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Address correspondence to Manjari Mazumdar, National Cancer Institute, National Institutes of Health, Bldg. 41, Rm. B 507, 41 Library Dr., Bethesda, MD 20892. Tel.: (301) 435-2672. Fax: (301) 496-4951. email: mazumdam{at}mail.nih.gov
Abstract
Accurate chromosome alignment at metaphase and subsequent segregation of condensed chromosomes is a complex process involving elaborate and only partially characterized molecular machinery. Although several spindle associated molecular motors have been shown to be essential for mitotic function, only a few chromosome armassociated motors have been described. Here, we show that human chromokinesin human HKIF4A (HKIF4A) is an essential chromosome-associated molecular motor involved in faithful chromosome segregation. HKIF4A localizes in the nucleoplasm during interphase and on condensed chromosome arms during mitosis. It accumulates in the mid-zone from late anaphase and localizes to the cytokinetic ring during cytokinesis. RNA interferencemediated depletion of HKIF4A in human cells results in defective prometaphase organization, chromosome mis-alignment at metaphase, spindle defects, and chromosome mis-segregation. HKIF4A interacts with the condensin I and II complexes and HKIF4A depletion results in chromosome hypercondensation, suggesting that HKIF4A is required for maintaining normal chromosome architecture. Our results provide functional evidence that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.
Key Words: chromokinesin; spindle; chromosome condensation; molecular motor
Introduction
Faithful segregation of the genome involves an elaborate macromolecular machine in which the mitotic spindle plays a central role. Defects in components that control spindle organization and function often lead to chromosome mis-segregation, aneuploidy, and cellular abnormalities (Pihan and Doxsey, 1999; Jallepalli and Lengauer, 2001). The dynamic nature of the spindle apparatus is believed to be maintained both by the dynamic instability of microtubules (MT) as well as several force producing MT motors (Scholey et al., 2003). Poleward and away from the pole forces balance each other during metaphase congression and are responsible for chromosome motility toward the poles (Marshall, 2002). Polar ejection forces may be generated either by dynamic MTs or by plus-enddirected motor including the chromokinesins, which associate with chromosome arms (McIntosh et al., 2002). Chromokinesins represent a family of chromosome arm-binding kinesins consisting of two distinct types of members: chromokinesins/KIF4 and the Kid homologues (Sekine et al., 1994; Vernos et al., 1995; Wang and Adler, 1995; Williams et al., 1995; Tokai et al., 1996; Yan and Wang, 1997; Antonio et al., 2000; Funabiki and Murray, 2000). Both types of chromokinesins are nuclear during interphase and localize on condensed chromosome arms during mitosis. In humans, two KIF4 members exist: HKIF4A and HKIF4B (Ha et al., 2000). Human KIF4A (HKIF4A) is a 140-kD protein that contains several conserved structural motifs including a kinesin-like motor domain, a long coiled-coil region, a nuclear localization signal, a DNA-binding motif and a cysteine-rich Zn fingerlike motif. The protein has been shown to interact with BRCA2-associated factor 35 and the DNA methyltransferase DNMT3B (Lee and Kim, 2003; Geiman et al., 2004). Although HKIF4A associates with chromosomes during mitosis, no information as to the function of the protein is available (Lee et al., 2001). Here, we show by RNA interference (RNAi) that HKIF4A is a novel multifunctional component of the chromosome condensation and segregation machinery.
Results and discussion
To gain insight into HKIF4A function, we raised a mouse mAb specific against the extreme COOH-terminal domain of human chromokinesin HKIF4A (Fig. 1 A; see Materials and methods). In Western blots of MRC-5 cell extracts, the HKIF4A antibody detected a single band of 140 kD (Fig. 1 A). In subcellular fractionation of nonsynchronized cells, the protein was highly enriched in the nuclear extract and only trace amounts were detected in the cytoplasmic fraction (Fig. 1 A). During mitosis HKIF4A associates along the entire arms of condensed chromosomes (Fig. 1 B). In addition to the chromosomal localization, the protein accumulates in the mid-zone from anaphase A to cytokinesis (Fig. 1 B). From anaphase B to cytokinesis HKIF4A is present in the mid-body as two distinct rings connecting the MTs from the two half spindles (Fig. 1 B, inset). In late cytokinesis, until the two daughter cells pinch off, the protein persists in the center of the mid-body (Fig. 1 B). Costaining of HKIF4A with tubulin shows partial colocalization at the spindle poles and at the central spindle (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200401142/DC1).
