Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation
2004; Springer Nature; Volume: 23; Issue: 16 Linguagem: Inglês
10.1038/sj.emboj.7600347
ISSN1460-2075
AutoresY. KURASAWA, William C. Earnshaw, Yuko Mochizuki, Naoshi Dohmae, Kazuo Todokoro,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoArticle5 August 2004free access Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation Yasuhiro Kurasawa Yasuhiro Kurasawa Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan Search for more papers by this author William C Earnshaw William C Earnshaw Wellcome Trust Center for Cell Biology, Institute for Cell and Molecular Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Yuko Mochizuki Yuko Mochizuki Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, JapanPresent address: Department of Internal Medicine, Division of Hematology, Washington University School of Medicine, St Louis, MO 63110, USA Search for more papers by this author Naoshi Dohmae Naoshi Dohmae Biomolecular Characterization Division, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan Search for more papers by this author Kazuo Todokoro Corresponding Author Kazuo Todokoro Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan Search for more papers by this author Yasuhiro Kurasawa Yasuhiro Kurasawa Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan Search for more papers by this author William C Earnshaw William C Earnshaw Wellcome Trust Center for Cell Biology, Institute for Cell and Molecular Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Yuko Mochizuki Yuko Mochizuki Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, JapanPresent address: Department of Internal Medicine, Division of Hematology, Washington University School of Medicine, St Louis, MO 63110, USA Search for more papers by this author Naoshi Dohmae Naoshi Dohmae Biomolecular Characterization Division, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan Search for more papers by this author Kazuo Todokoro Corresponding Author Kazuo Todokoro Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan Search for more papers by this author Author Information Yasuhiro Kurasawa1, William C Earnshaw2, Yuko Mochizuki1, Naoshi Dohmae3 and Kazuo Todokoro 1 1Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan 2Wellcome Trust Center for Cell Biology, Institute for Cell and Molecular Biology, University of Edinburgh, Edinburgh, UK 3Biomolecular Characterization Division, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan *Corresponding author. Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, 351-0198 Saitama, Japan. Tel: +81 48 462 4853; Fax: +81 48 462 4827; E-mail: [email protected] The EMBO Journal (2004)23:3237-3248https://doi.org/10.1038/sj.emboj.7600347 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A number of proteins accumulate in the anaphase spindle midzone, but the interaction and precise role of these proteins in midzone organization remain obscure. Here, we found that the microtubule-bundling protein PRC1 bound separately to the three motor proteins, KIF4, MKLP1 and CENP-E, but not to the chromosomal passenger proteins. In KIF4-deficient cells, the central spindle was disorganized, and all midzone-associated proteins including PRC1 failed to concentrate at the midline, instead being dispersed along the loosened microtubule bundles of the central spindle. This suggests that KIF4 is essential for the organization of central spindles and for midzone formation. In PRC1-deficient cells, no midzone was formed, KIF4 and CENP-E did not localize to the disconnected half-spindle, and MKLP1 and chromosomal passenger proteins localized to discrete subdomains near microtubule plus ends in the half-spindle. Thus, PRC1 is required for interaction of the two half-spindles and for localization of KIF4 and CENP-E. These results suggest that KIF4 and its binding partner PRC1 play essential roles in the organization of central spindles and midzone formation. Introduction In late mitotic anaphase, an organized central spindle midzone forms between the two separating