Cdk phosphorylation licenses Kif4A chromosome localization required for early mitotic progression

Cdk phosphorylation licenses Kif4A chromosome localization required for early mitotic progression Abstract The chromokinesin Kif4A controls proper chromosome condensation, congression/alignment, and cytokinesis to ensure faithful genetic inheritance. Here, we report that Cdk phosphorylation of human Kif4A at T1161 licenses Kif4A chromosomal localization, which, in turn, controls Kif4A early mitotic function. Phosphorylated Kif4A (Kif4AWT) or Cdk phospho-mimetic Kif4A mutant (Kif4ATE) associated with chromosomes and condensin I (non-SMC subunit CAP-G and core subunit SMC2) to regulate chromosome condensation, spindle morphology, and chromosome congression/alignment in early mitosis. In contrast, Cdk non-phosphorylatable Kif4A mutant (Kif4ATA) could neither localize on chromosomes nor associate with CAP-G and SMC2. Furthermore, Kif4ATA could not rescue defective chromosome condensation, spindle morphology, or chromosome congression/alignment in cells depleted of endogenous Kif4A, which activated a mitotic checkpoint and delayed early mitotic progression. However, targeting Kif4ATA to chromosomes by fusion of Kif4ATA with Histone H1 resulted in restoration of chromosome and spindle functions of Kif4A, similar to Kif4AWT and Kif4ATE, in cells depleted of endogenous Kif4A. Thus, our results demonstrate that Cdk phosphorylation-licensed chromosomal localization of Kif4A plays a critical role in regulating early mitotic functions of Kif4A that are important for early mitotic progression. Kif4A, chromokinesin, Cdk phosphorylation, mitosis Introduction Mitosis, the last event of the cell cycle, controls accurate segregation of chromosomes into two daughter cells. To accomplish this, sister chromatids precisely duplicated in interphase must be compacted with ‘chromosome scaffold’ proteins to organize higher order structures, a process known as chromosome condensation (Belmont, 2006; Vagnarelli, 2012; Jeppsson et al., 2014; Kakui and Uhlmann, 2018). Subsequently, a unique cellular apparatus, the mitotic spindle, is assembled to drive congression/alignment of the condensed chromosomes at the metaphase plate in early mitosis (McIntosh et al., 2002; Walczak and Heald, 2008; Vicente and Wordeman, 2015). After proper chromosome congression/alignment, sister chromatids are segregated faithfully by the elongated mitotic spindle in late mitosis, followed by cell division into two daughter cells, a process known as cytokinesis (Glotzer, 2001; Green et al., 2012; Nguyen et al., 2014; D’Avino et al., 2015). Large numbers of chromosome-associated and/or spindle-binding proteins are involved in regulating these processes, including chromosome scaffold proteins, spindle assembly/disassembly proteins, microtubule (MT) regulatory proteins (e.g. non-motor structural proteins, motor proteins, kinases, and phosphatases), and spindle checkpoint proteins (Mack and Compton, 2001; Goshima and Vale, 2005; Zhu et al., 2005; Zinchuk et al., 2007; Subramanian et al., 2010; Barr et al., 2011; Samejima et al., 2012; Foley and Kapoor, 2013; Barisic et al., 2014; London and Biggins, 2014; Wang et al., 2014; Vallardi and Saurin, 2015). Deregulation and dysfunction of these proteins result in chromosome mis-segregation and mitotic defects, leading to chromosome instability and aneuploidy, contributing to the development and progression of many genetic diseases including cancer (Holland and Cleveland, 2012; Ly and Cleveland, 2017). The chromokinesin Kif4A, a member of the kinesin-4 family, plays important roles in regulating mitosis, including chromosome condensation, spindle assembly/dynamics, and chromosome congression/alignment in early mitosis and spindle midzone formation, spindle elongation, and cytokinesis in late mitosis (Mazumdar et al., 2004; Zhu and Jiang, 2005; Castoldi and Vernos, 2006; Hu et al., 2011; Samejima et al., 2012; Wandke et al., 2012; Subramanian et al., 2013; Nguyen et al., 2014). In early mitosis, Kif4A associates with chromosome scaffold proteins, such as condensin core subunit SMC2 and condensin I non-SMC subunit CAP-G on chromosomes to promote lateral compaction of chromosomal arms (Mazumdar et al., 2004; Samejima et al., 2012; Takahashi et al., 2016). Chromosome-associated Kif4A also contributes to the regulation of chromosome-interacting MT dynamics and cooperates with another chromokinesin, Kid, to generate polar ejection forces (PEF) (Wandke et al., 2012). Together with the kinetochore motor proteins, CENP-E, dynein, and Kif18A, these chromokinesins drive chromosome congression/alignment in early mitosis (Stumpff et al., 2012; Barisic et al., 2014; Iemura and Tanaka, 2015). In late mitosis, Kif4A translocates to the spindle midzone/midbody, directly binding to the non-kinetochore interdigitating anti-parallel MT-bundling protein PRC1 to control spindle midzone formation and elongation (Kurasawa et al., 2004; Zhu and Jiang, 2005; Bieling et al., 2010). Kif4A delivers PRC1 to the midzone MTs plus-ends in a MT length-dependent manner and stabilizes midzone MTs plus-ends to control midzone elongation (Hu et al., 2011). In addition, Kif4A is phosphorylated at T799/S801 by Aurora B and AMPK at spindle midzone that promotes Kif4A interaction with PRC1 and stimulates ATPase activity of Kif4A to regulate spindle midzone dynamics (Nunes Bastos et al., 2013; Bastos et al., 2014; Li et al., 2018). Despite intensive efforts in elucidating the role of Kif4A in controlling mitotic processes, the spatiotemporal regulation of Kif4A chromosome/spindle distribution remains elusive. In this study, we report that Cdk phosphorylation licenses the chromosomal localization of Kif4A, enabling it to perform its crucial functions in early mitotic progression. Results Cdk phosphorylation of Kif4A in early mitosis To investigate the regulation of Kif4A distribution between chromosomes and the mitotic spindle, we arrested HeLa cells in early mitosis with taxol treatment. As shown in Figure 1A, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the Kif4A band was shifted in taxol-treated cells relative to asynchronous control cells, suggesting that Kif4A was modified in early mitosis. To identify the potential post-translational modification(s), Kif4A was immunoprecipitated from taxol-treated cells with anti-Kif4A antibodies (α-Kif4A), separated by SDS-PAGE and subjected to mass spectrometry (MS) (Figure 1A). MS analysis revealed that Kif4A was mainly phosphorylated at threonine 1161 (T1161) (Supplementary Figure S1), which is conserved across many species within a consensus proline-directed Cdk phosphorylation motif (Figure 1B). Figure 1 View largeDownload slide Identification of Cdk phosphorylation of Kif4A at T1161 in early mitosis. (A) Kif4A displayed mobility shift in mitotic HeLa cells. HeLa cells were treated with 30 nM taxol overnight and cell lysates were immunoprecipitated with polyclonal α-Kif4A. Immunoprecipitates were separated by SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. Arrow indicates 140 kDa Kif4A protein. (B) Multi-sequence alignment of Kif4A showed that Kif4A T1161 (asterisk) was conserved in several organisms with a consensus Cdk phosphorylation motif. (C) Kif4A T1161 was phosphorylated by Cdk1/Cyclin B1 in vitro. Bacterially expressed GST-tagged wild-type (WT) and T1161 non-phosphorylation mutant (TA) of 230 amino acids (C230) at C-terminus of Kif4A were incubated with baculovirus-expressed cyclin B1−Cdk1 complex in the presence of [γ-32P] ATP for in vitro kinase assay. The reaction was analyzed by CBB staining (right) followed by autoradiography (left). Asterisk indicates the GST-Kif4A C230 protein. (D) Characterization of the polyclonal α-Kif4Ap1161. Asynchronous or taxol-treated HeLa cell lysates were immunoblotted with phospho-peptide-treated, nonphospho-peptide-treated, or non-treated α-Kif4Ap1161, α-Kif4A, and anti-α-tubulin antibody. (E and F) Immunofluorescence images of Kif4A and T1161-phosphorylated Kif4A in HeLa cells. HeLa cells were stained with polyclonal α-Kif4A (E) or α-Kif4Ap1161 (F) (green). DNA was visualized by DAPI staining (red). White scale bar, 5 μm; yellow scale bar, 1 μm. Figure 1 View largeDownload slide Identification of Cdk phosphorylation of Kif4A at T1161 in early mitosis. (A) Kif4A displayed mobility shift in mitotic HeLa cells. HeLa cells were treated with 30 nM taxol overnight and cell lysates were immunoprecipitated with polyclonal α-Kif4A. Immunoprecipitates were separated by SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. Arrow indicates 140 kDa Kif4A protein. (B) Multi-sequence alignment of Kif4A showed that Kif4A T1161 (asterisk) was conserved in several organisms with a consensus Cdk phosphorylation motif. (C) Kif4A T1161 was phosphorylated by Cdk1/Cyclin B1 in vitro. Bacterially expressed GST-tagged wild-type (WT) and T1161 non-phosphorylation mutant (TA) of 230 amino acids (C230) at C-terminus of Kif4A were incubated with baculovirus-expressed cyclin B1−Cdk1 complex in the presence of [γ-32P] ATP for in vitro kinase assay. The reaction was analyzed by CBB staining (right) followed by autoradiography (left). Asterisk indicates the GST-Kif4A C230 protein. (D) Characterization of the polyclonal α-Kif4Ap1161. Asynchronous or taxol-treated HeLa cell lysates were immunoblotted with phospho-peptide-treated, nonphospho-peptide-treated, or non-treated α-Kif4Ap1161, α-Kif4A, and anti-α-tubulin antibody. (E and F) Immunofluorescence images of Kif4A and T1161-phosphorylated Kif4A in HeLa cells. HeLa cells were stained with polyclonal α-Kif4A (E) or α-Kif4Ap1161 (F) (green). DNA was visualized by DAPI staining (red). White scale bar, 5 μm; yellow scale bar, 1 μm. To determine whether Kif4A T1161 could be phosphorylated by mitotic Cdk, we performed an in vitro kinase assay using bacterially expressed GST-Kif4A C-terminal fusion protein (GST-Kif4AWT) or its non-phosphorylatable counterpart (GST-Kif4ATA) as substrates. These recombinant proteins were purified and incubated with purified baculovirus-expressed cyclin B1-Cdk1 complex in the presence of [γ-32P] ATP. As shown in Figure 1C, GST-Kif4AWT, but not GST-Kif4ATA, was phosphorylated efficiently by Cdk1/Cyclin B1, demonstrating that T1161 in Kif4A was a substrate for Cdk1/Cyclin B1 in vitro. We generated rabbit polyclonal phospho-specific antibodies against T1161 (α-Kif4Ap1161) and immunoblotting analysis indicated that α-Kif4Ap1161 specifically recognized Kif4A in lysates from taxol-treated cells but not in asynchronous control cells (Figure 1D and Supplementary Figure S2A). Furthermore, T1161 phospho-peptide competition or treatment with the Cdk1 inhibitor, roscovitine, abolished the immuno-reactivity of α-Kif4Ap1161 in taxol-treated cells (Figure 1D and Supplementary Figure S2A). As Kif4A could be phosphorylated at T1161 by Cdk1/Cyclin B1 in vitro, this suggests that Kif4A is a physiological mitotic Cdk substrate and phosphorylated at T1161 during early mitosis. Consistent with this hypothesis, immunoblotting analysis and co-immunofluorescence using affinity-purified α-Kif4Ap1161 and cyclin B1 antibodies revealed that phosphorylation of Kif4A at T1161 occurred in G2 phase cells when cyclin B1 was expressed, and diminished at the onset of anaphase, when cyclin B1 was degraded (Supplementary Figures S2B, S3A and B). In addition, immunofluorescence analysis using affinity-purified α-Kif4A showed that Kif4A was mainly localized on chromosomes, especially on chromosomal arms laterally, in early mitosis but translocated to the spindle midzone and midbody in late mitosis as we and others reported previously (Figure 1E and Supplementary Figure S3C) (Mazumdar et al., 2004; Zhu and Jiang, 2005). In contrast, α-Kif4Ap1161 only detected chromosomal arm-localized Kif4A in early mitosis, not spindle midzone- and midbody-localized Kif4A in late mitosis (Figure 1F and Supplementary Figure S3D). Taken together, these results demonstrated that chromosome-associated Kif4A was phosphorylated by mitotic Cdk in early mitosis. Regulation of Kif4A chromosomal localization and function by Cdk phosphorylation in early mitosis We investigated whether Cdk phosphorylation of Kif4A was involved in regulating Kif4A localization/function in early mitosis. We generated a set of mammalian expression vectors fusing green fluorescent protein (GFP) to wild-type Kif4A (GFP-Kif4AWT), the phosphorylation site mutant (Kif4ATA), or the phospho-mimetic mutant (Kif4ATE) and expressed these plasmids in HeLa cells. Fluorescence imaging showed that ectopically expressed GFP-Kif4AWT or GFP-Kif4ATE protein was mainly localized on chromosomal arms laterally in early mitosis similar to endogenous Kif4A protein (Figure 2A; Supplementary Figure S4A and C). In contrast, GFP-Kif4ATA protein was not detected on chromosomes but mainly localized in the cytoplasm in early mitosis (Figure 2A and Supplementary Figure S4B). When cells were pre-extracted with digitonin before fixation to remove cytoplasmic soluble proteins, fluorescence imaging revealed that, unlike GFP-Kif4AWT or GFP-Kif4ATE, GFP-Kif4ATA was not localized on chromosomes (Figure 2B). Immunoblotting analysis also showed that GFP-Kif4AWT or GFP-Kif4ATE but not GFP-Kif4ATA could be recognized by α-Kif4Ap1161 in early mitosis, confirming the specificity of this reagent (Supplementary Figure S4D). Furthermore, as previously reported that Kif4A and condensin core subunit SMC2 were colocalized on the chromosomes (Mazumdar et al., 2004; Samejima et al., 2012), co-fluorescence imaging and Pearson correlation coefficient (R-value) analysis revealed that GFP-Kif4AWT or GFP-Kif4ATE but not GFP-Kif4ATA was colocalized with SMC2 on chromosomal arms laterally (Figure 2C and D). Thus, these results demonstrated that phosphorylation of Kif4A by mitotic Cdk at T1161 regulated its chromosomal localization in early mitosis. Figure 2 View largeDownload slide Regulation of chromosomal localization of Kif4A by Cdk phosphorylation. (A) HeLa cells grown on coverslips were transfected with pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and stained with mouse anti-α-tubulin antibody (gray) and DAPI (red). Scale bar, 5 μm. More than 20 cells in three independent experiments were observed and representative images are shown. (B) HeLa cells were transfected with plasmids as in A and then treated with 5 μg/ml digitonin followed by formaldehyde fixation and fluorescent staining with mouse anti-α-tubulin antibody (gray) and DAPI (red). More than 20 cells in three independent experiments were observed and representative images are shown. (C) HeLa cells were transfected with plasmids as in A. Cells were fixed and stained with rabbit α-SMC2 (red) and DAPI (cyan). Scale bar, 5 μm. (D) The Pearson’s correlation coefficients (R-value) of GFP-Kif4A vs. SMC2 in C were determined as described in ‘Materials and methods’ section. Figure 2 View largeDownload slide Regulation of chromosomal localization of Kif4A by Cdk phosphorylation. (A) HeLa cells grown on coverslips were transfected with pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and stained with mouse anti-α-tubulin antibody (gray) and DAPI (red). Scale bar, 5 μm. More than 20 cells in three independent experiments were observed and representative images are shown. (B) HeLa cells were transfected with plasmids as in A and then treated with 5 μg/ml digitonin followed by formaldehyde fixation and fluorescent staining with mouse anti-α-tubulin antibody (gray) and DAPI (red). More than 20 