Why is metaphase necessary

These are prophase , metaphase , anaphase and telophase. Each of these phases is designated as I or II depending where it occurs, i. Metaphase II is the second stage in meiosis II. It follows prophase II , which primarily highlights the condensation of the chromosome s and the movement of centrosome s to polar regions of the cell. The cell is in metaphase II when the chromosomes align themselves along the metaphase plate through the facilitation of the spindle fibers.

The spindle fibers are now attached to the two kinetochores contained in the centromere of each chromosome. Similar to mitotic metaphase, the two kinetochores are bound to the spindle fibers rom opposite poles and they lie on the equatorial plane, readying for the chromosomal movement towards opposite poles in anaphase II.

See also:. Plants are characterized by having alternation of generations in their life cycles. Therefore, micronuclei formation is not necessarily a pathological condition that inevitably links mitotic errors to chromothripsis and tumorigenesis.

Nevertheless, it remains possible that, when combined with faulty surveillance mechanisms including p53 inactivation, a common feature in human cancers , mitotic errors leading to micronuclei formation promote genomic instability and rapid tumor evolution in mammals Fig. Further work will be necessary to explore these possibilities.

Chromosome alignment ensures anaphase synchrony to promote mitotic fidelity. Models illustrate the progression and fate for normal A and Kif18A-deficient B cells. Only a minor fraction of lagging chromosomes results in micronuclei, consistent with anaphase surveillance mechanisms e.

Cell proliferation in the presence of micronuclei is limited by p53, thereby ensuring genomic stability. Sign In or Create an Account. Advanced Search. User Tools. Sign In. Skip Nav Destination Article Navigation. Spotlight March 11 No chromosome left behind: The importance of metaphase alignment for mitotic fidelity Bernardo Orr , Bernardo Orr. This Site. Google Scholar. Helder Maiato Helder Maiato. Correspondence to Helder Maiato: maiato i3s. Author and Article Information.

Bernardo Orr. Online Issn: Horizon Framework Programme We thus postulate that a symmetric metaphase plate position is essential for symmetric cell divisions, explaining why it is conserved in all metazoans, plants, and many fungi. Control of this parameter is essential, since differences in cell size have been linked to cell fate Kiyomitsu and Cheeseman, Metaphase plate position may also play a crucial role in asymmetric cell divisions that depend on asymmetric spindles in anaphase, such as in embryonic D.

To form asymmetric spindles in a controlled and stereotypical manner, cells need an internal reference in space: breaking an existing symmetry, that is, a symmetric metaphase plate position, provides such a reference point. This is consistent with the progression of embryonic fly neuroblasts, which first align the metaphase plate in the middle of the spindle, before undergoing an asymmetric elongation of the spindle in anaphase.

Our results also shed light on the mechanisms controlling the position of the cytokinetic furrow. Original studies in sand dollar eggs showed that the position of the centrosomes is a key determinant of the cytokinetic furrow position Rappaport, ; later studies in C.

A role for chromosomes was, however, discarded in these two organisms, since midzone formation and cytokinesis did not require them. In contrast, in human cells, chromosomes stabilize microtubules of the midzone and thus favor the formation of a cytokinetic furrow Canman et al. Here, we show that cells only misplace the cytokinetic furrow in the presence of an asymmetric plate position in metaphase, implying that the position of the metaphase plate plays a crucial fine-tuning role in the positioning of the cytokinetic furrow.

Future studies will have to test whether the metaphase plate acts via the microtubules of the midzone, or as recently postulated, by influencing the cortical populations of Anillin and Myosin in anaphase in a Ran-GTP-dependent manner Kiyomitsu and Cheeseman, What might be these defects?

Kinetochores in cells bind a sufficient number of microtubules to form amphitelic attachments and stretch the two sister-kinetochores apart, but a number of kinetochores do not bind the full complement of stable microtubules required for SKAP loading. It is established that the SAC responds to detached kinetochores and is satisfied when kinetochores have bound the full set of microtubules.

Based on our results, we postulate that the SAC also responds if a kinetochore is only bound by a fraction of the full set of microtubules. This suggests a SAC that is more sensitive than a checkpoint that only senses detached kinetochores or kinetochores that become detached due to a tension defect. A SAC that detects such minor defects in kinetochore—microtubule occupancy caused by an imbalance of microtubule stability within the spindle would be able to indirectly probe for plate positioning, giving cells time to correct this imbalance and ensure a symmetric metaphase plate position.

Such graded response to microtubule occupancy within a kinetochore complements studies showing that the SAC acts in a graded manner when it comes to the number of unattached kinetochores Collin et al. Mad2 and Kid1 depletion had been previously validated in our laboratory Meraldi et al. For the cold-stable assay, cells were incubated in cold medium whilst placed on ice for 7 min. Cross-adsorbed secondary antibodies were used Invitrogen. Three-dimensional image stacks of mitotic cells were acquired in 0.