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To determine whether these mis-segregation and cytokinesis defects resulted in aneuploidy of daughter cells, metaphase chromosome spreads from mock or HKIF4A RNAi-depleted cells were prepared. 80% of spreads of HKIF4A-depleted cells were aneuploid. 53% of HKIF4A-depleted cells had lost one or more chromosomes and 25% of cells had gained one or more chromosomes (Fig. 3 D). Less than 1% of aneuploid spreads were found in control cells.
HKIF4A is required for maintaining normal metaphase chromosome morphology
Because HKIF4A is localized all along the condensed chromosome arms, we examined the consequences of HKIF4A depletion on the structural integrity of mitotic chromosomes. Metaphase chromosome spreads from mock-transfected or HKIF4A RNAi-transfected cells were prepared after 2 h of colcemid block and stained with DAPI (Fig. 4). RNAi-mediated depletion of HKIF4A induced significant hypercondensation and chromosomes from HKIF4A-depleted cells were dramatically shorter than chromosomes from mock-transfected cells (Fig. 4 A). The average length of control chromosomes was 4.88 µm with a range between 1.5 and 10 µm, reflecting the variable sizes of human chromosomes. The width of the chromosomes was on average 0.68 µm with a range of 0.50.8 µm (Fig. 4 B). In contrast, chromosomes from depleted cells were on average 3-µm long and 1.2-µm wide, with a range of 1.15.5 µm in length and 0.81.5 µm in width (Fig. 5 B). These differences were statistically significant at the P < 0.001 level. To rule out that the observed hypercondensation of chromosomes was caused by artifacts of chromosome spread preparation, and more importantly, to exclude the possibility that hypercondensation was caused by prolonged presence of chromosomes in mitosis, we analyzed chromosomes in intact cells. We followed progression of mitosis from nuclear envelope breakdown to telophase in single living HeLa cells stably expressing histone H2B-GFP that are either mock-transfected or RNAi-transfected (Fig. 4 C). In the majority of cells in the RNAi-treated population, chromosomes were more condensed compared with mock-transfected cells even before prometaphase (Fig. 4 C). As expected, cells containing hypercondensed chromosomes experienced a delay in mitotic progression (Fig. 4 C). Furthermore, chromosomes were already significantly more condensed in HKIF4A-depleted cells compared with control cells even before breakdown of the nuclear envelope in early prophase (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200401142/DC1).
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Although HKIF4 has previously been localized to mitotic chromosomes (Lee et al., 2001) our results extend these observations by demonstrating a functional role for HKIF4A in chromosome segregation, cytokinesis, and structural integrity of chromosomes.
A role of HKIF4A as a molecular motor is suggested by its close homology with the other KIF4 kinesin family members mouse KIF4 (Sekine et al., 1994), Xenopus Xklp1 (Vernos et al., 1995), and Drosophila KLP3A (Williams et al., 1995). In this function, it may contribute to generating an away from the pole force and cooperate with other plus- and minus-enddirected motors to create the force balance required for spindle bipolarity and chromosome alignment at metaphase (Goshima and Vale, 2003; Kwon et al., 2004; Bringmann et al., 2004). This interpretation is consistent with our observations because in the majority of HKIF4A-depleted cells, chromosomes were scattered along the length of the spindle, and a large number of aberrant spindle structures were generated. Furthermore, localization of HKIF4A on the cytokinetic mid-body is reminiscent of its Drosophila homologue KLP3A in cytokinesis during Drosophila male meiosis (Williams et al., 1995, 1997). Our observation that chromosome segregation is not completely blocked but continues at a low level until at least three cycles suggests that HKIF4A is redundant with the other chromokinesin Kid (Levesque and Compton, 2001) and kinetochore-associated plus-end motors (Yen and Schaar, 1996; Kapoor and Compton, 2002). Consistent with redundancy amongst these motors, KLP3A has been shown to be dispensable in Drosophila (Goshima and Vale, 2003; Kwon et al., 2004).
