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Research Article |
1 Division of Molecular Medicine, Wadsworth Center, New York State Department of
Health, P.O. Box 509, Albany, New York 12201-0509, USA
2 Department of Biomedical Sciences, State University of New York, Albany, New
York 12222, USA
* Author for correspondence (e-mail: Rieder{at}Wadsworth.org )
Accepted 5 May 2002
Although Drosophila larval neuroblasts are routinely used to
define mutations affecting mitosis, the dynamics of karyokinesis in this
system remain to be described. Here we outline a simple method for the
short-term culturing of neuroblasts, from Drosophila third instar
larvae, that allows mitosis to be followed by high-resolution multi-mode light
microscopy. At 24°C, spindle formation takes 7±0.5 minutes.
Analysis of neuroblasts containing various GFP-tagged proteins (e.g. histone,
fizzy, fizzy-related and 
-tubulin) reveals that attaching kinetochores
exhibit sudden, rapid pole-directed motions and that congressing and metaphase
chromosomes do not undergo oscillations. By metaphase, the arms of longer
chromosomes can be resolved as two chromatids, and they often extend towards a
pole. Anaphase A and B occur concurrently, and during anaphase A chromatids
move poleward at 3.2±0.1 µm/minute, whereas during anaphase B the
spindle poles separate at 1.6±01 µm/minute. In larger neuroblasts,
the spindle undergoes a sudden shift in position during midanaphase, after
which the centrally located centrosome preferentially generates a robust aster
and stops moving, even while the spindle continues to elongate. Together these
two processes contribute to an asymmetric positioning of the spindle midzone,
which, in turn, results in an asymmetric cytokinesis. Bipolar spindles form
predominately (83%) in association with the separating centrosomes. However,
in 17% of the cells, secondary spindles form around chromosomes without
respect to centrosome position: in most cases these spindles coalesce with the
primary spindle by anaphase, but in a few they remain separate and define
additional ectopic poles.
Key words: GFP imaging, Kinetochore, Centrosome, Spindle, Cytokinesis
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