cells in three independent experiments were observed and representative images are shown. (C) HeLa cells were transfected with plasmids as in A. Cells were fixed and stained with rabbit α-SMC2 (red) and DAPI (cyan). Scale bar, 5 μm. (D) The Pearson’s correlation coefficients (R-value) of GFP-Kif4A vs. SMC2 in C were determined as described in ‘Materials and methods’ section. We performed siRNA knockdown-rescue experiments to determine whether Cdk phosphorylation of Kif4A controlled its early mitotic functions. HeLa cells were transfected with Kif4A siRNA targeting the 3′-UTR of Kif4A mRNA together with vectors expressing siRNA-resistant GFP-Kif4AWT, GFP-Kif4ATA or GFP-Kif4ATE cDNAs lacking the 3′-UTR. Immunoblotting analysis showed that Kif4A siRNA could effectively ablate expression of endogenous Kif4A protein but did not affect ectopic expression of GFP-Kif4AWT, GFP-Kif4ATA, or GFP-Kif4ATE protein (Figure 3A). Analyses of mitotic index and different stages of early mitosis revealed that cells expressing GFP-Kif4AWT or GFP-Kif4ATE but lacking endogenous Kif4A displayed normal mitotic index and percentages of cells at different stages of early mitosis, similar to control cells (nonsence siRNA-transfected and GFP-expressing cells). In contrast, like cells depleted of Kif4A (Kif4A siRNA-transfected and GFP-expressing cells), cells expressing GFP-Kif4ATA but lacking endogenous Kif4A displayed increased mitotic index with accumulation of cells in prometaphase and metaphase (Figure 3B and C; Supplementary Table S1). Figure 3 View largeDownload slide Regulation of early mitotic progression by Cdk phosphorylation of Kif4A. (A) HeLa cells transfected with NS siRNA or Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were collected and lysed. Cell lysates were immunoblotted with the indicated antibodies. (B and C) HeLa cells were transfected with Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and stained with mouse anti-α-tubulin antibody and DAPI. Mitotic index (B) and the percent of early mitotic cells in whole mitotic cells (C) were counted and shown in histogram (mean + SD of three independent experiments; Student’s t-test, **P < 0.01; ns, not significant). (D−F) HeLa cells stably expressing CFP-Histone H2B were transfected with expression vectors encoding the indicated siRNA-resistant mCherry-Kif4A together with Kif4A siRNA for 36 h. Time-lapse images were collected every 1 min for 12 h. Duration of prometaphase (prometa: from nuclear envelope breakdown to chromosome alignment; red), metaphase (meta: from chromosome alignment to anaphase onset; blue), anaphase/telophase (ana/telo: from anaphase onset to chromosome decondensation; green), or aneuploidy (from anaphase onset to aneuploidy; magenta) is indicated in D. The data represent 25 cells for each category and the average duration of mitotic phase is shown in E. Results represent mean ± SD. Representative images of time-lapse microscopy are shown in F. Figure 3 View largeDownload slide Regulation of early mitotic progression by Cdk phosphorylation of Kif4A. (A) HeLa cells transfected with NS siRNA or Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were collected and lysed. Cell lysates were immunoblotted with the indicated antibodies. (B and C) HeLa cells were transfected with Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and stained with mouse anti-α-tubulin antibody and DAPI. Mitotic index (B) and the percent of early mitotic cells in whole mitotic cells (C) were counted and shown in histogram (mean + SD of three independent experiments; Student’s t-test, **P < 0.01; ns, not significant). (D−F) HeLa cells stably expressing CFP-Histone H2B were transfected with expression vectors encoding the indicated siRNA-resistant mCherry-Kif4A together with Kif4A siRNA for 36 h. Time-lapse images were collected every 1 min for 12 h. Duration of prometaphase (prometa: from nuclear envelope breakdown to chromosome alignment; red), metaphase (meta: from chromosome alignment to anaphase onset; blue), anaphase/telophase (ana/telo: from anaphase onset to chromosome decondensation; green), or aneuploidy (from anaphase onset to aneuploidy; magenta) is indicated in D. The data represent 25 cells for each category and the average duration of mitotic phase is shown in E. Results represent mean ± SD. Representative images of time-lapse microscopy are shown in F. We performed time-lapse microscopy with depletion of Kif4A and expression of mCherry-Kif4AWT or its mutant versions in HeLa cells stably expressing Histone H2B-CFP (Supplementary Figure S5A). Live-cell imaging revealed that mCherry-Kif4AWT or mCherry-Kif4ATE expressed in cells depleted of endogenous Kif4A localized to chromosomes in early mitosis (Supplementary Figure S5B). Similar to control cells, cells expressing mCherry-Kif4AWT or mCherry-Kif4ATE but lacking endogenous Kif4A exhibited normal chromosome congression in prometaphase and chromosome alignment in metaphase, progressing through early mitosis ~35 min (as measured from nuclear envelope breakdown to the onset of anaphase) (Figure 3D−F; Supplementary Movies S1, S3, and S4). In contrast, mCherry-Kif4ATA did not localize to chromosomes but was instead mainly cytoplasmic (Supplementary Figure S5B). Like cells depleted of Kif4A alone, cells expressing mCherry-Kif4ATA but lacking endogenous Kif4A displayed frequent chromosome oscillations and abnormal congression in prometaphase and nonaligned chromosomes with broader metaphase plates. These cells showed prolonged progression of early mitosis with significant delays around ~90 min (Figure 3D−F; Supplementary Movies S2 and S5). These results indicated that Cdk phosphorylation of Kif4A mediates its chromosomal localization, and is thus critical for the functions of Kif4A required for early mitotic progression. Effects on MT dynamics, spindle morphology, and chromosome congression/alignment by Cdk phosphorylation of Kif4A To determine in further detail how Cdk phosphorylation of Kif4A was involved in regulating its functions required for early mitotic progression, we examined spindle structures and chromosome congression/alignment in control cells and cells depleted of endogenous Kif4A with or without exogenous expression of GFP-Kif4AWT, GFP-Kif4ATA, or GFP-Kif4ATE. Immunofluorescence analysis indicated that depletion of Kif4A in HeLa cells (Kif4A siRNA-transfected and GFP-expressing cells) resulted in short and plump chromosomes with more barrel-like spindles in prometaphase and disorganized chromosome congression/alignment with broader metaphase plates in metaphase when compared with control cells (Supplementary Figure S6) (Wandke et al., 2012). GFP-Kif4AWT or GFP-Kif4ATE expressed in cells lacking endogenous Kif4A localized on chromosomes and rescued virtually all chromosome and spindle defects, including the defects of spindle and chromosome morphologies and abnormalities of chromosome congression/alignment in early mitosis (Supplementary Figure S6). In contrast, GFP-Kif4ATA expressed in cells lacking endogenous Kif4A did not localize to chromosomes in early mitosis and could not rescue the chromosome and spindle defects (Supplementary Figure S6). Consistent with these results, cells depleted of Kif4A alone or cells depleted of Kif4A and expressing GFP-Kif4ATA activated the mitotic spindle checkpoint persistently. Immunofluorescence analysis showed that the mitotic spindle checkpoint protein, BubR1, was detected in metaphase cells depleted of Kif4A alone or metaphase cells depleted of Kif4A and expressing GFP-Kif4ATA but not in control metaphase cells or metaphase cells depleted of Kif4A and expressing GFP-Kif4AWT or GFP-Kif4ATE (Supplementary Figure S7). Previous studies showed that the chromokinesins Kif4A and Kid independently controlled the positioning of chromosome arms and the dynamics of spindle MTs for chromosome congression/alignment by regulating PEF in early mitosis (Brouhard and Hunt, 2005; Wandke et al., 2012). It was proposed that while Kid regulated PEFs through the control of chromosome-associated MTs by stabilizing the orientation of chromosome arms (Funabiki and Murray, 2000; Levesque and Compton, 2001), Kif4A mainly contributed to PEFs through the control of chromosome-associated MT dynamics by limiting the elongation of MTs near chromatin (Bringmann et al., 2004; Hu et al., 2011). Consistent with these reports, we found that ablation of both Kif4A and Kid proteins with siRNAs simultaneously resulted in elongated spindles and broader metaphase plates with uncongressed and unaligned chromosomes in metaphase when compared with control cells or cells depleted of either Kif4A or Kid alone (Figure 4A−C; Supplementary Figure S8A and Table S1). However, cells with monopolar spindles, generated with inhibition of Eg5 with S-trityl-L-cysteine (STLC), revealed that although Kif4A and Kid were individually required for chromosome ejection from the monopole, only Kif4A was required for limitation of monopolar spindle MT length (Figure 4D−F; Supplementary Figure S8B and Table S1). Inhibition of MT dynamic instability of cells with monopolar spindles by low concentration of nocodazole abrogated monopolar spindle MT elongations in cells depleted of Kif4A alone or both Kif4A and Kid (Supplementary Figure S9 and Table S1). Thus, these results indicated that the chromokinesins, Kif4A and Kid, controlled the chromosome positioning independently and only Kif4A was involved in regulating chromosome-associated MT dynamics, which were required for spindle structures and chromosome congression/alignment in early mitosis. Figure 4 View largeDownload slide Regulation of MT dynamics, spindle morphology, and chromosome congression/alignment by Cdk phosphorylation of Kif4A. (A−C) HeLa cells were transfected with Kif4A or/and Kid siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and immunostained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in A. Scale bar, 5 μm. The biopolar spindle length (B) and widths of metaphase plates (C) were measured. (D−F) HeLa cells were transfected with Kif4A or/and Kid siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 32 h and treated with 5 μM STLC for 16 h. Cells were fixed and immunostained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in D. Scale bar, 5 μm. The monopolar spindle length (E) and the distance of kinetochore to pole (KT-to-pole) (F) were determined as described in ‘Materials and methods’ section. Results represent mean + SD from three independent experiments (student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001). Figure 4 View largeDownload slide Regulation of MT dynamics, spindle morphology, and chromosome congression/alignment by Cdk phosphorylation of Kif4A. (A−C) HeLa cells were transfected with Kif4A or/and Kid siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and immunostained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in A. Scale bar, 5 μm. The biopolar spindle length (B) and widths of metaphase plates (C) were measured. (D−F) HeLa cells were transfected with Kif4A or/and Kid siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 32 h and treated with 5 μM STLC for 16 h. Cells were fixed and immunostained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in D. Scale bar, 5 μm. The monopolar spindle length (E) and the distance of kinetochore to pole (KT-to-pole) (F) were determined as described in ‘Materials and methods’ section. Results represent mean + SD from three independent experiments (student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001). We expressed GFP-Kif4AWT, GFP-Kif4ATA, or GFP-Kif4ATE in cells depleted of both endogenous Kif4A and Kid in either a bipolar spindle or a monopolar configuration. Expression of GFP-Kif4AWT or GFP-Kif4ATE in cells depleted of both Kif4A and Kid rescued early mitotic spindle length, widths of metaphase plates, chromosome ejection, or monopolar spindle MT length significantly. Like cells depleted of Kid alone, cells depleted of both Kif4A and Kid and expressing GFP-Kif4AWT or GFP-Kif4ATE showed significant smaller bipolar spindles, thinner metaphase plates, stronger chromosome ejection, and shorter monopolar MT lengths when compared with cells depleted of Kif4A alone or both Kif4A and Kid (Figure 4A−F; Supplementary Figure S8A and Table S1). In contrast, expression of GFP-Kif4ATA in cells depleted of both Kif4A and Kid rescued neither abnormalities of spindle structure and chromosome congression/alignment in a bipolar configuration nor abnormal chromosome ejection and limitation of monopolar spindle MT lengths in a monopolar spindle configuration. Like cells depleted of both Kif4A and Kid, cells depleted of both Kif4A and Kid and expressing GFP-Kif4ATA exhibited elongated spindles, broader metaphase plates with uncongressed and unaligned chromosomes, weaker chromosome ejection and longer monopolar MT lengths when compared with control cells or cells depleted of Kid alone (Figure 4A−F; Supplementary Figure S8A and Table S1). Taken together, these results demonstrated that Cdk phosphorylation of Kif4A controlled Kif4A chromosomal localization to promote its chromosome-associated functions in regulating MT dynamics, spindle morphology, and chromosome congression/alignment required for mitotic progression in early mitosis. Effects on chromosome condensation by Cdk phosphorylation of Kif4A As a chromokinesin, Kif4A was also shown to be cooperated with condensins to supercoil and compact chromatid arms laterally (Mazumdar et al., 2004; Samejima et al., 2012). To further examine Cdk phosphorylation of Kif4A in regulating chromosome condensation, we performed chromosome spreading experiments, in which control cells, cells depleted of endogenous Kif4A, cells depleted of endogenous Kif4A but expressing GFP-Kif4AWT, GFP-Kif4ATA, or GFP-Kif4ATE were arrested in mitosis by colcemid treatment, and then chromosomes were spread on slides and stained with DAPI. Fluorescent intensities of spread chromosomes and widths of individual chromosome arms were analyzed under a fluorescent microscope. Short and plump chromosomes and decreased chromosome fluorescent intensities were detected in cells depleted of endogenous Kif4A and cells depleted of endogenous Kif4A but expressing GFP-Kif4ATA when compared with control cells and cells depleted of endogenous Kif4A but expressing GFP-Kif4AWT or GFP-Kif4ATE (Figure 5A, Supplementary Figure S10A). We measured the widths and fluorescent intensities of individual chromosome arms and found that cells depleted of endogenous Kif4A and cells depleted of endogenous Kif4A but expressing GFP-Kif4ATA displayed wider chromosome arms with low fluorescent intensities than control cells and cells depleted of endogenous Kif4A but expressing GFP-Kif4AWT or GFP-Kif4ATE (Figure 5B and C, Supplementary Table S1). These results indicated that Cdk phosphorylation of Kif4A was involved in regulating chromosome condensation in early mitosis. Figure 5 View largeDownload slide Regulation of chromosome condensation by Cdk phosphorylation of Kif4A. HeLa cells were transfected with NS siRNA or Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. (A−C) Cells were treated with 0.1 μg/ml of colcemid overnight and chromosomes were spread on coverslips followed by immunostaining with DAPI (red). Representative images are shown in A. Scale bar, 5 μm. Widths of individual chromosome arms (B) and fluorescence intensity of DAPI (C) were measured as described in ‘Materials and methods’ section. More than 100 chromosomes in three independent experiments were analyzed (student’s t-test, **P < 0.01; ns, not significant). (D) Cells were lysed and immunoprecipitated with mouse α-CAP-G, rabbit α-CAP-D3, or rabbit α-SMC2. The immunoprecipitates were then immunoblotted with antibodies as indicated. (E) Cells were fixed and stained with rabbit α-SMC2 and DAPI. The boxes (arrowhead) represent the region used to generate the line scans, which show the localization of SMC2 (red) relative to DNA (cyan). Scale bar, 5 μm. (F) Quantification of relative SMC2 staining intensity in E. Fluorescence intensities of SMC2 were determined at five chromosome arms (chromosomal) and five areas in the cytoplasm (cytoplasmic) in each 20 cells. Figure 5 View largeDownload slide Regulation of chromosome condensation by Cdk phosphorylation of Kif4A. HeLa cells were transfected with NS siRNA or Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. (A−C) Cells were treated with 0.1 μg/ml of colcemid overnight and chromosomes were spread on coverslips followed by immunostaining with DAPI (red). Representative images are shown in A. Scale bar, 5 μm. Widths of individual chromosome arms (B) and fluorescence intensity of DAPI (C) were measured as described in ‘Materials and methods’ section. More than 100 chromosomes in three independent experiments were analyzed (student’s t-test, **P < 0.01; ns, not significant). (D) Cells were lysed and immunoprecipitated with mouse α-CAP-G, rabbit α-CAP-D3, or rabbit α-SMC2. The immunoprecipitates were then immunoblotted with antibodies as indicated. (E) Cells were fixed and stained with rabbit α-SMC2 and DAPI. The boxes (arrowhead) represent the region used to generate the line scans, which show the localization of SMC2 (red) relative to DNA (cyan). Scale bar, 5 μm. (F) Quantification of relative SMC2 staining intensity in E. Fluorescence intensities of SMC2 were determined at five chromosome arms (chromosomal) and five areas in the cytoplasm (cytoplasmic) in each 20 cells. We determined whether Cdk phosphorylation of Kif4A regulated chromosome condensation by cooperation with condensin. Co-immunoprecipitation revealed that, in control cells, condensin core subunit SMC2 associated with endogenous Kif4A as previously reported (Supplementary Figure S10B) (Mazumdar et al., 2004; Samejima et al., 2012). As expected, Kif4A was not present in α-SMC2 immunoprecipitates from cells in which Kif4A was depleted by siRNA. Furthermore, GFP-Kif4AWT or GFP-Kif4ATE expressed in cells depleted of Kif4A were able to associate with condensin I subunit CAP-G and SMC2 but not condensin II subunit CAP-D3 (Figure 5D) (Takahashi et al., 2016). In contrast, GFP-Kif4ATA expressed in cells depleted of Kif4A did not associate with CAP-G, SMC2, and CAP-D3 (Figure 5D). Consistent with these results, immunofluorescence analysis showed that cells depleted of endogenous Kif4A displayed decreased chromosome localization of SMC2 when compared with control cells. Expression of GFP-Kif4AWT or GFP-Kif4ATE in cells depleted of endogenous Kif4A restored chromosomal localization of SMC2. However, cells depleted of endogenous Kif4A and expressing GFP-Kif4ATA still showed reduction of chromosomal localization of SMC2, similar to cells depleted of endogenous Kif4A (Figure 5E and F). Taken together, these results indicate that Cdk phosphorylation of Kif4A controls its localization and promotes its association with condensin I, which is required for chromosome condensation. Chromosomal localization rather than Cdk phosphorylation of Kif4A is crucial for early mitotic functions of Kif4A We asked whether Cdk phosphorylation of Kif4A is crucial for chromosome localization and its mitotic functions or whether it simply serves to license the chromosomal localization of Kif4A but is nonessential for its subsequent functioning. To distinguish between these two possibilities, we targeted non-phosphorylatable Kif4A (GFP-Kif4ATA) to chromosomes by fusing Kif4A with Histone H1. We generated a set of mammalian expression vectors expressing GFP-Kif4AWT, GFP-Kif4ATA, and GFP-Kif4ATE fused with Histone H1 protein at the C-terminus of Kif4A (GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, and GFP-Kif4ATE-H1) (Supplementary Figure S11A). As a control, we also generated a mammalian expression construct expressing GFP-Kif4AMD-H1, in which the Kif4A ATP-binding site was mutated (Kif4A motor-dead mutant; Zhu and Jiang, 2005). We expressed these plasmids in cells depleted of endogenous Kif4A. Immunoblotting analysis showed that GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, GFP-Kif4ATE-H1, and GFP-Kif4AMD-H1 were expressed in Kif4A depleted cells (Supplementary Figure S11B). As shown in Figure 6A, immunofluorescence analysis revealed that, like GFP-Kif4AWT and GFP-Kif4ATE, GFP-Kif4AWT-H1 and GFP-Kif4ATE-H1 were localized on chromosomes in early mitotic cells lacking endogenous Kif4A. However, unlike GFP-Kif4ATA, which was expressed mainly in the cytoplasm, GFP-Kif4ATA-H1 was localized on chromosomes in early mitotic cells lacking endogenous Kif4A. As expected, GFP-Kif4AMD-H1 was also localized on chromosomes in early mitotic cells lacking endogenous Kif4A. Figure 6 View largeDownload slide Chromosomal localization of Kif4A is crucial for Kif4A early mitotic functions. HeLa cells were transfected with Kif4A siRNA together with pEGFP-Kif4AWT-H1, pEGFP-Kif4ATA-H1, pEGFP-Kif4ATE-H1, or pEGFP-Kif4AMD-H1 plasmids for 48 h. (A and B) Cells were fixed and stained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in A. Scale bar, 5 μm. Widths of metaphase plates were measured (B). (C−E) Cells were treated with 0.1 μg/ml colcemid overnight and chromosomes were spread on coverslips followed by immunostaining with DAPI (red). Representative images are shown in C. Scale bar, 5 μm. Widths of individual chromosome arms (D) and fluorescence intensity of DAPI (E) were measured. More than 100 chromosomes in three independent experiments were analyzed (student’s t-test, **P < 0.01; ns, not significant). (F) Cells were fixed and stained with rabbit α-SMC2 (red) and DAPI (cyan). The boxes (arrowhead) represent the region used to generate the line scans, which show the localization of SMC2 (red) relative to DNA (cyan). Scale bar, 5 μm. (G) Quantification of relative SMC2 staining intensity in F. Fluorescence intensities of SMC2 were determined at five chromosome arms (chromosomal) and five areas in the cytoplasm (cytoplasmic) in each 20 cells. Figure 6 View largeDownload slide Chromosomal localization of Kif4A is crucial for Kif4A early mitotic functions. HeLa cells were transfected with Kif4A siRNA together with pEGFP-Kif4AWT-H1, pEGFP-Kif4ATA-H1, pEGFP-Kif4ATE-H1, or pEGFP-Kif4AMD-H1 plasmids for 48 h. (A and B) Cells were fixed and stained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in A. Scale bar, 5 μm. Widths of metaphase plates were measured (B). (C−E) Cells were treated with 0.1 μg/ml colcemid overnight and chromosomes were spread on coverslips followed by immunostaining with DAPI (red). Representative images are shown in C. Scale bar, 5 μm. Widths of individual chromosome arms (D) and fluorescence intensity of DAPI (E) were measured. More than 100 chromosomes in three independent experiments were analyzed (student’s t-test, **P < 0.01; ns, not significant). (F) Cells were fixed and stained with rabbit α-SMC2 (red) and DAPI (cyan). The boxes (arrowhead) represent the region used to generate the line scans, which show the localization of SMC2 (red) relative to DNA (cyan). Scale bar, 5 μm. (G) Quantification of relative SMC2 staining intensity in F. Fluorescence intensities of SMC2 were determined at five chromosome arms (chromosomal) and five areas in the cytoplasm (cytoplasmic) in each 20 cells. We analyzed chromosome condensation and congression/alignment in cells depleted of endogenous Kif4A and expressing GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, GFP-Kif4ATE-H1, or GFP-Kif4AMD-H1. As shown in Figure 6A and B, Supplementary Table S1, expression of chromosome-associated GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, or GFP-Kif4ATE-H1, but not GFP-Kif4AMD-H1, in cells depleted of endogenous Kif4A could rescue abnormalities of chromosome congression/alignment. Consistent with these results, the mitotic spindle checkpoint BubR1 was not detected in metaphase cells depleted of endogenous Kif4A but expressing GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, or GFP-Kif4ATE-H1. In contrast, BubR1 was still detected in metaphase cells depleted of endogenous Kif4A but expressing GFP-Kif4AMD-H1 (Supplementary Figure S12). Chromosome spreading experiments indicated that expression of GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, or GFP-Kif4ATE-H1, but not GFP-Kif4AMD-H1, in cells depleted of endogenous Kif4A could restore chromosome fluorescent intensities and widths of chromosome arms to the levels of control cells (Figure 6C−E, Supplementary Table S1). Consistent with these results, immunofluorescence analysis indicated that SMC2 was reloaded on chromosomes in cells depleted of endogenous Kif4A and expressing chromosome-associated GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, or GFP-Kif4ATE-H1, but not GFP-Kif4AMD-H1 (Figure 6F and G). These results indicated that targeting non-phosphorylatable Kif4ATA to chromosomes by Histone H1 was sufficient to restore Kif4A early mitotic function. Once Kif4A is localized to chromosomes, its Cdk-dependent phosphorylation is dispensable for carrying out its subsequent activities even though the ATP-dependent catalytic activity of Kif4A is still essential. Discussion Our study provides significant insights into the spatiotemporal regulation of the chromokinesin, Kif4A, in early mitosis. We show that Cdk phosphorylation of Kif4A licenses its chromosome localization. Kif4A, in turn, functions as a chromokinesin, regulates chromosome condensation, MT dynamics, spindle morphology and chromosome congression/alignment that are important for early mitotic progression. Cdk phosphorylation-licensed chromosome-associated Kif4A associates with CAP-G and SMC2 to promote chromosome condensation by participating in the regulation of mutual dependence of Kif4A and SMC2 on chromosomes for lateral compaction of chromosome arms (Figure 5) (Samejima et al., 2012). Moreover, Cdk phosphorylation-licensed chromosome-associated Kif4A is involved in regulating MT dynamics, spindle morphology, chromosome congression/alignment required for early mitotic progression. Chromosome-associated Kif4A coordinates with Kid to generate PEF and/or limit the elongation of chromosome-associated MTs in the vicinity of chromosomes (Figure 4) (Hu et al., 2011; Wandke et al., 2012). Targeting non-phosphorylatable Kif4ATA to chromosomes by fusion of Kif4ATA with Histone H1; however, could restore early mitotic functions of Kif4A in cells depleted of endogenous Kif4A (Figure 6). Thus, Cdk phosphorylation-licensed chromosome localization of Kif4A was crucial for early mitotic functions of Kif4A and early mitotic progression. Previous studies demonstrated that, as mitotic-promoting factors, mitotic Cdks phosphorylated multiple G2/M and early mitotic regulators, controlling their subcellular distribution, activities, and function that were critical for proper G2/M and early mitotic progression (Malik et al., 2009; Fisher et al., 2012). As a chromokinesin, Kif4A is involved in controlling chromosome condensation and chromosome congression/alignment in early mitosis. Our results reveal that Kif4A is phosphorylated by Cdk at its tail domain (T1161) and Cdk phosphorylation of Kif4A spatiotemporally regulates chromosome localization of Kif4A in early mitosis. Kif4A (and its cross-species homologs XKpl1 in Xenopus and Klp3A in Drosophila) contains two conserved DNA-binding motifs, a leucine zipper motif at its stalk region and a cysteine-rich motif at its tail domain, that were shown to be essential for chromatin-binding activity of Kif4A and subsequent chromosome localization of Kif4A in mitosis (Hu et al., 2011; Wandke et al., 2012). Our studies show that Cdk phosphorylation of Kif4A at its tail domain (T1161) only licenses chromosome localization of Kif4A but is not required for its chromosome-associated functions. As Kif4ATA-Histone H1 is fully functional, we propose that Cdk phosphorylation of Kif4A at T1161 might affect the total conformation of Kif4A molecule, depict the DNA-binding motifs of Kif4A to DNA/chromosomes and/or associate with condensin I (CAP-G/SMC2) (Takahashi et al., 2016), promoting Kif4A association with chromosomes in early mitosis. Once Kif4A localizes on chromosomes, chromosome-associated Kif4A, no matter whether T1161 is phosphorylated, would cooperate with condensin I to compact chromosome arms laterally for chromosome condensation. Meanwhile, chromosome condensation/chromosome arm compaction would reinforce Kif4A association with chromosome arms laterally. Lateral chromosomal arms-associated Kif4A would then function as a MT-dependent motor with MT plus-end ‘capping’ activity to regulate dynamics of chromosome-associated MTs. Chromosome-associated Kif4A could control dynamics of chromosome-associated MTs for proper formation and dynamics of mitotic spindle in early mitosis and/or coordinate with other mitotic motor proteins, such as Kid, Kif18A, CENP-E, and dynein, for chromosome congression/alignment (Stumpff et al., 2012; Wandke et al., 2012; Barisic et al., 2014; Iemura and Tanaka, 2015). Taken together, our results presented in this study demonstrate that Cdk phosphorylation licenses chromosomal localization of Kif4A and chromosomal localization of Kif4A, in turn, controls early mitotic functions of Kif4A important for proper early mitotic progression. Materials and methods Plasmids, siRNAs, and antibodies Mammalian expression plasmids of Kif4A and its point mutations were generated as described previously (Zhu and Jiang, 2005). The primers used in Kif4A T1161A point mutation were: sense 5′-tttaatcccgtctgtgccgcccccaatagcaagatcctg-3′ and antisense 5′-caggatcttgctattgggggcggcacagacgggattaaa-3′. The primers used in Kif4A T1161E point mutation were: sense 5′-tttaatcccgtctgtgccgaacccaatagcaagatcctg-3′ and antisense 5′-caggatcttgctattgggttcggcacagacgggattaaa-3′. Plasmids expressing Kif4A-Histone H1 fusion proteins were constructed by inserting Histone H1 cDNA at 3′ end of Kif4A cDNA before the stop codon in Kif4A expression plasmids. All constructs were fully sequenced. siRNA specific targeting to 3′-UTR of Kif4A (5′-GGAATGAGGTTGTGATCTT-3′) and siRNA specific targeting Kid cDNA coding region (5′-CAAGCUCACUCGCCUAUUGTT-3′) were synthesized by Genepharma. Polyclonal rabbit anti-Kif4ApT1161 antibodies (α-Kif4Ap1161) were developed against phospho-peptide of SFFNPVCA(pT)PNSKILKEMC. Polyclonal rabbit α-Kif4A were generated as previously described (Zhu and Jiang, 2005). Mouse anti-α-tubulin (#T5168) antibody was purchased from Sigma-Aldrich, rabbit α-SMC2 (#07-710) was purchased from Merk Millipore, mouse α-BubR 1 (#612503) was purchased from BD Biosciences, mouse α-cyclin B1 (#MAB3684) was purchased from Chemicon, mouse α-hCAP-G (#sc-515297) and goat α-Kid (#sc-30456) were purchased from Santa Cruz Biotechnology, and rabbit α-hCAP-D3 (A300-604A) was purchased from Bethyl Laboratories, Inc. All secondary antibodies were obtained from Life Technologies Inc. Cell culture, transfection, immunoprecipitation, immunoblotting, and immunofluorescence HeLa cells were cultured in DMEM containing 10% fetal calf serum (Invitrogen) at 37°C and 5% CO2. For transfection of plasmids and siRNAs, HeLa cells were cultured in 6-well plates and transfected with 50 nM siRNA and/or 0.1–0.5 μg of plasmid(s) using Lipofectamine 3000 (Thermo Fisher Scientific). Two to three days after transfection, cells were harvested or fixed for immunoprecipitation, immunoblotting, or immunofluorescence analysis as previously described (Jiang et al., 1998; Zhu and Jiang, 2005). For peptide competition assay, polyclonal rabbit antibodies, α-Kif4Ap1161, were incubated with Kif4A phospho-peptide or nonphospho-peptide at 4°C overnight. Control and taxol-treated mitotic cells were lysed in 1× sample buffer and subjected to SDS-PAGE followed by immunoblotting with peptide pre-treated α-Kif4Ap1161. Pearson’s correlation coefficients (R-value) were calculated using Image-Pro Plus 7.0 (Zinchuk et al., 2007). In brief, we generated coordinated datasets of fluorescence intensities at each pixel from 10 chromosome arms/cell and calculated Pearson correlation coefficients (R-values) for all pairwise combinations (total positive correlation appears as 1, total negative correlation as −1, and no correlation as 0). The datasets were obtained from 10 cells in three independent experiments. Cell cycle synchronization and drug treatments HeLa cells were treated with 2 mM thymidine (Sigma-Aldrich) for 16 h, released into fresh medium for 6 h, and then blocked with 40 ng/ml nocodazole (Sigma-Aldrich) for 12 h as described (Zhu and Jiang, 2005). To collected early mitotic cells, 30 nM taxol (Cytoskeleton) was added to the media and cells were collected after 16 h. To inhibit Cdk1 activity, 5 mg/ml roscovitine (Selleck Chemicals) was added to the media and cells were collected after 2 h. To stabilize MTs, 9 nM nocodazole (Sigma-Aldrich) was added to the cell culture media 3 h before fixation. 