Mitotic chromosomes move dynamically along the spindle microtubules using the forces generated by motor proteins such as chromokinesin Kid (also known as KIF22). Kid generates a polar ejection force and contributes to alignment of the chromosome arms during prometaphase and metaphase, whereas during anaphase, Kid contributes to chromosome compaction. How Kid is regulated and how this regulation is important for chromosome dynamics remains unclear. Here, we address these questions by expressing mutant forms of Kid in Kid-deficient cells. We demonstrate that Cdk1-mediated phosphorylation of Thr463 is required to generate the polar ejection force on Kid-binding chromosomes, whereas dephosphorylation of Thr463 prevents generation of the ejection force on such chromosomes. In addition to activation of the second microtubule-binding domain through dephosphorylation of Thr463, the coiled-coil domain is essential in suspending generation of the polar ejection force, preventing separated chromosomes from becoming recongressed during anaphase. We propose that phosphorylation of Thr463 switches the mitotic chromosome movement from an anti-poleward direction to a poleward direction by converting the Kid functional mode from polar-ejection-force-ON to -OFF during the metaphase–anaphase transition, and that both the second microtubule-binding domain and the coiled-coil domain are involved in this switching process.
The accuracy of chromosome segregation during mitosis is guaranteed by strictly regulated processes. Failures in orchestrating these processes can result in aneuploidy, tumorigenesis or cell death. The dynamic movement of mitotic chromosomes along the spindle is achieved by forces generated by microtubule dynamics and several motor proteins, including members of the cytoplasmic dynein and kinesin superfamilies (Cross and McAinsh, 2014). Forces acting on the kinetochores through the attached microtubules are the primary and indispensable driving forces for chromosome movement throughout mitosis. In addition, the forces generated on the chromosome arms contribute to the congression and alignment of the chromosomes during prometaphase and metaphase. The chromokinesin Kid (also known as KIF22) is a plus end-directed motor protein characterized by a DNA-binding domain (Tokai et al., 1996). During prometaphase and metaphase, Kid is implicated in chromosome congression and alignment through generation of a polar ejection force (PEF) on the chromosome arms (Brouhard and Hunt, 2005), although the degree of the physiological importance of Kid-mediated PEF varies among species and cell types. Kid in Xenopus has been shown to be essential for metaphase chromosome alignment in Xenopus egg extract; however, there is no evidence of the involvement of Kid in chromosome congression in mouse oocytes and zygotes (Funabiki and Murray, 2000; Antonio et al., 2000; Ohsugi et al., 2008; Kitajima et al., 2011). Moreover, in somatic cell lines, mammalian Kid generates the PEF and contributes to driving chromosome movement toward the spindle equator during prometaphase (Levesque and Compton, 2001; Tokai-Nishizumi et al., 2005; Magidson et al., 2011; Iemura and Tanaka, 2015).
At the metaphase–anaphase transition, loss of cohesion between sister chromatids leads to a change in direction toward the spindle poles. In addition to the loss of sister chromatid cohesion, the change of direction of chromosome movement requires a reduction of the PEF, which occurs through degradation of Xenopus Kid degradation in Xenopus egg extract (Funabiki and Murray, 2000). In contrast, the majority of Kid is maintained during anaphase and contributes to the tight clustering of anaphase chromosomes (anaphase chromosome compaction), which prevents the formation of blastomeres with multiple nuclei in mouse zygotes (Ohsugi et al., 2008). Therefore, the effects of reduced PEF in anaphase chromosome segregation in mammalian cells remains unclear; if a reduction in PEF does occur, it must be achieved by means other than through protein degradation.
Although previous studies have revealed different functions of Kid, how transitions between these multiple functions are regulated remains unclear. One plausible regulatory mechanism is Cdk1–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 (MTBD) between the motor and coiled-coil (CC) domains (Shiroguchi et al., 2003). We have previously shown that the microtubule-binding activity of Kid mediated by the MTBD is regulated negatively by phosphorylation on Thr463 (Ohsugi et al., 2003). Amino acid substitution of Thr463 to Ala increases the affinity of Kid for microtubules, leading to its sequestration to spindle microtubules and a failure in chromosomal localization of the protein during prometaphase and metaphase. This indicates that phosphorylation on Thr463 is essential for the proper localization of Kid on chromosomes (Ohsugi et al., 2003). However, whether phosphorylation of Thr463 is essential for Kid to generate PEF, and, conversely, whether dephosphorylation of Thr463 is sufficient to suppress PEF remain unproven. Many kinesin-family members form oligomers through the CC domain when they move along the microtubules. However, Kid is purified as a monomer from cells arrested in prometaphase (Shiroguchi et al., 2003) and is known to be motile as a monomer (Yajima et al., 2003). The CC domain of Kid has been shown to be important for the regulation of spindle length (Tokai-Nishizumi et al., 2005), but its involvement in PEF generation has remained obscure.
