable of complementing the Plk (Cdc5) functions in Saccharomyces cerevisiae, thus raising the question of how it may function in the trypanosome with cytokinesis dissociated from mitosis. Depletion of TbPLK in the procyclic form of T. brucei by RNA interference resulted in growth arrest with accumulation of multiple nuclei, kinetoplasts, basal bodies, and flagella in approximately equal numbers among individual cells. There were, however, few zoids detectable, indicating inhibited cytokinesis with unblocked mitosis and kinetoplast segregation. TbPLK is thus apparently involved only in initiating cytokinesis in T. brucei. Overexpression of TbPLK in the trypanosome did not affect cell growth, but 13% of the resulting population was in the zoid form, suggesting runaway cytokinesis. An immunofluorescence assay indicated that TbPLK was localized in a chain of likely flagellum attachment zones in the cytoskeleton. In a dividing cell, a new line of such zones appeared closely paralleling the existing one, which could constitute the cleavage furrow. An exposed region of TbPLK at the anterior tip of the cell may provide the trigger of cytokinesis. Taken together, our results revealed a novel mechanism of cytokinesis initiation in the trypanosome that may serve as a useful model for further in-depth investigations.     

Identification of a novel chromosomal passenger complex and its unique localization during cytokinesis in Trypanosoma brucei

Aurora B kinase is a key component of the chromosomal passenger complex (CPC), which regulates chromosome segregation and cytokinesis. An ortholog of Aurora B was characterized in Trypanosoma brucei (TbAUK1), but other conserved components of the complex have not been found. Here we identified four novel TbAUK1 associated proteins by tandem affinity purification and mass spectrometry. Among these four proteins, TbKIN-A and TbKIN-B are novel kinesin homologs, whereas TbCPC1 and TbCPC2 are hypothetical proteins without any sequence similarity to those known CPC components from yeasts and metazoans. RNAi-mediated silencing of each of the four genes led to loss of spindle assembly, chromosome segregation and cytokinesis. TbKIN-A localizes to the mitotic spindle and TbKIN-B to the spindle midzone during mitosis, whereas TbCPC1, TbCPC2 and TbAUK1 display the dynamic localization pattern of a CPC. After mitosis, the CPC disappears from the central spindle and re-localizes at a dorsal mid-point of the mother cell, where the anterior tip of the daughter cell is tethered, to start cell division toward the posterior end, indicating a most unusual CPC-initiated cytokinesis in a eukaryote.     

Mitosis and cytokinesis in androgonidia ofVolvox carteri f.weismannia

Summary Reproductive cells (androgonidia) ofVolvox carteri f.weismannia divide to form packets of 64 or 128 sperm cells. The androgonidium morphology, stages of mitosis, and cytokinesis were examined by electron microscopy. The biflagellate androgonidium loses its flagella before mitosis but the flagellar bases at the anterior end of the cell are retained. Two additional basal bodies are formed and the nucleus migrates from its central position to the area of the basal bodies before mitosis begins. A five-layered kinetochore is present on the chromosomes and remnant nucleolar material persists during mitosis. A furrow at the chloroplast end of the cell and the formation of phycoplast microtubules and vesicles signal the beginning of cytokinesis at early telophase. The cells maintain cytoplasmic connections until after the packet of sperm cells completes its development.     

Microtubule polarity and dynamics in the control of organelle positioning,segregation, and cytokinesis in the trypanosome cell cycle

Trypanosoma brucei has a precisely ordered microtubule cytoskeleton whose morphogenesis is central to cell cycle events such as organelle positioning, segregation, mitosis, and cytokinesis. We have defined microtubule polarity and show the + ends of the cortical microtubules to be at the posterior end of the cell. Measurements of organelle positions through the cell cycle reveal a high degree of coordinate movement and a relationship with overall cell extension. Quantitative analysis of the segregation of the replicated mitochondrial genome (the kinetoplast) by the flagellar basal bodies identifies a new G2 cell cycle event marker. The subsequent mitosis then positions one "daughter" nucleus into the gap between the segregated basal bodies/kinetoplasts. The anterior daughter nucleus maintains its position relative to the anterior of the cell, suggesting an effective yet cryptic nuclear positioning mechanism. Inhibition of microtubule dynamics by rhizoxin results in a phenomenon whereby cells, which have segregated their kinetoplasts yet are compromised in mitosis, cleave into a nucleated portion and a flagellated, anucleate, cytoplast. We term these cytoplasts "zoids" and show that they contain the posterior (new) flagellum and associated basal-body/kinetoplast complex. Examination of zoids suggests a role for the flagellum attachment zone (FAZ) in defining the position for the axis of cleavage in trypanosomes. Progression through cytokinesis, (zoid formation) while mitosis is compromised, suggests that the dependency relationships leading to the classical cell cycle check points may be altered in trypanosomes, to take account of the need to segregate two unit genomes (nuclear and mitochondrial) in this cell.     

The ultrastructure of mitosis inChroomonas salina (Cryptophyceae)

Summary A detailed account of the ultrastructure of mitosis in a member of theCryptophyceae is given for the first time. The initial indication of mitosis is the duplication of the flagellar bases. The nucleus migrates towards the anterior of the cell and its envelope and nucleolus break down. The chromatin which at interphase is in the form of scattered clumps, condenses into a solid mass through which run narrow tunnels. Each tunnel allows the passage of one to four microtubules. At metaphase the dense plate of chromatin is situated on the equator and the spindle has a rectangular shape. Individual chromosomes cannot be recognized and no morphologically differentiated kinetochores have been observed. The flagella remain functional, their bases stay at the anterior side of the nucleus and do not move to the poles. At anaphase two plates of chromatin separate and these move apart until they come to lie against the ER sheath surrounding the chloroplasts. The new nuclear envelope starts to form on the opposite side of the daughter nucleus. Cytokinesis may commence early in mitosis and consists of a constriction of the parent cell, starting from the posterior end, followed by separation of the two daughters. The present work supports earlier views that only one chromosome is evident during the nuclear division of these organisms. The mitosis is completely different from that of theDinophyceae with which theCryptophyceae were formerly linked.     