5 μM S-trityl-L-cysteine (STLC; Sigma-Aldrich) was added to the cell culture media 16 h before fixation to induce monopolar spindles by inhibiting Eg5. Mass spectrometry For mass spectrometry, cells were treated with 30 nM Taxol overnight. Cells were lysed and immunoprecipitated with α-Kif4A. The precipitates were separated by SDS/PAGE and stained with Coomassie blue. Specific Kif4A proteins in SDS-Gel were isolated and cut into pieces followed by digestion with trypsin. The digested samples were analyzed with a matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance mass spectrometer (Bruker Daltonics) and the phosphorylated peptides were further analyzed with automated nanoflow liquid chromatography/tandem mass spectrometry. In vitro kinase assay For in vitro kinase assay, 1 μg of bacterially expressed GST–Kif4AC230WT (230 amino acids at caboxyl terminus of Kif4A) or GST–Kif4AC230TA (Threonine 1161 was substituted by Alanine) mutant protein was incubated with 0.1 μg of baculovirus-expressed Cyclin B1–Cdk1 protein complex in kinase reaction as previously described (Jiang et al., 1998). The kinase reactions were terminated by adding an equal volume of 2× sample buffer. The reaction products were separated by SDS-PAGE and stained with Coomassie brilliant blue. The SDS-gel was then dried prior to autoradiography. Time-lapse microscopy HeLa cells stably expressing CFP-Histone H2B were cultured on 35-mm glass-bottom microwell dishes (Nest Biotechnology, China) and transfected with 0.2 μg of pmCherry-Kif4A plasmid together with 50 nM of Kif4A siRNA using Lipofectamine 3000 (Thermo Fisher Scientific). Thirty-six hours after transfection, cell culture dishes were transferred to a heated stage (37°C) on a Nikon HSJ Ti E-PFS microscope. Phase-contrast and fluorescence images of live cells were collected at 1-min intervals for 12 h and processed by using NIKON NIS-Elements BR software and Image J. Monopolar spindles assay HeLa cells were transfected with siRNA targeting Kid or/and Kif4A together with indicated plasmids, and treated with STLC for 16 h. Formaldehyde fixed cells were stained with mouse anti-α-tubulin antibody, human anti-CREST serum and DAPI. Monopolar spindles were imaged as a 3D z-stack using a 100× NA 1.35 oil objective on a fluorescence microscope (NIKON) and deconvolved using NIKON NIS-Elements AR software (NIKON). The cell center was defined as pole and the position was saved. The distance between kinetochores and pole (KT-to-pole Distance) and monopolar spindle length were measured using NIKON NIS-Elements AR software and calculated in Excel (Microsoft). Chromosome spreading analysis Cells were treated with 0.1 μg/ml of colcemid overnight and then hypotonically swollen in 75 mM KCl. Cells were fixed in ice-cold methanol/acetic acid and dropped on slides to spread chromosomes. The spreading chromosomes were stained with DAPI and images were acquired and processed under a Nikon Ts-100FL microscope. The widths and the fluorescent intensities of chromosome arms were measured using NIKON NIS-Elements AR software and Image-Pro Plus 7.0. Statistical analysis Student’s test was used to calculate the statistical significance of the experimental data. The level of significance was set as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. Acknowledgements We thank Drs Joel Leverson (Oncology Development Department, AbbVie Inc.) and Gary Chiang (Discovery, Global Pharmaceutical Research and Development, AbbVie Inc.) for critical reading of the manuscript. Funding This work was supported by CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-12M-1-001), the National Natural Science Foundation of China (31171299, 31271485, and 31301138), the National Basic Research Program of China (2012CB910703), and Tianjin Research Program of Application Foundation and Advanced Technology (12JC2DJC21400). C.Z. is supported by Program for New Century Excellent Talents in University in China (NCET-11-1066). Conflict of interest none declared. Author contributions Z.D., C.Z., and W.J. designed research; Z.D. and C.Z. performed research; Q.Z. contributed reagents; Z.D., C.Z., and W.J. analyzed data; and Z.D., C.Z., and W.J. wrote the paper. References Barisic , M. , Aguiar , P. , Geley , S. , et al. . ( 2014 ). Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces . Nat. Cell Biol. 16 , 1249 – 1256 . Google Scholar Crossref Search ADS PubMed Barr , F.A. , Elliott , P.R. , and Gruneberg , U. ( 2011 ). Protein phosphatases and the regulation of mitosis . J. Cell Sci. 124 , 2323 – 2334 . Google Scholar Crossref Search ADS PubMed Bastos , R.N. , Cundell , M.J. , and Barr , F.A. ( 2014 ). KIF4A and PP2A-B56 form a spatially restricted feedback loop opposing Aurora B at the anaphase central spindle . J. Cell Biol. 207 , 683 – 693 . Google Scholar Crossref Search ADS PubMed Belmont , A.S. ( 2006 ). Mitotic chromosome structure and condensation . Curr. Opin. Cell Biol. 18 , 632 – 638 . Google Scholar Crossref Search ADS PubMed Bieling , P. , Telley , I.A. , and Surrey , T. ( 2010 ). A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps . Cell 142 , 420 – 432 . Google Scholar Crossref Search ADS PubMed Bringmann , H. , Skiniotis , G. , Spilker , A. , et al. . ( 2004 ). A kinesin-like motor inhibits microtubule dynamic instability . Science 303 , 1519 – 1522 . Google Scholar Crossref Search ADS PubMed Brouhard , G.J. , and Hunt , A.J. ( 2005 ). Microtubule movements on the arms of mitotic chromosomes: polar ejection forces quantified in vitro . Proc. Natl Acad. Sci. USA 102 , 13903 – 13908 . Google Scholar Crossref Search ADS Castoldi , M. , and Vernos , I. ( 2006 ). Chromokinesin Xklp1 contributes to the regulation of microtubule density and organization during spindle assembly . Mol. Biol. Cell 17 , 1451 – 1460 . Google Scholar Crossref Search ADS PubMed D’Avino , P.P. , Giansanti , M.G. , and Petronczki , M. ( 2015 ). Cytokinesis in animal cells . Cold. Spring Harb. Perspect. Biol. 7 , a015834 . Google Scholar Crossref Search ADS PubMed Fisher , D. , Krasinska , L. , Coudreuse , D. , et al. . ( 2012 ). Phosphorylation network dynamics in the control of cell cycle transitions . J. Cell Sci. 125 , 4703 – 4711 . Google Scholar Crossref Search ADS PubMed Foley , E.A. , and Kapoor , T.M. ( 2013 ). Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore . Nat. Rev. Mol. Cell Biol. 14 , 25 – 37 . Google Scholar Crossref Search ADS PubMed Funabiki , H. , and Murray , A.W. ( 2000 ). The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement . Cell 102 , 411 – 424 . Google Scholar Crossref Search ADS PubMed Glotzer , M. ( 2001 ). Animal cell cytokinesis . Annu. Rev. Cell Dev. Biol. 17 , 351 – 386 . Google Scholar Crossref Search ADS PubMed Goshima , G. , and Vale , R.D. ( 2005 ). Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells . Mol. Biol. Cell 16 , 3896 – 3907 . Google Scholar Crossref Search ADS PubMed Green , R.A. , Paluch , E. , and Oegema , K. ( 2012 ). Cytokinesis in animal cells . Annu. Rev. Cell Dev. Biol. 28 , 29 – 58 . Google Scholar Crossref Search ADS PubMed Holland , A.J. , and Cleveland , D.W. ( 2012 ). Losing balance: the origin and impact of aneuploidy in cancer . EMBO Rep. 13 , 501 – 514 . Google Scholar Crossref Search ADS PubMed Hu , C.K. , Coughlin , M. , Field , C.M. , et al. . ( 2011 ). KIF4 regulates midzone length during cytokinesis . Curr. Biol. 21 , 815 – 824 . Google Scholar Crossref Search ADS PubMed Iemura , K. , and Tanaka , K. ( 2015 ). Chromokinesin Kid and kinetochore kinesin CENP-E differentially support chromosome congression without end-on attachment to microtubules . Nat. Commun. 6 , 6447 . Google Scholar Crossref Search ADS PubMed Jeppsson , K. , Kanno , T. , Shirahige , K. , et al. . ( 2014 ). The maintenance of chromosome structure: positioning and functioning of SMC complexes . Nat. Rev. Mol. Cell Biol. 15 , 601 – 614 . Google Scholar Crossref Search ADS PubMed Jiang , W. , Jimenez , G. , Wells , N.J. , et al. . ( 1998 ). PRC1: a human mitotic spindle-associated CDK substrate protein required for cytokinesis . Mol. Cell 2 , 877 – 885 . Google Scholar Crossref Search ADS PubMed Kakui , Y. , and Uhlmann , F. ( 2018 ). SMC complexes orchestrate the mitotic chromatin interaction landscape . Curr. Genet. 64 , 335 – 339 . Google Scholar Crossref Search ADS PubMed Kurasawa , Y. , Earnshaw , W.C. , Mochizuki , Y. , et al. . ( 2004 ). Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation . EMBO J. 23 , 3237 – 3248 . Google Scholar Crossref Search ADS PubMed Levesque , A.A. , and Compton , D.A. ( 2001 ). The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles . J. Cell Biol. 154 , 1135 – 1146 . Google Scholar Crossref Search ADS PubMed Li , Q.R. , Yan , X.M. , Guo , L. , et al. . ( 2018 ). AMPK regulates anaphase central spindle length by phosphorylation of KIF4A . J. Mol. Cell Biol. 10 , 2 – 17 . Google Scholar Crossref Search ADS PubMed London , N. , and Biggins , S. ( 2014 ). Signalling dynamics in the spindle checkpoint response . Nat. Rev. Mol. Cell Biol. 15 , 736 – 747 . Google Scholar Crossref Search ADS PubMed Ly , P. , and Cleveland , D.W. ( 2017 ). Rebuilding chromosomes after catastrophe: emerging mechanisms of chromothripsis. Trends Cell Biol. 27 , 917 – 930 . Google Scholar Crossref Search ADS PubMed Mack , G.J. , and Compton , D.A. ( 2001 ). Analysis of mitotic microtubule-associated proteins using mass spectrometry identifies astrin, a spindle-associated protein . Proc. Natl Acad. Sci. USA 98 , 14434 – 14439 . Google Scholar Crossref Search ADS Malik , R. , Lenobel , R. , Santamaria , A. , et al. . ( 2009 ). Quantitative analysis of the human spindle phosphoproteome at distinct mitotic stages . J. Proteome Res. 8 , 4553 – 4563 . Google Scholar Crossref Search ADS PubMed Mazumdar , M. , Sundareshan , S. , and Misteli , T. ( 2004 ). Human chromokinesin KIF4A functions in chromosome condensation and segregation . J. Cell Biol. 166 , 613 – 620 . Google Scholar Crossref Search ADS PubMed McIntosh , J.R. , Grishchuk , E.L. , and West , R.R. ( 2002 ). Chromosome-microtubule interactions during mitosis . Annu. Rev. Cell Dev. Biol. 18 , 193 – 219 . Google Scholar Crossref Search ADS PubMed Nguyen , P.A. , Groen , A.C. , Loose , M. , et al. . ( 2014 ). Spatial organization of cytokinesis signaling reconstituted in a cell-free system . Science 346 , 244 – 247 . Google Scholar Crossref Search ADS PubMed Nunes Bastos , R. , Gandhi , S.R. , Baron , R.D. , et al. . ( 2013 ). Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A . J. Cell Biol. 202 , 605 – 621 . Google Scholar Crossref Search ADS PubMed Samejima , K. , Samejima , I. , Vagnarelli , P. , et al. . ( 2012 ). Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα . J. Cell Biol. 199 , 755 – 770 . Google Scholar Crossref Search ADS PubMed Stumpff , J. , Wagenbach , M. , Franck , A. , et al. . ( 2012 ). Kif18A and chromokinesins confine centromere movements via microtubule growth suppression and spatial control of kinetochore tension . Dev. Cell 22 , 1017 – 1029 . Google Scholar Crossref Search ADS PubMed Subramanian , R. , Ti , S.C. , Tan , L. , et al. . ( 2013 ). Marking and measuring single microtubules by PRC1 and kinesin-4 . Cell 154 , 377 – 390 . Google Scholar Crossref Search ADS PubMed Subramanian , R. , Wilson-Kubalek , E.M. , Arthur , C.P. , et al. . ( 2010 ). Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein . Cell 142 , 433 – 443 . Google Scholar Crossref Search ADS PubMed Takahashi , M. , Wakai , T. , and Hirota , T. ( 2016 ). Condensin I-mediated mitotic chromosome assembly requires association with chromokinesin KIF4A . Genes Dev. 30 , 1931 – 1936 . Google Scholar Crossref Search ADS PubMed Vagnarelli , P. ( 2012 ). Mitotic chromosome condensation in vertebrates . Exp. Cell Res. 318 , 1435 – 1441 . Google Scholar Crossref Search ADS PubMed Vallardi , G. , and Saurin , A.T. ( 2015 ). Mitotic kinases and phosphatases cooperate to shape the right response . Cell Cycle 14 , 795 – 796 . Google Scholar Crossref Search ADS PubMed Vicente , J.J. , and Wordeman , L. ( 2015 ). Mitosis, microtubule dynamics and the evolution of kinesins . Exp. Cell Res. 334 , 61 – 69 . Google Scholar Crossref Search ADS PubMed Walczak , C.E. , and Heald , R. ( 2008 ). Mechanisms of mitotic spindle assembly and function . Int. Rev. Cytol. 265 , 111 – 158 . Google Scholar Crossref Search ADS PubMed Wandke , C. , Barisic , M. , Sigl , R. , et al. . ( 2012 ). Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis . J. Cell Biol. 198 , 847 – 863 . Google Scholar Crossref Search ADS PubMed Wang , G. , Jiang , Q. , and Zhang , C. ( 2014 ). The role of mitotic kinases in coupling the centrosome cycle with the assembly of the mitotic spindle . J. Cell Sci. 127 , 4111 – 4122 . Google Scholar Crossref Search ADS PubMed Zhu , C. , and Jiang , W. ( 2005 ). Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis . Proc. Natl Acad. Sci. USA 102 , 343 – 348 . Google Scholar Crossref Search ADS Zhu , C. , Zhao , J. , Bibikova , M. , et al. . ( 2005 ). Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference . Mol. Biol. Cell 16 , 3187 – 3199 . Google Scholar Crossref Search ADS PubMed Zinchuk , V. , Zinchuk , O. , and Okada , T. ( 2007 ). Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena . Acta Histochem. Cytochem. 40 , 101 – 111 . Google Scholar Crossref Search ADS PubMed © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

Cdk phosphorylation licenses Kif4A chromosome localization required for early mitotic progression

Loading next page...
 
/lp/ou_press/cdk-phosphorylation-licenses-kif4a-chromosome-localization-required-AY0CObJh1J
Publisher
Oxford University Press
Copyright
© The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
ISSN
1674-2788
eISSN
1759-4685
D.O.I.
