Summary of key activities carried out by the Programme

  1. We have previously shown that centrosomal Aurora A kinase interacts with TPX2 in a RanGTP dependent manner after nuclear envelope breakdown resulting in kinase activation. Using the Xenopus egg extract system we found last year that Aurora A works though different mechanisms to promote MT assembly both at the centrosome and the acentrosomal pathway (Sardon et al, 2008). In collaboration with Prof Giannis (Leipzig, Germany) we identified one compound that shows specificity for Aurora A inhibition in cells suggesting that it could be useful for basic and applied research (Sardon et al, 2009).

  2. We continued studying the kinase Nek9 in centrosome maturation and spindle assembly using tissue culture cells and Xenopus egg extracts.

  3. Centrosome separation is essential for the establishment of spindle bipolarity. The kinesin Eg5 has a predominant and essential role in centrosome separation and bipolar spindle establishment. However, we found that Eg5 inhibition does not compromise the stability of the bipolar spindle at metaphase and that another kinesin, Hklp2 becomes essential under these conditions for spindle stability. Hklp2 localizes to the spindle microtubules and the chromosomes and promotes the switch from the monopolar to the bipolar configuration, stabilizing spindle bipolarity in metaphase. Our data provided an additional understanding of the mechanism driving the initial establishment and subsequent maintenance of the bipolar spindle at metaphase, and reveals the existence of a novel specific mechanism that stabilizes bipolar spindles and may be essential to prevent mitotic defects (Vanneste et al, 2009).

  4. We are trying to understand how TACC3 interacts with XMAP215 and Aurora A at the centrosome.

Functional facility

  1. Purification of centrosomes:
    The protocol for the purification of centrosomes from Drosophila embryos was improved by testing various approaches:

    1. Immunopurification of centrosomes with polystyrene Sphero proteinG beads for direct checking by EM.

    2. Elution of centrosomes from beads with gamma-tubulin peptide.

    3. These two approaches were unsuccessful.

    4. Pooling the peak fractions of several gradients and using long and thin tubes for another gradient. This approach resulted in the obtention of highly concentrated preps at 108 centrosomes/ml. Negative stain revealed some structures similar to centrosomes observed in previous reports. Some regular shaped contaminants were also visible. As DNA was found in the preps, a treatment with DNAse was performed and indeed found to improve the purity of the centrosome preps.

    5. To test whether these contaminants could come from the parasite Wolbachia that is commonly found in Drosophila populations, the fly colony of the CRG was checked. Indeed the flies are infected by this parasite. A test treatment to erase it form the flies was performed and found to efficiently eliminate the parasite.

  2. Preparation of specific antibodies for selected centrosomal proteins
    To obtain specific antibodies against 40 centrosomal proteins (human and Xenopus) synthetic peptides (selected using a program developed by the group of Luis Serrano) were used for immunogenization of rabbits. The titer of the resulting sera was determined by ELISA. Some of them were affinity purified and characterized by Western blot and immunofluorescence analysis. A database with all the results has been setup.

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