The Microtubule-Binding and Coiled-Coil Domains of Kid Are
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© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 3609-3619 doi:10.1242/jcs.189969 RESEARCH ARTICLE The microtubule-binding and coiled-coil domains of Kid are required to turn off the polar ejection force at anaphase Shou Soeda1,2,3,4,*, Kaori Yamada-Nomoto1,5,* and Miho Ohsugi1,2,3,*,‡ ABSTRACT throughout mitosis. In addition, the forces generated on the Mitotic chromosomes move dynamically along the spindle chromosome arms contribute to the congression and alignment of microtubules using the forces generated by motor proteins such as the chromosomes during prometaphase and metaphase. The chromokinesin Kid (also known as KIF22). Kid generates a polar chromokinesin Kid (also known as KIF22) is a plus end-directed ejection force and contributes to alignment of the chromosome arms motor protein characterized by a DNA-binding domain (Tokai et al., during prometaphase and metaphase, whereas during anaphase, 1996). During prometaphase and metaphase, Kid is implicated in Kid contributes to chromosome compaction. How Kid is regulated chromosome congression and alignment through generation of a and how this regulation is important for chromosome dynamics remains polar ejection force (PEF) on the chromosome arms (Brouhard and unclear. Here, we address these questions by expressing mutant Hunt, 2005), although the degree of the physiological importance of forms of Kid in Kid-deficient cells. We demonstrate that Cdk1-mediated Kid-mediated PEF varies among species and cell types. Kid in Xenopus phosphorylation of Thr463 is required to generate the polar ejection has been shown to be essential for metaphase chromosome Xenopus force on Kid-binding chromosomes, whereas dephosphorylation alignment in egg extract; however, there is no evidence of of Thr463 prevents generation of the ejection force on such the involvement of Kid in chromosome congression in mouse chromosomes. In addition to activation of the second microtubule- oocytes and zygotes (Funabiki and Murray, 2000; Antonio et al., binding domain through dephosphorylation of Thr463, the coiled-coil 2000; Ohsugi et al., 2008; Kitajima et al., 2011). Moreover, in domain is essential in suspending generation of the polar ejection force, somatic cell lines, mammalian Kid generates the PEF and contributes preventing separated chromosomes from becoming recongressed to driving chromosome movement toward the spindle equator during during anaphase. We propose that phosphorylation of Thr463 switches prometaphase (Levesque and Compton, 2001; Tokai-Nishizumi the mitotic chromosome movement from an anti-poleward direction to et al., 2005; Magidson et al., 2011; Iemura and Tanaka, 2015). – a poleward direction by converting the Kid functional mode from At the metaphase anaphase transition, loss of cohesion between polar-ejection-force-ON to -OFF during the metaphase–anaphase sister chromatids leads to a change in direction toward the spindle transition, and that both the second microtubule-binding domain and poles. In addition to the loss of sister chromatid cohesion, the the coiled-coil domain are involved in this switching process. change of direction of chromosome movement requires a reduction of the PEF, which occurs through degradation of Xenopus Kid KEY WORDS: Cell division, Chromosome dynamics, Kinesin, degradation in Xenopus egg extract (Funabiki and Murray, 2000). In Phosphorylation status contrast, the majority of Kid is maintained during anaphase and contributes to the tight clustering of anaphase chromosomes INTRODUCTION (anaphase chromosome compaction), which prevents the The accuracy of chromosome segregation during mitosis is formation of blastomeres with multiple nuclei in mouse zygotes guaranteed by strictly regulated processes. Failures in orchestrating (Ohsugi et al., 2008). Therefore, the effects of reduced PEF in these processes can result in aneuploidy, tumorigenesis or cell death. anaphase chromosome segregation in mammalian cells remains The dynamic movement of mitotic chromosomes along the spindle is unclear; if a reduction in PEF does occur, it must be achieved by achieved by forces generated by microtubule dynamics and several means other than through protein degradation. motor proteins, including members of the cytoplasmic dynein and Although previous studies have revealed different functions of kinesin superfamilies (Cross and McAinsh, 2014). Forces acting on Kid, how transitions between these multiple functions are regulated the kinetochores through the attached microtubules are the primary remains unclear. One plausible regulatory mechanism is Cdk1– and indispensable driving forces for chromosome movement cyclin-B-mediated phosphorylation of Thr463 on Kid (Ohsugi et al., 2003). Besides the motor domain (the first microtubule- binding domain), Kid has a second microtubule-binding domain 1 Division of Oncology, Institute of Medical Science, The University of Tokyo, Minato- (MTBD) between the motor and coiled-coil (CC) domains ku, Tokyo 108-8639, Japan. 