10.1093/jmcb/mjy033
Publisher site
See Article on Publisher Site

Abstract

Abstract The chromokinesin Kif4A controls proper chromosome condensation, congression/alignment, and cytokinesis to ensure faithful genetic inheritance. Here, we report that Cdk phosphorylation of human Kif4A at T1161 licenses Kif4A chromosomal localization, which, in turn, controls Kif4A early mitotic function. Phosphorylated Kif4A (Kif4AWT) or Cdk phospho-mimetic Kif4A mutant (Kif4ATE) associated with chromosomes and condensin I (non-SMC subunit CAP-G and core subunit SMC2) to regulate chromosome condensation, spindle morphology, and chromosome congression/alignment in early mitosis. In contrast, Cdk non-phosphorylatable Kif4A mutant (Kif4ATA) could neither localize on chromosomes nor associate with CAP-G and SMC2. Furthermore, Kif4ATA could not rescue defective chromosome condensation, spindle morphology, or chromosome congression/alignment in cells depleted of endogenous Kif4A, which activated a mitotic checkpoint and delayed early mitotic progression. However, targeting Kif4ATA to chromosomes by fusion of Kif4ATA with Histone H1 resulted in restoration of chromosome and spindle functions of Kif4A, similar to Kif4AWT and Kif4ATE, in cells depleted of endogenous Kif4A. Thus, our results demonstrate that Cdk phosphorylation-licensed chromosomal localization of Kif4A plays a critical role in regulating early mitotic functions of Kif4A that are important for early mitotic progression. Kif4A, chromokinesin, Cdk phosphorylation, mitosis Introduction Mitosis, the last event of the cell cycle, controls accurate segregation of chromosomes into two daughter cells. To accomplish this, sister chromatids precisely duplicated in interphase must be compacted with ‘chromosome scaffold’ proteins to organize higher order structures, a process known as chromosome condensation (Belmont, 2006; Vagnarelli, 2012; Jeppsson et al., 2014; Kakui and Uhlmann, 2018). Subsequently, a unique cellular apparatus, the mitotic spindle, is assembled to drive congression/alignment of the condensed chromosomes at the metaphase plate in early mitosis (McIntosh et al., 2002; Walczak and Heald, 2008; Vicente and Wordeman, 2015). After proper chromosome congression/alignment, sister chromatids are segregated faithfully by the elongated mitotic spindle in late mitosis, followed by cell division into two daughter cells, a process known as cytokinesis (Glotzer, 2001; Green et al., 2012; Nguyen et al., 2014; D’Avino et al., 2015). Large numbers of chromosome-associated and/or spindle-binding proteins are involved in regulating these processes, including chromosome scaffold proteins, spindle assembly/disassembly proteins, microtubule (MT) regulatory proteins (e.g. non-motor structural proteins, motor proteins, kinases, and phosphatases), and spindle checkpoint proteins (Mack and Compton, 2001; Goshima and Vale, 2005; Zhu et al., 2005; Zinchuk et al., 2007; Subramanian et al., 2010; Barr et al., 2011; Samejima et al., 2012; Foley and Kapoor, 2013; Barisic et al., 2014; London and Biggins, 2014; Wang et al., 2014; Vallardi and Saurin, 2015). Deregulation and dysfunction of these proteins result in chromosome mis-segregation and mitotic defects, leading to chromosome instability and aneuploidy, contributing to the development and progression of many genetic diseases including cancer (Holland and Cleveland, 2012; Ly and Cleveland, 2017). The chromokinesin Kif4A, a member of the kinesin-4 family, plays important roles in regulating mitosis, including chromosome condensation, spindle assembly/dynamics, and chromosome congression/alignment in early mitosis and spindle midzone formation, spindle elongation, and cytokinesis in late mitosis (Mazumdar et al., 2004; Zhu and Jiang, 2005; Castoldi and Vernos, 2006; Hu et al., 2011; Samejima et al., 2012; Wandke et al., 2012; Subramanian et al., 2013; Nguyen et al., 2014). In early mitosis, Kif4A associates with chromosome scaffold proteins, such as condensin core subunit SMC2 and condensin I non-SMC subunit CAP-G on chromosomes to promote lateral compaction of chromosomal arms (Mazumdar et al., 2004; Samejima et al., 2012; Takahashi et al., 2016). Chromosome-associated Kif4A also contributes to the regulation of chromosome-interacting MT dynamics and cooperates with another chromokinesin, Kid, to generate polar ejection forces (PEF) (Wandke et al., 2012). Together with the kinetochore motor proteins, CENP-E, dynein, and Kif18A, these chromokinesins drive chromosome congression/alignment in early mitosis (Stumpff et al., 2012; Barisic et al., 2014; Iemura and Tanaka, 2015). In late mitosis, Kif4A translocates to the spindle midzone/midbody, directly binding to the non-kinetochore interdigitating anti-parallel MT-bundling protein PRC1 to control spindle midzone formation and elongation (Kurasawa et al., 2004; Zhu and Jiang, 2005; Bieling et al., 2010). Kif4A delivers PRC1 to the midzone MTs plus-ends in a MT length-dependent manner and stabilizes midzone MTs plus-ends to control midzone elongation (Hu et al., 2011). In addition, Kif4A is phosphorylated at T799/S801 by Aurora B and AMPK at spindle midzone that promotes Kif4A interaction with PRC1 and stimulates ATPase activity of Kif4A to regulate spindle midzone dynamics (Nunes Bastos et al., 2013; Bastos et al., 2014; Li et al., 2018). Despite intensive efforts in elucidating the role of Kif4A in controlling mitotic processes, the spatiotemporal regulation of Kif4A chromosome/spindle distribution remains elusive. In this study, we report that Cdk phosphorylation licenses the chromosomal localization of Kif4A, enabling it to perform its crucial functions in early mitotic progression. Results Cdk phosphorylation of Kif4A in early mitosis To investigate the regulation of Kif4A distribution between chromosomes and the mitotic spindle, we arrested HeLa cells in early mitosis with taxol treatment. As shown in Figure 1A, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the Kif4A band was shifted in taxol-treated cells relative to asynchronous control cells, suggesting that Kif4A was modified in early mitosis. To identify the potential post-translational modification(s), Kif4A was immunoprecipitated from taxol-treated cells with anti-Kif4A antibodies (α-Kif4A), separated by SDS-PAGE and subjected to mass spectrometry (MS) (Figure 1A). MS analysis revealed that Kif4A was mainly phosphorylated at threonine 1161 (T1161) (Supplementary Figure S1), which is conserved across many species within a consensus proline-directed Cdk phosphorylation motif (Figure 1B). Figure 1 View largeDownload slide Identification of Cdk phosphorylation of Kif4A at T1161 in early mitosis. (A) Kif4A displayed mobility shift in mitotic HeLa cells. HeLa cells were treated with 30 nM taxol overnight and cell lysates were immunoprecipitated with polyclonal α-Kif4A. Immunoprecipitates were separated by SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. Arrow indicates 140 kDa Kif4A protein. (B) Multi-sequence alignment of Kif4A showed that Kif4A T1161 (asterisk) was conserved in several organisms with a consensus Cdk phosphorylation motif. (C) Kif4A T1161 was phosphorylated by Cdk1/Cyclin B1 in vitro. Bacterially expressed GST-tagged wild-type (WT) and T1161 non-phosphorylation mutant (TA) of 230 amino acids (C230) at C-terminus of Kif4A were incubated with baculovirus-expressed cyclin B1−Cdk1 complex in the presence of [γ-32P] ATP for in vitro kinase assay. The reaction was analyzed by CBB staining (right) followed by autoradiography (left). Asterisk indicates the GST-Kif4A C230 protein. (D) Characterization of the polyclonal α-Kif4Ap1161. Asynchronous or taxol-treated HeLa cell lysates were immunoblotted with phospho-peptide-treated, nonphospho-peptide-treated, or non-treated α-Kif4Ap1161, α-Kif4A, and anti-α-tubulin antibody. (E and F) Immunofluorescence images of Kif4A and T1161-phosphorylated Kif4A in HeLa cells. HeLa cells were stained with polyclonal α-Kif4A (E) or α-Kif4Ap1161 (F) (green). DNA was visualized by DAPI staining (red). White scale bar, 5 μm; yellow scale bar, 1 μm. Figure 1 View largeDownload slide Identification of Cdk phosphorylation of Kif4A at T1161 in early mitosis. (A) Kif4A displayed mobility shift in mitotic HeLa cells. HeLa cells were treated with 30 nM taxol overnight and cell lysates were immunoprecipitated with polyclonal α-Kif4A. Immunoprecipitates were separated by SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. Arrow indicates 140 kDa Kif4A protein. (B) Multi-sequence alignment of Kif4A showed that Kif4A T1161 (asterisk) was conserved in several organisms with a consensus Cdk phosphorylation motif. (C) Kif4A T1161 was phosphorylated by Cdk1/Cyclin B1 in vitro. Bacterially expressed GST-tagged wild-type (WT) and T1161 non-phosphorylation mutant (TA) of 230 amino acids (C230) at C-terminus of Kif4A were incubated with baculovirus-expressed cyclin B1−Cdk1 complex in the presence of [γ-32P] ATP for in vitro kinase assay. The reaction was analyzed by CBB staining (right) followed by autoradiography (left). Asterisk indicates the GST-Kif4A C230 protein. (D) Characterization of the polyclonal α-Kif4Ap1161. Asynchronous or taxol-treated HeLa cell lysates were immunoblotted with phospho-peptide-treated, nonphospho-peptide-treated, or non-treated α-Kif4Ap1161, α-Kif4A, and anti-α-tubulin antibody. (E and F) Immunofluorescence images of Kif4A and T1161-phosphorylated Kif4A in HeLa cells. HeLa cells were stained with polyclonal α-Kif4A (E) or α-Kif4Ap1161 (F) (green). DNA was visualized by DAPI staining (red). White scale bar, 5 μm; yellow scale bar, 1 μm. To determine whether Kif4A T1161 could be phosphorylated by mitotic Cdk, we performed an in vitro kinase assay using bacterially expressed GST-Kif4A C-terminal fusion protein (GST-Kif4AWT) or its non-phosphorylatable counterpart (GST-Kif4ATA) as substrates. These recombinant proteins were purified and incubated with purified baculovirus-expressed cyclin B1-Cdk1 complex in the presence of [γ-32P] ATP. As shown in Figure 1C, GST-Kif4AWT, but not GST-Kif4ATA, was phosphorylated efficiently by Cdk1/Cyclin B1, demonstrating that T1161 in Kif4A was a substrate for Cdk1/Cyclin B1 in vitro. We generated rabbit polyclonal phospho-specific antibodies against T1161 (α-Kif4Ap1161) and immunoblotting analysis indicated that α-Kif4Ap1161 specifically recognized Kif4A in lysates from taxol-treated cells but not in asynchronous control cells (Figure 1D and Supplementary Figure S2A). Furthermore, T1161 phospho-peptide competition or treatment with the Cdk1 inhibitor, roscovitine, abolished the immuno-reactivity of α-Kif4Ap1161 in taxol-treated cells (Figure 1D and Supplementary Figure S2A). As Kif4A could be phosphorylated at T1161 by Cdk1/Cyclin B1 in vitro, this suggests that Kif4A is a physiological mitotic Cdk substrate and phosphorylated at T1161 during early mitosis. Consistent with this hypothesis, immunoblotting analysis and co-immunofluorescence using affinity-purified α-Kif4Ap1161 and cyclin B1 antibodies revealed that phosphorylation of Kif4A at T1161 occurred in G2 phase cells when cyclin B1 was expressed, and diminished at the onset of anaphase, when cyclin B1 was degraded (Supplementary Figures S2B, S3A and B). In addition, immunofluorescence analysis using affinity-purified α-Kif4A showed that Kif4A was mainly localized on chromosomes, especially on chromosomal arms laterally, in early mitosis but translocated to the spindle midzone and midbody in late mitosis as we and others reported previously (Figure 1E and Supplementary Figure S3C) (Mazumdar et al., 2004; Zhu and Jiang, 2005). In contrast, α-Kif4Ap1161 only detected chromosomal arm-localized Kif4A in early mitosis, not spindle midzone- and midbody-localized Kif4A in late mitosis (Figure 1F and Supplementary Figure S3D). Taken together, these results demonstrated that chromosome-associated Kif4A was phosphorylated by mitotic Cdk in early mitosis. Regulation of Kif4A chromosomal localization and function by Cdk phosphorylation in early mitosis We investigated whether Cdk phosphorylation of Kif4A was involved in regulating Kif4A localization/function in early mitosis. We generated a set of mammalian expression vectors fusing green fluorescent protein (GFP) to wild-type Kif4A (GFP-Kif4AWT), the phosphorylation site mutant (Kif4ATA), or the phospho-mimetic mutant (Kif4ATE) and expressed these plasmids in HeLa cells. Fluorescence imaging showed that ectopically expressed GFP-Kif4AWT or GFP-Kif4ATE protein was mainly localized on chromosomal arms laterally in early mitosis similar to endogenous Kif4A protein (Figure 2A; Supplementary Figure S4A and C). In contrast, GFP-Kif4ATA protein was not detected on chromosomes but mainly localized in the cytoplasm in early mitosis (Figure 2A and Supplementary Figure S4B). When cells were pre-extracted with digitonin before fixation to remove cytoplasmic soluble proteins, fluorescence imaging revealed that, unlike GFP-Kif4AWT or GFP-Kif4ATE, GFP-Kif4ATA was not localized on chromosomes (Figure 2B). Immunoblotting analysis also showed that GFP-Kif4AWT or GFP-Kif4ATE but not GFP-Kif4ATA could be recognized by α-Kif4Ap1161 in early mitosis, confirming the specificity of this reagent (Supplementary Figure S4D). Furthermore, as previously reported that Kif4A and condensin core subunit SMC2 were colocalized on the chromosomes (Mazumdar et al., 2004; Samejima et al., 2012), co-fluorescence imaging and Pearson correlation coefficient (R-value) analysis revealed that GFP-Kif4AWT or GFP-Kif4ATE but not GFP-Kif4ATA was colocalized with SMC2 on chromosomal arms laterally (Figure 2C and D). Thus, these results demonstrated that phosphorylation of Kif4A by mitotic Cdk at T1161 regulated its chromosomal localization in early mitosis. Figure 2 View largeDownload slide Regulation of chromosomal localization of Kif4A by Cdk phosphorylation. (A) HeLa cells grown on coverslips were transfected with pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and stained with mouse anti-α-tubulin antibody (gray) and DAPI (red). Scale bar, 5 μm. More than 20 cells in three independent experiments were observed and representative images are shown. (B) HeLa cells were transfected with plasmids as in A and then treated with 5 μg/ml digitonin followed by formaldehyde fixation and fluorescent staining with mouse anti-α-tubulin antibody (gray) and DAPI (red). More than 20 cells in three independent experiments were observed and representative images are shown. (C) HeLa cells were transfected with plasmids as in A. Cells were fixed and stained with rabbit α-SMC2 (red) and DAPI (cyan). Scale bar, 5 μm. (D) The Pearson’s correlation coefficients (R-value) of GFP-Kif4A vs. SMC2 in C were determined as described in ‘Materials and methods’ section. Figure 2 View largeDownload slide Regulation of chromosomal localization of Kif4A by Cdk phosphorylation. (A) HeLa cells grown on coverslips were transfected with pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and stained with mouse anti-α-tubulin antibody (gray) and DAPI (red). Scale bar, 5 μm. More than 20 cells in three independent experiments were observed and representative images are shown. (B) HeLa cells were transfected with plasmids as in A and then treated with 5 μg/ml digitonin followed by formaldehyde fixation and fluorescent staining with mouse anti-α-tubulin antibody (gray) and DAPI (red). More than 20 cells in three independent experiments were observed and representative