Apart from its possible role as a molecular motor, our observations suggest that HKIF4A might also have an additional, and possibly complementary, function as a critical component in chromosome condensation. We find that HKIF4A interacts with both condensin I and II complexes and the depletion of the protein in vivo leads to hypercondensation of chromosomes. Similar to condensin I and II complexes, topoisomerase II and some condensin subunits, HKIF4A localizes in an alternating, punctate pattern along the metaphase chromosome axis (Maeshima and Laemmli, 2003; Ono et al., 2003). Depletion of HKIF4A from chromosomes appeared to partially delocalize condensin subunits from the chromosome axis, which is consistent with their physical interaction. We speculate that HKIF4A might function as a molecular linker and/or spacer between chromosome condensation proteins and DNA to contribute to higher order organization of metaphase chromosomes. Its depletion might thus be expected to result in a collapse of the chromosome fiber, giving rise to the observed hypercondensation phenotype. HKIF4A may, together with condensin and other nonhistone proteins, form the structural framework of the metaphase chromosome (Earnshaw and Laemmli, 1983; Hudson et al., 2003; Swedlow and Hirano, 2003; Gassmann et al., 2004; Strick et al., 2004). Consistent with such a role of HKIF4A, we find multiple defects both in chromosome structure and mitotic spindle organization. Similar phenotypes including formation of anaphase bridges have recently been observed in studies in which the function of components of chromosome condensation machinery have been inhibited (Saka et al., 1994; Steffensen et al., 2001; Kaitna et al., 2002; Lavoie et al., 2002; Chang et al., 2003; Coelho et al., 2003; Hagstrom and Meyer, 2003; Hudson et al., 2003; Somma et al., 2003; Wignall et al., 2003; Ono et al., 2004). The sum of these results suggests a functional link between chromosome condensation and subsequent steps of chromosome segregation.
Materials and methods
Cell lines
MRC-5 human fetal lung fibroblast cells (CCL-171; American Type Culture Collection) were grown in DME (GIBCO BRL) supplemented with 10% FBS, L-glutamate, and penicillin-streptomycin.
Antibodies
The human chromokinesin HKIF4A monoclonal mouse antibody was generated as described previously (Geiman et al., 2004). The culture supernatant was used at 1:50 for Western blots or undiluted for immunoprecipitation reactions. Antibodies for immunofluorescence were goat antimouse or antirabbit IgG conjugated with Alexa 488 or Alexa 568 (Molecular Probes) and donkey antirat IgG conjugated with cy3 (Jackson Labs)
RNAi depletion of KIF4A in MRC-5 cells
Two siRNA duplexes (HKIF4A RNA1, 5'-GCAATTGATTACCCAGTTA-3'; HKIF4A RNA2, 5'-GAAAGATCCTGGCTCAAGA-3') targeting HKIF4A were obtained from SMARTPOOL (Dharmacon Research) and gave identical results. Cells were transfected with 100 nM RNAi duplexes using Oligofectamine (Invitrogen). Cells were transfected for a second time 24 h after the first transfection (Elbashir et al., 2002). For protein analyses, the transfected cells were washed twice with PBS and extracted with SDS sample buffer. Cells on coverslips were fixed at different time points after transfection up to 55 h.
Immunofluorescent staining of MRC-5 cells and chromosomes
Immunofluorescence was performed as described previously (Misteli and Spector, 1996). For double staining with tubulin, cells were pre-extracted with 0.5% Triton X-100 before fixation. For spindle staining, anti-HKIF4A was coincubated with rat anti-tubulin antibody (YL/2; Sera Lab) at a dilution of 1:200.
In situ chromosome and metaphase chromosome spreads were prepared and subjected to immunofluorescent staining as described previously (Ono et al., 2003) except that the chromosomes were treated with 0.056 M of hypotonic solution and chromosome spreads were prepared by vertically dropping the cell suspension with a Pasteur pipette onto the slide. Image analysis was performed using either an Eclipse microscope (Nikon) fitted with a cooled CCD camera (Micromax) or a 510 LSM META confocal microscope (Carl Zeiss MicroImaging, Inc.).
Coimmunoprecipitation and Western blotting
Nuclear extracts were prepared and coimmunoprecipitations were performed essentially as described previously (Nielsen et al., 1999). Mitotic extracts from HeLa S3 were prepared as described previously (Gaglio et al., 1995). Precipitated proteins were separated by 7.5% SDS-PAGE and analyzed by Western blotting. Immunoblots were blocked with 5% Carnation nonfat milk in TBST (20 mM Tris, pH 7.5, 137 mM NaCl, 0.1% Tween 20). Primary and secondary antibodies were diluted in 1% blocking solution. Immunoreactive bands of proteins were detected using ECL (Amersham Biosciences).
Online supplemental materials
Fig. S1 A shows the colocalization of HKIF4A with MTs at different phases of the cell cycle. Fig. S1 B shows microinjection of HKIF4A antibody into prometaphase cells causes mis-alignment of chromosomes and mitotic delay. Fig. S2 shows HKIF4A depletion hypercondenses chromosomes even before nuclear envelope breakdown. Fig. S3 shows immunoprecipitation controls. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200401142/DC1.
Acknowledgments
We thank T. Hirano, K. Yokomori, and M. Bustin for reagents, L. Parada for insightful discussion, T. Cheutin and T. Karpova for help with imaging, and A. Karande for help with raising the mAbs. Imaging was performed at the NCI Fluorescence Imaging Facility.