In the current study, we expressed a series of Kid mutants in Kid-deficient cells to analyze the importance of each domain and the phosphorylation status of Thr463 for Kid functions, as well as in switching the direction of chromosome movement at the metaphase–anaphase transition.
Thr463 phosphorylation, but not the CC domain of Kid, is essential for generating PEF
In order to clarify whether phosphorylation of Thr463 (Fig. 1A) of Kid was required for the generation of PEF as well as for its proper chromosomal localization, we determined whether a mutant of Kid in which Thr463 was replaced by Ala (Kid-T463A) generated PEF. In the absence of Kid, chromosomes on a monopolar spindle are centered (Levesque and Compton, 2001). Taking advantage of this effect, we expressed GFP, GFP–Kid-WT (GFP-tagged wild-type construct) or GFP–Kid-T463A in Kid−/− mouse embryonic fibroblasts and cultured these cells with or without an inhibitor of Eg5 (also known as kinesin-5 and KIF11) (Fig. 1B and C). Chromosome distributions on monopolar spindles in Eg5-inhibitor-treated cells were examined by examining Hoechst signal intensities along a 12-µm line centered on a spindle pole in GFP-positive monopolar mitotic cells (Fig. 1D). In Kid−/− cells expressing GFP alone or GFP–Kid-T463A, which was preferentially localized on a spindle (Fig. 1B and C), chromosomes were centered on a monopolar spindle. In contrast, chromosomes were ejected from a pole of a monopolar spindle in cells that could be rescued with GFP–Kid-WT (Fig. 1B and E). Because Kid also contributes to the maintenance of spindle size during metaphase (Tokai-Nishizumi et al., 2005), we also compared spindle sizes in these cells and found no significant differences (Fig. 1F). These results indicate that differences in chromosome distribution were not due to differences in the spindle length, and that phosphorylation at Thr463 is essential for Kid to generate PEF during prometaphase.
We next sought to examine the involvement of the MTBD and CC domain in PEF generation. To this end, we constructed Kid mutants that lacked part of the MTBD (amino acids 445–451, ΔMTBD) or CC domain (amino acids 471–496, ΔCC). Deletion of the MTBD led to preferential localization on chromosomes, even with the T463A mutation (Fig. 1B and C). This finding is consistent with our previous results that, in the absence of Thr463 phosphorylation, the MTBD enhances the affinity of Kid for microtubules (Ohsugi et al., 2003; Shiroguchi et al., 2003), whereas GFP–Kid-ΔCC showed a similar localization to that of GFP–Kid-WT (Fig. 1B and C). Chromosomes were ejected from the pole of monopolar spindles under expression of either protein (Fig. 1G). These results indicate that the CC domain is dispensable for Kid-mediated PEF generation, and that PEF generation is also dependent on the suppression of the MTBD by phosphorylation of Thr463.
Activation of the MTBD through dephosphorylation of Thr463 is required for chromosome segregation in anaphase
The results described above raise the possibility that the MTBD and CC domain are essential for Kid to function during or after the onset of anaphase. To address this possibility, we used mouse oocytes, which offer several advantages. First, oocytes are arrested at metaphase of the second meiosis (metaphase II), and the metaphase–anaphase transition can be induced artificially through parthenogenetic activation. Second, the importance of Kid during anaphase and telophase is prominent (Ohsugi et al., 2008). Moreover, exogenous protein expression is achieved easily in metaphase-II-arrested oocytes by the injection of mRNA that has been transcribed in vitro.