sets of chromatids. This consists of a dense network of overlapping antiparallel microtubules (Mastronarde et al, 1993). The spindle midzone plays an essential role in the initiation and completion of cell cleavage, and is the binding site for a number of proteins that play a part in cytokinesis (Cao and Wang, 1996; Wheatley and Wang, 1996; Glotzer, 1997; Robinson and Spudich, 2000). Proteins that have been reported to accumulate at the midzone during late mitosis include microtubule-bundling protein PRC1 (Jiang et al, 1998), chromosomal passenger proteins, inner centromere protein (INCENP) (Cooke et al, 1987), Survivin (Skoufias et al, 2000; Uren et al, 2000), Aurora B (Terada et al, 1998), TD-60 (Andreassen et al, 1991; Mollinari et al, 2003) and Borealin (Gassmann et al, 2004), protein kinase Plk (Carmena et al, 1998), Rho regulatory factors, CYK4/RhoGAP (Jantsch-Plunger et al, 2000; Mishima et al, 2002) and ECT2/RhoGEF (Prokopenko et al, 1999; Tatsumoto et al, 1999), and microtubule motor proteins MKLP1 (Sellitto and Kuriyama, 1988; Nislow et al, 1992), CENP-E (Yen et al, 1991) and Rab6-KIFL/MKLP2 (Hill et al, 2000; Neef et al, 2003). The mechanism of central spindle assembly and the functional roles of these midzone-associated proteins during late mitosis are still largely unknown. Ase1 is a protein first identified in the budding yeast that binds and bundles microtubules and is required for anaphase spindle elongation. Overexpression of Ase1 induces spindle elongation in S phase (Pellman et al, 1995; Juang et al, 1997; Schuyler et al, 2003). Ase1 was proposed to form cross-bridges within the spindle midzone that are essential for maintenance of anaphase spindle integrity. PRC1, the mammalian homolog of Ase1 (Jiang et al, 1998), accumulates in the central spindle midzone during anaphase and midbody during telophase (Mollinari et al, 2002). Overexpressed PRC1 extensively bundles interphase microtubules, and a phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Suppression of PRC1 by siRNA causes a failure of microtubule interdigitation between half-spindles and the absence of a spindle midzone (Mollinari et al, 2002). The mechanism by which PRC1 is concentrated at the spindle midline during cytokinesis remains to be determined. The kinesin superfamily proteins (KIFs) are a family of 45 (in human) microtubule-based motor proteins that transport membranous organelles, protein complexes and other cargoes to specific destinations in a microtubule- and ATP-dependent manner (Hirokawa, 1998; Miki et al, 2001, Schliwa and Woehlke, 2003). Chromokinesins are a subfamily of KIFs with amino-terminal motor domains that bind to chromosome arms. The chromokinesins identified to date can be divided into two main functional categories. Kid/Nod family proteins are required for chromosome alignment (Murphy and Karpen, 1995; Antonio et al, 2000; Funabiki and Murray, 2000), chromosome orientation and oscillation of chromosome arms (Levesque and Compton, 2001). A second chromokinesin subfamily is composed of Xenopus Xklp1 (Vernos et al, 1995), Drosophila Klp3A (Williams et al, 1995) and mammalian KIF4 (Sekine et al, 1994). Xenopus Xklp1 is required for chromosome positioning and bipolar spindle stabilization in egg extracts (Vernos et al, 1995), and chromatin–microtubule interactions in vitro (Walczak et al, 1998). Many functions of Drosophila Klp3A have been suggested. In spermatocytes these include central spindle formation, initiation of cytokinesis (Williams et al, 1995) and assembly of the actin-based contractile ring (Giansanti et al, 1998). Klp3A has been implicated in chromosome congression to the metaphase plate (Goshima and Vale, 2003) and spindle pole separation (Kwon et al, 2004) in S2 cells. KIF4 has multiple functions in membrane trafficking and cell division. In neurons, KIF4 is an anterograde motor that transports specific organelles involved in nerve cell morphogenesis (Sekine et al, 1994). Remarkably, KIF4 is also a chromokinesin that is associated with chromosome arms (Wang and Adler, 1995). The role of KIF4 