Correspondence to: Alexey Khodjakov, Wadsworth Center, P.O. Box 509, Albany, NY 12201-0509. Tel:(518) 486-5339 Fax:(518) 486-4901 E-mail:khodj{at}wadsworth.org.
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Abstract |
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When centrosomes are destroyed during prophase by laser microsurgery, vertebrate somatic cells form bipolar acentrosomal mitotic spindles (Khodjakov, A., R.W. Cole, B.R. Oakley, and C.L. Rieder. 2000. Curr. Biol. 10:5967), but the fate of these cells is unknown. Here, we show that, although these cells lack the radial arrays of astral microtubules normally associated with each spindle pole, they undergo a normal anaphase and usually produce two acentrosomal daughter cells. Relative to controls, however, these cells exhibit a significantly higher (3050%) failure rate in cytokinesis. This failure correlates with the inability of the spindle to properly reposition itself as the cell changes shape. Also, we destroyed just one centrosome during metaphase and followed the fate of the resultant acentrosomal and centrosomal daughter cells. Within 72 h, 100% of the centrosome-containing cells had either entered DNA synthesis or divided. By contrast, during this period, none of the acentrosomal cells had entered S phase. These data reveal that the primary role of the centrosome in somatic cells is not to form the spindle but instead to ensure cytokinesis and subsequent cell cycle progression.
Key Words: centrosome, cell cycle progression, cytokinesis, mitosis, vertebrates
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Introduction |
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Despite over a century of study, the function(s) of the centrosome remain vague, and recent papers on this topic have succeeded more in defining what this organelle does not do than revealing its true functions (for reviews see
The fact that there are no viable vertebrate somatic cells lacking centrosomes reveals that this organelle is normally essential. As a result, the only way to determine how the absence of a centrosome affects cell behavior is to remove it at defined points in the cell cycle. In an early effort,
We have developed a method to selectively destroy the centrosome. The basis for this approach was originally defined by Berns (for review see
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Materials and Methods |
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Cell Culture
Culture conditions and isolation of CVG-2 and PtKG-23 have been reported previously (
Laser Microsurgery
Centrosome ablation by laser microsurgery has been described previously (0.5 µm in the specimen plane (
-tubulin/GFP fluorescence is completely abolished. This typically takes
10 s and requires two to three series of 2030 laser pulses.
Images were captured by a MicroMax 5 Hz cooled charge-coupled device camera (Princeton Instruments) and saved as 8-bit TIFF files. The imaging system is driven by Image Pro software (Media Cybernetic).
Long-Term Imaging
After laser microsurgery, the position of the experimental cell was marked on the coverslip, and the culture was transferred to a phasecontrast microscope equipped with a Rose chamber heater (80 h using a video-rate charge-coupled device camera (model 100; Paultek Imaging), and the media was changed every 24 h by perfusion. Illumination was obtained from a 100W Tungsten filament, filtered to remove UV (GG400) and infrared (KG5) components, made monochromatic (GIF 546), and shuttered (UniBlitz Electronics) between exposures (1 s/image).
Immunofluorescence Microscopy
Immunofluorescence staining and imaging were conducted as previously described (-tubulin (clone YL1/2; gift of Dr. J.V. Kilmartin, Medical Research Council, Cambridge, UK) at 1:100, and anti
-tubulin (number T6557; Sigma-Aldrich) at 1:300. BrdU labeling and visualization was conducted using a BrdU staining kit (number 93-3943; Zymed) according to manufacturer's instructions.
Some images (see Fig 1 and Fig 2) were collected as a Z-series (200-nm steps) and deconvolved using Delta Vision 2.1 deconvolution software (Applied Precision), and they are presented as maximal intensity projections.
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Online Supplemental Material
Time-lapse movies (Supplemental Videos 14) of the all cells presented in the published figures are available at http://www.jcb.org/cgi/content/full/153/1/237/DC1. Additionally, an example of a CVG cell (not shown as a printed figure), which formed multiple asynchronous cleavage furrows, is available as a movie.
