Ncd-GFP in Mitosis

Fig. 1 Mitotic spindles visualized with Ncd-GFP* in a cleavage stage Drosophila embryo
The time-lapse images show a cycle 9 division in an ncd-gfp* embryo. During interphase, Ncd-GFP is associated with centrosomes and is also present in the cytoplasm. Centrosomes increase in brightness at nuclear envelope breakdown, remain bright during metaphase and late metaphase/early anaphase, and diminish in brightness in mid- and late anaphase and telophase. Spindle fibers are bright with Ncd-GFP in metaphase and anaphase, and Ncd-GFP is redistributed to the developing midbody in late anaphase and telophase.

Fig. 2 Ncd-GFP distribution in metaphase spindles differs from that of tubulin
Double images were collected from an ncd-gfp* embryo injected with rhodamine-tubulin to label the microtubules. The time-lapse series shows spindles during a cycle 10 division. The Ncd-GFP fluorescence shows spindle fibers that extend from pole to pole. In contrast, the rhodamine-tubulin fluorescence is diminished in the region of the metaphase plate. The color icon is a difference overlay with coincident Ncd-GFP and tubulin fluorescence in purple, and Ncd-GFP alone in green.

Fig. 3 Spindle fibers associated with Ncd-GFP extend across the chromosomes in metaphase
Double images of Ncd-GFP and rhodamine-labelled chromosomes were collected from ncd-gfp* embryos injected with rhodamine-conjugated histones to visualize the chromosomes. The time-lapse series shows Ncd-GFP decorated metaphase spindles and rhodamine-labelled chromosomes during a cycle 9 cleavage division. The Ncd-GFP fluorescence shows spindle fibers that extend from pole to pole and extend across the chromosomes aligned on the plate during metaphase.

Fig. 4 Embryos mutant for ncd-gfp* show spindle abnormalities in mitosis
Images were collected from a cycle 10 embryo carrying a mutant ncd fused to gfp*. Microtubule spurs form on the spindles in metaphase and touch other spindles, forming bridges. The spindle spurs can also extend, then retract later in anaphase. The spurs are associated with mis-segregating chromosomes (see Fig. 5).

Fig. 5 Chromosome mis-segregation and loss in an ncd-gfp* mutant embryo
The sequence shows a spurred spindle associated with a mis-segregating chromosome in mid-metaphase and early anaphase of cycle 9. The ncd-gfp* mutant embryo has been injected with rhodamine-histones to label the chromosomes. The spindle spur is associated with a chromosome that has partially detached from the metaphase plate. The chromosome subsequently reattaches, but lags behind the other chromosomes in moving poleward in early anaphase. Meanwhile, one of the small chromosomes 4 (top arrowhead) mis-segregates along the spindle spur and moves off the spindle, while the other moves towards a pole (bottom arrowhead).

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