during mitosis has not yet been determined. In order to begin to dissect the mechanism of spindle midzone formation, we have identified proteins that bind to PRC1 during late mitosis. Immunoprecipitation with an anti-PRC1-specific antibody revealed a specific interaction between KIF4 and PRC1. We therefore studied the function of KIF4 and PRC1 during late mitosis in relation to other midzone-associated proteins including the two motor proteins MKLP1 and CENP-E and the chromosomal passenger proteins Aurora B, INCENP and Survivin. Our studies have revealed that both KIF4 and PRC1 have important roles in the formation of an organized spindle midzone and midbody. Results KIF4 is bound to PRC1 in vivo To study the precise role of PRC1 during spindle midzone formation, we used immunoprecipitation to identify proteins that specifically associated with it in vivo. Affinity-purified anti-human PRC1-specific rabbit antibody was used to immunoprecipitate the endogenous protein from HeLa cell extracts at late mitosis. These immunoprecipitates contained a prominent band with an apparent molecular weight of 155 kDa in SDS–PAGE (Figure 1A, lane 2). Two lower molecular weight bands corresponded to PRC1 and the antibody used (Figure 1A, lane 2). No abundant polypeptides were seen in the immunoprecipitates with preimmune serum (Figure 1A, lane 1). Amino-acid sequence analysis of the potential PRC1 binding protein revealed it to be the chromokinesin KIF4. The other two bands of 175 and 110 kDa turned out not to be specific PRC1 binding proteins (data not shown). Figure 1.PRC1 specifically binds to KIF4 during late mitosis. (A) Immunoaffinity chromatography. HeLa cell extracts at late mitosis were fractionated by affinity chromatography using anti-PRC1-specific antibody, and bound proteins were separated by SDS–PAGE and silver-stained. (B) Immunoprecipitates with anti-PRC1 antibody contain KIF4. HeLa cell extracts at late mitosis were subjected to immunoprecipitation, with preimmune sera, anti-PRC1 or anti-KIF4, fractionated by SDS–PAGE and immunoblotted with anti-KIF4-specific antibody. The common band in the immunoprecipitates with preimmune serum and anti-KIF4 antibody is antibody-derived protein. (C) Immunoprecipitates with anti-KIF4 antibody include PRC1. (D–E) Pull-down experiments with a series of PRC1 fragments fused to GST. Schematic of PRC1 fragments used for pull-down experiments (D) and their SDS–PAGE pattern (E). The PRC1 fragments were incubated with HeLa cell extracts at late mitosis, and bound proteins separated by SDS–PAGE were probed with anti-KIF4, anti-MKLP1 and CENP-E antibodies (F). Download figure Download PowerPoint This in vivo interaction between endogenous KIF4 and PRC1 was confirmed by immunoprecipitation using a rat serum specific for KIF4. Immunoblot analysis with this antiserum detected KIF4 as a single band of 155 kDa in HeLa cell extracts at late mitosis and in immunoprecipitates with anti-PRC1 antibody (Figure 1B). Furthermore, PRC1 was also readily detected in immunoprecipitates with the anti-KIF4 antiserum (Figure 1C). In controls, each protein was detected in immunoprecipitates with its cognate serum, but not in control immunoprecipitates with preimmune sera (Figure 1B and C). We conclude from these experiments that PRC1 is specifically associated with KIF4 in vivo during late mitosis. The amino-terminal half of PRC1 interacts with itself and the carboxyl-terminal half of KIF4 To further analyze the interaction between PRC1 and KIF4, we performed in vitro binding assays with KIF4 from HeLa late mitotic cell extracts and full-length or a series of truncated forms of human PRC1 (Figure 1D) fused to glutathione S-transferase (GST) and expressed in Escherichia coli. Figure 1E shows the SDS–PAGE patterns of the GST-fused PRC1 fragments used for binding assays. Both full-length PRC1 (Figure 1F, lane 1) and the amino-terminal half of the protein (amino acids 1–303) (Figure 1F, lane 4) bound to KIF4. The amino-terminal one-third of PRC1 weakly bound to KIF4 (Figure 1F, lane 2), but the other PRC1 fragments