Mitotic spindle organization by a plus-end-directed microtubule motor

Abstract

Intracellular microtubule motor proteins may direct the motile properties and/or morphogenesis of the mitotic spindle (reviewed in ref. 3). The recent identification of kinesin-like proteins important for mitosis or meiosis indicates that kinesin-related proteins may play a universal role in eukaryotic cell division, but the precise function of such proteins in mitosis remains unknown. Here we use an in vitro assay for spindle assembly, derived from Xenopus egg extracts, to investigate the role of Eg5, a kinesin-like protein in Xenopus eggs. Eg5 is localized along spindle microtubules, and particularly enriched near spindle poles. Immunodepletion of Eg5 from egg extracts markedly reduces the extent of spindle formation in extracts, as does direct addition of anti-Eg5 antibodies. We also demonstrate that Eg5 is a plus-end-directed microtubule motor in vitro. Our results suggest a novel mechanism for the dynamic self-organization of spindle poles in mitosis.

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).…”

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

“…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. (Saunders and Hoyt, 1992;Sawin et al, 1992;Heck et al, 1993;Saunders et al, 1997;Straight et al, 1998;Walczak et al, 1998;Sharp et al, 2000a;Brust-Mascher and Scholey, 2002;Brust-Mascher et al, 2004;Cheerambathur et al, 2007). It is clear that this activity is deployed differently in different systems, however.…”

“…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.…”

“…These motility events are driven by molecular-scale forces generated by mitotic kinesins and dyneins, together with dynamic microtubules (MTs), whose activities are controlled by a network of regulatory proteins, e.g., mitotic kinases, phosphatases, and proteolytic enzymes (Sharp et al, 2000c; BettencourtDias et al, 2004;Maiato and Sunkel, 2004;Rogers et al, 2005;Goshima et al, 2007). Among these mitotic proteins, the kinesin-5 motor is thought to play a key role (Cottingham et al, 1999;Valentine et al, 2006a;Civelekoglu-Scholey and Scholey, 2007).Purified kinesin-5 is a slow, modestly processive, plusend-directed bipolar homotetramer capable of cross-linking adjacent MTs and sliding apart antiparallel MTs in motility assays (Sawin et al, 1992;Cole et al, 1994;Kashina et al, 1996a;Kapitein et al, 2005;Tao et al, 2006;Valentine et al, 2006b;Krzysiak et al, 2008;Van den Wildenberg 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).…”

A good molecular target for prostate cancer chemotherapy

“…Eg5, a member of the kinesin-5 motor proteins, 11 is critical for bipolar spindle formation and chromosome separation during mitosis. 12,13 Inhibition of Eg5 by S(MeO)TLC blocks cell division leading to cell death thus providing an option to microtubule-targeted cancer chemotherapy.…”