LIVE ANALYSIS OF FREE CENTROSOMES IN NORMAL AND APHIDICOLIN-TREATED DROSOPHILA EMBRYOS

In a number of embryonic systems, centrosomes that have lost their ass ociation with the nuclear envelope and spindle maintain their ability to duplicate and induce astral microtubules, To identify additional ac tivities of free centrosomes, we monitored astral microtubule dynamics by injecting living syncytial Drosophila embryos with fluorescently l abeled tubulin, Our recordings follow multiple rounds of free centroso me duplication and separation during the cortical divisions, The rate and distance of free sister centrosome separation corresponds well wit h the initial phase of associated centrosome separation, However, the later phase of separation observed for centrosomes associated with a s pindle (anaphase B) does not occur. Free centrosome separation regular ly occurs on a plane parallel to the plasma membrane, While previous w ork demonstrated that centrosomes influence cytoskeletal dynamics, thi s observation suggests that the cortical cytoskeleton regulates the or ientation of centrosome separation, Although free centrosomes do not f orm spindles, they display relatively normal cell cycle-dependent modu lations of their astral microtubules. In addition, free centrosome dup lication, separation, and modulation of microtubule dynamics often occ ur in synchrony with neighboring associated centrosomes, These observa tions suggest that free centrosomes respond normally to local nuclear division signals. Disruption of the cortical nuclear divisions with ap hidicolin supports this conclusion; large numbers of abnormal nuclei r ecede into the interior while their centrosomes remain on the cortex, Following individual free centrosomes through multiple focal planes fo r 45 min after the injection of aphidicolin reveals that they do not u ndergo 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 activiti es are disrupted and centrosome duplication is extensively delayed. Th is indicates the presence of a feedback mechanism that creates a depen dency relationship between the cortical nuclear cycles and the centros ome cycles.