RPDOC - Retour Post-Doctorants

Adaptive Response of the Cell for Coordination of Chromosome Segregation with Cell Division – ARC2-ChromSCeD


Adaptive Response of the Cell for Coordination of Chromosome Segregation with Cell Division

Cell cycle regulation to prevent aneuploidy

Mitosis is the last step of the cell cycle during which errors in chromosome transmission are irreversible. Failure to properly transmit the genetic material produces daughter cells with an inappropriate genome content also called aneuploid cells. Aneuploidy can have deleterious consequences for the cell and the organism. In fact, aneuploidy is a hallmark of cancerous cells. Recent studies have shown that aneuploidy can promote tumorigenesis but can also antagonize the development of tumors depending of the cellular type and the genetic context. Therefore it is crucial to decipher all the mechanisms that the cell employs to prevent aneuploidy while going through multiple rounds of division. <br />One fundamental aspect of mitosis that is largely uncovered is how chromosome segregation is coordinated with cleavage furrow ingression. This coordination is essential for proper transmission of the genetic material into daughter cells. During anaphase, sister chromatids are pulled by the microtubules toward opposite poles via their kinetochore, which, in most organisms, is located on one small region of the chromosome. Therefore, during the pulling process, the kinetochore moves first and the chromatid arms lag behind. Meanwhile the cytokinetic apparatus is set up at the equator of the cell to prepare for cleavage. How do cells sense that chromatids have cleared the midzone (also called cleavage site) before completion of cytokinesis? Failure to timely coordinate chromatid segregation with cell division can lead to a situation where a chromatid arm still at the midzone is trapped and cut by the contractile ring during cytokinesis completion. This will be detrimental for the organism as it produces aneuploid daughter cells. <br />

To probe the mechanism by which chromosome segregation is coordinated with cleavage furrow ingression, previous studies have monitored the effect of abnormally long chromosomes on cell division. The rational for using long chromosomes is that this assay does not impair spindle morphology and, thus, should not affect the rate of sister chromatid segregation. However, abnormally long sister chromatids will take a longer time to clear the midzone, hence, exacerbating the mechanism that coordinates chromosome separation with cell division if it exists.
In Drosophila, the ability to segregate correctly long chromatids varies with cell type: errors in segregation occur frequently during the syncytial embryonic divisions, while larval neuroblasts divide correctly via an unknown mechanism.
We have identified a novel mechanism by which drosophila neuroblasts coordinate chromosome segregation with cell division. Cells adapt to a dramatic increase in chromatid length by transiently elongating during anaphase/telophase. This increase in cell length is concomitant with the spreading of cortical myosin rings without compromising cytokinesis. This response is mediated by the Rho Guanine-nucleotide exchange factor, Pebble (Pbl). The crosstalk between chromatid and cortical myosin ensures the clearance of the chromatid arms from the midzone before completion of cytokinesis. The challenge of my proposal is to elucidate the mechanism by which the trailing chromatid arm triggers Pbl activation, myosin ring formation and, thus, cell elongation. To do so, I will address the following questions:
- Which cytoskeletal proteins are involved in this mechanism?
- Is Aurora-B an actor of this pathway?
- Do microtubules play a role in this mechanism?
- What is the role of chromatin in this pathway?
- Is this pathway specific to neural stem cell asymmetric division?
- Can I identify novel regulators of this pathway?

Concerning the role of cytoskeletal protein, I recently observed that other proteins such as anillin and septin 2 are also recruited along with myosin on the extra rings. Furthermore, looking more closely at myosin dynamics, I was able to detect a enrichment of myosin in patches at the polar cortex of both control cells or cells carrying hyper long chromosomes after anaphase onset. However, these patches disappear after 2 to 3 minutes in control cells whereas myosin continues to accumulate in the presence of lagging chromosomes, and this period of enrichment is concomittant with the period of deformation of the cell. This result suggests that there is a signal triggering the depolymerisation of cortical myosin that is delayed in presence of lagging chromosomes. I am thus investigating what is the nature of this mitotic exit signal that can be delayed.
To decipher whether AuroraB is involved in this adaptive elongation, I am creating some tools carrying a mutation in Subito gene. This gene encodes for a protein required for the translocation of AuroraB to the centralspindle in anaphase. When cells start to elongate, AuroraB localises on the centralspindle, so this experiment will allow us to know whether AuroraB at the centralspindle is required for the elongation process.
Concerning the role of chromatin in this pathway, I recently checked whether the Ran GTPase gradient that form around the chromosomes is necessary to trigger the cell elongation. Preliminary results suggest that this gradient is necessary since removing one of its activator, a protein called RCC1, most of cells carrying long chromosomes do not elongate anymore.These results suggest that, like Pebble, Ran is a key player in this signalling pathway.
To determine whether this response is specific to neural stem cells, I recently looked at other Drosophila cell types (Sensory Organ Precursors) and observed also that they can elongate showing that this process is not specific to neural stem cells.