The role of pre- and post-anaphase microtubules in the cytokinesis phase of the cell cycle

The cytokinesis phase, or C phase, of the cell cycle results in the separation of one cell into two daughter cells after the completion of mitosis. Although it is known that microtubules are required for proper positioning of the cytokinetic furrow [1] [2], the role of pre-anaphase microtubules in cytokinesis has not been clearly defined for three key reasons. First, inducing microtubule depolymerization or stabilization before the onset of anaphase blocks entry into anaphase and cytokinesis via the spindle checkpoint [3]. Second, microtubule organization changes rapidly at anaphase onset as the mitotic kinase, Cdc2-cyclin B, is inactivated [4]. Third, the time between the onset of anaphase and the initiation of cytokinesis is very short, making it difficult to unambiguously alter microtubule polymer levels before cytokinesis, but after inactivation of the spindle checkpoint. Here, we have taken advantage of the discovery that microinjection of antibodies to the spindle checkpoint protein Mad2 (mitotic arrest deficient) in prometaphase abrogates the spindle checkpoint, producing premature chromosome separation, segregation, and normal cytokinesis [5] [6]. To test the role of pre-anaphase microtubules in cytokinesis, microtubules were disassembled in prophase and prometaphase cells, the cells were then injected with anti-Mad2 antibodies and recorded through C phase. The results show that exit from mitosis in the absence of microtubules triggered a 50 minute period of cortical contractility that was independent of microtubules. Furthermore, upon microtubule reassembly during this contractile C-phase period, approximately 30% of the cells underwent chromosome poleward movement, formed a midzone microtubule complex, and completed cytokinesis.     

The Chromosomal Passenger Complex and a Mitotic Kinesin Interact with the Tousled-Like Kinase in Trypanosomes to Regulate Mitosis and Cytokinesis

Aurora B kinase plays essential roles in mitosis and cytokinesis in eukaryotes. In the procyclic form of Trypanosoma brucei, the Aurora B homolog TbAUK1 regulates mitosis and cytokinesis, phosphorylates the Tousled-like kinase TbTLK1, interacts with two mitotic kinesins TbKIN-A and TbKIN-B and forms a novel chromosomal passenger complex (CPC) with two novel proteins TbCPC1 and TbCPC2. Here we show with time-lapse video microscopy the time course of CPC trans-localization from the spindle midzone in late anaphase to the dorsal side of the cell where the anterior end of daughter cell is tethered, and followed by a glide toward the posterior end to divide the cell, representing a novel mode of cytokinesis in eukaryotes. The three subunits of CPC, TbKIN-B and TbTLK1 interact with one another suggesting a close association among the five proteins. An ablation of TbTLK1 inhibited the subsequent trans-localization of CPC and TbKIN-B, whereas a knockdown of CPC or TbKIN-B disrupted the spindle pole localization of TbTLK1 during mitosis. In the bloodstream form of T. brucei, the five proteins also play essential roles in chromosome segregation and cytokinesis and display subcellular localization patterns similar to that in the procyclic form. The CPC in bloodstream form also undergoes a trans-localization during cytokinesis similar to that in the procyclic form. All together, our results indicate that the five-protein complex CPC-TbTLK1-TbKIN-B plays key roles in regulating chromosome segregation in the early phase of mitosis and that the highly unusual mode of cytokinesis mediated by CPC occurs in both forms of trypanosomes.     

Polo-like kinase is expressed in S/G2/M phase and associated with the flagellum attachment zone in both procyclic and bloodstream forms of Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of African sleeping sickness, divides into insect (procyclic) and bloodstream forms. These two forms are subject to distinct cell cycle regulations, with cytokinesis controlled primarily by basal body/kinetoplast segregation in the procyclic form but by mitosis in the bloodstream form. Polo-like kinases (PLKs), known to play essential roles in regulating both mitosis and cytokinesis among eukaryotes, have a homologue in T. brucei, TbPLK, which regulates only cytokinesis. In our previous study, overexpressed triply hemagglutinin-tagged TbPLK (TbPLK-3HA) in the procyclic form localized to a mid-dorsal point and the anterior tip of the cell along the flagellum attachment zone (FAZ). In our current study, TbPLK-3HA expressed at the endogenous level was identified at the same dorsal location of both procyclic and bloodstream forms, albeit it was no longer detectable at the anterior tip of the cell. Endogenously expressed TbPLK fused with an enhanced yellow fluorescent protein (EYFP) localized to the same dorsal location along the FAZs in living procyclic and bloodstream cells. Fluorescence-activated cell sorter analysis of hydroxyurea-synchronized procyclic cells revealed that TbPLK-EYFP emerges during S phase, persists through G(2)/M phase, and vanishes in G(1) phase. An indicated TbPLK-EYFP association with the FAZs of G(2)/M cells may thus represent a timely localization to a potential initiation site of cytokinesis, which agrees with the recognized role of TbPLK in cytokinetic initiation.