A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone
A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone
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一种新的免疫荧光方法，用于可视化后期和末期中区微管加端反平行重叠中的微管
DOI：
10.1016/j.yexcr.2017.09.025
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发表时间：
2017
影响因子：
3.7
通讯作者：
Nakayama Yuji
中科院分区：
文献类型：
--
作者：
Ifuji Aya;Kuga Takahisa;Kaibori Yuichiro;Saito Youhei;Nakayama Yuji
细胞分裂是细胞增殖过程中最具活力的事件，其中重复的染色体被分成两个子细胞。染色体运动主要由微管提供动力，微管在形态上不同，并根据有丝分裂进程组织成特征结构。在有丝分裂后期，反平行微管形成纺锤体中间区，中间区的不规则形成往往导致胞质分裂的失败，从而引起染色体的不均匀分离。然而，这是很难分析这些微管的形态，因为微管的反平行重叠的微管加在中间区的结束是嵌入在高电子密度的矩阵，阻碍了访问的抗微管蛋白抗体在免疫荧光染色的表位。在这里，我们开发了一种新的方法来可视化选择性反平行微管重叠的中间区。当细胞在固定前风干时，观察到对齐的α-微管蛋白染色，并与PRC 1共定位于后期和末期细胞的中间区的中心，表明可以通过该方法观察到反平行微管重叠。在空气干燥的细胞中，mCherry-α-微管蛋白荧光和β-微管蛋白染色显示与中间区的α-微管蛋白染色几乎相同的模式，表明空气干燥的细胞中反向平行微管重叠的选择性可视化不是由于α-微管蛋白抗原性的改变。紫杉醇处理延长了空气干燥细胞中中间区的微管丝，而诺考达唑处理相反地减少了微管的数量，这表明在空气干燥方法中不稳定的微管被解聚。值得注意的是，空气干燥方法能够分别检测Aurora B和Plk 1抑制后中间区的破坏和过早的中间区形成。这些结果表明，空气干燥的方法是适合于可视化微管的反平行重叠的微管加端的中间区，并检测其对中间区形成的影响。
Cell division, in which duplicated chromosomes are separated into two daughter cells, is the most dynamic event during cell proliferation. Chromosome movement is powered mainly by microtubules, which vary in morphology and are organized into characteristic structures according to mitotic progression. During the later stages of mitosis, antiparallel microtubules form the spindle midzone, and the irregular formation of the midzone often leads to failure of cytokinesis, giving rise to the unequal segregation of chromosomes. However, it is difficult to analyze the morphology of these microtubules because microtubules in the antiparallel overlaps of microtubule-plus ends in the midzone are embedded in highly electron-dense matrices, impeding the access of anti-tubulin antibodies to their epitopes during immunofluorescence staining. Here, we developed a novel method to visualize selectively antiparallel microtubule overlaps in the midzone. When cells are air-dried before fixation, aligned α-tubulin staining is observed and colocalized with PRC1 in the center of the midzone of anaphase and telophase cells, suggesting that antiparallel microtubule overlaps can be visualized by this method. In air-dried cells, mCherry-α-tubulin fluorescence and β-tubulin staining show almost the same pattern as α-tubulin staining in the midzone, suggesting that the selective visualization of antiparallel microtubule overlaps in air-dried cells is not attributed to an alteration of the antigenicity of α-tubulin. Taxol treatment extends the microtubule filaments of the midzone in air-dried cells, and nocodazole treatment conversely decreases the number of microtubules, suggesting that unstable microtubules are depolymerized during the air-drying method. It is of note that the air-drying method enables the detection of the disruption of the midzone and premature midzone formation upon Aurora B and Plk1 inhibition, respectively. These results suggest that the air-drying method is suitable for visualizing microtubules in the antiparallel overlaps of microtubule-plus ends of the midzone and for detecting their effects on midzone formation.