To visualize chromosomes, mRFP-fused histone H2B was expressed in oocytes from mice wild-type for Kid or in which Kid had been knocked out (Kid-KO mice). In addition to H2B-mRFP, GFP-tagged wild-type or mutant forms of Kid were expressed in Kid-KO oocytes. The expression levels of GFP-tagged Kid proteins were almost equivalent to each other (Fig. S1A). Oocytes were subjected to live-cell imaging immediately after parthenogenetic activation. In wild-type oocytes, one set of segregated sister chromatids was extruded from oocytes as a second polar body, and the other formed a single female pronucleus (fPN) (Fig. 2A and B; Movie 1). In contrast, although chromosomes were segregated and a polar body was emitted, about 75% of the Kid-KO oocytes exhibited fragmentation of the fPN (Fig. 2A and C), as reported previously (Ohsugi et al., 2008). Hereafter, we refer to this as the multi-fPN phenotype. Exogenously expressed GFP–Kid-WT almost completely rescued the multi-fPN phenotype of Kid-KO oocytes, whereas the Kid mutant lacking the DNA-binding domain (GFP–Kid-ΔDBD), which failed to localize on anaphase chromosomes, did not (Fig. 2A,D and E). This demonstrated that GFP-tagged human Kid protein compensates for the lack of endogenous mouse Kid protein in oocytes.
We examined the importance of dephosphorylation of Thr463 in the post-metaphase processes by expressing phosphorylation-defective (T463A) or phosphorylation-mimetic (T463D) mutants of Kid in Kid-KO oocytes. GFP–Kid-T463A accumulated on anaphase chromosomes and rescued the multi-fPN phenotype as effectively as GFP–Kid-WT (Fig. 3A and B). This suggested that phosphorylation of Thr463 is not necessary for Kid function after the onset of anaphase. In contrast, when Kid-KO oocytes expressing GFP–Kid-T463D entered anaphase, chromosomes initially moved toward each pole, but then a portion of or all separated chromosomes moved back to the spindle equator (Fig. 3A and C; Movie 2). When one or more anaphase chromosomes moved toward the spindle equator, we classified the oocytes as undergoing ‘recongression’. The anti-poleward movement of anaphase chromosomes was never observed in Kid-KO oocytes, suggesting that this chromosome-recongression phenotype was distinct from fragmentation of the fPN caused by the loss of Kid. Furthermore, the expression of GFP–Kid-ΔMTBD in Kid-KO oocytes resulted in a recongression of anaphase chromosomes, even when Thr463 was replaced with Ala (Fig. 3A,D and E; Movie 3). These results suggest that dephosphorylation of Thr463 activates the MTBD, which is essential for chromosome segregation during anaphase.
The phosphorylation status of Thr463 acts as a switch to convert Kid between the PEF-ON and PEF-OFF modes
We further characterized the anaphase chromosome phenotypes induced by GFP–Kid-T463D and GFP–Kid-ΔMTBD. Despite the fact that Kid generates PEF on prometaphase–metaphase chromosomes in somatic cells, Kid is dispensable for chromosome alignment during oocyte meiosis (Ohsugi et al., 2008; Kitajima et al., 2011). Expression of neither the GFP-tagged Kid-WT nor the mutant forms caused the redistribution of already aligned metaphase-II chromosomes, suggesting that the recongression phenotype caused by GFP–Kid-T463D or GFP–Kid-ΔMTBD was not due to their influence on the chromosomes during the phases of meiosis that occur before anaphase II. We also addressed the possibility that the recongression phenotype had resulted from defective spindle elongation during anaphase caused by the Kid mutants. To this end, we expressed mCherry–α-tubulin in oocytes, and measured the pole-to-pole distance of the spindle during metaphase II and anaphase II. In this assay, oocytes were treated with an inhibitor of actin polymerization to prevent the anaphase spindle from being rotated and bent during chromosome segregation and polar body emission. Under this condition, the spindles elongated rapidly, reached their maximum length within 10 min after the onset of anaphase, and then shortened gradually (Fig. S1B). The spindle-length kinetics were not significantly affected by the expression of Kid mutants (Fig. S1B and C), indicating that the recongression phenotype could not be attributed to defective anaphase spindle elongation. Upon the onset of anaphase, the chromosomes were pulled apart toward the poles as the spindle elongated. However, the chromosomes kept moving and became fully segregated in Kid-KO oocytes expressing Kid-WT, whereas the chromosomes in oocytes expressing Kid-T463D or Kid-ΔMTBD traveled only a short distance and then began to move back to the spindle equator as the central microtubules were beginning to develop between the segregated chromosomes (Fig. S1D). These results suggest that the chromosomes were pulled apart by the forces generated by the spindle; however, they were then recongressed by Kid-T463D or Kid-ΔMTBD along the central spindle. When motor activity was suppressed by introducing a rigor-inducing mutation (Thr34 to Asn substitution) into Kid-ΔMTBD (Meluh and Rose, 1990; Ohsugi et al., 2003), the resulting GFP–Kid-Rigor-ΔMTBD did not induce the anaphase chromosome-recongression phenotype (Fig. 3A and F). This result supports the model that the chromosome-recongressing force is driven by Kid-ΔMTBD motor activity, although we cannot exclude the possibility that enhanced microtubule-binding affinity resulting from the rigor-inducing mutation bypasses the requirement for the MTBD. The expression of GFP–Kid-T463D in Kid-KO zygotes also resulted in the anaphase chromosome-recongression phenotype at the first cleavage division (Fig. S2A). Furthermore, the anaphase chromosome-recongression phenotype was rarely observed in wild-type oocytes that expressed Kid-T463D or Kid-ΔMTBD (Fig. S2B). This suggests that, during anaphase, dephosphorylated Kid not only halts the generation of PEF but also antagonizes the PEF generated by these Kid mutants.