I am checking whether these other cytoskeletal proteins, Anillin and Septin, which localise like myosin on the extra rings, are also required for the shape changes of the cell in presence of long chromosomes. Furthermore, to identify the signal trigerring the depolymerisation of myosin, that is delayed in presence of long chromosomes, I will compare the rate of chromosome decondensation and also the timing of nuclear envelope reassembly of those cells versus control cells.
To check whether AuroraB is involved in this process, flies carrying the Subito gene mutation will soon be available to carry the experiments on their neuroblasts in presence of hyper long chromosomes.
As for the signal emanating from chromatin, I need to confirm the results and I would like to check whether RCC1 mutant adults display morphological defects similar to those observed in Pbl mutant adults. If so, this would mean that apopotosis probably occurs during the development of the organism. To prove that, I will check whether I can detect apoptotic cells at the larval stage in presence of long chromosomes in tissues showing morphological defects at the adult stage such as wing discs.
Concerning the specificity of this adaptive elongation, the Sensory Organ Precursors, like the neuroblasts, divide asymmetrically. Therefore, I will follow the division of other cell types such as cells from the wing discs in presence of long chromosomes. These cells divide symmetrically and this should let us know whether the adaptive elongation process is specific to asymmetric division.

We published part of our results in the Journal of Cell Biology in November 2012 (Vol.199, N°5) just before the ANR grant started and I am sharing the first authorship of this paper, the last author being Anne Royou who welcomes me in her laboratory and who initiated the project while she was at Santa Cruz University in California as a post-doc fellow.
Since the beginning of my ANR RPDoc, I went to two meetings: one in Montpellier (France, 3rd Cell Cycle and Cancer Meeting, 2nd to 5th of April 2013) and the other one in Breukelen (The Netherlands, Chromosome Segregation and Aneuploidy EMBO workshop, 22nd to 26th of June 2013) to present a poster with my data. My results were well received by scientists in the field and I won the poster prize in Montpellier allowing me to subsidise my registration for the meeting in Breukelen. Moreover, I have been recently selected to give an oral communication at Totnes meeting (UK, EMBO meeting on Drosophila Cell Division Cycle Workshop, 12th to 16th of September 2013).

Cell division is a fundamental process that sustains life. By this process, unicellular organisms create an entire new organism, and for multicellular organisms, it is a requirement for development, growth and tissue repair. Cell division is the process by which a mother-cell gives rise to two daughter cells. This process is an ordered set of events, which comprises two major phases called interphase (comprising G1, S and G2 phases), a growth phase during which DNA is replicated simulteanously to centrosomes duplication, and mitosis, itself a five-stage process (prophase, prometaphase, metaphase, anaphase and telophase) during which nuclear division occurs subsequently followed by cytokinesis, the physical separation of the daughter cells.

The progression through this cell cycle must be tightly regulated in order to ensure that the two daughter cells are genetically identical. To do so, cells have developed checkpoints to monitor the progression through the cell cycle. To date, four main checkpoints have been described in the literature occurring at the G1/S, G2/M, Metaphase/Anaphase transitions and during cytokinesis. Progress throught the cell cycle can be halted at these checkpoints if conditions for successful cell division are not met. These mechanisms ensure that each daughter cell inherits the correct number of chromosomes.

We recently unraveled a novel mechanism that coordinates chromosome segregation with cell cleavage during asymmetric division of Drosophila neuroblasts. Cells adapt to a dramatic increase in chromatid length by transiently elongating during anaphase/telophase. This increase in cell length is concomitant with the spreading of cortical myosin rings without compromising cytokinesis. This response is mediated by the Rho Guanine-nucleotide exchange factor, Pebble. This study reveals a novel chromatin to cortical myosin signalling pathway that ensures that all chromatid arms have cleared the cleavage plane prior to the completion of cytokinesis. My research project aims at deciphering this molecular pathway that prevents the formation of aneuploidy daughter cells. On one hand, I propose to explore the mechanisms by which cytoskeletal proteins mediate the shape changes in anaphase/telophase. On the other hand, the signalling pathway emanating from chromatin at the origin of this elongation of the cell will be deciphered. By means of screens, I should identify the different actors implicated in the communication between chromatin and cortical myosin. Finally, studying the behaviour of different cell types will improve my knowledge on the specificity and conservation of this mechanism preventing aneuploidy.

Aneuploidy, an abnormality in gene copy number, is the most prevalent genomic alteration identified in human solid tumors and can contribute to the acquisition of a genetic instability state, a hallmark of cancerous cells, confirming Boveri’s theory, back in 1914, that tumors may become malignant as the result of abnormal chromosome numbers. Our understanding of a novel mechanism that prevent aneuploidy may be relevant for the improvement of existing cancer therapies and the development of new cancer therapy.

Project coordinator

Madame Emilie MONTEMBAULT (Institut Européen de Chimie et Biologie, Bordeaux (Emilie Montembault)) – e.montembault@iecb.u-bordeaux.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


CNRS (UMR5095) Institut Européen de Chimie et Biologie, Bordeaux (Emilie Montembault)

Help of the ANR 240,958 euros
Beginning and duration of the scientific project: January 2013 - 36 Months

Useful links

Explorez notre base de projets financés



ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter