3-DIMENSIONAL STRUCTURAL CHARACTERIZATION OF CENTROSOMES FROM EARLY DROSOPHILA EMBRYOS

An understanding of the mechanism and structure of microtubule (MT)-nu cleating sites within the pericentriolar material (PCM) of the centros ome has been elusive. This is partly due to the difficulty in obtainin g large quantities of centrosomes for analysis, as well as to the prob lem of attaining interpretable structural data with conventional EM te chniques, We describe a protocol for isolating a large quantity of fun ctional centrosomes from early Drosophila embryos. Using automated ele ctron tomography, we have begun a three-dimensional structural charact erization of these intact centrosomes with and without regrown MTs. Re constructions of the centrosomes to similar to 6-8 nm resolution revea led no large structures at the minus ends of MTs, suggesting that if M T-nucleating material physically contacts the MTs, it must conform clo sely to the shape of the minus end. While many MTs originate near the centrioles, MT minus ends were found throughout the PCM, and even clos e to its outer boundary. The MTs criss-crossed the PCM, suggesting tha t nucleating sites are oriented in many different directions. Reconstr uctions of centrosomes without MTs suggest that there is a reorganizat ion of the PCM upon MT regrowth; moreover, ring-like structures that h ave a similar diameter as MTs are apparent in the PCM of centrosomes w ithout MTs, and may be MT-nucleating sites.