images are shown. (C) HeLa cells were transfected with plasmids as in A. Cells were fixed and stained with rabbit α-SMC2 (red) and DAPI (cyan). Scale bar, 5 μm. (D) The Pearson’s correlation coefficients (R-value) of GFP-Kif4A vs. SMC2 in C were determined as described in ‘Materials and methods’ section. We performed siRNA knockdown-rescue experiments to determine whether Cdk phosphorylation of Kif4A controlled its early mitotic functions. HeLa cells were transfected with Kif4A siRNA targeting the 3′-UTR of Kif4A mRNA together with vectors expressing siRNA-resistant GFP-Kif4AWT, GFP-Kif4ATA or GFP-Kif4ATE cDNAs lacking the 3′-UTR. Immunoblotting analysis showed that Kif4A siRNA could effectively ablate expression of endogenous Kif4A protein but did not affect ectopic expression of GFP-Kif4AWT, GFP-Kif4ATA, or GFP-Kif4ATE protein (Figure 3A). Analyses of mitotic index and different stages of early mitosis revealed that cells expressing GFP-Kif4AWT or GFP-Kif4ATE but lacking endogenous Kif4A displayed normal mitotic index and percentages of cells at different stages of early mitosis, similar to control cells (nonsence siRNA-transfected and GFP-expressing cells). In contrast, like cells depleted of Kif4A (Kif4A siRNA-transfected and GFP-expressing cells), cells expressing GFP-Kif4ATA but lacking endogenous Kif4A displayed increased mitotic index with accumulation of cells in prometaphase and metaphase (Figure 3B and C; Supplementary Table S1). Figure 3 View largeDownload slide Regulation of early mitotic progression by Cdk phosphorylation of Kif4A. (A) HeLa cells transfected with NS siRNA or Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were collected and lysed. Cell lysates were immunoblotted with the indicated antibodies. (B and C) HeLa cells were transfected with Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and stained with mouse anti-α-tubulin antibody and DAPI. Mitotic index (B) and the percent of early mitotic cells in whole mitotic cells (C) were counted and shown in histogram (mean + SD of three independent experiments; Student’s t-test, **P < 0.01; ns, not significant). (D−F) HeLa cells stably expressing CFP-Histone H2B were transfected with expression vectors encoding the indicated siRNA-resistant mCherry-Kif4A together with Kif4A siRNA for 36 h. Time-lapse images were collected every 1 min for 12 h. Duration of prometaphase (prometa: from nuclear envelope breakdown to chromosome alignment; red), metaphase (meta: from chromosome alignment to anaphase onset; blue), anaphase/telophase (ana/telo: from anaphase onset to chromosome decondensation; green), or aneuploidy (from anaphase onset to aneuploidy; magenta) is indicated in D. The data represent 25 cells for each category and the average duration of mitotic phase is shown in E. Results represent mean ± SD. Representative images of time-lapse microscopy are shown in F. Figure 3 View largeDownload slide Regulation of early mitotic progression by Cdk phosphorylation of Kif4A. (A) HeLa cells transfected with NS siRNA or Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were collected and lysed. Cell lysates were immunoblotted with the indicated antibodies. (B and C) HeLa cells were transfected with Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and stained with mouse anti-α-tubulin antibody and DAPI. Mitotic index (B) and the percent of early mitotic cells in whole mitotic cells (C) were counted and shown in histogram (mean + SD of three independent experiments; Student’s t-test, **P < 0.01; ns, not significant). (D−F) HeLa cells stably expressing CFP-Histone H2B were transfected with expression vectors encoding the indicated siRNA-resistant mCherry-Kif4A together with Kif4A siRNA for 36 h. Time-lapse images were collected every 1 min for 12 h. Duration of prometaphase (prometa: from nuclear envelope breakdown to chromosome alignment; red), metaphase (meta: from chromosome alignment to anaphase onset; blue), anaphase/telophase (ana/telo: from anaphase onset to chromosome decondensation; green), or aneuploidy (from anaphase onset to aneuploidy; magenta) is indicated in D. The data represent 25 cells for each category and the average duration of mitotic phase is shown in E. Results represent mean ± SD. Representative images of time-lapse microscopy are shown in F. We performed time-lapse microscopy with depletion of Kif4A and expression of mCherry-Kif4AWT or its mutant versions in HeLa cells stably expressing Histone H2B-CFP (Supplementary Figure S5A). Live-cell imaging revealed that mCherry-Kif4AWT or mCherry-Kif4ATE expressed in cells depleted of endogenous Kif4A localized to chromosomes in early mitosis (Supplementary Figure S5B). Similar to control cells, cells expressing mCherry-Kif4AWT or mCherry-Kif4ATE but lacking endogenous Kif4A exhibited normal chromosome congression in prometaphase and chromosome alignment in metaphase, progressing through early mitosis ~35 min (as measured from nuclear envelope breakdown to the onset of anaphase) (Figure 3D−F; Supplementary Movies S1, S3, and S4). In contrast, mCherry-Kif4ATA did not localize to chromosomes but was instead mainly cytoplasmic (Supplementary Figure S5B). Like cells depleted of Kif4A alone, cells expressing mCherry-Kif4ATA but lacking endogenous Kif4A displayed frequent chromosome oscillations and abnormal congression in prometaphase and nonaligned chromosomes with broader metaphase plates. These cells showed prolonged progression of early mitosis with significant delays around ~90 min (Figure 3D−F; Supplementary Movies S2 and S5). These results indicated that Cdk phosphorylation of Kif4A mediates its chromosomal localization, and is thus critical for the functions of Kif4A required for early mitotic progression. Effects on MT dynamics, spindle morphology, and chromosome congression/alignment by Cdk phosphorylation of Kif4A To determine in further detail how Cdk phosphorylation of Kif4A was involved in regulating its functions required for early mitotic progression, we examined spindle structures and chromosome congression/alignment in control cells and cells depleted of endogenous Kif4A with or without exogenous expression of GFP-Kif4AWT, GFP-Kif4ATA, or GFP-Kif4ATE. Immunofluorescence analysis indicated that depletion of Kif4A in HeLa cells (Kif4A siRNA-transfected and GFP-expressing cells) resulted in short and plump chromosomes with more barrel-like spindles in prometaphase and disorganized chromosome congression/alignment with broader metaphase plates in metaphase when compared with control cells (Supplementary Figure S6) (Wandke et al., 2012). GFP-Kif4AWT or GFP-Kif4ATE expressed in cells lacking endogenous Kif4A localized on chromosomes and rescued virtually all chromosome and spindle defects, including the defects of spindle and chromosome morphologies and abnormalities of chromosome congression/alignment in early mitosis (Supplementary Figure S6). In contrast, GFP-Kif4ATA expressed in cells lacking endogenous Kif4A did not localize to chromosomes in early mitosis and could not rescue the chromosome and spindle defects (Supplementary Figure S6). Consistent with these results, cells depleted of Kif4A alone or cells depleted of Kif4A and expressing GFP-Kif4ATA activated the mitotic spindle checkpoint persistently. Immunofluorescence analysis showed that the mitotic spindle checkpoint protein, BubR1, was detected in metaphase cells depleted of Kif4A alone or metaphase cells depleted of Kif4A and expressing GFP-Kif4ATA but not in control metaphase cells or metaphase cells depleted of Kif4A and expressing GFP-Kif4AWT or GFP-Kif4ATE (Supplementary Figure S7). Previous studies showed that the chromokinesins Kif4A and Kid independently controlled the positioning of chromosome arms and the dynamics of spindle MTs for chromosome congression/alignment by regulating PEF in early mitosis (Brouhard and Hunt, 2005; Wandke et al., 2012). It was proposed that while Kid regulated PEFs through the control of chromosome-associated MTs by stabilizing the orientation of chromosome arms (Funabiki and Murray, 2000; Levesque and Compton, 2001), Kif4A mainly contributed to PEFs through the control of chromosome-associated MT dynamics by limiting the elongation of MTs near chromatin (Bringmann et al., 2004; Hu et al., 2011). Consistent with these reports, we found that ablation of both Kif4A and Kid proteins with siRNAs simultaneously resulted in elongated spindles and broader metaphase plates with uncongressed and unaligned chromosomes in metaphase when compared with control cells or cells depleted of either Kif4A or Kid alone (Figure 4A−C; Supplementary Figure S8A and Table S1). However, cells with monopolar spindles, generated with inhibition of Eg5 with S-trityl-L-cysteine (STLC), revealed that although Kif4A and Kid were individually required for chromosome ejection from the monopole, only Kif4A was required for limitation of monopolar spindle MT length (Figure 4D−F; Supplementary Figure S8B and Table S1). Inhibition of MT dynamic instability of cells with monopolar spindles by low concentration of nocodazole abrogated monopolar spindle MT elongations in cells depleted of Kif4A alone or both Kif4A and Kid (Supplementary Figure S9 and Table S1). Thus, these results indicated that the chromokinesins, Kif4A and Kid, controlled the chromosome positioning independently and only Kif4A was involved in regulating chromosome-associated MT dynamics, which were required for spindle structures and chromosome congression/alignment in early mitosis. Figure 4 View largeDownload slide Regulation of MT dynamics, spindle morphology, and chromosome congression/alignment by Cdk phosphorylation of Kif4A. (A−C) HeLa cells were transfected with Kif4A or/and Kid siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and immunostained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in A. Scale bar, 5 μm. The biopolar spindle length (B) and widths of metaphase plates (C) were measured. (D−F) HeLa cells were transfected with Kif4A or/and Kid siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 32 h and treated with 5 μM STLC for 16 h. Cells were fixed and immunostained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in D. Scale bar, 5 μm. The monopolar spindle length (E) and the distance of kinetochore to pole (KT-to-pole) (F) were determined as described in ‘Materials and methods’ section. Results represent mean + SD from three independent experiments (student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001). Figure 4 View largeDownload slide Regulation of MT dynamics, spindle morphology, and chromosome congression/alignment by Cdk phosphorylation of Kif4A. (A−C) HeLa cells were transfected with Kif4A or/and Kid siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. Cells were fixed and immunostained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in A. Scale bar, 5 μm. The biopolar spindle length (B) and widths of metaphase plates (C) were measured. (D−F) HeLa cells were transfected with Kif4A or/and Kid siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 32 h and treated with 5 μM STLC for 16 h. Cells were fixed and immunostained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in D. Scale bar, 5 μm. The monopolar spindle length (E) and the distance of kinetochore to pole (KT-to-pole) (F) were determined as described in ‘Materials and methods’ section. Results represent mean + SD from three independent experiments (student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001). We expressed GFP-Kif4AWT, GFP-Kif4ATA, or GFP-Kif4ATE in cells depleted of both endogenous Kif4A and Kid in either a bipolar spindle or a monopolar configuration. Expression of GFP-Kif4AWT or GFP-Kif4ATE in cells depleted of both Kif4A and Kid rescued early mitotic spindle length, widths of metaphase plates, chromosome ejection, or monopolar spindle MT length significantly. Like cells depleted of Kid alone, cells depleted of both Kif4A and Kid and expressing GFP-Kif4AWT or GFP-Kif4ATE showed significant smaller bipolar spindles, thinner metaphase plates, stronger chromosome ejection, and shorter monopolar MT lengths when compared with cells depleted of Kif4A alone or both Kif4A and Kid (Figure 4A−F; Supplementary Figure S8A and Table S1). In contrast, expression of GFP-Kif4ATA in cells depleted of both Kif4A and Kid rescued neither abnormalities of spindle structure and chromosome congression/alignment in a bipolar configuration nor abnormal chromosome ejection and limitation of monopolar spindle MT lengths in a monopolar spindle configuration. Like cells depleted of both Kif4A and Kid, cells depleted of both Kif4A and Kid and expressing GFP-Kif4ATA exhibited elongated spindles, broader metaphase plates with uncongressed and unaligned chromosomes, weaker chromosome ejection and longer monopolar MT lengths when compared with control cells or cells depleted of Kid alone (Figure 4A−F; Supplementary Figure S8A and Table S1). Taken together, these results demonstrated that Cdk phosphorylation of Kif4A controlled Kif4A chromosomal localization to promote its chromosome-associated functions in regulating MT dynamics, spindle morphology, and chromosome congression/alignment required for mitotic progression in early mitosis. Effects on chromosome condensation by Cdk phosphorylation of Kif4A As a chromokinesin, Kif4A was also shown to be cooperated with condensins to supercoil and compact chromatid arms laterally (Mazumdar et al., 2004; Samejima et al., 2012). To further examine Cdk phosphorylation of Kif4A in regulating chromosome condensation, we performed chromosome spreading experiments, in which control cells, cells depleted of endogenous Kif4A, cells depleted of endogenous Kif4A but expressing GFP-Kif4AWT, GFP-Kif4ATA, or GFP-Kif4ATE were arrested in mitosis by colcemid treatment, and then chromosomes were spread on slides and stained with DAPI. Fluorescent intensities of spread chromosomes and widths of individual chromosome arms were analyzed under a fluorescent microscope. Short and plump chromosomes and decreased chromosome fluorescent intensities were detected in cells depleted of endogenous Kif4A and cells depleted of endogenous Kif4A but expressing GFP-Kif4ATA when compared with control cells and cells depleted of endogenous Kif4A but expressing GFP-Kif4AWT or GFP-Kif4ATE (Figure 5A, Supplementary Figure S10A). We measured the widths and fluorescent intensities of individual chromosome arms and found that cells depleted of endogenous Kif4A and cells depleted of endogenous Kif4A but expressing GFP-Kif4ATA displayed wider chromosome arms with low fluorescent intensities than control cells and cells depleted of endogenous Kif4A but expressing GFP-Kif4AWT or GFP-Kif4ATE (Figure 5B and C, Supplementary Table S1). These results indicated that Cdk phosphorylation of Kif4A was involved in regulating chromosome condensation in early mitosis. Figure 5 View largeDownload slide Regulation of chromosome condensation by Cdk phosphorylation of Kif4A. HeLa cells were transfected with NS siRNA or Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. (A−C) Cells were treated with 0.1 μg/ml of colcemid overnight and chromosomes were spread on coverslips followed by immunostaining with DAPI (red). Representative images are shown in A. Scale bar, 5 μm. Widths of individual chromosome arms (B) and fluorescence intensity of DAPI (C) were measured as described in ‘Materials and methods’ section. More than 100 chromosomes in three independent experiments were