朱顶红生殖细胞分裂过程中微管超微结构的变化

用透射电镜的方法,对朱顶红（Ａｍ ａｒｙｌｌｉｓｖｉｔｔａｔａ Ａｉｔ．）花粉管中生殖细胞的分裂过程中微管分布和结构形态变化进行了观察,获得如下主要的结果：有丝分裂前期,微管的数量较分裂前减少并变短,靠近细胞核分布。分裂前中期,微管出现于原来的核区并与染色体发生联系,形成着丝点微管。分裂中期,染色体排列于赤道面上形成赤道板,微管构成纺锤体。分裂后期,染色体分成两群,被缩短的着丝点微管拉向两极。在纺锤体两极的微管汇聚。后期的晚期,当极的微管尚未消失时,在赤道区域出现丰富的成膜体微管,在成膜体中央,细胞板前体物聚集。分裂末期,极微管和着丝点微管消失,成膜体微管在新形成的核膜和细胞板间扩展并穿过细胞板     

黄蝉花生殖细胞有丝分裂期间微管的动态

用微管免疫荧光方法观察了黄蝉花生殖细胞在花粉管中进行有丝分裂时的微管动态。微管在不同分裂期的分布情形很不一样。当生殖细胞由花粉进入花粉管后,细胞便立刻开始分裂进入早前期,在这阶段微管以一个紧密微管网笼子形式存在生殖细胞内。之后,细胞进入中前期,在此阶段细胞核扩大,染色体变粗,而存在细胞内的微管网逐渐变为疏松散漫状,跟着细胞进入晚前期,而微管笼子则由网状变为纵向排列状。分裂进入早中期微管变细并呈波浪状,微管由笼子结构过渡到纺锤体结构。进入中期,纺锤体全部形成,在纺锤体内可以清楚地看到两种不同类型的微管束,一种附着在染色体上,而另一种则从一极延伸至另一极。跟着细胞进入早后期,在这一阶段姊妹染色体分开并分别移向两极,在赤道板位置微管明显减少。之后,细胞进入晚后期,姊妹染色体集中在两极,极端有新微管出现。在两个染色体团之间又汇集了许多类似成膜体微管的微管。细胞进入分裂末期,存在赤道板位置的微管又再次减少,而在中央部位则新形成一“成膜体联接区”,把两个新形成的精子连接着。     

百合生殖细胞分裂过程中微管分布的显微荧光观察

用抗微管蛋白抗体和荧光标记技术,观察了百合生殖细胞经有丝分裂形成精细胞过程中微管的变化。生殖细胞在分裂的前期,存在于核外围以及细胞两端胞质内的微管大都以微管束的形式沿细胞长轴方向平行排列。在靠近核的部位,有些微管有时会斜向排列。分裂进入中期后,染色体集中排列在赤道面。在染色体周围可以见到有多束与细胞长轴平行排列着的微管,但这些微管束是在分裂中期时新形成的或是在前期已存在,尚难以断定。这些微管束有一个特点,就是当它们延伸至赤道板部位时,在每一条微管束上都有一个无荧光的小圆区;这个小圆区可能代表着丝粒的位置。细胞分裂进入后期,姊妹染色单体分别向两极移动形成两组染色体。在它们之间近赤道板位置出现了一个具有强烈荧光的区域,显示在这一部位,微管相当浓密。从这一强烈荧光区向两极分别伸出多条微管束。因此,在这一强烈荧光区内可能有多个微管束重叠。到细胞分裂末期,在这一强烈的荧光区的中央出现了一条横向的无荧光区。这一区域有可能为胞质完成分裂后新形成的细胞板所在的部位。     

