The Bipolar Kinesin, KLP61F, Cross-links Microtubules within Interpolar Microtubule Bundles of Drosophila Embryonic Mitotic Spindles

Abstract

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (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 contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a “sliding filament mechanism” during the formation and function of the mitotic spindle.

Bipolar, anastral spindle development in artificially activated sea urchin eggs

“…8) is in agreement with the crucial role that the Kinesin-5 family plays in bipolar spindle development and dynamics (Sawin et al, 1992;Miyamoto et al, 2004). In addition, the intranuclear localization of KRP 170 reflects a similar localization pattern previously demonstrated for the Drosophila Kinesin-5 member KLP61F in both S2 cells (Goshima and Vale, 2005) and embryos (Sharp et al, 1999). Several mitotic kinesins have been shown to be sequestered in the nucleus during interphase and to be released for mitotic functions following nuclear envelope breakdown (Goshima and Vale, 2005).…”

Kinesin-5–dependent Poleward Flux and Spindle Length Control inDrosophilaEmbryo Mitosis

“…Accordingly antibodies were raised against a tail domain fragment in which the Thr-933 residue was changed to an aspartate phosphomimic by site-directed mutagenesis (Materials and Methods). These antibodies react with a single polypeptide of appropriate molecular weight on immunoblots of embryonic extracts and have been shown to stain mitotic spindles by immunofluorescence (our unpublished results and SilvermanGavrila and Wilde, 2006), consistent with our previous results (Sharp et al, 1999a;Cheerambathur et al, 2008). As reported previously, the microinjection of affinity-purified antibody into the syncytial blastoderm embryo blocked mitosis and produced monoastral arrays of microtubules (Sharp et al, 1999b(Sharp et al, , 2000a similar to those seen in fixed loss-of-function klp61f mutants after depletion of the maternal load in larvae (Heck et al, 1993; see below).…”

“…In yeast cells the homotetrameric structure of kinesin-5 appears to be essential for mitosis (Hildebrandt et al, 2006), and in Drosophila embryos KLP61F displays dynamic properties consistent with an association with spindle MTs (Cheerambathur et al, 2008) and forms presumptive MT-MT cross-bridges (Sharp et al, 1999a). These results suggest that ensembles of multiple kinesin-5 motors could serve as dynamic cross-links that organize spindle MTs into bundles and drive a sliding filament mechanism that pushes apart antiparallel spindle MTs (Sharp et al, 1999a;Brust-Mascher et al, 2004), although alternative mechanisms of action for kinesin-5 motors have also been proposed (Kapoor and Mitchison, 2001;Tsai et al, 2006;Johansen and Johansen, 2007;Gardner et al, 2008).…”

“…In yeast cells the homotetrameric structure of kinesin-5 appears to be essential for mitosis (Hildebrandt et al, 2006), and in Drosophila embryos KLP61F displays dynamic properties consistent with an association with spindle MTs (Cheerambathur et al, 2008) and forms presumptive MT-MT cross-bridges (Sharp et al, 1999a). These results suggest that ensembles of multiple kinesin-5 motors could serve as dynamic cross-links that organize spindle MTs into bundles and drive a sliding filament mechanism that pushes apart antiparallel spindle MTs (Sharp et al, 1999a;Brust-Mascher et al, 2004), although alternative mechanisms of action for kinesin-5 motors have also been proposed (Kapoor and Mitchison, 2001;Tsai et al, 2006;Johansen and Johansen, 2007;Gardner et al, 2008).The most obvious and frequently observed consequence of loss of activity of kinesin-5 motors, induced by loss-offunction mutation, antibody inhibition, small molecule inhibition, or RNA interference, is the formation of abnormal monoastral spindles (Enos and Morris, 1990;Saunders and Hoyt, 1992;Sawin et al, 1992;Heck et al, 1993;Saunders et al, 1997;Cottingham et al, 1999;Mayer et al, 1999;Sharp et al, 1999b;Sharp et al, 2000a;Goshima and Vale, 2003). This suggests that kinesin-5 may normally contribute to spindle bipolarity by sliding apart interpolar (ip) MTs to drive spindle pole separation during mitotic spindle assembly, elongation and function.…”

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

“…To determine the mechanical and kinetic requirements for Eg5-promoted spindle assembly [47][48][49], a stable Eg5 dimer was developed [11••]. Eg5-513 promoted robust plus-end-directed microtubule gliding at a rate similar to that of native tetramers [11••,50].…”