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キーワード:
Centrosome; Drosophila; Microtubule; MTOC; SEM; TEM; Whole-mount preparation
要旨:
Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 μm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50–300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of γ-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition.
