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

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.

Use of dextran beads for live analysis of the nuclear division and nuclear envelope breakdown/reformation cycles in the Drosophila embryo.

We employ fluorescently labeled dextran beads to follow cycles of nuclear division and nuclear envelope breakdown in living Drosophila embryos. When injected into syncytial Drosophila embryos, 3000 mol wt fluorescently labeled dextran beads concentrate in the interphase nucleus. Through confocal microscopy, undisrupted multiple cycles of nuclear division are readily followed. In contrast, 40,000 mol wt fluorescently labeled dextran beads concentrate in the cytoplasm and enter the nucleus only after nuclear envelope breakdown during prophase. Once the nuclear envelope reforms during telophase, these large dextran beads are again excluded from the nuclei. The complementary behavior of the large and small dextran beads makes them applicable to a broad range of cellular studies. We employ the 40,000 mol wt fluorescein-labeled beads, in combination with rhodamine-labeled histones, to demonstrate that nuclear envelope breakdown occurs about 3.5-4.0 minutes prior to the initiation of anaphase.

The Drosophila maternal-effect mutation grapes causes a metaphase arrest at nuclear cycle 13.

grapes (grp) is a second chromosome (36A-B) maternal-effect lethal mutation in Drosophila melanogaster. We demonstrate that the syncytial nuclear divisions of grp-derived embryos are normal through metaphase of nuclear cycle 12. However, as the embryos progress into telophase of cycle 12, the microtubule structures rapidly deteriorate and midbodies never form. Immediately following the failure of midbody formation, sister telophase products collide and form large tetraploid nuclei. These observations suggest that the function of the midbody in the syncytial embryo is to maintain separation of sister nuclei during telophase of the cortical divisions. After an abbreviated nuclear cycle 13 interphase, these polyploid nuclei progress through prophase and arrest in metaphase. The spindles associated with the arrested nuclei are stable for hours even though the microtubules are rapidly turning over. The nuclear cycle 13 anaphase separation of sister chromatids never occurs and the chromosomes, still encompassed by spindles, assume a telophase conformation. Eventually neighboring arrested spindles begin to associate and form large clusters of spindles and nuclei. To determine whether this arrest was the result of a disruption in normal developmental events that occur at this time, both grp-derived and wild-type embryos were exposed to X-irradiation. Syncytial wild-type embryos exhibit a high division error rate, but not a nuclear-cycle arrest after exposure to low doses of X-irradiation. In contrast, grp-derived embryos exhibit a metaphase arrest in response to equivalent doses of X-irradiation. This arrest can be induced even in the early syncytial divisions prior to nuclear migration. These results suggest that the nuclear cycle 13 metaphase arrest of unexposed grp-derived embryos is independent of the division-cycle transitions that also occur at this stage. Instead, it may be the result of a previously unidentified feedback mechanism.