Structure and microtubule-nucleation activity of isolated Drosophila embryo centrosomes characterized by whole mount scanning and transmission electron microscopy |
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Authors: | B M H Lange G Kirfel I Gestmann V Herzog C González |
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Institution: | (1) Present address: Max-Planck Institute for Molecular Genetics, Vertebrate Genomics, Ihnestr. 73, 14195 Berlin, Germany;(2) Institute for Cell Biology, Ulrich-Haberland-Strasse 61a, 53121 Bonn, Germany;(3) Present address: ICREA and IRBB, Parc Científic de Barcelona, C/Josep Samitier 1-5, 08028 Barcelona, Spain |
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Abstract: | 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. |
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Keywords: | Centrosome Drosophila Microtubule MTOC SEM TEM Whole-mount preparation |
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