Regulation of cell cycle by the anaphase spindle midzone
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A number of proteins accumulate in the spindle midzone and midbody of dividing animal cells. Besides proteins essential for cytokinesis, there are also components essential for interphase functions, suggesting that the spindle midzone and/or midbody may play a role in regulating the following cell cycle.
We microsurgically severed NRK epithelial cells during anaphase or telophase, such that the spindle midzone/midbody was associated with only one of the daughter cells. Time-lapse recording of cells severed during early anaphase indicated that the cell with midzone underwent cytokinesis-like cortical contractions and progressed normally through the interphase, whereas the cell without midzone showed no cortical contraction and an arrest or substantial delay in the progression of interphase. Similar microsurgery during telophase showed a normal progression of interphase for both daughter cells with or without the midbody. Microsurgery of anaphase cells treated with cytochalasin D or nocodazole indicated that interphase progression was independent of cortical ingression but dependent on microtubules.
We conclude that the mitotic spindle is involved in not only the separation of chromosomes but also the regulation of cell cycle. The process may involve activation of components in the spindle midzone that are required for the cell cycle, and/or degradation of components that are required for cytokinesis but may interfere with the cell cycle.
KeywordsDaughter Cell Nocodazole NRK52E Cell Sister Cell Microtubule Bundle
Microtubules undergo striking reorganization during anaphase and telophase. During anaphase, antiparallel, interdigitating microtubules and many associated proteins become organized into discrete bundles in the spindle midzone , the region between separated chromosomes. As the cell enters cytokinesis, these midzone microtubule bundles merge into a single compact, electron-dense structure called midbody. It is generally recognized that, at least for cultured animal cells, midzone microtubules play a major role in cytokinesis. For example, cleavage furrows, both normal and ectopic, are associated with similar microtubule bundles [2, 3, 4], while regions physically blocked from midzone microtubules by micromanipulation are unable to undergo cytokinesis . Moreover, continuous interactions of midzone microtubules with the cell cortex are required for sustaining the cytokinesis of cultured animal cells .
Recent progress suggested that, in addition to cytokinesis, the spindle midzone might be involved in additional functions. For example, midzone and midbody microtubules are associated with many regulatory proteins apparently unrelated to cortical contraction, such as the DNA replication initiator Orc6 , the inhibitor of apoptosis survivin , and the tyrosine kinase binder Nir2 . In addition, mother centrioles were found to migrate to the midbody during telophase before returning to their interphase position, possibly activating some centrosomal components for cell cycle progression . Furthermore, treatment of dividing cells with dihydrocytochalasin B, an inhibitor of cytokinesis, caused not only the inhibition of cytokinesis but also G1 arrest after mitosis , raising the possibility that ploidy, cortical contraction, and/or activation/deactivation of proteins during cytokinesis, may play a role in the regulation of the following cell cycle.
To address this possibility, we severed cells at anaphase or telophase by microsurgery, to bypass the normal mechanism of cytokinesis. The functional role of the spindle midzone or midbody in the following cell cycle was then tested by manipulating the position of the microsurgery or by applying pharmacological agents. Using extended time-lapse microscopy, we found that anaphase midzone microtubules play an important role in the progression of the subsequent interphase. However, there was no evidence of the involvement of telophase midbody in cell cycle progression.
Results and discussion
Duration of interphase for daughter cells with and without the spindle midzone or midbody#
spindle midzone (8)
721 ± 66
1444 ± 236**
spindle midzone (5)*
671 ± 28
1724 ± 301**
610 ± 92
611 ± 16
Duration of interpahse for cells severed in the presence of cytoskeletal inhibitors#
599 ± 49 (5)
587 ± 24 (10)
892 ± 71 (7)
621 ± 30 (14)
Some of major mitotic regulators are degraded by anaphase promoting complex/cyclosome (APC) during anaphase . Degradation of the polo-like kinase (Plk1) and aurora A by APC occurred while they were localized along midzone microtubules , raising the possibility that interphase progression may require the degradation of some mitotic/cytokinetic proteins, which may then cause activation of downstream components crucial for cell cycle. In addition, some molecules associated with midzone microtubules may be directly involved in cell cycle events such as DNA synthesis, as suggested by the chromosomal passenger protein-like dynamics of a DNA replication initiating factor, Orc6 during cell division .