The CC domain is required to convert unphosphorylated Kid to the PEF-OFF mode
Next, we examined the importance of the CC domain after the onset of anaphase. Kid-KO oocytes expressing either the Kid mutant lacking the CC domain (Kid-ΔCC) or with a disrupted CC domain (Kid-8D or 7S, in which Val475, Glu476, Glu477, Lys478, Glu491, Lys492, Glu493, Ala494' were replaced with Asp or `Val475, Glu476, Glu477, Lys478, Lys492, Glu493, Ala494' were replaced with Ser residues, respectively; see Fig. S3; Lupas, 1996) entered anaphase. However, the segregated chromosomes congressed again to the spindle equator, similar to in Kid-KO oocytes that expressed Kid-T463D or Kid-ΔMTBD (Fig. 4A,B; Movie 4, Fig. S3A–D). The rigor-inducing mutation also bypassed the requirement of the CC domain in order to arrest PEF generation (Fig. 4A and C). These results indicate that the CC domain of Kid is required to halt PEF generation during anaphase.
Because Thr463 is located close to the CC domain, it is possible that loss of the CC structure interferes with dephosphorylation of Thr463 and that the mutants act as constitutively phosphorylated forms, similar to Kid-T463D. To test this possibility, we constructed and expressed a phosphorylation-defective form of Kid-ΔCC (Kid-T463A-ΔCC) in Kid-KO oocytes. Although GFP–Kid-ΔCC showed no chromosomal accumulation during anaphase, GFP–T463A-ΔCC showed preferred localization to the chromosomes rather than to the microtubules (Fig. 4B and D). The difference in localization between these two mutants suggests that the deletion of the CC domain affects T463 dephosphorylation. However, the anaphase chromosome-recongression phenotype was still observed in oocytes expressing Kid-T463A-ΔCC (Fig. 4A and D), indicating that the recongression phenotype caused by Kid-ΔCC was not dependent on Thr463 phosphorylation. This suggests that, in addition to the MTBD, the CC domain is essential for unphosphorylated Kid to be set to the PEF-OFF mode.
Immunofluorescence analysis demonstrated that while Kid-WT, Kid-T463D, and Kid-ΔMTBD showed a preferential localization on anaphase chromosomes, Kid-ΔCC localized on both central spindle microtubules and chromosomes (Fig. S3E). To examine whether Kid-ΔCC used MTBD to ectopically localize on the spindle, the double deletion mutant Kid-ΔMTBD-ΔCC was constructed and expressed in Kid-KO oocytes. GFP-Kid-ΔMTBD-ΔCC showed similar localization as Kid-ΔCC and caused the recongression phenotype (Fig. 4B and E). Furthermore, introducing the rigor-type mutation, which enhances the affinity for microtubules, restored the localization of Kid-ΔCC (Fig. 4C). These results suggest that the mislocalization of Kid-ΔCC was not due to enhanced affinity for microtubules. Together, these results indicate that the CC domain may facilitate or maintain Kid accumulation on anaphase chromosomes by controlling the association between the DNA-binding domain of Kid and the chromosomes, thereby enabling dephosphorylated Kid to function in anaphase chromosome compaction but not in PEF generation.
