Department of Biological Science, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2012;7(4):e34763. doi: 10.1371/journal.pone.0034763. Epub 2012 Apr 6.
Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.
嗜热四膜虫（Naegleria pringsheimi）的有丝分裂为无中心体和封闭的（核膜不会破裂）。占据核体积约 20%的大中央核仁在整个细胞周期中都存在。在有丝分裂过程中，核仁分裂并与染色体一起移动到两极。有丝分裂纺锤体的结构及其与核仁的关系尚不清楚。为了确定有丝分裂纺锤体的起源和结构，以及它与核仁的关系，并进一步了解持久核仁对嗜热四膜虫等无中心体生物细胞分裂的影响，使用共聚焦显微镜对有丝分裂纺锤体和核仁进行了三维重建。使用针对三个不同核仁区域和α-微管蛋白的单克隆抗体来对核仁及有丝分裂纺锤体进行成像。微管仅限于核仁，最早的早前期纺锤体微管开始出现。早期纺锤体微管被视为核仁表面的短杆。纺锤体微管的伸长导致核仁周围形成粗糙的微管笼。在中期，有丝分裂纺锤体完全嵌入核仁中形成宽带。随着纺锤体的伸长，核仁分裂成两个离散的体，由密集的微管带连接。在末期，有丝分裂纺锤体的远端仍然完全嵌入子核仁内。微管蛋白和核仁蛋白荧光的逐点比较显示，早前期 70%或更多的微管蛋白与核仁蛋白共定位。这些观察结果表明了一种模型，即微管的特定核仁结合位点允许有丝分裂纺锤体的形成和附着。核仁物质在染色体之前的大量存在表明，纺锤体的伸长驱动核仁的分裂。