T. Misteli is a Fellow of the Keith R. Porter Endowment for Cell Biology.
Submitted: 29 January 2004
Accepted: 16 July 2004
Antonio, C., I. Ferby, H. Wilhelm, M. Jones, E. Karsenti, A.R. Nebreda, and I. Vernos. 2000. Xkid, a chromokinesin required for chromosome alignment on the metaphase plate. Cell. 102:425435.[Medline]
Bringmann, H., G. Skiniotis, A. Spilker, S. Kandels-Lewis, I. Vernos, and T. Surrey. 2004. A kinesin-like motor inhibits microtubule dynamic instability. Science. 303:15191522.
Chang, C.J., S. Goulding, W.C. Earnshaw, and M. Carmena. 2003. RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate. J. Cell Sci. 116:47154726.
Coelho, P.A., J. Queiroz-Machado, and C.E. Sunkel. 2003. Condensin-dependent localization of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis. J. Cell Sci. 116:47634776.
Earnshaw, W.C., and U.K. Laemmli. 1983. Architecture of metaphase chromosomes and chromosome scaffolds. J. Cell Biol. 96:8493.[Abstract]
Elbashir, S.M., J. Harborth, K. Weber, and T. Tuschl. 2002. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 26:199213.[CrossRef][Medline]
Funabiki, H., and A.W. Murray. 2000. The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement. Cell. 102:411424.[Medline]
Gaglio, T., A. Saredi, and D.A. Compton. 1995. NuMA is required for the organization of microtubules into aster-like mitotic arrays. J. Cell Biol. 131:693708.[Abstract]
Gassmann, R., P. Vagnarelli, D. Hudson, and W.C. Earnshaw. 2004. Mitotic chromosome formation and the condensin paradox. Exp. Cell Res. 296:3542.[CrossRef][Medline]
Geiman, T.M., U.T. Sankpal, A.K. Robertson, Y. Chen, M. Mazumdar, J.T. Heale, J.A. Schmiesing, W. Kim, K. Yokomori, Y. Zhao, and K.D. Robertson. 2004. Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery. Nucleic Acids Res. 32:27162729.
Goshima, G., and R.D. Vale. 2003. The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. J. Cell Biol. 162:10031016.
Ha, M.J., J. Yoon, E. Moon, Y.M. Lee, H.J. Kim, and W. Kim. 2000. Assignment of the kinesin family member 4 genes (KIF4A and KIF4B) to human chromosome bands Xq13.1 and 5q33.1 by in situ hybridization. Cytogenet. Cell Genet. 88:4142.[Medline]
Hagstrom, K.A., and B.J. Meyer. 2003. Condensin and cohesin: more than chromosome compactor and glue. Nat. Rev. Genet. 4:520534.[CrossRef][Medline]
Hudson, D.F., P. Vagnarelli, R. Gassmann, and W.C. Earnshaw. 2003. Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev. Cell. 5:323336.[CrossRef][Medline]
Jallepalli, P.V., and C. Lengauer. 2001. Chromosome segregation and cancer: cutting through the mystery. Nat. Rev. Cancer. 1:109117.[CrossRef][Medline]
Kaitna, S., P. Pasierbek, M. Jantsch, J. Loidl, and M. Glotzer. 2002. The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis. Curr. Biol. 12:798812.[CrossRef][Medline]
Kapoor, T.M., and D.A. Compton. 2002. Searching for the middle ground: mechanisms of chromosome alignment during mitosis. J. Cell Biol. 157:551556.
Kwon, M., S. Morales-Mulia, I. Brust-Mascher, G.C. Rogers, D.J. Sharp, and J.M. Scholey. 2004. The chromokinesin, KLP3A, drives mitotic spindle pole separation during prometaphase and anaphase, and facilitates chromatid motility. Mol Biol Cell. 15:219233.
Lavoie, B.D., E. Hogan, and D. Koshland. 2002. In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin. J. Cell Biol. 156:805815.
Lee, Y.M., and W. Kim. 2003. Association of human kinesin superfamily protein member 4 with BRCA2-associated factor 35. Biochem. J. 374:497503.[CrossRef][Medline]
Lee, Y.M., S. Lee, E. Lee, H. Shin, H. Hahn, W. Choi, and W. Kim. 2001. Human kinesin superfamily member 4 is dominantly localized in the nuclear matrix and is associated with chromosomes during mitosis. Biochem. J. 360:549556.[CrossRef][Medline]
Levesque, A.A., and D.A. Compton. 2001. The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles. J. Cell Biol. 154:11351146.