2Department of Life Science, Graduate school of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan. (Shiroguchi et al., 2003). We have previously shown that the 3Department of Biophysics and Biochemistry, Graduate school of Science, The microtubule-binding activity of Kid mediated by the MTBD is University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. 4Japan Society for the Promotion of Science Research Fellow, Chiyoda-Ku, Tokyo 102-0083, Japan. regulated negatively by phosphorylation on Thr463 (Ohsugi et al., 5Department of Obstetrics and Gynecology, The University of Toyama, Toyama-shi, 2003). Amino acid substitution of Thr463 to Ala increases the Toyama 930-0194, Japan. affinity of Kid for microtubules, leading to its sequestration to *Present address: Department of Life Science, Graduate school of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, 153-8902, Japan. spindle microtubules and a failure in chromosomal localization of the protein during prometaphase and metaphase. This indicates that ‡ Author for correspondence ([email protected]) phosphorylation on Thr463 is essential for the proper localization of M.O., 0000-0003-0288-3428 Kid on chromosomes (Ohsugi et al., 2003). However, whether phosphorylation of Thr463 is essential for Kid to generate PEF, and, Received 30 March 2016; Accepted 17 August 2016 conversely, whether dephosphorylation of Thr463 is sufficient to Journal of Cell Science 3609 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3609-3619 doi:10.1242/jcs.189969 suppress PEF remain unproven. Many kinesin-family members of anaphase. To address this possibility, we used mouse oocytes, form oligomers through the CC domain when they move along the which offer several advantages. First, oocytes are arrested at microtubules. However, Kid is purified as a monomer from cells metaphase of the second meiosis (metaphase II), and the arrested in prometaphase (Shiroguchi et al., 2003) and is known to metaphase–anaphase transition can be induced artificially through be motile as a monomer (Yajima et al., 2003). The CC domain of parthenogenetic activation. Second, the importance of Kid during Kid has been shown to be important for the regulation of spindle anaphase and telophase is prominent (Ohsugi et al., 2008). length (Tokai-Nishizumi et al., 2005), but its involvement in PEF Moreover, exogenous protein expression is achieved easily in generation has remained obscure. metaphase-II-arrested oocytes by the injection of mRNA that has In the current study, we expressed a series of Kid mutants in Kid- been transcribed in vitro. deficient cells to analyze the importance of each domain and the To visualize chromosomes, mRFP-fused histone H2B was phosphorylation status of Thr463 for Kid functions, as well as in expressed in oocytes from mice wild-type for Kid or in which Kid switching the direction of chromosome movement at the had been knocked out (Kid-KO mice). In addition to H2B-mRFP, metaphase–anaphase transition. GFP-tagged wild-type or mutant forms of Kid were expressed in Kid-KO oocytes. The expression levels of GFP-tagged Kid proteins RESULTS were almost equivalent to each other (Fig. S1A). Oocytes were Thr463 phosphorylation, but not the CC domain of Kid, is subjected to live-cell imaging immediately after parthenogenetic essential for generating PEF activation. In wild-type oocytes, one set of segregated sister In order to clarify whether phosphorylation of Thr463 (Fig. 1A) of chromatids was extruded from oocytes as a second polar body, and Kid was required for the generation of PEF as well as for its proper the other formed a single female pronucleus (fPN) (Fig. 2A and B; chromosomal localization, we determined whether a mutant of Kid Movie 1). In contrast, although chromosomes were segregated and a in which Thr463 was replaced by Ala (Kid-T463A) generated PEF. polar body was emitted, about 75% of the Kid-KO oocytes In the absence of Kid, chromosomes on a monopolar spindle are exhibited fragmentation of the fPN (Fig. 2A and C), as reported centered (Levesque and Compton, 2001). Taking advantage of this previously (Ohsugi et al., 2008). Hereafter, we refer to this as the effect, we expressed GFP, GFP–Kid-WT (GFP-tagged wild-type multi-fPN phenotype. Exogenously expressed GFP–Kid-WT construct) or GFP–Kid-T463A in Kid−/− mouse embryonic almost completely rescued the multi-fPN phenotype of Kid-KO fibroblasts and cultured these cells with or without an inhibitor of oocytes, whereas the Kid mutant lacking the DNA-binding