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

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.

Mars, aDrosophilaprotein related to vertebrate HURP, is required for the attachment of centrosomes to the mitotic spindle during syncytial nuclear divisions

“…The free centrosomes retained their capacity to nucleate microtubules and continued to duplicate and separate, resulting in numerous microtubule asters detached from nuclei. Similar observations have been reported for other situations that result in the formation of free centrosomes (Raff and Glover, 1988;Gonzalez et al, 1990;Yasuda et al, 1991;Debec et al, 1996). Most likely as a secondary consequence of the centrosome detachment, different types of mitotic defects accumulated in mars mutant embryos, including monopolar spindles with circular condensed chromosomes, multipolar spindles and short anastral spindles that were probably organized by the nucleation of microtubules around chromosomes.…”

Onset of the DNA Replication Checkpoint in the Early Drosophila Embryo

“…A gradual attainment of full checkpoint function is also supported by the fact that aph. injections before cycle 11 can stall/ delay the nuclear cycle, but not the centrosomal cycle (Raff and Glover 1988;Debec et al 1996;Wakefield et al 2000;B. Edgar, personal communication;and our unpublished observations).…”

“…Emphasis was placed upon fixed material, however, because many embryos are processed under the same conditions as well as at different time points and with different genotypes. Furthermore, present data can be compared with previously published data using two cycB embryos (Raff and Glover 1988;Debec et al 1996;Yu et al 2000).…”

“…at 60 min AED (cycle 6) and incubated the embryos for 30 min. Failure of checkpoint regulation was scored by chromosomal abnormalities, such as bridges and large and small nuclei, as well as abnormally condensed nuclei, all of which indicate that M-phase was induced before DNA replication was completed (Raff and Glover 1988;Debec et al 1996;Yu et al 2000). Normal interphase or prophase nuclear morphology scored 30 min after injection indicated a delay of M-phase, implicating some checkpoint effect.…”

Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest inDrosophila melanogaster

“…In the Drosophila syncytial embryo, global perturbations such as blocking DNA replication, inducing extensive DNA damaged, or blocking spindle assembly are known to arrest or delay mitosis throughout the syncytium (Callaini et al, 1994;Debec et al, 1996;Fogarty et al, 1997;Raff and Glover, 1988). Interestingly, embryonic expression of a non-degradable form of cyclin B or increase maternal cyclin B contribution can affect the global behaviour of nuclear cycles (Ji et al, 2002;Su et al, 1998).…”