川百合精细胞的超微结构

川百合与朱顶红花粉管中的生殖细胞分裂行为非常不同。诸如：染色体行为微管的组织形式和分布包括着丝点微管形成的时间,纺锤体的形状及间期周质微管网络在生殖细胞分裂过程中消失与否等,但这两种细胞具有共性,包括在有丝分裂前期缺乏早前期带微管（PPB）,未其形成细胞板等,这两种植物精细胞的结构应有较大差异,我们曾报道了朱顶红精细胞的超微结构,本文详细从超微结构方面描述了川百合精细胞的特征。川百合花粉管的萌发采用半离体－活体培养方式,11－18小时后,DNA荧光染料Hoechst33258和醋酸地衣红染色检查花分管中生殖细胞和精细胞发育时期。切取含有分裂的生细胞和精细细胞的花柱部分,按曾报道的方法固定、包埋、切片、染色及观察。在所有检查的花粉管中,两精子均前后排列（Fig.1-3),营养核前导并靠近花粉管顶端（Fig.,3)。H33258染色可见两精核间以DNA联系（Fig.3)。两个新形成的精核彼此分离（Fig.1),后来又相互造近,并维持一定距离（Fig.3)偶尔一对精子与营养核靠近（Fig.2)。两精细胞被一共同的细胞壁连接,他们不仅被自己的质膜也被营养细胞的质膜包围构成周质。周质平坦光滑。共同壁横向、弯曲、网状具胞质通道（Fig.4),厚度明显大于周质。色质凝集的程度更大些（Fig.5),可能意味着一个精子发育的早些。精细胞质中具有线粒体、内质网、高尔基体、脂体和大量核糖体。无质体。线粒体具有发育完好的精细胞中,微管呈纵向束排列于随精细胞的继续发育,共同壁消失了。与朱顶红等植物的染色体行为遵循典型有丝分裂方式不同,川百合生殖细胞与紫露草相同,它的染色体在有丝分裂中期沿细胞长轴分布,胞质分裂时没有细胞板出现。可以认为：川百合象烟草一样是介于朱顶红和紫露草之间的中间类型。雄性生殖单位（MGU）在三细胞和二细胞花粉中普遍存在。尽管本工作观察到营养核与精细胞紧密联系,以及两精子与DNA联系的例子,但MGU在超薄切片中并未见到,有可能MGU是一个动态的和时间上的暂时结构。另一方面,MGU的建立是以性细胞（生殖细胞或精细胞）的突起和营养核的裂瓣相互环绕为基础的,而性细胞中的细胞骨架（即：微管）可能对维持其与营养核的附着起重要作用。缺乏微管,可能是川百合精细胞不存在MGU的原因之一。     

川百合精细胞的超微结构(英文)

川百合与朱顶红花粉管中的生殖细胞分裂行为非常不同。诸如：染色体行为、微管的组织形式和分布、包括着丝点、微管形成的时间,纺锤体的形状及间期周质微管网络在生殖细胞分裂过程中消失与否等。但这两种细胞具有某些共性,包括在有丝分裂前期缺乏早前期带微管（ＰＰＢ）,末期形成细胞板等。这两种植物精细胞的结构应有较大差异。我们曾报道了朱顶红精细胞的超微结构,本文详细从超微结构方面描述了川百合精细胞的特征。川百合花粉管的萌发采用半离体活体培养方式。１１～１８小时后,ＤＮＡ荧光染料Ｈｏｅｃｈｓｔ３３２５８和醋酸地衣红染色检查花粉管中生殖细胞和精细胞发育时期。切取含有分裂的生殖细胞和精细胞的花柱部分,按曾报道的方法固定、包埋、切片、染色及观察。在所有检查的花粉管中,两精子均前后排列（Ｆｉｇ．１～３）,营养核前导并靠近花粉管顶端（Ｆｉｇ．２,３）。Ｈ３３２５８染色可见两精核间以ＤＮＡ联系（Ｆｉｇ．３）。两个新形成的精核彼此分离（Ｆｉｇ．１）,后来又相互靠近,并维持一定距离（Ｆｉｇ．３）。偶尔一对精子与营养核靠近（Ｆｉｇ．２）。两精细胞被一共同的细胞壁连接,他们不仅被自己的质膜也被营养细胞的质膜包围构成周质。周质平坦光滑。共同壁横向     

