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Title: A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone

Abstract

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 notmore » 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. - Highlights: • A novel method to visualize antiparallel microtubule overlaps is developed. • Unstable microtubules are depolymerized during an air-drying method. • This method can detect the effect of compounds on antiparallel microtubule overlaps.« less

Authors:
; ; ; ;
Publication Date:
OSTI Identifier:
22738190
Resource Type:
Journal Article
Journal Name:
Experimental Cell Research
Additional Journal Information:
Journal Volume: 360; Journal Issue: 2; Other Information: Copyright (c) 2017 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0014-4827
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CELL PROLIFERATION; CHROMOSOMES; MICROTUBULES; MITOSIS

Citation Formats

Ifuji, Aya, Kuga, Takahisa, Kaibori, Yuichiro, Saito, Youhei, and Nakayama, Yuji. A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone. United States: N. p., 2017. Web. doi:10.1016/J.YEXCR.2017.09.025.
Ifuji, Aya, Kuga, Takahisa, Kaibori, Yuichiro, Saito, Youhei, & Nakayama, Yuji. A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone. United States. doi:10.1016/J.YEXCR.2017.09.025.
Ifuji, Aya, Kuga, Takahisa, Kaibori, Yuichiro, Saito, Youhei, and Nakayama, Yuji. Wed . "A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone". United States. doi:10.1016/J.YEXCR.2017.09.025.
@article{osti_22738190,
title = {A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone},
author = {Ifuji, Aya and Kuga, Takahisa and Kaibori, Yuichiro and Saito, Youhei and Nakayama, Yuji},
abstractNote = {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. - Highlights: • A novel method to visualize antiparallel microtubule overlaps is developed. • Unstable microtubules are depolymerized during an air-drying method. • This method can detect the effect of compounds on antiparallel microtubule overlaps.},
doi = {10.1016/J.YEXCR.2017.09.025},
journal = {Experimental Cell Research},
issn = {0014-4827},
number = 2,
volume = 360,
place = {United States},
year = {2017},
month = {11}
}