Dhonukshe Pankaj, Vischer Norbert, Gadella Theodorus W J
Section of Molecular Cytology and Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
J Cell Sci. 2006 Aug 1;119(Pt 15):3193-205. doi: 10.1242/jcs.03048.
The spindle occupies a central position in cell division as it builds up the chromosome-separating machine. Here we analysed the dynamics of spindle formation in acentrosomal plant cells by visualizing microtubules labelled with GFP-EB1, GFP-MAP4 and GFP-alpha-tubulin and chromosomes marked by the vital dye SYTO82. During prophase, few microtubules penetrate the nuclear area, followed by nuclear envelope disintegration. During prometaphase, microtubules invading the nuclear space develop a spindle axis from few bipolar microtubule bundles, which is followed by spindle assembly. Using a novel quantitative kymograph analysis based on Fourier transformation, we measured the microtubule growth trajectories of the entire dynamic metaphase spindle. Microtubules initiating from spindle poles either pass through the metaphase plate to form interpolar microtubule bundles or grow until they reach chromosomes. We also noticed a minor fraction of microtubules growing away from the chromosomes. Microtubules grow at 10 microm/minute both at the spindle equator and at the spindle poles. Photobleached marks created on metaphase and anaphase spindles revealed a poleward tubulin flux. During anaphase, the velocity of tubulin flux (2 microm/minute) equals the speed of chromatid-separation. With these findings we identified spatially coordinated microtubule growth dynamics and microtubule flux-based chromosome-separation as important facets of plant spindle operation.
纺锤体在细胞分裂中占据中心位置，因为它构建了染色体分离机制。在这里，我们通过观察用绿色荧光蛋白-EB1、绿色荧光蛋白-MAP4和绿色荧光蛋白-α微管蛋白标记的微管以及用活性染料SYTO82标记的染色体，分析了无中心体植物细胞中纺锤体形成的动态过程。在前期，很少有微管穿透核区域，随后核膜解体。在前中期，侵入核空间的微管从少数双极微管束形成纺锤体轴，随后进行纺锤体组装。使用基于傅里叶变换的新型定量波形图分析，我们测量了整个动态中期纺锤体的微管生长轨迹。从纺锤体极发出的微管要么穿过中期板形成极间微管束，要么生长直到到达染色体。我们还注意到一小部分微管远离染色体生长。微管在纺锤体赤道和纺锤体极的生长速度均为每分钟10微米。在中期和后期纺锤体上产生的光漂白标记显示了微管蛋白向极的流动。在后期，微管蛋白流动的速度（每分钟2微米）等于染色单体分离的速度。通过这些发现，我们确定了空间协调的微管生长动态和基于微管蛋白流动的染色体分离是植物纺锤体运作的重要方面。
