Summary of key activities carried out by the Programme

During this year the group has been working on the characterization of two centrosome protein/complexes:

  1. The Par complex (Drosophila melanogaster). Previous experiments showed that the expression and purification of the individual members of the complex resulted in insoluble protein. Therefore, we decided to try a co-expression approach in Escherichia coli in collaboration with Dr. Montoya. Different constructs were built for the members of the complex, although we focused our attention on the complex core: aPKC-Par6-Bazooka. We were able to obtain soluble fractions of the complex containing the three members and perform a preliminary analysis of the sample by negative staining. Currently, we have a preliminary 3D model; to improve it we are optimizing the purification protocol and the purification steps. We are also considering of isolating the complex from a natural source as Drosophila embryos. Transgenic flies were obtained by the group of A. Wodarz (University of Göttingen) with a GFP tagged Bazooka gene. Preliminary analysis suggests that we could isolate the whole complex using a GFP trap approach. However we are experiencing many problems in terms of scaling up the purification and introducing new steps.

  2. Miranda protein (Drosophila melanogaster). We were working with samples provided by Dr. Coll. The sample resulted very heterogeneous for an EM analysis and different techniques used in terms of sample stabilization didn’t work. So far, we are not thinking in continuing with this line.

  3. Recently, we established collaboration with Dr. Sharples (Albert Einstein College of Medicine, NY) for the structural characterization of Cep192, a well-known centrosomal protein. First attempts in the cloning procedure resulted not to work very well and other strategies are being considered at the moment.

  4. Concerning the tomographic part significant advances have been made on the two projects:

  5. The thymus preparation has been optimized. During Veronique Chevrier's visit and with her regular help, the purification protocol was set-up at the CNB. Enriched solutions of isolated thymocyte centrosome have been produced, firstly from young calf, then from young to almost new born lambs and kids. The quality of the preparations was checked by immunofluorescence, negative stain after centrifugation (Chretien et al. 1997 protocol) and inclusion of sedimented centrosomes followed by resin cutting. The sample purity was reproducibly assessed compatible with cryo-tomography. After comparing centrosomes from the different animal species showing comparable coplanar geometry and reduced amount of PCM, lambs were chosen as the more convenient source.
    During different attempts at different ages the thymocyte preservation was checked by inclusion EM. Optimization of the cryo preparation for tomography was with the help of Montserrat Barcena from the Koster’s lab. We will try to increase the number of centrosomes per grid, if necessary also by an improvement of the centrosome solution concentration.

  6. Regarding centrosome from drosophila embryo, we tried to improve the purity of preparations. Due to an important DNA contamination, Maria Sanz from the Dr. Vernos’s functional facility tried a drastic increase of the DNAse treatment. As suggested by Pr. Jordan Raff and Monica Bettencourt-Diaz, we investigated the hypothesis of the relation between the presence of a well structured contaminant (studied by negative stain, as previously reported) and a bacterial contamination in the drosophila cultures used by the purification facility.

  7. We obtained significant decrease of the contaminant presence after a tetracycline treatment that must be continued until elimination of the parasite. Combining the two approaches it is expected to obtain rather pure preparations in a reasonable amount of time. (??? Ask Jose Mari).

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