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。     

朱顶红精细胞超微结构观察

精细胞是双受精作用的直接参与者,是生殖生物学中的重点研究对象之一。以往的研究表明,应用连续超薄切片和计算机辅助三维重组技术,结合免疫荧光定位,发现两个精细胞在体积和细胞器含量上存着差异,即精子的二型性,而且与营养细胞核三者构成紧密功能单位却雄性生殖单位（MGU）。微管对精细胞的性状的确定、运动和维持MGU的动态结构稳定具有重要的作用。本文应用透射电镜。详细观察了朱顶红花粉管中细胞的超微结构,并着重微管结构及其分布的观察。朱顶红成熟花粉为两细胞型。成熟花粉于26℃、黑暗条件下,在液体培养基（含10％蔗糖和100ppm硼酸）中培养13－18小时,然后收集花粉管,固定,供电观察并照相。朱顶红成熟花粉培养13小时后,生殖细胞在花粉管中完成核分裂和胞质分裂等两个过程。形成两个精细胞。形成的两个精细胞前后排列,营养核前导并靠近花粉管顶端。领头的精细胞的细胞质以很大的表面与营养核相互融合（图版1－1,2）,有时营养核与两个精细胞彼此穿插、缠绕（图版1－3）。两精细胞之间的壁上具有多胞质通道和含均质电子密度中等的基质（图版II－4）。精细胞质在核与共同壁之间的区域染色较深,经高倍放大,观察到此处含丰富的微管, 自由分布,但以纵向或斜向为主（图版II－5,6）。所有的微管构成松散的桶状网络存在于两精核之间,除此之外,其他区域无微管分布（图版II－7）。培养18小时之后,两精细胞的共同呈网状（图版II－8）,此时微管均成纵向排列,平行于细胞长轴,构成筐状结构包围状精核,但不构成紧密的束状,证明了前人的免疫荧光观察结果。从以上观察结果我们可以得出如下的结论,朱顶红花粉为两细胞型,因此在花粉管中形成的两个精细胞一般前后排列,跟在营养细胞后面,这种线状排列方式在其他植物中也观察到,可能有利于三者作为一个结构功能单位在花粉管中移动和对花粉管狭窄空间的进化适应。MGU形成得较晚,在生殖细胞和营养核进入花粉管后才形成,并一直维持到精核形成,与其他报道不同。精细胞发育过程中,微管的分布方式变化显著。精细胞中微管的分布仅限于共同的细胞壁和靠近茎核之间的区域,总体构成一个松散的桶状结构。精细胞发育后期,微管均成纵向排列,包围着精核,极似生殖细胞筐状的微管结构形式。     

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     

烟草小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察了烟草小孢子母细胞减数分裂过程中微管的分布变化。在减数分裂前期,小孢子母细胞中的微管较短,随机分散在细胞质中。在减数分裂中期,细胞质中微管形成纺锤体,控制染色体的分布。进入减数分裂I后期,部分纺锤体微管将两组染色体拉向两级。在减数分裂Ⅱ中期,细胞中的微管又形成两个纺锤体。在减数分裂Ⅱ后期,纺锤体微管解聚为微管蛋白分散在细胞质中。胞质分裂发生在四个细胞核形成之后,通过细胞核之间的质膜向内缢缩分隔四个细胞核,产生四个小孢子。     

黄蝉离体生殖细胞微管的共焦激光扫描镜观察

用共焦激光扫描光学显微镜观察了从黄蝉花粉管中分离出来的生殖细胞微管骨架的结构变化。当生殖细胞刚从花粉管释出时，大多数呈纺锤形，细胞内的微管束与长轴平等排列着，形成一个笼子结构。生殖细胞在培养液内培养大约５－１０分种后，便由纺锤形变为椭圆形和圆球形，而微管结构也从平行排列状变为疏散状，形成许多随机分布的小向管束。之后，有些微管束消失，但有些则集结在靠近核的部位，形成一圈特亮的荧光区。以后，这特亮的荧     

Changes of Microtubular Skeleton in the Generative Cell in Pollen Tube During Mitosis of Lilium davidii

Previous observations indicated that division of the generative cell (GC) in some plant genura such as Lilium and Tradescantia is characterized by several unusual features, including persistence of surrounding microtubule (MT) bundles during mitosis, lacking a matephase plate, the cytokinesis is completed with furrow. The authors have further studied the changes of MT organizations and the chromosome (CHs) behavior in the GC during mitosis using electron microscopy and method of tubulin localizations. No MTs in the GC before GC division and during prophase was seen under electron microscopy. However, there was tubulin in the GC with antitubulin staining. During promatephase to matephase, the CHs appeared and arranged in a complexed tangled pattern lengthwise along the cell. Correspond- ingly, transverse pairs of kinetochores were located along the length and depth of the cell. They stacked successively like the rungs of a ladder. In this phase, a large mount of MTs appeared in the GC, which distributed in the cortex of the cell and among the CHs and along the whole length of the CHs. In the beginning, one or two kinetochore pairs changed from transversely to longitudinally situated in each cell. MTs ended on the kinetochore to form kinetochore MTs (KMTs). With the electron microscopy, authors did not find the image of lateral connection between the MTs and the kinetochores as previous reported with immunofluorescent method. As karyokinesis proceeded, more transverse kinetochore pairs gradually became longitudinal, and KMTs gradually increased in number. However, a distinct spindle was not evidenced. During anaphase, CHs seperation started at various positions along the length of the cell. The distribution of MTs in the GC was similar to that of promatephase to matephase. In late anaphase, the CHs segregated as two groups. Most MTs disappeared but only some remained in the polar regions and the interzone. Authors also measured and compared the lengths of the CHs and indirectly identified the existing anaphase B. During late tolephase, the MTs increased in number gradually in the region between the two newly formed sperm nuclei. The region might be the MT interdigitating zone visualized with antitubulin localization. The MTs disappeared after the cell plate (CP) appeared.     