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Results and Discussion |
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Astral Microtubules Rapidly Disappear upon Centrosome Destruction
The only noticeable difference between spindles formed in the presence or absence of centrosomes is that the latter lack the radial arrays of astral Mts normally associated with mitotic centrosomes (-tubulin/GFP confirmed this conclusion and revealed that the disappearance of astral Mts actually occurs within 23 min (not shown).
Destroying both Centrosomes during Metaphase Does Not Prevent Anaphase or Formation of the Cleavage Furrow
To determine if acentrosomal spindles produce acentrosomal daughter cells, we destroyed both centrosomes during prometaphase or metaphase in 10 PtKG-23 and 10 CVG-2 cells, and then followed them by time-lapse microscopy. We found that the outcome was always the same regardless of when the centrosomes were destroyed: after all chromosomes had achieved an equatorial alignment, the bipolar acentrosomal and anastral spindle subsequently entered anaphase, during which time, the chromatids moved towards the ends of the spindle.
In most cases (7/10 PtKGs and 5/10 CVGs), ablating both centrosomes during metaphase did not affect the later stages of mitosis. The cell exhibited normal chromatid separation and cytokinesis, and eventually severed the midbody to form two acentrosomal daughter cells (Fig 2). At the light microscopy level, the morphology of these cells (Fig 2 I) was the same as surrounding centrosome-containing cells, and they also appeared to contain normal numbers of Mts (Fig 2 J). The only feature distinguishing acentrosomal cells from controls was that they lacked the sharp Mt focus normally associated with the centrosome (Fig 2 J, arrows).
In the remaining cases (3/10 PtKGs and 5/10 CVGs), cytokinesis failed, and a binucleated cell was produced lacking a centrosome. This failure rate was significantly higher than in nonirradiated control cells in which cytokinesis fails <5% of the time. In controls, the mitotic spindle is positioned near the geometric center of the cell with the spindle axis parallel to the long axis of the cell. As the shape of the cell changes during mitosis, the spindle rotates to maintain its proper orientation (
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Cytokinesis in the acentrosomal cells could also fail due to abnormalities in the formation and/or propagation of the furrow. Unilateral furrows were commonly formed only on one side of the spindle (Fig 3, FH, arrowhead) that wandered through the cytoplasm, could impact chromosomes, and could turn, but that ultimately relaxed (Fig 3). In some cells, such furrows formed well removed from the spindle equator, and in others multiple bilateral furrows were formed, all of which ultimately relaxed (time-lapse videos available at http://www.jcb.org/cgi/content/full/153/1/237/DC1).
The fact that most of our experimental cells exited mitosis and formed two daughters reveals that centrosomes, per se, are not required for progression through mitosis, as might be expected, for example, from the observation that cyclin B degradation starts in the spindle poles (
Until recently, the putative primary function of centrosomes was to define the poles of the mitotic spindle and to orchestrate its formation. However, it is now clear that bipolar spindles can be formed via acentrosomal pathway(s), even in cell types that normally possess centrosomes (for reviews see
Our data also reveal that acentrosomal spindles are no longer able to reposition themselves in response to ensuing changes in cell shape, and this is correlated with defects in the formation and/or propagation of cleavage furrows. Overall, these types of abnormalities support the hypothesis that astral Mts orient the spindle (
Acentrosomal Cells Arrest during G1
The fact that our laser microsurgery approach can be used to reproducibly generate cells that begin a new cycle in the absence of a centrosome allowed us to address the question of whether centrosomes are required for cell cycle progression. For these experiments, we ablated only one of the two centrosomes during metaphase when the centrosomes were maximally separated from the chromosomes. This approach minimized possible collateral damage to the DNA, which is known to impede cell cycle progression (
We used CVG cells for these experiments because, as typical fibroblasts, they grow individually. PtKG, on the other hand, are typical epithelia in which progression of an individual cell through the cycle depends on many variables including, for example, whether it is located within or at the periphery of a cell sheeta fact that complicates individual cell analyses. To determine if the progeny of our operation underwent DNA synthesis during the observation period, we perfused them with media containing BrdU immediately after the operation, and the media was replenished with fresh medium containing BrdU every 24 h. All cultures were then fixed 72 h after operation.