analyzed (student’s t-test, **P < 0.01; ns, not significant). (D) Cells were lysed and immunoprecipitated with mouse α-CAP-G, rabbit α-CAP-D3, or rabbit α-SMC2. The immunoprecipitates were then immunoblotted with antibodies as indicated. (E) Cells were fixed and stained with rabbit α-SMC2 and DAPI. The boxes (arrowhead) represent the region used to generate the line scans, which show the localization of SMC2 (red) relative to DNA (cyan). Scale bar, 5 μm. (F) Quantification of relative SMC2 staining intensity in E. Fluorescence intensities of SMC2 were determined at five chromosome arms (chromosomal) and five areas in the cytoplasm (cytoplasmic) in each 20 cells. Figure 5 View largeDownload slide Regulation of chromosome condensation by Cdk phosphorylation of Kif4A. HeLa cells were transfected with NS siRNA or Kif4A siRNA together with pEGFP, pEGFP-Kif4AWT, pEGFP-Kif4ATA, or pEGFP-Kif4ATE plasmids for 48 h. (A−C) Cells were treated with 0.1 μg/ml of colcemid overnight and chromosomes were spread on coverslips followed by immunostaining with DAPI (red). Representative images are shown in A. Scale bar, 5 μm. Widths of individual chromosome arms (B) and fluorescence intensity of DAPI (C) were measured as described in ‘Materials and methods’ section. More than 100 chromosomes in three independent experiments were analyzed (student’s t-test, **P < 0.01; ns, not significant). (D) Cells were lysed and immunoprecipitated with mouse α-CAP-G, rabbit α-CAP-D3, or rabbit α-SMC2. The immunoprecipitates were then immunoblotted with antibodies as indicated. (E) Cells were fixed and stained with rabbit α-SMC2 and DAPI. The boxes (arrowhead) represent the region used to generate the line scans, which show the localization of SMC2 (red) relative to DNA (cyan). Scale bar, 5 μm. (F) Quantification of relative SMC2 staining intensity in E. Fluorescence intensities of SMC2 were determined at five chromosome arms (chromosomal) and five areas in the cytoplasm (cytoplasmic) in each 20 cells. We determined whether Cdk phosphorylation of Kif4A regulated chromosome condensation by cooperation with condensin. Co-immunoprecipitation revealed that, in control cells, condensin core subunit SMC2 associated with endogenous Kif4A as previously reported (Supplementary Figure S10B) (Mazumdar et al., 2004; Samejima et al., 2012). As expected, Kif4A was not present in α-SMC2 immunoprecipitates from cells in which Kif4A was depleted by siRNA. Furthermore, GFP-Kif4AWT or GFP-Kif4ATE expressed in cells depleted of Kif4A were able to associate with condensin I subunit CAP-G and SMC2 but not condensin II subunit CAP-D3 (Figure 5D) (Takahashi et al., 2016). In contrast, GFP-Kif4ATA expressed in cells depleted of Kif4A did not associate with CAP-G, SMC2, and CAP-D3 (Figure 5D). Consistent with these results, immunofluorescence analysis showed that cells depleted of endogenous Kif4A displayed decreased chromosome localization of SMC2 when compared with control cells. Expression of GFP-Kif4AWT or GFP-Kif4ATE in cells depleted of endogenous Kif4A restored chromosomal localization of SMC2. However, cells depleted of endogenous Kif4A and expressing GFP-Kif4ATA still showed reduction of chromosomal localization of SMC2, similar to cells depleted of endogenous Kif4A (Figure 5E and F). Taken together, these results indicate that Cdk phosphorylation of Kif4A controls its localization and promotes its association with condensin I, which is required for chromosome condensation. Chromosomal localization rather than Cdk phosphorylation of Kif4A is crucial for early mitotic functions of Kif4A We asked whether Cdk phosphorylation of Kif4A is crucial for chromosome localization and its mitotic functions or whether it simply serves to license the chromosomal localization of Kif4A but is nonessential for its subsequent functioning. To distinguish between these two possibilities, we targeted non-phosphorylatable Kif4A (GFP-Kif4ATA) to chromosomes by fusing Kif4A with Histone H1. We generated a set of mammalian expression vectors expressing GFP-Kif4AWT, GFP-Kif4ATA, and GFP-Kif4ATE fused with Histone H1 protein at the C-terminus of Kif4A (GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, and GFP-Kif4ATE-H1) (Supplementary Figure S11A). As a control, we also generated a mammalian expression construct expressing GFP-Kif4AMD-H1, in which the Kif4A ATP-binding site was mutated (Kif4A motor-dead mutant; Zhu and Jiang, 2005). We expressed these plasmids in cells depleted of endogenous Kif4A. Immunoblotting analysis showed that GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, GFP-Kif4ATE-H1, and GFP-Kif4AMD-H1 were expressed in Kif4A depleted cells (Supplementary Figure S11B). As shown in Figure 6A, immunofluorescence analysis revealed that, like GFP-Kif4AWT and GFP-Kif4ATE, GFP-Kif4AWT-H1 and GFP-Kif4ATE-H1 were localized on chromosomes in early mitotic cells lacking endogenous Kif4A. However, unlike GFP-Kif4ATA, which was expressed mainly in the cytoplasm, GFP-Kif4ATA-H1 was localized on chromosomes in early mitotic cells lacking endogenous Kif4A. As expected, GFP-Kif4AMD-H1 was also localized on chromosomes in early mitotic cells lacking endogenous Kif4A. Figure 6 View largeDownload slide Chromosomal localization of Kif4A is crucial for Kif4A early mitotic functions. HeLa cells were transfected with Kif4A siRNA together with pEGFP-Kif4AWT-H1, pEGFP-Kif4ATA-H1, pEGFP-Kif4ATE-H1, or pEGFP-Kif4AMD-H1 plasmids for 48 h. (A and B) Cells were fixed and stained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in A. Scale bar, 5 μm. Widths of metaphase plates were measured (B). (C−E) Cells were treated with 0.1 μg/ml colcemid overnight and chromosomes were spread on coverslips followed by immunostaining with DAPI (red). Representative images are shown in C. Scale bar, 5 μm. Widths of individual chromosome arms (D) and fluorescence intensity of DAPI (E) were measured. More than 100 chromosomes in three independent experiments were analyzed (student’s t-test, **P < 0.01; ns, not significant). (F) Cells were fixed and stained with rabbit α-SMC2 (red) and DAPI (cyan). The boxes (arrowhead) represent the region used to generate the line scans, which show the localization of SMC2 (red) relative to DNA (cyan). Scale bar, 5 μm. (G) Quantification of relative SMC2 staining intensity in F. Fluorescence intensities of SMC2 were determined at five chromosome arms (chromosomal) and five areas in the cytoplasm (cytoplasmic) in each 20 cells. Figure 6 View largeDownload slide Chromosomal localization of Kif4A is crucial for Kif4A early mitotic functions. HeLa cells were transfected with Kif4A siRNA together with pEGFP-Kif4AWT-H1, pEGFP-Kif4ATA-H1, pEGFP-Kif4ATE-H1, or pEGFP-Kif4AMD-H1 plasmids for 48 h. (A and B) Cells were fixed and stained with mouse anti-α-tubulin antibody (red), human anti-CREST serum (gray), and DAPI (cyan). Representative images are shown in A. Scale bar, 5 μm. Widths of metaphase plates were measured (B). (C−E) Cells were treated with 0.1 μg/ml colcemid overnight and chromosomes were spread on coverslips followed by immunostaining with DAPI (red). Representative images are shown in C. Scale bar, 5 μm. Widths of individual chromosome arms (D) and fluorescence intensity of DAPI (E) were measured. More than 100 chromosomes in three independent experiments were analyzed (student’s t-test, **P < 0.01; ns, not significant). (F) Cells were fixed and stained with rabbit α-SMC2 (red) and DAPI (cyan). The boxes (arrowhead) represent the region used to generate the line scans, which show the localization of SMC2 (red) relative to DNA (cyan). Scale bar, 5 μm. (G) Quantification of relative SMC2 staining intensity in F. Fluorescence intensities of SMC2 were determined at five chromosome arms (chromosomal) and five areas in the cytoplasm (cytoplasmic) in each 20 cells. We analyzed chromosome condensation and congression/alignment in cells depleted of endogenous Kif4A and expressing GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, GFP-Kif4ATE-H1, or GFP-Kif4AMD-H1. As shown in Figure 6A and B, Supplementary Table S1, expression of chromosome-associated GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, or GFP-Kif4ATE-H1, but not GFP-Kif4AMD-H1, in cells depleted of endogenous Kif4A could rescue abnormalities of chromosome congression/alignment. Consistent with these results, the mitotic spindle checkpoint BubR1 was not detected in metaphase cells depleted of endogenous Kif4A but expressing GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, or GFP-Kif4ATE-H1. In contrast, BubR1 was still detected in metaphase cells depleted of endogenous Kif4A but expressing GFP-Kif4AMD-H1 (Supplementary Figure S12). Chromosome spreading experiments indicated that expression of GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, or GFP-Kif4ATE-H1, but not GFP-Kif4AMD-H1, in cells depleted of endogenous Kif4A could restore chromosome fluorescent intensities and widths of chromosome arms to the levels of control cells (Figure 6C−E, Supplementary Table S1). Consistent with these results, immunofluorescence analysis indicated that SMC2 was reloaded on chromosomes in cells depleted of endogenous Kif4A and expressing chromosome-associated GFP-Kif4AWT-H1, GFP-Kif4ATA-H1, or GFP-Kif4ATE-H1, but not GFP-Kif4AMD-H1 (Figure 6F and G). These results indicated that targeting non-phosphorylatable Kif4ATA to chromosomes by Histone H1 was sufficient to restore Kif4A early mitotic function. Once Kif4A is localized to chromosomes, its Cdk-dependent phosphorylation is dispensable for carrying out its subsequent activities even though the ATP-dependent catalytic activity of Kif4A is still essential. Discussion Our study provides significant insights into the spatiotemporal regulation of the chromokinesin, Kif4A, in early mitosis. We show that Cdk phosphorylation of Kif4A licenses its chromosome localization. Kif4A, in turn, functions as a chromokinesin, regulates chromosome condensation, MT dynamics, spindle morphology and chromosome congression/alignment that are important for early mitotic progression. Cdk phosphorylation-licensed chromosome-associated Kif4A associates with CAP-G and SMC2 to promote chromosome condensation by participating in the regulation of mutual dependence of Kif4A and SMC2 on chromosomes for lateral compaction of chromosome arms (Figure 5) (Samejima et al., 2012). Moreover, Cdk phosphorylation-licensed chromosome-associated Kif4A is involved in regulating MT dynamics, spindle morphology, chromosome congression/alignment required for early mitotic progression. Chromosome-associated Kif4A coordinates with Kid to generate PEF and/or limit the elongation of chromosome-associated MTs in the vicinity of chromosomes (Figure 4) (Hu et al., 2011; Wandke et al., 2012). Targeting non-phosphorylatable Kif4ATA to chromosomes by fusion of Kif4ATA with Histone H1; however, could restore early mitotic functions of Kif4A in cells depleted of endogenous Kif4A (Figure 6). Thus, Cdk phosphorylation-licensed chromosome localization of Kif4A was crucial for early mitotic functions of Kif4A and early mitotic progression. Previous studies demonstrated that, as mitotic-promoting factors, mitotic Cdks phosphorylated multiple G2/M and early mitotic regulators, controlling their subcellular distribution, activities, and function that were critical for proper G2/M and early mitotic progression (Malik et al., 2009; Fisher et al., 2012). As a chromokinesin, Kif4A is involved in controlling chromosome condensation and chromosome congression/alignment in early mitosis. Our results reveal that Kif4A is phosphorylated by Cdk at its tail domain (T1161) and Cdk phosphorylation of Kif4A spatiotemporally regulates chromosome localization of Kif4A in early mitosis. Kif4A (and its cross-species homologs XKpl1 in Xenopus and Klp3A in Drosophila) contains two conserved DNA-binding motifs, a leucine zipper motif at its stalk region and a cysteine-rich motif at its tail domain, that were shown to be essential for chromatin-binding activity of Kif4A and subsequent chromosome localization of Kif4A in mitosis (Hu et al., 2011; Wandke et al., 2012). Our studies show that Cdk phosphorylation of Kif4A at its tail domain (T1161) only licenses chromosome localization of Kif4A but is not required for its chromosome-associated functions. As Kif4ATA-Histone H1 is fully functional, we propose that Cdk phosphorylation of Kif4A at T1161 might affect the total conformation of Kif4A molecule, depict the DNA-binding motifs of Kif4A to DNA/chromosomes and/or associate with condensin I (CAP-G/SMC2) (Takahashi et al., 2016), promoting Kif4A association with chromosomes in early mitosis. Once Kif4A localizes on chromosomes, chromosome-associated Kif4A, no matter whether T1161 is phosphorylated, would cooperate with condensin I to compact chromosome arms laterally for chromosome condensation. Meanwhile, chromosome condensation/chromosome arm compaction would reinforce Kif4A association with chromosome arms laterally. Lateral chromosomal arms-associated Kif4A would then function as a MT-dependent motor with MT plus-end ‘capping’ activity to regulate dynamics of chromosome-associated MTs. Chromosome-associated Kif4A could control dynamics of chromosome-associated MTs for proper formation and dynamics of mitotic spindle in early mitosis and/or coordinate with other mitotic motor proteins, such as Kid, Kif18A, CENP-E, and dynein, for chromosome congression/alignment (Stumpff et al., 2012; Wandke et al., 2012; Barisic et al., 2014; Iemura and Tanaka, 2015). Taken together, our results presented in this study demonstrate that Cdk phosphorylation licenses chromosomal localization of Kif4A and chromosomal localization of Kif4A, in turn, controls early mitotic functions of Kif4A important for proper early mitotic progression. Materials and methods Plasmids, siRNAs, and antibodies Mammalian expression plasmids of Kif4A and its point mutations were generated as described previously (Zhu and Jiang, 2005). The primers used in Kif4A T1161A point mutation were: sense 5′-tttaatcccgtctgtgccgcccccaatagcaagatcctg-3′ and antisense 5′-caggatcttgctattgggggcggcacagacgggattaaa-3′. The primers used in Kif4A T1161E point mutation were: sense 5′-tttaatcccgtctgtgccgaacccaatagcaagatcctg-3′ and antisense 5′-caggatcttgctattgggttcggcacagacgggattaaa-3′. Plasmids expressing Kif4A-Histone H1 fusion proteins were constructed by inserting Histone H1 cDNA at 3′ end of Kif4A cDNA before the stop codon in Kif4A expression plasmids. All constructs were fully sequenced. siRNA specific targeting to 3′-UTR of Kif4A (5′-GGAATGAGGTTGTGATCTT-3′) and siRNA specific targeting Kid cDNA coding region (5′-CAAGCUCACUCGCCUAUUGTT-3′) were synthesized by Genepharma. Polyclonal rabbit anti-Kif4ApT1161 antibodies (α-Kif4Ap1161) were developed against phospho-peptide of SFFNPVCA(pT)PNSKILKEMC. Polyclonal rabbit α-Kif4A were generated as previously described (Zhu and Jiang, 2005). Mouse anti-α-tubulin (#T5168) antibody was purchased from Sigma-Aldrich, rabbit α-SMC2 (#07-710) was purchased from Merk Millipore, mouse α-BubR 1 (#612503) was purchased from BD Biosciences, mouse α-cyclin B1 (#MAB3684) was purchased from Chemicon, mouse α-hCAP-G (#sc-515297) and goat α-Kid (#sc-30456) were purchased from Santa Cruz Biotechnology, and rabbit α-hCAP-D3 (A300-604A) was purchased from Bethyl Laboratories, Inc. All secondary antibodies were obtained from Life Technologies Inc. Cell culture, transfection, immunoprecipitation, immunoblotting, and immunofluorescence HeLa cells were cultured in DMEM containing 10% fetal calf serum (Invitrogen) at 37°C and 5% CO2. For transfection of plasmids and siRNAs, HeLa cells were cultured in 6-well plates and transfected with 50 nM siRNA and/or 0.1–0.5 μg of plasmid(s) using Lipofectamine 3000 (Thermo Fisher Scientific). Two to three days after transfection, cells were harvested or fixed for immunoprecipitation, immunoblotting, or immunofluorescence analysis as previously described (Jiang et al., 1998; Zhu and Jiang, 2005). For peptide competition assay, polyclonal rabbit antibodies, α-Kif4Ap1161, were incubated with Kif4A phospho-peptide or nonphospho-peptide at 4°C overnight. Control and taxol-treated mitotic cells were lysed in 1× sample buffer and subjected to SDS-PAGE followed by immunoblotting with peptide pre-treated α-Kif4Ap1161. Pearson’s correlation coefficients (R-value) were calculated using Image-Pro Plus 7.0 (Zinchuk et al., 2007). In brief, we generated coordinated datasets of fluorescence intensities at each pixel from 10 chromosome arms/cell and calculated Pearson correlation coefficients (R-values) for all pairwise combinations (total positive correlation appears as 1, total negative correlation as −1, and no correlation as 0). The datasets were obtained from 10 cells in three independent experiments. Cell cycle synchronization and drug treatments HeLa cells were treated with 2 mM thymidine (Sigma-Aldrich) for 16 h, released into fresh medium for 6 h, and then blocked with 40 ng/ml nocodazole (Sigma-Aldrich) for 12 h as described (Zhu and Jiang, 2005). To collected early mitotic cells, 30 nM taxol (Cytoskeleton) was added to the media and cells were collected after 16 h. To inhibit Cdk1 activity, 5 mg/ml roscovitine (Selleck Chemicals) was added to the media and cells were collected after 2 h. To stabilize MTs, 9 nM nocodazole (Sigma-Aldrich) was added to the cell culture media 3 h before fixation. 