Condensin and Repo-Man–PP1 co-operate in the regulation of chromosome architecture during mitosisCondensation observed during mitosis and its reversible nature is one of the more important dilemmas faced by cell biologists. It has been determined in a number of different studies that the condensin complex serves an important role in securing successful chromosome segregation during the anaphase stage of mitosis. Cells lacking in condensin complex have been observed to lead to a delay in chromosome condensation.
Kinetochores are also affected when there is a lack in condensin. In such cells, the heterochromatin of the kinetochores appear to have reduced rigidity. This affects the behavior of the kinetochores.
In some condensin-depleted cells, the result is not only a delay in condensation but a complete failure in the mitotic process. Whenever a condensin-depleted cell hits the anaphase stage, segregation of chromosomes is almost always a failure. The relative lack of the condensin complex leads to formation of chromatin bridges in the cell.
The chromosome segregation failure observed in these cells is not thought to be attributable to problems in cohesin dynamics. The failure is attributed more to losses in DNA topoisomerase II (topo II) function especially since the aforementioned functions are affected in condensin-depleted cells. However upon closer study utilizing physiological sites, it was concluded that DNA topo II does not, in fact, require the condensing complex to continue normal activity. The contradiction between findings is brought about by the fact that previous studies utilized Drosophilia for their study while the newer findings were based on obseravations from vertebrate cells. This raises the problem, therefore, that the particular event affected in condensin-depleted cells still has not been identified.
In the study by Vagnarelli et al. (2006) the above mentioned problem was investigated. The possibility that another factor, Repo-Man-PP1 and not DNA topo II, was also involved in the failed mitosis of condensin-depleted cells was examined.
The aims, methodology, and findings of the Vagnarelli et al. (2006) study are presented in this paper in summary.Aims The main objective of the Vagnarelli et al. (2006) study was to identify the specific activity affected in condensing-depleted cells. The researchers aimed to pinpoint the particular critical event that became inactivated during the anaphase stage. Basically, the study hoped to find out what goes wrong in a condensin-depleted cell, when it goes wrong, and why.
The focus of the research was on activities possibly inactivated by Repo-Man-PP1 during anaphase.Methods Vagnarelli et al. (2006) examined the activities that took place during anaphase in the absence of condensin. Live-cell analysis was conducted by the group in order to best observe the said activities.
SMC2 conditional-knockout (SMC2 ON/OFF) chicken DT40 cells were utilized for the experiment. The cells were grown without doxycycline and were, therefore, SMC2ON at the beginning. Upon introduction of the doxycycline, the cells became SMC2OFF. Cells that have lost their SMC2 were also noted to have lost other condensin subunits. The researchers were thus able to examine the activity of condensin-depleted cells.
For better observation of the events occurring during anaphase in the condensin-depleted cell culture, the researchers opted to use cells expressing functional CENP-H–GFP. These were produced by targeting GFP into the CENP-H gene21. The onset of anaphase was thus more accurately determined by the labeled kinetochores. Chromosome behavior in the cell culture was observed via labeling of the chromatin with H2B-mRFP.
SMC2ON/OFF cells expressing H2B-RFP were produced by cotransfection of the H2B–RFP vector with a ?-actin-blasticidin resistant construct The researchers defined chromatin compaction “changes in the volume occupied by chromosomes” (Vagnarelli, 2006: 1133). This was quantified by measuring the chromatin per unit volume in chromosomes. Chromosome condensation, on the other hand, was defined as “formation of individual compact chromosomes during mitosis” (Vagnarelli, 2006:1133) and thus needed no quantification. Chromosome compaction, chromosome condensation, and chromosome architecture were all observed in the cell cultures. Analysis of the said variables allowed the researchers to reach certain conclusions about the behavior of condensin-depleted cells. Because of the findings of the initial observation, a second live-cell analysis was conducted. This time, the focus was on the second system believed to be responsible for the stabilization of condensed chromatin architecture.
The second system was designated as RCA. The second experiment required the exogenous expression of GFP-cyclin B3 in SMC2OFF cells. Expression of GFP-cyclin B3 prolonged the early stages of mitosis, during which RCA was most active. This allowed the researchers to observe the activities of RCA in mitosis. RCA was believed by the researchers to be positively regulated by CDK activity.