did not bind to KIF4. Yeast two-hybrid analyses confirmed that the amino-terminal half of PRC1 (amino acids 1–303) interacted both with PRC1 and with the carboxyl-terminal half of KIF4 (amino acids 663–1232) (Supplementary Figure 1). Taken together, these results suggest that PRC1 associates with KIF4 through interaction between its amino-terminal half and the carboxyl-terminal half of KIF4. Colocalization of PRC1 and KIF4 during late mitosis Immunofluorescence analysis of HeLa cells with anti-KIF4-specific antibody showed that KIF4 was localized on the chromosomes throughout mitosis, and additionally to the spindle midzone during anaphase and the midbody during telophase and cytokinesis (Figure 2A). KIF4 was nuclear during interphase (data not shown). Figure 2.Colocalization of PRC1 and KIF4 during late mitosis. (A) Subcellular localization of KIF4 during mitosis. Immunofluorescence analyses of HeLa cells show KIF4 (green), α-tubulin (red) and DNA (blue). Scale bar, 5 μm. (B) PRC1 and KIF4 colocalize in the central spindle during late mitosis. HeLa cells at indicated mitotic stages were fixed with paraformaldehyde, and KIF4 (green), PRC1 (blue) and tubulin (red) were stained. Staining of KIF4 in chromosomes was weak due to the paraformaldehyde fixation. Scale bar, 5 μm. (C) The rectangular regions of (B) are enlarged. Scale bar, 5 μm. Download figure Download PowerPoint As predicted by the biochemical analysis, PRC1 and KIF4 colocalize in late mitosis. This experiment was complicated by the fact that detection of PRC1 requires fixation with formaldehyde under conditions where the detection of KIF4 on chromosomes is relatively inefficient. PRC1 was distributed diffusely around the chromosomes and spindles until metaphase. PRC1 colocalized with KIF4 from anaphase B to telophase (Figure 2B). PRC1 localized to the spindle midzone at anaphase A, but KIF4 localized to the midzone only later, during anaphase B (see below). When KIF4 reached the midzone, it accumulated in tight ball-like foci that appeared to link microtubule bundles from the two half-spindles. These KIF4 foci were surrounded by PRC1 in anaphase B, and completely coincided with PRC1 in telophase (Figure 2C). In late cytokinesis, KIF4 was localized in the center of the midbody (Flemming body), while some PRC1 was present on the flanking bundles of microtubules. These data revealed that PRC1 shows significant colocalization with KIF4 during late mitosis. KIF4 is localized at the spindle midzone at anaphase B but not at anaphase A At anaphase in normal cells, PRC1 preceded KIF4 localization to the midzone (Figure 2B). To examine the timing of localization of KIF4 to the midzone in greater detail, cells at anaphase A and anaphase B were co-stained with KIF4, MKLP1 and Aurora B. At anaphase A in normal cells, KIF4 had not yet relocated to the central spindle, at a time when Aurora B and MKLP1 had already done so (Figure 3A—the blue signal represents KIF4 on chromatid arms). KIF4 subsequently localized to the midzone in anaphase B, at which point it colocalized with MKLP1 and was flanked by Aurora B on either side (Figure 3B). Therefore, KIF4 does not immediately relocate to the central spindle at the onset of anaphase. KIF4 and Aurora B also colocalized near the plus ends of microtubules at the periphery of normal cells at anaphase B. At these sites, MKLP1 localized to an extended region proximal to the plus ends (Figure 3C). Figure 3.KIF4 is localized to the spindle midline at anaphase B but not at anaphase A. (A, B) Staining of KIF4 (blue), MKLP1 (green) and Aurora B (red) in normal cells at anaphase A (A) and at anaphase B (B). Scale bar, 5 μm. The rectangular regions are enlarged and shown in the right panels. (C) The peripheral region of the central spindle of a normal cell at anaphase B is enlarged in the right panels. Scale bar, 5 μm. Download figure Download PowerPoint These data suggest that Aurora B and MKLP1 relocate directly to the spindle midzone, while KIF4 localizes first near the plus ends of the microtubules and then to the midzone as