5 μM S-trityl-L-cysteine (STLC; Sigma-Aldrich) was added to the cell culture media 16 h before fixation to induce monopolar spindles by inhibiting Eg5. Mass spectrometry For mass spectrometry, cells were treated with 30 nM Taxol overnight. Cells were lysed and immunoprecipitated with α-Kif4A. The precipitates were separated by SDS/PAGE and stained with Coomassie blue. Specific Kif4A proteins in SDS-Gel were isolated and cut into pieces followed by digestion with trypsin. The digested samples were analyzed with a matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance mass spectrometer (Bruker Daltonics) and the phosphorylated peptides were further analyzed with automated nanoflow liquid chromatography/tandem mass spectrometry. In vitro kinase assay For in vitro kinase assay, 1 μg of bacterially expressed GST–Kif4AC230WT (230 amino acids at caboxyl terminus of Kif4A) or GST–Kif4AC230TA (Threonine 1161 was substituted by Alanine) mutant protein was incubated with 0.1 μg of baculovirus-expressed Cyclin B1–Cdk1 protein complex in kinase reaction as previously described (Jiang et al., 1998). The kinase reactions were terminated by adding an equal volume of 2× sample buffer. The reaction products were separated by SDS-PAGE and stained with Coomassie brilliant blue. The SDS-gel was then dried prior to autoradiography. Time-lapse microscopy HeLa cells stably expressing CFP-Histone H2B were cultured on 35-mm glass-bottom microwell dishes (Nest Biotechnology, China) and transfected with 0.2 μg of pmCherry-Kif4A plasmid together with 50 nM of Kif4A siRNA using Lipofectamine 3000 (Thermo Fisher Scientific). Thirty-six hours after transfection, cell culture dishes were transferred to a heated stage (37°C) on a Nikon HSJ Ti E-PFS microscope. Phase-contrast and fluorescence images of live cells were collected at 1-min intervals for 12 h and processed by using NIKON NIS-Elements BR software and Image J. Monopolar spindles assay HeLa cells were transfected with siRNA targeting Kid or/and Kif4A together with indicated plasmids, and treated with STLC for 16 h. Formaldehyde fixed cells were stained with mouse anti-α-tubulin antibody, human anti-CREST serum and DAPI. Monopolar spindles were imaged as a 3D z-stack using a 100× NA 1.35 oil objective on a fluorescence microscope (NIKON) and deconvolved using NIKON NIS-Elements AR software (NIKON). The cell center was defined as pole and the position was saved. The distance between kinetochores and pole (KT-to-pole Distance) and monopolar spindle length were measured using NIKON NIS-Elements AR software and calculated in Excel (Microsoft). Chromosome spreading analysis Cells were treated with 0.1 μg/ml of colcemid overnight and then hypotonically swollen in 75 mM KCl. Cells were fixed in ice-cold methanol/acetic acid and dropped on slides to spread chromosomes. The spreading chromosomes were stained with DAPI and images were acquired and processed under a Nikon Ts-100FL microscope. The widths and the fluorescent intensities of chromosome arms were measured using NIKON NIS-Elements AR software and Image-Pro Plus 7.0. Statistical analysis Student’s test was used to calculate the statistical significance of the experimental data. The level of significance was set as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. Acknowledgements We thank Drs Joel Leverson (Oncology Development Department, AbbVie Inc.) and Gary Chiang (Discovery, Global Pharmaceutical Research and Development, AbbVie Inc.) for critical reading of the manuscript. Funding This work was supported by CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-12M-1-001), the National Natural Science Foundation of China (31171299, 31271485, and 31301138), the National Basic Research Program of China (2012CB910703), and Tianjin Research Program of Application Foundation and Advanced Technology (12JC2DJC21400). C.Z. is supported by Program for New Century Excellent Talents in University in China (NCET-11-1066). Conflict of interest none declared. Author contributions Z.D., C.Z., and W.J. designed research; Z.D. and C.Z. performed research; Q.Z. contributed reagents; Z.D., C.Z., and W.J. analyzed data; and Z.D., C.Z., and W.J. wrote the paper. References Barisic , M. , Aguiar , P. , Geley , S. , et al. . ( 2014 ). Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces . Nat. Cell Biol. 16 , 1249 – 1256 . Google Scholar Crossref Search ADS PubMed Barr , F.A. , Elliott , P.R. , and Gruneberg , U. ( 2011 ). Protein phosphatases and the regulation of mitosis . J. Cell Sci. 124 , 2323 – 2334 . Google Scholar Crossref Search ADS PubMed Bastos , R.N. , Cundell , M.J. , and Barr , F.A. ( 2014 ). KIF4A and PP2A-B56 form a spatially restricted feedback loop opposing Aurora B at the anaphase central spindle . J. Cell Biol. 207 , 683 – 693 . Google Scholar Crossref Search ADS PubMed Belmont , A.S. ( 2006 ). Mitotic chromosome structure and condensation . Curr. Opin. Cell Biol. 18 , 632 – 638 . Google Scholar Crossref Search ADS PubMed Bieling , P. , Telley , I.A. , and Surrey , T. ( 2010 ). A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps . Cell 142 , 420 – 432 . Google Scholar Crossref Search ADS PubMed Bringmann , H. , Skiniotis , G. , Spilker , A. , et al. . ( 2004 ). A kinesin-like motor inhibits microtubule dynamic instability . Science 303 , 1519 – 1522 . Google Scholar Crossref Search ADS PubMed Brouhard , G.J. , and Hunt , A.J. ( 2005 ). Microtubule movements on the arms of mitotic chromosomes: polar ejection forces quantified in vitro . Proc. Natl Acad. Sci. USA 102 , 13903 – 13908 . Google Scholar Crossref Search ADS Castoldi , M. , and Vernos , I. ( 2006 ). Chromokinesin Xklp1 contributes to the regulation of microtubule density and organization during spindle assembly . Mol. Biol. Cell 17 , 1451 – 1460 . Google Scholar Crossref Search ADS PubMed D’Avino , P.P. , Giansanti , M.G. , and Petronczki , M. ( 2015 ). Cytokinesis in animal cells . Cold. Spring Harb. Perspect. Biol. 7 , a015834 . Google Scholar Crossref Search ADS PubMed Fisher , D. , Krasinska , L. , Coudreuse , D. , et al. . ( 2012 ). Phosphorylation network dynamics in the control of cell cycle transitions . J. Cell Sci. 125 , 4703 – 4711 . Google Scholar Crossref Search ADS PubMed Foley , E.A. , and Kapoor , T.M. ( 2013 ). Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore . Nat. Rev. Mol. Cell Biol. 14 , 25 – 37 . Google Scholar Crossref Search ADS PubMed Funabiki , H. , and Murray , A.W. ( 2000 ). The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement . Cell 102 , 411 – 424 . Google Scholar Crossref Search ADS PubMed Glotzer , M. ( 2001 ). Animal cell cytokinesis . Annu. Rev. Cell Dev. Biol. 17 , 351 – 386 . Google Scholar Crossref Search ADS PubMed Goshima , G. , and Vale , R.D. ( 2005 ). Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells . Mol. Biol. Cell 16 , 3896 – 3907 . Google Scholar Crossref Search ADS PubMed Green , R.A. , Paluch , E. , and Oegema , K. ( 2012 ). Cytokinesis in animal cells . Annu. Rev. Cell Dev. Biol. 28 , 29 – 58 . Google Scholar Crossref Search ADS PubMed Holland , A.J. , and Cleveland , D.W. ( 2012 ). Losing balance: the origin and impact of aneuploidy in cancer . EMBO Rep. 13 , 501 – 514 . Google Scholar Crossref Search ADS PubMed Hu , C.K. , Coughlin , M. , Field , C.M. , et al. . ( 2011 ). KIF4 regulates midzone length during cytokinesis . Curr. Biol. 21 , 815 – 824 . Google Scholar Crossref Search ADS PubMed Iemura , K. , and Tanaka , K. ( 2015 ). Chromokinesin Kid and kinetochore kinesin CENP-E differentially support chromosome congression without end-on attachment to microtubules . Nat. Commun. 6 , 6447 . Google Scholar Crossref Search ADS PubMed Jeppsson , K. , Kanno , T. , Shirahige , K. , et al. . ( 2014 ). The maintenance of chromosome structure: positioning and functioning of SMC complexes . Nat. Rev. Mol. Cell Biol. 15 , 601 – 614 . Google Scholar Crossref Search ADS PubMed Jiang , W. , Jimenez , G. , Wells , N.J. , et al. . ( 1998 ). PRC1: a human mitotic spindle-associated CDK substrate protein required for cytokinesis . Mol. Cell 2 , 877 – 885 . Google Scholar Crossref Search ADS PubMed Kakui , Y. , and Uhlmann , F. ( 2018 ). SMC complexes orchestrate the mitotic chromatin interaction landscape . Curr. Genet. 64 , 335 – 339 . Google Scholar Crossref Search ADS PubMed Kurasawa , Y. , Earnshaw , W.C. , Mochizuki , Y. , et al. . ( 2004 ). Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation . EMBO J. 23 , 3237 – 3248 . Google Scholar Crossref Search ADS PubMed Levesque , A.A. , and Compton , D.A. ( 2001 ). The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles . J. Cell Biol. 154 , 1135 – 1146 . Google Scholar Crossref Search ADS PubMed Li , Q.R. , Yan , X.M. , Guo , L. , et al. . ( 2018 ). AMPK regulates anaphase central spindle length by phosphorylation of KIF4A . J. Mol. Cell Biol. 10 , 2 – 17 . Google Scholar Crossref Search ADS PubMed London , N. , and Biggins , S. ( 2014 ). Signalling dynamics in the spindle checkpoint response . Nat. Rev. Mol. Cell Biol. 15 , 736 – 747 . Google Scholar Crossref Search ADS PubMed Ly , P. , and Cleveland , D.W. ( 2017 ). Rebuilding chromosomes after catastrophe: emerging mechanisms of chromothripsis. Trends Cell Biol. 27 , 917 – 930 . Google Scholar Crossref Search ADS PubMed Mack , G.J. , and Compton , D.A. ( 2001 ). Analysis of mitotic microtubule-associated proteins using mass spectrometry identifies astrin, a spindle-associated protein . Proc. Natl Acad. Sci. USA 98 , 14434 – 14439 . Google Scholar Crossref Search ADS Malik , R. , Lenobel , R. , Santamaria , A. , et al. . ( 2009 ). Quantitative analysis of the human spindle phosphoproteome at distinct mitotic stages . J. Proteome Res. 8 , 4553 – 4563 . Google Scholar Crossref Search ADS PubMed Mazumdar , M. , Sundareshan , S. , and Misteli , T. ( 2004 ). Human chromokinesin KIF4A functions in chromosome condensation and segregation . J. Cell Biol. 166 , 613 – 620 . Google Scholar Crossref Search ADS PubMed McIntosh , J.R. , Grishchuk , E.L. , and West , R.R. ( 2002 ). Chromosome-microtubule interactions during mitosis . Annu. Rev. Cell Dev. Biol. 18 , 193 – 219 . Google Scholar Crossref Search ADS PubMed Nguyen , P.A. , Groen , A.C. , Loose , M. , et al. . ( 2014 ). Spatial organization of cytokinesis signaling reconstituted in a cell-free system . Science 346 , 244 – 247 . Google Scholar Crossref Search ADS PubMed Nunes Bastos , R. , Gandhi , S.R. , Baron , R.D. , et al. . ( 2013 ). Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A . J. Cell Biol. 202 , 605 – 621 . Google Scholar Crossref Search ADS PubMed Samejima , K. , Samejima , I. , Vagnarelli , P. , et al. . ( 2012 ). Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα . J. Cell Biol. 199 , 755 – 770 . Google Scholar Crossref Search ADS PubMed Stumpff , J. , Wagenbach , M. , Franck , A. , et al. . ( 2012 ). Kif18A and chromokinesins confine centromere movements via microtubule growth suppression and spatial control of kinetochore tension . Dev. Cell 22 , 1017 – 1029 . Google Scholar Crossref Search ADS PubMed Subramanian , R. , Ti , S.C. , Tan , L. , et al. . ( 2013 ). Marking and measuring single microtubules by PRC1 and kinesin-4 . Cell 154 , 377 – 390 . Google Scholar Crossref Search ADS PubMed Subramanian , R. , Wilson-Kubalek , E.M. , Arthur , C.P. , et al. . ( 2010 ). Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein . Cell 142 , 433 – 443 . Google Scholar Crossref Search ADS PubMed Takahashi , M. , Wakai , T. , and Hirota , T. ( 2016 ). Condensin I-mediated mitotic chromosome assembly requires association with chromokinesin KIF4A . Genes Dev. 30 , 1931 – 1936 . Google Scholar Crossref Search ADS PubMed Vagnarelli , P. ( 2012 ). Mitotic chromosome condensation in vertebrates . Exp. Cell Res. 318 , 1435 – 1441 . Google Scholar Crossref Search ADS PubMed Vallardi , G. , and Saurin , A.T. ( 2015 ). Mitotic kinases and phosphatases cooperate to shape the right response . Cell Cycle 14 , 795 – 796 . Google Scholar Crossref Search ADS PubMed Vicente , J.J. , and Wordeman , L. ( 2015 ). Mitosis, microtubule dynamics and the evolution of kinesins . Exp. Cell Res. 334 , 61 – 69 . Google Scholar Crossref Search ADS PubMed Walczak , C.E. , and Heald , R. ( 2008 ). Mechanisms of mitotic spindle assembly and function . Int. Rev. Cytol. 265 , 111 – 158 . Google Scholar Crossref Search ADS PubMed Wandke , C. , Barisic , M. , Sigl , R. , et al. . ( 2012 ). Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis . J. Cell Biol. 198 , 847 – 863 . Google Scholar Crossref Search ADS PubMed Wang , G. , Jiang , Q. , and Zhang , C. ( 2014 ). The role of mitotic kinases in coupling the centrosome cycle with the assembly of the mitotic spindle . J. Cell Sci. 127 , 4111 – 4122 . Google Scholar Crossref Search ADS PubMed Zhu , C. , and Jiang , W. ( 2005 ). Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis . Proc. Natl Acad. Sci. USA 102 , 343 – 348 . Google Scholar Crossref Search ADS Zhu , C. , Zhao , J. , Bibikova , M. , et al. . ( 2005 ). Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference . Mol. Biol. Cell 16 , 3187 – 3199 . Google Scholar Crossref Search ADS PubMed Zinchuk , V. , Zinchuk , O. , and Okada , T. ( 2007 ). Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena . Acta Histochem. Cytochem. 40 , 101 – 111 . Google Scholar Crossref Search ADS PubMed © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Journal of Molecular Cell BiologyOxford University Press

Published: Aug 1, 2018

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off