This confirmed that loss of a single daughter centriole can directly affect force generation at sister-kinetochores, consistent with our findings that Sasdepleted cells show defects in the quality of kinetochore—microtubule attachments. We also conclude that an acute laser-ablation of a daughter centriole in metaphase has a more severe effect on the forces acting on kinetochore than Sas-6 depletion.
Possible explanations for this difference could be that Sasdepleted cells might have more time to adapt to the lack of a missing centriole as they progressively build up the spindle, or that the laser-ablation destroys not just a centriole but also part of enzymatic activities in the vicinity of the centriole, such as minus-end depolymerases, which in normal cells are known to exert a pulling force on kinetochore-fibers Meunier and Vernos, Note the asymmetric metaphase plate position after the ablation of a single centriole.
To confirm the presence of partially destabilized kinetochore—microtubule attachments in siSas-6 -treated cells and test whether they are the cause of the SAC response, we used the Aurora-B inhibitor ZM1. Aurora-B inhibition does not overcome a SAC-dependent mitotic arrest caused by unattached kinetochores nocodazole treatment , but overcomes a mitotic arrest caused by insufficient tension at sister-kinetochores in monopolar spindles monastrol treatment , as Aurora-B inhibition stabilizes kinetochore—microtubules and prevents loss of kinetochore—microtubule attachment Figure 5A ; Ditchfield et al.
We therefore tested whether the depletion of MCAK alone in cells is sufficient to suppress the mitotic delay. As this was not the case, we conclude that depletion of both MT-depolymerases is necessary to overcome the instability of kinetochore—microtubules in cells Figure 5—figure supplement 1C.
Data from Figure 1E without Aurora-B inhibition are shown for comparison. This suggested that an asymmetric metaphase plate position leads to asymmetric cell division. A recent study, however, demonstrated that asymmetric position of the entire spindle can also lead to asymmetric cell divisions in HeLa cells Kiyomitsu and Cheeseman, To discriminate between the two possibilities, we quantified the position of the spindle center in relationship to the cell center at anaphase onset, which was often not centered in the middle of the cell at anaphase onset, as has been previously reported Figure 6D ; Collins et al.
We conclude that entry into anaphase with an asymmetric position of the metaphase plate results in an asymmetric cell division.
Phase contrast images were used to quantify the ratio of the two daughter cell sizes, which was plotted as a histogram in C. See also Videos 5, 6. Shown is the ratio of the two daughter cell sizes. See also Videos 7, 8. Note how the cell divides in a symmetric manner. Note how the cell divides in an asymmetric manner. Here, we show that an equatorial position of the metaphase plate in the middle of the spindle is necessary for symmetric cell divisions and demonstrate that cells actively center the metaphase plate before anaphase onset.
Metaphase plate centering requires the SAC, which provides cells with enough time to correct metaphase plate position. The SAC responds to subtle defects in kinetochore—microtubule stability that arise in cells with an asymmetric plate position and an imbalance of centrioles, implying that the SAC is more sensitive than previously assumed. Recent studies have shown that proper positioning of the spindle ensures symmetric cell divisions, and that, deviations from a symmetric position are corrected by dynein-dependent cortical forces and membrane elongation during anaphase Kiyomitsu and Cheeseman, Here, we find that this external cortical correction mechanism in anaphase is complemented in metaphase by an internal centering mechanism that ensures a symmetric position of the metaphase plate within the spindle with the help of the SAC.
This centering mechanism is particularly visible in cells with an asymmetric distribution of centrioles cells , but it also acts in wild-type cells, indicating that it is active in every cell division. 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. 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. Metaphase is a stage in the cell cycle where all the genetic material is condensing into chromosomes. These chromosomes then become visible.
During this stage, the nucleus disappears and the chromosomes appear in the cytoplasm of the cell. During this stage in human cells, the chromosomes then become visible under the microscope. As metaphase continues, the cells partition into the two daughter cells.
The kinetochore microtubules exert tension on the chromosomes, which move back and forth in rapid erratic motion as a result, and the entire spindle-chromosome complex is now ready for the next event, separation of the daughter chromatids.
Metaphase, one of the most critical stages in mitosis, occupies a substantial portion of the division cycle. The primary reason for this extended interval is that dividing cells pause until all of their chromosomes are completely aligned at the metaphase plate. This sets the stage for chromosome separation in the next stage of mitosis, termed anaphase.
Anaphase - Almost immediately after the metaphase chromosomes are aligned at the metaphase plate, the two halves of each chromosome are pulled apart by the spindle apparatus and migrate to the opposite spindle poles Figure 1 d. The kinetochore microtubules shorten as the chromosomes are pulled toward the poles, while the polar microtubules elongate to assist in the separation.
Anaphase typically is a rapid process that lasts only a few minutes. When the chromosomes have completely migrated to the spindle poles, the kinetochore microtubules begin to disappear, although the polar microtubules continue to elongate. This is the junction between late anaphase and early telophase , the last stage in chromosome division. In photomicrographs of the process, polar microtubules are in a clearly formed network and the synthesis of a new cell membrane is initiated in the cytoplasm between the two spindle poles.
Telophase - In telophase, the daughter chromosomes arrive at the spindle poles and are eventually redistributed into chromatin. The process of cytokinesis , where the cytoplasm is divided by cleavage, also starts sometime in late anaphase and continues through telophase see Figure 1 e. After complete separation of the chromosomes and their extrusion to the spindle poles, the nuclear membrane begins to reform around each group of chromosomes at the opposite ends of the cell.
The nucleoli also reappear in what will eventually become the two new cell nuclei. When telophase is complete and the new cell membrane or cell wall in the case of higher plants is being formed, the nuclei have almost matured to the pre-mitotic state. The final steps in telophase involve the initiation of plasma membrane cleavage between each of the new daughter cells to ultimately yield two separate cells during cytokinesis, the next phase of cell division.
Cytokinesis - The final stage in the process of cell division is known as cytokinesis, which usually begins during late anaphase or early telophase before mitosis ends as the nuclear envelope and nucleoli are reforming and the chromosomes are de-condensing see Figure 1 f.
During cytokinesis, the cytoplasm divides by a process termed cleavage , driven by the tightening of a contractile ring composed of actin and myosin protein subunits. As the ring of cytoskeletal proteins contracts, a cleavage furrow is formed perpendicular to the mitotic spindle and gradually splits the cytoplasm and its contents into two daughter cells. John D. Griffin , Nathan S. Claxton , and Michael W.
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