Our results suggest that anaphase midzone not only play a role in the stimulation of cytokinesis in cultured cells, but also provide a scaffold for the activation/deactivation of factors essential for the progression of subsequent cell cycle.
Cell culture, microscopy, and image processing
Normal Rat Kidney epithelial cells (NRK-52E; American Type Culture Collection, Rockville, MD) were cultured in Kaighn's modified F12 (F12K) medium supplemented with 10% FBS (JRH Bioscience, Lenexa, KS), 50 U/ml penicillin, and 50 μg/ml streptomycin, on glass chamber dishes as previously described . The cells were maintained at 37°C in a stage incubator built on top of a Zeiss Axiovert S100TV or an Axiovert 35 inverted microscope (Carl Zeiss, Thornwood, NY), and viewed with 10X, NA 0.25 Achrostigmat, 40X, NA 0.75 Plan-Neofluor or 100X, NA 1.30 Plan-Neofluor lens. All images were acquired with a cooled charge-coupled device camera (ST133 controller and CCD57 chip; Roper Scientific, Trenton, NJ) and processed with custom software for background subtraction.
Microsurgery and drug treatment
Glass needles for microsurgery were prepared with a David-Kopf Model 700 vertical puller. The tip of the needle was melted and elongated into a fine fiber with a Narishige microforge (Model MF900). Microsurgery of the cells was achieved by carefully lowering a fiber onto the target cell followed by slow dragging with a micromanipulator (Leica, Deerfield, IL).
Cytochalasin D (Sigma, St. Louis, MO) were stored at -20°C as 2.5 mM stock in DMSO, and diluted into warm medium before application to cells. We found that treatment of early anaphase cells with cytochalasin D for 30 min completely inhibited cytokinesis even upon removal of the drug, similar to what was reported with dihydrocytochalasin B . Thus, early anaphase cells were treated with cytochalasin D at a final concentration of 2 μM for 10 min before microsurgery. The cells were then incubated for additional 20 min and washed at least twice with fresh medium. Nocodazole (Sigma, St. Louis, MO) was stored at -20°C as 10 mM stocks in DMSO and was diluted with warmed medium before use. Early anaphase cells were incubated with nocodazole for 6–13 min and then cut into two daughter cells with microsurgery. Immediately after microsurgery or following 50 min incubation, the daughter cells were released from nocodazole by incubating with two changes of fresh medium for at least 5 min each. The significance of these results was assessed using analysis of variance (ANOVA) and t-test in Microsoft Excel.
Transfection and immunofluorescence
Aurora B-GFP was constructed and transfected into NRK cells as described previously . Immunofluorescence of tubulin and reconstruction of microtubules images were carried out as described previously . For immunofluorescence, cells were rinsed with warm cytoskeleton buffer and fixed with 4% paraformaldehyde (EM Science, Gibbstown, NJ) in warm cytoskeleton buffer for 10 min . They were then rinsed thoroughly in the cytoskeleton buffer and permeablized by incubation with 0.5% Triton X-100 in cytoskeleton buffer for 5 min. Fixed cells were rinsed with the cytoskeleton buffer, blocked for 10 min with 1% BSA (Boehringer Mannheim, Indianapolis, IN) in PBS, then incubated with anti-AIM-1 monoclonal antibodies (BD Biosciences, San Jose, CA) at a dilution of 1:200 in PBS with 1% BSA for 45 min at 37°C. After washing with PBS/BSA thoroughly, cells were incubated with Alexa 546-conjugated goat anti-mouse antibodies (Molecular Probes, Eugene, OR) at a dilution of 1:100 for 30 min at 37°C.
This study was supported by NIH grant GM-32476 to YLW, GM-30758 to GS, and intramural funds from the Temasek Life Sciences Laboratory to MMH.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.