> Stages de M2 > Liste des stages proposés pour l’année 2013-2014 > Acentrosomal spindle pole shaping in oocytes

Acentrosomal spindle pole shaping in oocytes

proposé par Marie-Hélène Verlhac, Collège de France, Paris

Projet de stage : In most animal cells, bipolar mitotic spindle formation relies on centrosomes acting as major microtubule organizing centers. At mitosis onset, duplicated centrosomes rapidly promote spindle bipolarization (Toso 2009), defining spindle poles as well as the spindle axis along which chromosome attachment and segregation will take place (for review Tanenbaum & Medema 2011). Chromosome segregation in female meiosis I is unusual in that meiotic spindle poles do not have centrioles, are not anchored to the cortex via astral microtubules, therefore lack canonical centrosomes. Such an atypical organization raises the important question of how force transmission, essential for correct chromosome segregation, is achieved inside the female meiotic spindle. Oocytes excepted, most healthy cells possess one or two canonical centrosomes, depending on their progression in the cell cycle (centrosome duplication takes place in S-phase). However, many solid tumours harbour an excess of centrosomes, which correlates with an increased rate of aneuploidy. In drosophila, the presence of extra-centrosomes induces tumorigenesis (Basto 2008). Yet the presence of extra-centrosomes challenges tumour development : multipolar mitosis induces high levels of aneuploidy putting cell viability at risk (Ganem 2009). How can cancer cells achieve to maintain a balance between extra-centrosome number and a certain degree of aneuploidy, yet keep dividing ? It appears that cancer cells cluster their extra-centrosomes prior to division (Quintyne 2005). We have shown that proper spindle assembly in oocytes also requires the sorting and coalescence of multiple acentriolar MTOCs (Breuer 2010), a process reminiscent of extra-centrosomes clustering. We would like to study this process in greater details in the oocyte. We will notably test the role of a minus-end directed motor Kinesin-14 (HSET). This kinesin has been involved in the process of extra-centrosome clustering (Kwon 2008). Hurp knock-out, as well as HSET knock-down by siRNA, do not affect mitosis. Therefore both proteins are good therapeutic targets to specifically slow down cancer cell, but not somatic cell, division (cancer cells that do not cluster their extra-centrosomes will eventually die). A previous study, using anti-HSET antibody injection, has suggested that this kinesin is only involved in meiosis II spindle assembly (Mountain 1999). More specific tools are now available to re-investigate HSET function in meiosis (siRNA, Rescue, Gain of function..). This study will be done in collaboration with Claire Walczak lab (Indiana University, Bloomington), which provided all the tools to address HSET function in meiosis. We will also assess HSET localisation and function in Hurp knock-out mice as well as in oocytes mutant for NuMA (Kolano 2012). This way we can test potential interactions between these two MAPs and the HSET motor in meiotic spindle assembly and MTOCs sorting to the poles.

Techniques mises en œuvre par le stagiaire : oocyte collection and culture, Immunocytochemistry, Immunotransfer, molecular biology, in vivo imaging using spinning disk confocal microscopy.

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