Maeshima, K., and U.K. Laemmli. 2003. A two-step scaffolding model for mitotic chromosome assembly. Dev. Cell. 4:467480.[Medline]
Marshall, W.F. 2002. Polar wind left flapping in the breeze? Trends Cell Biol. 12:9.
McIntosh, J.R., E.L. Grishchuk, and R.R. West. 2002. Chromosome-microtubule interactions during mitosis. Annu. Rev. Cell Dev. Biol. 18:193219.[CrossRef][Medline]
Misteli, T., and D.L. Spector. 1996. Serine/threonine phosphatase 1 modulates the subnuclear distribution of pre-mRNA splicing factors. Mol. Biol. Cell. 7:15591572.[Abstract]
Nielsen, A.L., J.A. Ortiz, J. You, M. Oulad-Abdelghani, R. Khechumian, A. Gansmuller, P. Chambon, and R. Losson. 1999. Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family. EMBO J. 18:63856395.
Ono, T., A. Losada, M. Hirano, M.P. Myers, A.F. Neuwald, and T. Hirano. 2003. Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell. 115:109121.[CrossRef][Medline]
Ono, T., Y. Fang, D.L. Spector, and T. Hirano. 2004. Spatial and temporal regulation of condensins I and II in mitotic chromosome assembly in human cells. Mol Biol Cell. 15:32963308.
Pihan, G.A., and S.J. Doxsey. 1999. The mitotic machinery as a source of genetic instability in cancer. Semin. Cancer Biol. 9:289302.[CrossRef][Medline]
Saka, Y., T. Sutani, Y. Yamashita, S. Saitoh, M. Takeuchi, Y. Nakaseko, and M. Yanagida. 1994. Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J. 13:49384952.[Abstract]
Scholey, J.M., I. Brust-Mascher, and A. Mogilner. 2003. Cell division. Nature. 422:746752.[CrossRef][Medline]
Sekine, Y., Y. Okada, Y. Noda, S. Kondo, H. Aizawa, R. Takemura, and N. Hirokawa. 1994. A novel microtubule-based motor protein (KIF4) for organelle transports, whose expression is regulated developmentally. J. Cell Biol. 127:187201.[Abstract]
Somma, M.P., B. Fasulo, G. Siriaco, and G. Cenci. 2003. Chromosome condensation defects in barren RNA-interfered Drosophila cells. Genetics. 165:16071611.
Steffensen, S., P.A. Coelho, N. Cobbe, S. Vass, M. Costa, B. Hassan, S.N. Prokopenko, H. Bellen, M.M. Heck, and C.E. Sunkel. 2001. A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis. Curr. Biol. 11:295307.[CrossRef][Medline]
Strick, T.R., T. Kawaguchi, and T. Hirano. 2004. Real-time detection of single-molecule DNA compaction by condensin I. Curr. Biol. 14:874880.[CrossRef][Medline]
Swedlow, J.R., and T. Hirano. 2003. The making of the mitotic chromosome: modern insights into classical questions. Mol. Cell. 11:557569.[Medline]
Tokai, N., A. Fujimoto-Nishiyama, Y. Toyoshima, S. Yonemura, S. Tsukita, J. Inoue, and T. Yamamota. 1996. Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle. EMBO J. 15:457467.[Abstract]
Vernos, I., J. Raats, T. Hirano, J. Heasman, E. Karsenti, and C. Wylie. 1995. Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning. Cell. 81:117127.[Medline]
Wang, S.Z., and R. Adler. 1995. Chromokinesin: a DNA-binding, kinesin-like nuclear protein. J. Cell Biol. 128:761768.[Abstract]
Wignall, S.M., R. Deehan, T.J. Maresca, and R. Heald. 2003. The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts. J. Cell Biol. 161:10411051.
Williams, B.C., M.F. Riedy, E.V. Williams, M. Gatti, and M.L. Goldberg. 1995. The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis. J. Cell Biol. 129:709723.[Abstract]
Williams, B.C., A.F. Dernburg, J. Puro, S. Nokkala, and M.L. Goldberg. 1997. The Drosophila kinesin-like protein KLP3A is required for proper behavior of male and female pronuclei at fertilization. Development. 124:23652376.
Yan, R.T., and S.Z. Wang. 1997. Increased chromokinesin immunoreactivity in retinoblastoma cells. Gene. 189:263267.[CrossRef][Medline]
Yen, T.J., and B.T. Schaar. 1996. Kinetochore function: molecular motors, switches and gates. Curr. Opin. Cell Biol. 8:381388.[CrossRef][Medline]
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