Spindle Pole Regulation by a Discrete Eg5-Interacting Domain in TPX2

Eckerdt, Frank; Eyers, Patrick A.; Lewellyn, Andrea L.; Prigent, Claude; Maller, James L.

doi:10.1016/j.cub.2008.02.077

Cited by 69 publications

(88 citation statements)

References 19 publications

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“…Moreover, it is also possible that force-induced unbinding from the microtubule that appears to be typical for Eg5 (Valentine et al, 2006), in contrast to the force-induced stall that is characteristic for example for Kinesin-1 (Svoboda and Block, 1994), is an important requirement for spindle maintenance. Finally, the motor domain of Eg5 might also specifically interact with other binding partners, as was recently proposed (Eckerdt et al, 2008).…”

Section: Introductionmentioning

confidence: 67%

Motile microtubule crosslinkers require distinct dynamic properties for correct functioning during spindle organization inXenopusegg extract

Cahu

Surrey

2009

Journal of Cell Science

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Results and DiscussionWe generated chimeric motor constructs of Kinesin-5 by domain swapping. Our goal was to change the conserved properties of its motor domain without, however, changing its directionality, and to preserve the overall architecture of Kinesin-5, which is required for its crosslinking activity. Therefore, we replaced the motor domain of Xenopus laevis Kinesin-5 (also called Eg5) by the motor domains of kinesins from other subfamilies, leaving intact the Eg5 backbone, which is required for its tetrameric oligomerization state (Tao et al., 2006). We chose the Xenopus chromokinesin Kid (a Kinesin-10) (Antonio et al., 2000;Funabiki and Murray, 2000) as a source for an unprocessive motor that is a little faster than Eg5 (Brouhard and Hunt, 2005;Yajima et al., 2003), and Drosophila Kinesin-1 (Kin1) (Saxton et al., 1988) as a source for a very processive motor that is much faster than Eg5 (Block et al., 1990;Hancock and Howard, 1998). The replacement of the mechanically active part of Eg5 included, in addition to the motor domain, the neck-linker, a flexible stretch of amino acids that connect the motor domain with the backbone of the molecule and that is known to be important for the stepping mechanism of kinesins (Kalchishkova and Bohm, 2008;Tomishige et al., 2006;Vale and Milligan, 2000). The exact position for the exchange was determined from an alignment of multiple kinesin sequences (Fig. 1A). Both chimeric constructs were additionally tagged with a C-terminal green fluorescent protein (GFP), generating the two chimeric constructs Kid-Eg5-GFP and Kin1-Eg5-GFP. These constructs were expressed in insect cells and purified (Fig. 1B).The organization of the microtubule cytoskeleton depends crucially on crosslinking motors that arrange microtubules in space. Kinesin-5 is such an essential motile crosslinker. It is unknown whether its organizing capacity during bipolar spindle formation depends on its characteristic kinetic properties, or whether simply crosslinking combined with any plus-enddirected motility is sufficient for its function in a physiological context. To address this question, we replaced the motor domain of Xenopus Kinesin-5 by motor domains of kinesins belonging to other kinesin subfamilies, without changing the overall architecture of the molecule. This generated novel microtubule crosslinkers with altered kinetic properties. The chimeric crosslinkers mislocalized in spindles and consequently caused spindle collapse into tightly bundled microtubule arrays. This demonstrates that plus-end directionality and microtubule crosslinking are not the only characteristics required for proper functioning of Kinesin-5 during spindle assembly in Xenopus egg extract. Instead, its motor domain properties appear to be fine-tuned for the specific function of this kinesin.

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Section: Introductionmentioning

confidence: 67%

Motile microtubule crosslinkers require distinct dynamic properties for correct functioning during spindle organization inXenopusegg extract

Cahu

Surrey

2009

Journal of Cell Science

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“…43 Cell culture, transfection, and microscopy analysis Xenopus S3 cell culture, transfection of DsRed-Gwl, and cell imaging by confocal microscopy were performed as previously described. 44 Xenopus S3 GFP-a-tubulin-expressing cells were a kind gift from Dr. Gary Gorbsky (Oklahoma Medical Research Foundation). In general, cells were transfected in suspension immediately after trypsinization using Lipofectamine LTX in combination with PLUS reagent (Invitrogen) according to the manufacturer's instructions.…”

Section: Immunoprecipitation Immunoblotting and Immunodepletionmentioning

confidence: 99%

Regulation of Greatwall kinase by protein stabilization and nuclear localization

et al. 2014

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“…TPX2 binds to and activates the mitotic kinase Aurora A and localizes it to spindle microtubules (Bayliss et al, 2003). TPX2 also interacts with and regulates the activity of the bipolar mitotic kinesin Eg5 (Eckerdt et al, 2008;Ma et al, 2011) and kinesin-12 motors (Wittmann et al, 2000;Tanenbaum et al, 2009;Vanneste et al, 2009). Thus, TPX2 functions in part as a scaffold to bring mitotic regulators to spindle microtubules.…”

Section: Introductionmentioning

confidence: 99%

Suppression of microtubule assembly kinetics by the mitotic protein TPX2

Reid

Schuster

Mann

et al. 2016

Journal of Cell Science

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TPX2 is a widely conserved microtubule-associated protein that is required for mitotic spindle formation and function. Previous studies have demonstrated that TPX2 is required for the nucleation of microtubules around chromosomes; however, the molecular mechanism by which TPX2 promotes microtubule nucleation remains a mystery. In this study, we found that TPX2 acts to suppress tubulin subunit off-rates during microtubule assembly and disassembly, thus allowing for the support of unprecedentedly slow rates of plus-end microtubule growth, and also leading to a dramatically reduced microtubule shortening rate. These changes in microtubule dynamics can be explained in computational simulations by a moderate increase in tubulin-tubulin bond strength upon TPX2 association with the microtubule lattice, which in turn acts to reduce the departure rate of tubulin subunits from the microtubule ends. Thus, the direct suppression of tubulin subunit off-rates by TPX2 during microtubule growth and shortening could provide a molecular mechanism to explain the nucleation of new microtubules in the presence of TPX2.

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Spindle Pole Regulation by a Discrete Eg5-Interacting Domain in TPX2

Cited by 69 publications

References 19 publications

Motile microtubule crosslinkers require distinct dynamic properties for correct functioning during spindle organization inXenopusegg extract

Motile microtubule crosslinkers require distinct dynamic properties for correct functioning during spindle organization inXenopusegg extract

Regulation of Greatwall kinase by protein stabilization and nuclear localization

Suppression of microtubule assembly kinetics by the mitotic protein TPX2

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