Previous in vitro assays showed that the presence of the MTBD and CC domain enhanced the affinity of Kid for the same microtubule along which the motor domain moved, but did not reduce the velocity (Shiroguchi et al., 2003). Therefore, it is likely that Thr463-unphosphorylated Kid retains motility but cannot generate sufficient force on the binding chromosomes to transport them along the microtubule. Together, these findings suggest that the MTBD and CC domain are the key domains for switching Kid from the PEF-ON to the PEF-OFF mode upon the onset of anaphase in response to the phosphorylation status of Thr463.
The motor domain but not motor activity is essential for Kid to ensure formation of a single fPN
When GFP–Kid-Rigor-ΔMTBD or GFP–Kid-Rigor-ΔCC was expressed, only ∼44% of Kid-KO parthenogenetic zygotes showed the multi-fPN phenotype (Figs 3A and 4A). This indicates that the multi-fPN phenotype was rescued partially by Kid-Rigor-ΔMTBD [P<0.001 (Fig.3A) with control (−) in Kid-KO oocytes in Kid-KO oocytes (Fig. 2A), chi-squared test] or Kid-Rigor-ΔCC [P<0.01 (Fig. 4A) with control (−) in Kid-KO oocytes (Fig. 2A), chi-squared test]. To clarify the contribution of the MTBD, CC and motor domains to Kid-mediated fPN formation, we expressed GFP-tagged Kid-Rigor (containing only the rigor-inducing mutation) or Kid-ΔMot (lacking the Kid motor domain) constructs into Kid-KO oocytes. Despite similar localization on anaphase chromosomes, oocytes expressing GFP–Kid-ΔMot eventually formed multi-fPNs at a high frequency (73%) (Fig. 5A and B) [P=0.84 with control (−) BAF in Kid-KO oocytes (Fig. 2A), chi-squared test]. In contrast, expression of GFP–Kid-Rigor in oocytes reduced the formation of multi-fPNs to 37% (Fig. 5A and C) [P<0.001 with control (−) BAF in Kid-KO oocytes (Fig. 2A), chi-squared test]. These results indicate that the motor domain, but not motor activity, is essential for Kid to ensure formation of a single fPN. Because there were no significant differences in the formation of multi-fPNs among zygotes expressing Kid-Rigor, Kid-Rigor-ΔMTBD or Kid-Rigor-ΔCC (P=0.72, chi-squared test), the significance of the MTBD and CC domain in Kid-mediated anaphase chromosome compaction remain unclear.
In conclusion, the results of the current study show that both the MTBD and CC domain are dispensable for PEF generation but are essential for halting PEF generation after the onset of anaphase. The phosphorylation status of Thr463 of Kid functions as a switching mechanism to alter the functional modes (PEF-ON and PEF-OFF) of Kid on mitotic chromosomes (Fig. 5D). Based on our results, and previous knowledge, we propose the following model (Fig. 5D). (1) During prometaphase and metaphase, Cdk1–cyclin-B phosphorylates Kid on Thr463. This phosphorylation suppresses the MTBD-mediated affinity of Kid for microtubules, and enables Kid to localize and generate PEF on the chromosome arms. (2) Upon the onset of anaphase, Thr463 is dephosphorylated and the MTBD becomes functional. (3) Then, the MTBD together with the CC domain functions so that Kid can no longer generate PEF but rather resist this force.
In this study, we provide evidence that Cdk1–cyclin-B-mediated phosphorylation on Thr463 of Kid is essential for generating the PEF on the chromosome arms in mammalian cells during prometaphase and metaphase. Furthermore, phosphorylation-mimetic substitution of Thr463 to Asp resulted in ectopic generation of PEF during anaphase. This is consistent with a previous report that shows that anaphase chromosomes are pulled back to the spindle equator in a Kid-dependent manner when cells retain Cdk1 activity owing to the expression of non-degradable cyclin B1, (Wolf et al., 2006). Our data further indicate that among the numerous sites on many proteins that would be phosphorylated by Cdk1–cyclin-B, phosphorylation on Kid Thr463 is sufficient to generate ectopic PEF on anaphase chromosomes and realign them at the spindle equator. Based on these results, we propose that the phosphorylation status of Thr463 functions as a switch for Kid-mediated PEF generation, and that dephosphorylation promotes the dynamic directional change of mitotic chromosome movement from anti-poleward to poleward upon the metaphase–anaphase transition.