To determine if something produced within the cell by the laser operation inhibits progression through the cell cycle, we irradiated four CVG metaphase spindles next to one of the centrosomes. In all cases, all of the resultant eight daughter cells replicated their DNA during our 72-h observation period, and two divided.
We completely destroyed one of the two centrosomes in 25 metaphase CVG cells, 16 of which subsequently underwent cytokinesis to produce two daughter cells. Of the 16 centrosome-containing control cells generated during this study, 3 underwent the next mitosis during the 72-h observation period (not shown). The other 13 centrosome-containing controls remained in interphase during this time, but all had incorporated BrdU and duplicated their centrosomes by the time of fixation (Fig 4). By contrast, during this same period, 3 of the acentrosomal cells rounded and died, after extensive blebbing (not shown) characteristic of apoptosis (
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Except for the 3 cells that died, the remaining 13 acentrosomal cells could not be distinguished morphologically from surrounding centrosome-containing controls: each exhibited a similar behavior (polarization, locomotion, etc.) and distribution of mitochondria and Golgi apparatus (not shown). However, at the end of 72 h, acentrosomal cells contained no discrete -tubulincontaining structures, and their Mt arrays lacked a sharp focus, i.e., there was no evidence of centrosome reformation (Fig 4 M). In a separate set of experiments, we analyzed 10 PtKG and 10 CVG cells by serial section electron microscopy, 2448 h after ablating the centrosome, and never found any evidence of centriole/centrosome regeneration (not shown).
These data reveal that acentrosomal daughter cells, produced during mitosis from cells containing an acentrosomal spindle pole, invariably arrest in interphase. This general conclusion is consistent with that of 26 h. Under these conditions, those acentrosomal cells that incorporated BrdU (11/14) could have been near the G1/S transition, or even in S, when the centrosome was removed. By contrast, in our experiments, the centrosomes were destroyed during mitosis, before initiating the next cell cyclein essence, before the cell was born. The fact that none of our acentrosomal cells incorporated BrdU clearly reveals that the centrosome is required during early G1 for cell cycle progression, such as, passage through START. However, at some point after this time, it may be dispensable for cell cycle progression.
All of our centrosome-containing control cells either incorporated BrdU or divided during our 72-h observational period. The reason why more of these cells did not enter another mitosis is unknown, but it likely means that our filming conditions are not optimal. Cell cycle progression in most mammalian cells, and in particular the G2/M transition, is light sensitive and easily inhibited or delayed by the illumination used during microscopy (
Morphologically our acentrosomal cells can only be distinguished from their centrosomal sisters by their lack of a sharp cytoplasmic Mt focal point. This raises the issue of whether the cell cycle arrest we see in acentrosomal cells is due to this abnormal organization of Mts or to the lack of the centrosome as an organelle. Current evidence favors the later idea: when CHO cells synchronized in mitosis are replated in the presence of nocodazole, they appear to progress through the cell cycle with normal kinetics (
Finally, we never observed centrosome regeneration. In contrast, when antibodies to polyglutamylated tubulin are loaded into cells, the centrosome as a structural and functional entity disappears, but as the antibody concentration drops, the centrosome reassembles (
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: GFP, green fluorescent protein; Mt, microtubule.
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Acknowledgements |
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We thank Mr. R. Cole for assistance with laser microsurgery and Drs. M. Koonce and G. Sluder for stimulating discussions and critical comments on the manuscript. We also acknowledge use of the Wadsworth Center's Video Light Microscopy core facilities.
This work was supported by National Institutes of Health grants 59363 (A. Khodjakov) and 40198 (C.L. Rieder).
Note Added in Proof. The observation that acentrosomal cells become arrested during G1 also has been reported recently by Hinchcliffe et al. (Hinchcliffe, E.D., F.J. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:15471550).
Submitted: 27 November 2000
Revised: 30 January 2001
Accepted: 31 January 2001
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References |
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