Images were mounted as figures using Adobe Illustrator. To monitor the polar ejection force, the distance between centrosomes and kinetochores was measured as described Wandke et al. To monitor cell contours, cells were illuminated with white light and recorded by phase-contrast microscopy.

Time-lapse videos were visualized in Softworx to quantify mitotic timing and to detect rotating spindles. The tracking assay was also used to quantify the length of the two half-spindles: the tracking assay estimates the metaphase plate by fitting a plane to the calculated kinetochore positions; metaphase plate position relative to the spindle poles was calculated using a custom MATLAB function that detects centrioles and calculates plate position as the intersection of the fitted plane with the spindle axis.

The earliest time point data of each cell imaged was used for plate position and inter-kinetochore distance analysis to ensure that data come from early metaphase cells. To measure plate position at anaphase and to better visualize the centering mechanisms, we used a temporal resolution of 30 s and applied our combined kinetochore and centrosome tracking analysis.

Videos were manually screened for the presence of chromosome segregation errors. To determine spindle positions within cells, we used the centrosome positions to determine the center of the spindle equidistant to both centrosomes and compared it to the cell center, which was determined using phase contrast images point on the spindle axis that is equidistant to both cell cortexes. Three-dimensional image stacks of fixed cells were subjected to the kinetochore tracking assay for sister—kinetochore pair identification.

The pulse width was 8 ns and the pulse energy used was 1. A more detailed description of the laser-microsurgery unit can be found in Pereira et al. Statistical analyses were performed in R 2. Unpaired t-tests with Welch's correction and Mann—Whitney U tests against cells were carried out to check for the statistical significance of normal and non-normal distributed data, respectively. Count data were analyzed using the Fisher's Exact test.

Graphs were plotted in R using the ggplot2 package and mounted in Adobe Illustrator. We thank J Pines Univ. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Competing interests The authors declare that no competing interests exist.

PM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent see review process.

Similarly, the author response typically shows only responses to the major concerns raised by the reviewers. Your article has been favorably evaluated by Tony Hunter Senior editor , a Reviewing editor, and three reviewers.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. There is a general consensus that this paper is interesting and worthy of publication in eLife but a number of concerns must first be addressed.

In particular, the authors should:. The authors should also show an R vs. The authors should also show data to clarify that SKAP loading is specifically reduced on the 1-centriole side, instead of sometimes being reduced on the 1-centriole side, and sometimes on the 2-centriole side. In addition to these concerns the authors should comment on possible differences between laser ablation and Sas-6 depletion, and more clearly explain how they compute asymmetry.

The authors should also make it explicit that the delay in mitosis observed in cells whose metaphase plate is off-centre is a consequence of an imbalance in microtubule forces resulting from kinetochore-microtubule occupancy that is monitored by the SAC. We knew from previous studies that this temporal resolution was necessary to catch record and analyze chromosome movements in metaphase Jaqaman et al. Cell Biol. To avoid photo-toxicity we were however forced to work with low intensities and to limit our movies to 5 minutes.

Our representation of R is therefore built on a population analysis of cells either early in metaphase or just before anaphase. This population analysis shows that just before anaphase onset, cells have on average a metaphase plate that is located more precisely in the middle of the spindle. These experiments directly visualize and validate the existence of a centering mechanism, providing a clearer representation to the reader.

This suggests that the difference in microtubule stability is an important factor in the asymmetry of the plate, but certainly not the only one. Second, to further support our hypothesis of a differential minus-end stability that causes the asymmetric plate positioning in cells, we measured with a cold-stable assay minus-end stability in cells after a 1 hour MG treatment.

Since MG treatment leads to a symmetric position of the metaphase plate, our hypothesis predicted an attenuated difference in minus end stability between the 1- and 2-centriole pole.

This is exactly what we found, as now shown in the novel Figure 3J. As shown in the novel Figure 3N , taxol treatment largely corrects the metaphase plate position, validating our hypothesis that the asymmetric plate position is caused by differences in microtubule stability.

Our interpretation of SKAP as a marker for microtubule stability is not our claim, but a conclusion of Schmidt et al. To fully validate this hypothesis we now in addition briefly treated cells with 10 nM taxol and stained for SKAP.

With regard to the second question, we find that SKAP is not systemically absent on the 1-centriole-side or the 2-centriole-side, but that the frequency of the absence is the same for both sides. We do not think that this invalidates our model, as the SKAP read-out just indicates a reduced stability and higher dynamics of kinetochore-microtubules on both sides of the sister-kinetochores, consistent with the fact that these kinetochores are over time moving and correcting their position towards the middle of the spindle.

To provide a better overview of our laser ablation experiment, we now show the individual traces of R over time before and after ablation for 11 cells, in which a single centriole was ablated, and we plot the median of this cell population novel Figure 4H.