Our study provides further support for the concept that, in addition to sister chromatid disjunction, Kid-mediated PEF must be suspended when cells enter anaphase in order to ensure chromosome segregation, despite the fact that Kid-mediated PEF is dispensable for metaphase chromosome alignment in mammalian cells (Levesque and Compton, 2001; Tokai-Nishizumi et al., 2005; Ohsugi et al., 2008). Expression of a constitutively PEF-generating mutant such as Kid-T463D, Kid-ΔMTBD or Kid-ΔCC in Kid-deficient oocytes induced anaphase chromosome recongression in more than 70% of the cells, whereas only 26% or fewer cells showed anaphase chromosome recongression when expressed in wild-type oocytes (Fig. S2B). These results suggest that the cells can tolerate the presence of some phosphorylated Kid after the onset of anaphase. Although termination of PEF is achieved by the anaphase-promoting complex (APC/C)-proteasome-mediated degradation of Xenopus Kid in Xenopus egg extracts (Funabiki and Murray, 2000), in mammalian cells, this termination is achieved indirectly by the APC/C proteasome through degradation of cyclin B followed by dephosphorylation of Kid, contributing to the formation of proper daughter nuclei (Ohsugi et al., 2008). Thus, APC/C-proteasome-mediated protein degradation both triggers and maintains the poleward movement of anaphase chromosomes by eliminating both sister chromatid cohesion and Kid-mediated PEF. Because a substantial amount of Xenopus Kid has been reported to remain on anaphase chromosomes in a Xenopus somatic cell line (Antonio et al., 2000) and the amino acid sequences of the MTBD and CC domain, as well as Thr463, are highly conserved between Xenopus Kid and mammalian Kid (Shiroguchi et al., 2003), the dephosphorylation-mediated PEF-OFF mechanism could also be used in Xenopus during non-embryonic cell division.
In addition to the Kid-T463A mutant, other Kid mutants that lacked the MTBD or the CC domain also exhibited the chromosome-recongression phenotype. Although we cannot completely exclude the possibility that the chromosome recongression was due to defects in other functions of Kid rather than the (re-)activation of PEF, we believe that the most reasonable interpretation of the results is that these mutations afford constitutive PEF generation even after anaphase onset, resulting in the recongression phenotype. A previous study has demonstrated that bacterially expressed, and thus unphosphorylated, Kid515GSdC, in which the C-terminal region containing the DNA-binding domain of Kid is replaced with a portion of the gelsolin protein, is motile and transports an actin fragment along the microtubules (Shiroguchi et al., 2003). An in vitro study has further demonstrated that the presence or absence of the amino acid region 442–515, which includes both the MTBD and CC domain, as well as artificial dimerization of the Kid motor domain does not affect the velocity (Shiroguchi et al., 2003; Yajima et al., 2003). Therefore, it is unlikely that the MTBD and CC domain act simply as a brake system when Thr463 is dephosphorylated. The failure of PEF generation by Kid-T463A in prometaphase cells could be explained by the reduction of Kid protein on the chromosome arms due to sequestration to the spindle microtubules (Fig. 1; Ohsugi et al., 2003). However, despite the predominant localization on anaphase chromosomes, Kid-WT and Kid-T463A arrested PEF and mediated anaphase chromosome compaction, whereas Kid-T463D and Kid-ΔMTBD ectopically generated PEF (Figs 2B, 3B–D; Fig. S3E). These results imply that MTBD-mediated switching between the ON and OFF modes of Kid-mediated PEF is not accomplished simply through association with and dissociation from the chromosomes by changing the affinity for microtubules, but rather by changing the mode of action of Kid.
The CC domain of Kid is shorter than those of other kinesin-family motor proteins that function as oligomers (Verhey and Hammond, 2009). Although we cannot exclude the possibility that Kid forms an oligomer under certain conditions, such as a high concentration on chromosomes or microtubules, there is no evidence of CC-mediated oligomerization both in vivo and in vitro (Ohsugi et al., 2003; Shiroguchi et al., 2003). Unlike the majority of other motile kinesin-family proteins, the CC domain appears to be dispensable for Kid to transport its cargo (mitotic chromosome) along microtubules. Instead, the CC domain, as well as the MTBD, was required to halt the congressing force on chromosomes after anaphase onset. In addition to the failure in turning off PEF, deletion of the CC domain resulted in the failure of chromosomal accumulation. Instead, even in anaphase cells, Kid-ΔCC localized on both the chromosomes and spindles, as in prometaphase–metaphase cells. Further, because Kid-ΔMTBD-ΔCC showed anaphase spindle localization and Kid-Rigor, which shows increased microtubule-binding activity (Ohsugi et al., 2003), did not induce preferred localization on anaphase spindles (Fig. 5C; Ohsugi et al., 2003), the mislocalization caused by ΔCC was probably due to decreased affinity for anaphase chromosomes rather than increased affinity for microtubules. Therefore, we speculate that the CC domain is involved in anaphase chromosome accumulation by changing the mode of association with chromosomes, from binding them as cargo to binding them in order to cluster them, after anaphase onset, leading to a difference in the mobility of chromosome-binding Kid between metaphase and anaphase (Tahara et al., 2008). We speculate that the chromosome-bound fraction of Kid-ΔCC generates PEF, similar to the action of the chromosome-bound fraction of Kid-WT on prometaphase–metaphase chromosomes.
