Three-dimensional structural characterization of centrosomes from early drosophila embryos

Abstract. An understanding of the mechanism and structure of microtubule (MT)-nucleating sites within the pericentriolar material (PCM) of the centrosome has been elusive. This is partly due to the difficulty in obtaining large quantities of centrosomes for analysis, as well as to the problem of attaining interpretable structural data with conventional EM techniques. We describe a protocol for isolating a large quantity of functional centrosomes from early Drosophila em-bryos. Using automated electron tomography, we have begun a three-dimensional structural characterization of these intact centrosomes with and without regrown MTs. Reconstructions of the centrosomes to ~6-8 nm resolution revealed no large structures at the minus