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Title: Cell cycle-dependent changes of the pericentriolar material
Author: Fant, Xavier
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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The centrosome is the microtubule organising centre of animal cells and is composed of a pair of centrioles surrounded by an electron-dense mass of proteins, called the pericentriolar material (PCM). Several proteins of the PCM exhibit a dynamic association with the centrosome in a cell cycle-dependent manner. The centrosome duplicates during S-phase of the cell cycle and may play an essential role during the transition from G1 to S phase of the cell cycle. In order to better understand the mechanism of centrosome duplication and the role of the centrosome at the G1/S transition, we decided to investigate the overall changes occurring in the PCM between G1 and S phase by comparing the composition of the PCM at these two cell cycle stages. Therefore, we isolated centrosomes from unsynchronised (mainly in G1) Jurkat cells and from cells arrested in early S phase. Centrosomes were further fractionated by salt extraction with potassium iodide into soluble and insoluble material. Comparative gel electrophoresis of the soluble fractions in G1 and S phase allowed use to detect several differences in the protein composition. Using MALDI-tof mass spectrometry, we identified eleven different proteins specifically accumulating in either phase. Among those accumulating in S-phase, some were known proteins whereas others were novel uncharacterised proteins. Thus, our results led to the identification of new proteins potentially recruited to the centrosome in S phase where they may play a specific role in cell cycle-dependent centrosome functions. We started the study of HCA66, one of the novel proteins. We cloned its full-length cDNA by RT-PCR from Jurkat cells, expressed a recombinant portion of the protein in bacteria and raised a polyclonal antibody. A protein of 66kDa corresponding to the predicted molecular weight of HCA66 was enriched in the PCM from S phase centrosomes. Thus, we laid the basis for further investigation on the function of HCA66 at the centrosome. The protein 4.1R, previously characterised as a component of the plasma membrane skeleton, is also localised at the centrosome. We investigated the role of this protein at the centrosome and found that the carboxy terminus of 4.1R bears similarities to a short motif in β-tubulin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available