Mitosis in primary cultures of Drosophila melanogaster larval neuroblasts

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 degrees C, spindle formation takes 7+/-0.5 minutes. Analysis of neuroblasts containing various GFP-tagged proteins (e.g. histone, fizzy, fizzy-related and alpha-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 microm/minute, whereas during anaphase B the spindle poles separate at 1.6+/-01 microm/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.