Live analysis of free centrosomes in normal and aphidicolin-treated Drosophila embryos.

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Abstract
In a number of embryonic systems, centrosomes that have lost their association with the nuclear envelope and spindle maintain their ability to duplicate and induce astral microtubules. To identify additional activities of free centrosomes, we monitored astral microtubule dynamics by injecting living syncytial Drosophila embryos with fluorescently labeled tubulin. Our recordings follow multiple rounds of free centrosome duplication and separation during the cortical division. The rate and distance of free sister centrosome separation corresponds well with the initial phase of associated centrosome separation. However, the later phase of separation observed for centrosomes associated with a spindle (anaphase B) does not occur. Free centrosome separation regularly occurs on a plane parallel to the plasma membrane. While previous work demonstrated that centrosomes influence cytoskeletal dynamics, this observation suggests that the cortical cytoskeleton regulates the orientation of centrosome separation. Although free centrosomes do not form spindles, they display relatively normal cell cycle-dependent modulations of their astral microtubules. In addition, free centrosome duplication, separation, and modulation of microtubule dynamics often occur in synchrony with neighboring associated centrosomes. These observations suggest that free centrosomes respond normally to local nuclear division signals. Disruption of the cortical nuclear divisions with aphidicolin supports this conclusion; large numbers of abnormal nuclei recede into the interior while their centrosomes remain on the cortex. Following individual free centrosomes through multiple focal planes for 45 min after the injection of aphidicolin reveals that they do not undergo normal modulation of their astral dynamics nor do they undergo multiple rounds of duplication and separation. We conclude that in the absence of normally dividing cortical nuclei many centrosome activities are disrupted and centrosome duplication is extensively delayed. This indicates the presence of a feedback mechanism that creates a dependency relationship between the cortical nuclear cycles and the centrosome cycles.
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