The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles

Previous genetic and biochemical studies have led to the hypothesis that th e essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtu bules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM), We show that Drosophila embryonic spindles at metaphase and anaphase conta in abundant bundles of MTs running between the spindle poles. These interpo lar MT bundles are parallel near the poles and antiparallel. in the midzone , We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B co nsensus domain within the BimC box (BCB), localize along the length of thes e interpolar MT bundles, being concentrated in the midzone region. Nonphosp horylated KLP61F motors, in contrast, are excluded from the spindle and dis play a cytoplasmic localization. Immunoelectron microscopy further suggeste d that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of or ganizing parallel MTs into bundles within half spindles and sliding antipar allel MTs apart in the spindle midzone. Thus we propose that bipolar kinesi n motors and MTs interact by a "sliding filament mechanism" during the form ation and function of the mitotic spindle.