Although the essential difference of conformation of microtubule-Kid-chromosome complex between the PEF-ON and PEF-OFF modes of Kid remains unclear, Kid might bind to mitotic chromosomes and/or microtubules in distinct ways during metaphase and anaphase as a result of changes in its affinity for microtubules and mitotic chromosomes through the MTBD and the CC domain, respectively. Further exploration of the function of the CC domain will help to establish the precise mechanism underlying the functional switching of Kid. Our data also revealed that motility along the microtubule, and thus the PEF generation, was not essential for Kid function in ensuring nucleus formation in the mouse zygote. Further research should address whether the PEF-OFF state of Kid is coupled to Kid-mediated anaphase chromosome compaction.
MATERIALS AND METHODS
Cell culture and retrovirus infection
Kid+/+ and Kid−/− mouse embryonic fibroblasts (MEFs) obtained from embryos at embryonic day (E)14 (Ohsugi et al., 2008) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and serially passaged more than 28 times according to the 3T3 protocol (Todaro and Green, 1963), and were then used for experiments. Retrovirus-mediated expression of Kid mutants was performed as described previously (Ohsugi et al., 2003). For retrovirus production, pMX-puro vector (Onishi et al., 1996) encoding GFP, GFP–Kid-WT, GFP–Kid-T463A, GFP–Kid-ΔMTBD, GFP–Kid-ΔMTBD-T463A or GFP–Kid-ΔCC was transfected into PlatE packaging cells. Cells were incubated for 24 h in retrovirus-containing DMEM, treated with 100 nM Eg5 inhibitor III (Calbiochem) for an additional 24 h post transfection and then fixed and subjected to immunofluorescent staining.
Collection of oocytes and mRNA injection
Female Kid+/+ and Kid−/− mice having BalbC/A×C57BL6/J F1 background (6 weeks–6 months old) (Ohsugi et al., 2008) were induced to superovulate with intraperitoneal injections of 5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chronic gonadotropin (hCG) at 48-h intervals. The unfertilized oocytes (MII oocytes) were collected 16–17 h post hCG administration. The cumulus cells were removed through exposure to M2 medium (Sigma-Aldrich) containing 100 μg/ml hyaluronidase for less than 5 min, followed by pipetting into fresh M2 medium. The oocytes were cultured in M16 medium (Sigma-Aldrich) at 37°C under 5% CO2 before and after mRNA injection. The mRNAs were in vitro transcribed using RiboMax™ Large Scale RNA Production System-T7 (Promega) supplemented with Ribo m7G Cap Analog (Promega), as described previously (Yamagata et al., 2005). The template plasmids for in vitro mRNA production were constructed by inserting DNA fragments coding GFP–Kid mutants, GFP–BAF, GFP–α-tubulin or H2B–mRFP into pcDNA3.1-polyA vector, which was a gift from Kazuo Yamagata (Kinki University, Kinokawa, Wakayama, Japan). mRNAs (10–100 ng/μl) were injected into oocytes at metaphase II and cultured for 4 h before the oocytes were subjected to live-cell imaging or fixation. The experiments with animals were performed in accordance with the guidelines for animal use issued by the Committee of Animal Experiments, Institute of Medical Science, The University of Tokyo.