As a negative control we also plot the median R over time for control-ablated cells laser pulse in the cytoplasm. Figure 4H shows how R increases after destruction of a single centriole, leading to an asymmetric position of the metaphase plate, consistent with our Sas-6 depletion experiments.

Moreover, we now provide a novel Supplementary movie 4, visualizing how the ablation of a single centriole creates an asymmetric position of the metaphase plate. Since such laser-ablation experiments are technically very challenging, and not easy to combine with a second assay, we used mainly Sas-6 depletion to investigate how the removal of a single centriole affects spindle length, showing how it leads to an imbalance of microtubule dynamics see also point 2 for the additional experiments.

Importantly, when Mps1 was inhibited in late metaphase cells, when the plate has reached a symmetric plate position, we only observed symmetric cell division. Since these cells are, apart from the plate position, identical to the cells that divide asymmetrically, we concluded that the plate position is a key determinant for the a symmetry of cell division.

To fully confirm this claim, we have now performed a second back-up experiment, in which we created cells by laser-ablation to create an asymmetric plate position , before forcing cells into anaphase by abrogating the spindle assembly checkpoint with an Mps1 inhibitor. We found that 4 out of 6 laser-ablated cells underwent an asymmetric cell division ratio between daughter cell size of more than 1.

In contrast 6 out of 6 control-ablated cells had a ratio smaller than 1. Despite the low numbers — laser-ablation experiments are technically very challenging — these experiments validate our initial claim.

While our initial manuscript was based mainly on functional validations e. Moreover, the reviewers were worried about potential siRNA off-target effects, reason for which they asked for siRNA rescue experiments. Since such rescue experiments are technically extremely challenging when analyzing double or triple siRNAs and very difficult to analyze in a conclusive manner, we have instead performed a number of complementary experiments for each siRNA or provide additional explanations.

However, this is not possible, since such an off-target effect would also be present in cells that have been subjected to the same siRNA. Moreover, we confirm the effects of Sas-6 depletion on spindle positioning and asymmetric cell division using laser-ablation, as an alternative tool to create cells e.

Instead of performing a rescue experiment, which is very difficult since mitotic timing is sensitive to minor changes in Mad2 levels, we repeated the Sas-6 depletion in the presence of an Mps1 inhibitor to inhibit the spindle assembly checkpoint in an independent manner. These experiments validated our Mad2 depletion experiments Figure 2—figure supplement 2.

Kid siRNA: this siRNA was used as a positive control for a disruption of the polar ejection force, based on previous literature. It has been previously validated in our and other laboratories Wandke et al. In contrast, we find that Sas-6 depletion has no effect on the polar ejection force. Therefore, we cannot test for an off-target effect. The novel aspect of our manuscript comes from co-depletion of Sas-6 siRNA in cells.

While we appreciate that a siRNA rescue experiment would be informative, we believe that a triple siRNA rescue experiment is nearly impossible, particularly since the overexpression of either of these microtubule depolymerases is toxic. Moreover, we also validated in the same Figure the new siRNA treatment by immunoblotting. Since this is not the case, we conclude that KIF2a co-depletion is essential to overcome the mitotic delay.

Our claim that stabilization of kinetochore microtubules in cells overcomes the spindle assembly checkpoint delay and allows anaphase entry with an asymmetric plate position is confirmed by the Aurora-B inhibition experiment;. We envisage two potential reasons for the stronger phenotype seen in cells obtained through laser ablation as compared to Sas-6 depletion.

First, since Sas-6 depleted cells lose a centriole in interphase, they might be better able to partially compensate for this loss as they gradually build up the mitotic spindle. Second, it is likely that the laser pulse on a mitotic spindle pole not only impairs the centriole, but that it also disrupts biochemical activities associated with the mitotic spindle pole, such as microtubule-depolymerases, which are able to exert a pulling force on k-fibers Meunier et al. Cell Biol, Finally, we now more explicitly link the occupancy of kinetochores with the activation of the spindle assembly checkpoint in cells see Discussion, fourth paragraph.

National Center for Biotechnology Information , U. Journal List eLife v. Published online Jul Author information Article notes Copyright and License information Disclaimer. Received Oct 11; Accepted Jul This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.

This article has been cited by other articles in PMC. Abstract Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell divisions. Research organism: human. Introduction During mitosis, chromosomes are bound to microtubules emanating from both poles of the mitotic spindle via sister-kinetochores and aligned on the metaphase plate precisely in the middle of the spindle.

Results Cells center the metaphase plate position before anaphase onset To monitor the relative position of the metaphase plate in the spindle over time, we recorded by time-lapse imaging HeLa cells stably expressing eGFP-centrin1 centriole marker and eGFP-CENPA kinetochore marker and automatically tracked centrosomes and the metaphase plate using an in-house developed software Jaqaman et al.