This means that RCA requires a high level of CDK activity in order to function. This was also tested by the researchers. The utilized cells were treated with a selective inhibitor called roscovitine. Roscovitine is able to inactivate the activity of Cdk1 when there are high levels of GFP-cyclin B3. In order to insure the inactivation of Cdk1 for this part of the experiment, Roscovitine was introduced to the cell culture for 10 and 20 minutes prior to fixation.Findings Observation of the chicken DT40 cells with SMC2 conditional-knockout subunit revealed that chromosomes that lack condensin lose compactness during anaphase and thus form chromatin bridges.
The anaphase stage of the said cells are atypical. The chromosomes were observed to lose compact architecture yet these still continued to move towards the poles. Only 2 to 3 minutes after the beginning of anaphase, chromatin bridges were observed in the majority of the cells. These findings were noted despite the fact that there was no early decondensation of chromatin. Although the chromosomes exhibited less chromatin compaction at the beginning of anaphase, recompaction occurred later on as the chromatins continued to move towards the poles. This resulted in the same relaxation-recompaction activity observed in SMCON cells although SMCOFF cells were less compact.
These observations led to the conclusion that condensin is most important in stabilizing the architecture of chromosomes during mitosis. It also led to the conclusion that there is a different system ensuring successful chromosome condensation. Condensin-depleted cells are not a product of a mere failure in decatenation of sister chromatids. Rather, it shows a complete loss of individual chromosome architecture in the anaphase stage.A second system must then be present to regulate chromosome condensation. This same system must also be responsible, therefore, for the architecture of condensed chromosome in the SMCOFF cells. Vagnarelli et al. (2006) labeled this second system as RCA.
Observation of the cell’s activities during RCA was made possible by the exogenous expression of GFP-cyclin B3. During this part of the experiment, the researchers observed the correction of previously defective anaphase stage chromosome architecture. No chromatin bridges were observed for these cells. Separated chromatids were also observed to have remained stably condensed at the spindle poles.Another important finding of the study is that the second system, RCA, is highly dependent on CDK activity. Without the protection of CDK, RCA cannot perform well enough to salvage the condensation at anaphase. When the cells were treated with rocovitine, Cdk1 was inactivated in the presence of GFP-cyclin B3.
The findings of this part of the experiment revealed an inability of the mitotic process to produce a stabilized chromosome architecture during anaphase. Prominent chromatin bridges were again observed in the cells.The fact that RCA is reliant on CDK activity may be attributable to the targeting of protein phosphatase PP1 to chromosomes. A subunit called Repo-Man is able to recruit a pool of PP1? to chromatid arms during the beginning of anaphase. Repo-Man is able to do this without condensing. This event thus occurs in SMC2OFF cells. The result is a mutation in Repo-Man that eventually leads to apoptotic cell death.It can be seen, therefore, that targeting of PP1 to anaphase chromatin can lead to a failure in condensation because of a loss of chromosome architecture.
It is only CDK activity that protects PP1 from being targeted by Repo-Man. Repo-Man is thus negatively regulated by CDK activity.In cells with condensin, chromatins are still observed to decondense when CDK activity is lowered.
Introduction of roscovitine in condensin-containing cells causes lowered CDK activity. Decondensation resulted from increased Repo-Man activity as it could no longer be regulated by the decreased CDK activity. This then stresses the importance of protecting PP1 from being targeted in order to achieve successful condensation during mitosis.Vagnarelli et al. (2006) were able to develop a working model for the regulation of mitotic chromosome architecture. This model accounted the regulatory activity of both condensing and RCA.
Conclusion SMC2OFF cells that are condensin-depleted only have the RCA system functional. It is only this system that can be used for regulation of chromatin architecture during condensation. However, when CDK levels are lowered during anaphase, Repo-Man binds to chromatin and promotes the dephosphorylation of the RCA system. This occurs through PP1.
The dephosphorylated RCA is the inactivated form. With the RCA system inactive, there is no longer a regulatory system available to maintain stability of the condensed chromosomes. Thus normal architecture is lost and chromatin bridges begin to form. The formation of chromatin bridges result in the failure of the anaphase stage of mitosis.
In conclusion, it can be said that RCA is able to stabilize the architecture of condensed chromosomes even when condensin is absent. This is, however, only possible Repo-Man is not able to target PP? to chromosomes. Thus CDK activity must be maintained in order to both negatively regulate Repo-Man and to positively regulate RCA.Condensation has been observed by many earlier studies. The failure of condensation in the absence of condensin has also been studied before.
Vagnarelli et al. (2006), however, are at the forefront of the field of cell biology in the proposition that there is a secondary system, RCA, which regulates the process. The observation and analysis of this second system has led to the conclusion that condensing may truly only be responsible in stabilizing condensed chromosomes while RCA holds the main role in the process.