For parthenogenetic activation, oocytes were transferred into activation medium (ActM; M16 medium containing 5 mM SrCl2 and 5 mM EGTA) with or without 0.25 ng/ml cytochalasin B (Sigma-Aldrich) 4 h after mRNA injection. They were then subjected to live-cell imaging or were cultured at 37°C under 5% CO2 until subjected to immunofluorescent staining.
mRNA-injected oocytes were transferred into a parthenogenetic activation medium drop and covered with mineral oil in a glass-bottomed dish and observed using a fluorescent microscope (IX70, OLUMPUS, 20×0.85 NA oil objective lens, CoolSNAP HQ camera, Roper Scientific) that was controlled using Delta Vision SoftWorx (Applied Precision) (Fig. 2A; Fig. S2A), or a laser spinning disc confocal microscopy system utilizing a Yokogawa CSU22 instrument (IX71, OLUMPUS, 40×1.30 NA oil or 20×0.85 NA oil objective lens, iXon DU897E-CSO-#BV camera, ANDOR) controlled by Metamorph Software (Universal Imaging) (all figures except Fig. 2A and Fig. S2A ) equipped with a CO2 microscope stage incubator. Samples were scanned from their bottom to their top (21 optical sections, 4 μm optical section spacing, ActM imaging medium).
MEFs were fixed with ice-cold methanol for 10 min at −20°C. The mRNA-injected oocytes were denuded of their zona pellucida with acidic Tyrode's solution, washed with 0.5% polyvinyl pyrrolidone in PBS and fixed with ice-cold methanol for 10 min at −20°C. Fixed samples were incubated with a primary antibody solution and a secondary antibody solution sequentially. Observations were performed at 25°C with a fluorescent microscope (IX70, OLUMPUS, 100×1.35 NA oil objective lens, CoolSNAP HQ camera, Roper Scientific) that was controlled using Delta Vision SoftWorx (Applied Precision). GFP-positive MEF samples were scanned from their bottom to their top (60 optical sections, 0.2 μm optical section spacing, 80% glycerol with 1% N-propyl gallate imaging medium). For DNA distribution measurements, non-deconvolved image stacks were quick-projected, and the projected images were analyzed with ImageJ software. For quantification of the GFP signal, images were deconvolved, and the image stacks were quick-projected. Oocyte samples were scanned from the bottom of their spindles to the top (60 optical sections, 0.2 μm optical section spacing, 0.1% bovine serum albumin in PBS imaging medium). Images were deconvolved, and the image stacks were quick-projected. In the projected images of α-tubulin, the areas with higher signals than the mean value were judged as spindle microtubule regions. The diameters of these regions were measured as monopolar spindle diameters.
Antibodies and staining reagents
Primary antibodies against α-tubulin (Santa Cruz, YL1/2, 1:500 or Sigma-Aldrich, T9026, 1:500), β-tubulin (Sigma-Aldrich, 2-28-33; 1:500) and GFP (MBL, 598, 1:500 or BioAcademia, 60-001, 1:500) and secondary goat antibodies against mouse IgG (conjugated to Alexa-Fluor-555) (Invitrogen, A-21428, 1:800), rat IgG (conjugated to TRITC) (Invitrogen, A18870, 1:800), rat IgG (conjugated to Alexa-Fluor-488) (Invitrogen, A-21208, 1:800) and rabbit IgG (conjugated to Alexa-Fluor-488) (Invitrogen, A-11008, 1:800) were used for immunofluorescent staining. For DNA staining, Hoechst 33342 dye (Invitrogen) was used.
We thank K. Yamagata for materials and kind advice on the time-lapse observations of mouse embryos. We also thank Hiroshi Kimura (Tokyo Institute of Technology, Yokohama, Kanagawa, Japan) for the cDNA encoding histone H2B, Toshio Kitamura (The Institute of Medical Sciences, The University Tokyo, Minato-Ku, Tokyo, Japan) for the retrovirus vector systems, and N. Tokai-Nishizumi and T. Yamamoto for helpful discussions.
The authors declare no competing or financial interests.
S.S. and M.O. designed the research. K.Y.-N. performed expression experiments in Kid-KO oocytes. S.S. performed all other experiments and data analysis. S.S. and M.O. wrote the manuscript.
This work was supported by Japan Society for the Promotion of Science KAKENHI [grant number 14J10315 to S.S.]; Ministry of Education, Culture, Sports, Science, and Technology KAKENHI [grant numbers 20055004, 20570161, 26116505 and 15H05971 to M.O.]; the Ministry of Education, Culture, Sports, Science and Technology Grant Research Program of Innovative Cell Biology by Innovative Technology; and the Precursory Research for Embryonic Science and Technology program by Japan Science and Technology Agency.
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.189969.supplemental
- Received March 30, 2016.
- Accepted August 17, 2016.
- © 2016. Published by The Company of Biologists Ltd