Ultrastructural Changes of the Microtubule in the Generative Cell of Amaryllis vittata Ait. During Mitosis

Structural changes of microtubules (MTs) in the generative cell (GC) of Amaryllis vittara Alt. during mitosis in pollen tube have been investigated with electron microscopy. The division cycle was completed approximately within 12 h. During prophase, the MTs bundles distributed in the cortex of the GC, they were less and shorter than that before mitosis, some of which beginning to be near the nucleus. When the chromatin condensed and the GC entered metaphase, the MTs increased in number and distributed among the chromosomes (CHs) in the original nuclear zone, but they were not arranged in distinct bundlesed. Some of them connected with the CHs to form kinetochore MTs (KMTs), where as the cortical MTs in prophase still remained there. During metaphase, the CHs were arranged on the equartor forming a metaphase plate, and all the MTs formed a diffuse spindle. When the GC entered anaphase, the KMTs were shortened and they were involved in the segregation of the CHs into two groups. The MTs were much more and focused in the two polar regions. In late anaphase, while the MTs still existed at the poles, rich phragmoplast MTs appeared in the equator zone and the precusors of cell plate (CP) aggregated in the middle of the phragmoplast. When the GC entered telophase, the CHs diffused as chromatin, and phragmoplast MTs extended between the two newly formed nuclear envelops and even through the CP While the polar MTs and KMTs disappeared, the MTs in the newly formed sperm cells were different from that of the GC.     

Dynamics of spindle microtubule organization: kinetochore fiber microtubules of plant endosperm

Organization of kinetochore fiber microtubules (MTs) throughout mitosis in the endosperm of Haemanthus katherinae Bak. has been analysed using serial section reconstruction from electron micrographs. Accurate and complete studies have required careful analysis of individual MTs in precisely oriented serial sections through many (45) preselected cells. Kinetochore MTs (kMTs) and non-kinetochore MTs (nkMTs) intermingle within the fiber throughout division, undergoing characteristic, time- dependent, organizational changes. The number of kMTs increases progressively throughout the kinetochore during prometaphase-metaphase. Prometaphase chromosomes which were probably moving toward the pole at the time of fixation have unequally developed kinetochores associated with many nkMTs. The greatest numbers of kMTs (74-109/kinetochore), kinetochore cross-sectional area, and kMT central density all occur at metaphase. Throughout anaphase and telophase there is a decrease in the number of kMTs and, in the kinetochore cross-sectional area, an increased obliquity of kMTs and increased numbers of short MTs near the kinetochore. Delayed kinetochores possess more kMTs than do kinetochores near the poles, but fewer kMTs than chromosomes which have moved equivalent distances in other cells. The frequency of C-shaped proximal MT terminations within kinetochores is highest at early prometaphase and midtelophase, falling to zero at midanaphase. Therefore, in Haemanthus, MTs are probably lost from the periphery of the kinetochore during anaphase in a manner which is related to both time and position of the chromosome along the spindle axis. The complex, time-dependent organization of MTs in the kinetochore region strongly suggests that chromosome movement is accompanied by continual MT rearrangement and/or assembly/disassembly.     

Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding

We have isolated chromosomes from Chinese hamster ovary cells arrested in mitosis with vinblastine and examined the interactions of their kinetochores with purified tubulin in vitro. The kinetochores nucleate microtubule (MT) growth with complex kinetics. After an initial lag phase, MTs are continuously nucleated with both plus and minus ends distally localized. This mixed polarity seems inconsistent with the formation of an ordered, homopolar kinetochore fiber in vivo. As isolated from vinblastine-arrested cells, kinetochores contain no bound tubulin. The kinetochores of chromosomes isolated from colcemid-arrested cells or of chromosomes incubated with tubulin in vitro are brightly stained after anti-tubulin immunofluorescence. This bound tubulin is probably not in the form of MTs. It is localized to the corona region by immunoelectron microscopy, where it may play a role in MT nucleation in vitro.     

Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to- pole motion

During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.     

Phallacidin stains the kinetochore region in the mitotic spindle of the green algaeOedogonium spp.

Summary We found previously that in living cells ofOedogonium cardiacum andO. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (<2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore ofOedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.Abbreviations CD cytochalasin D - EM electron microscopy - MBS m-maleimidobenzoyl N-hydroxysuccinimide ester - MTs microtubules     

Calmodulin is associated with microtubules forming in PTK1 cells upon release from nocodazole treatment

To investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of CaM and tubulin were examined in cells in which the normal spindle organization had been altered. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM-TRITC) and a dichlorotriazinyl aminofluorescein conjugate with tubulin (tubulin-DTAF) were injected into cells that had been treated with the MT inhibitor nocodazole. With moderate nocodazole concentration (0.3 micrograms/ml, 37 degrees C, 4 h) in live cells, CaM-TRITC and tubulin-DTAF concentrated identically on or near the centrosomes and kinetochores. In serial sections of these cells, small MT segments were observed by transmission electron microscopy (TEM) in the regions where fluorescent protein had concentrated. When a higher drug concentration was used (3.0 micrograms/ml, 37 degrees C, 4 h), no regions of CaM-TRITC or tubulin-DTAF localization were observed, and no MTs were observed when serial sections were examined by TEM. However, following release from the high-concentration nocodazole block, CaM-TRITC colocalized with newly formed MTs at the kinetochores and centrosomes. Later in the recovery period, when chromosome-to-pole fibers had formed, CaM association with kinetochores diminished, ultimately attaining its normal pole-proximal association with kinetochore MTs in cells that progressed through mitosis. We interpret these observations as supporting the hypothesis that in the MA, CaM attains a physical association with kinetochore MTs and suggest that CaM-associated MTs may be inherently more stable.     

Microtubule dynamics investigated by microinjection of Paramecium axonemal tubulin: lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase

Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of incorporation of the axonemal tubulin were localized using a specific antibody which does not react with vertebrate cytoplasmic tubulin (Adoutte, A., M. Claisse, R. Maunoury, and J. Beisson. 1985. J. Mol. Evol. 22:220-229), followed by immunogold labeling, Nanovid microscopy, and ultrastructural observation of the same cells. We confirm data from microinjection of labeled tubulins in other cell types (Soltys, B. J., and G. G. Borisy. 1985. J. Cell Biol. 100:1682-1689; Mitchison, T., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527; Schulze, E., and M. Kirschner. 1986. J. Cell Biol. 102:1020-1031). In agreement with the dynamic instability model (Mitchison, T., and M. Kirschner. 1984. Nature (Lond.). 312:237-242), during interphase, fast (2.6 microns/min) distal growth of MTs occurs, together with new centrosomal nucleation. Most of the cytoplasmic MT complex is replaced within 15-30 min. During mitosis, astral MTs display the same pattern of renewal, but the turnover of the MT system is much faster (approximately 6 min). We have concentrated on the construction of the kinetochore fibers during prometaphase and observe that (a) incorporation of tubulin in the vicinity of the kinetochores is not seen during prophase and early prometaphase as long as the kinetochores are not yet connected to a pole by MTs; (b) proximal time-dependent incorporation occurs only into preexisting kinetochore MTs emanating from centrosomes. Consequently, in undisturbed prometaphase cells, the kinetochores probably do not act as independent nucleation sites. This confirms a model in which, at prometaphase, fast probing centrosomal MTs are grabbed by the kinetochores, where tubulin incorporation then takes place.     

Visualizing kinetochore architecture

Kinetochores are large macromolecular assemblies that link chromosomes to spindle microtubules (MTs) during mitosis. Here we review recent advances in the study of core MT-binding kinetochore complexes using electron microcopy methods in vitro and nanometer-accuracy fluorescence microscopy in vivo. We synthesize these findings in novel three-dimensional models of both the budding yeast and vertebrate kinetochore in different stages of mitosis. There is a growing consensus that kinetochores are highly dynamic, supra-molecular machines that undergo dramatic structural rearrangements in response to MT capture and spindle forces during mitosis.