microtubules from opposite half-spindles are drawn together into bundles. KIF4 depletion causes formation of aberrant midzone and midbody We next analyzed KIF4 function during mitosis by using siRNA to deplete the protein in HeLa cells. Expression of KIF4 was effectively suppressed by 100 nM of KIF4 siRNA in transfection medium (Figure 4A), and KIF4 was either greatly reduced or absent from the chromosomes or midzone/midbody of the siRNA-transfected cells (Figures 4B and D (panels d and g) and 5A–G (panel c)). In all subsequent siRNA experiments, cells transfected with single-stranded sense RNA alone are shown as controls. The single-stranded KIF4 RNA had no effect on mitosis in control cells (Figures 4B and D (panel a) and 5A–G (panel a)). Figure 4.KIF4 deficiency results in aberrant midzone and midbody formation. (A) Immunoblot analysis of HeLa cells treated with various concentrations of KIF4 siRNA (0–100 nM) or with single-stranded sense RNA alone (100 nM). Cell extracts were probed with anti-KIF4 antibody and anti-tubulin antibody as an internal control. (B, D) Phenotypes of KIF4-deficient cells at anaphase (B) and telophase (D). HeLa cells were treated with single-stranded sense KIF4 RNA (panels a–c; control) or with KIF4 siRNA (panels d–i) for 48–55 h, and KIF4 (green), α-tubulin (red) and DNA (blue) were stained. Scale bar, 5 μm. (C) Percentage of each mitotic stage in KIF4-deficient cells (white) and control cells (black). (E) Plots of distance between the poles versus cell size in KIF4-deficient cells (open circles) and control cells (closed circles) at anaphase. Download figure Download PowerPoint Figure 5.KIF4-deficient cells exhibit dispersed localization of PRC1, CENP-E, MKLP1, Aurora B, Survivin and INCENP. HeLa cells were treated with single-stranded sense KIF4 RNA (panels a and b; control) or with KIF4 siRNA (panels c and d), and cells at anaphase (A, C–G) and late telophase (B). (A, B) Cells were stained with anti-KIF4 (green), anti-PRC1 (blue) and tubulin (red) antibodies. (C–G) Cells were stained with anti-CENP-E (C, red), anti-MKLP1 (D, red), anti-Aurora B (E, red), anti-Survivin (F, red), anti-INCENP (G, red) antibodies, and with anti-KIF4 antibody (green) and DNA (blue). Scale bar, 5 μm. (H) Quantification of the lack of focus in the central spindle in KIF4-deficient cells. The graph shows the width of the region stained by PRC1, CENP-E, MKLP1 and Aurora B in KIF4-deficient cells (open circles), and normal cells (closed circles) are shown. Download figure Download PowerPoint In KIF4-deficient cells, spindle formation, chromosome congression to a metaphase plate, sister chromatid segregation, interpolar microtubule elongation and cleavage furrow formation occurred normally, but the progression of these cells appeared to be delayed compared with normal cell division. The mitotic index of control cells was 5.2% and that of KIF4-deficient cells was 5.8%. Figure 4C shows the percentage of each mitotic stage in KIF4-deficient and control cells. The percentage of prophase, prometaphase and cytokinesis was about the same for both populations, but that of metaphase, anaphase and, especially, telophase was significantly increased. Although the phenotypes of KIF4-deficient cells at anaphase, telophase and cytokinesis were abnormal (see below), these cells clearly progressed through the late mitotic stages, albeit more slowly. Immunofluorescence analysis with anti-α-tubulin antibody showed that the central spindle was disorganized at anaphase in KIF4-depleted cells (Figure 4B, panels e and h), in comparison with the organized central spindle in control cells (Figure 4B, panel b). We observed a tight correlation between the level of KIF4 and the degree of microtubule bundling. No organized spindle midzone was formed when KIF4 was strongly suppressed and could not be detected in equatorial regions (Figure 4B, panels g–i). In KIF4-depleted late-anaphase cells, the separated sister chromatids were localized abnormally close to the plasma membrane (Figure 4B, panels f and i). The effect of KIF4 depletion on the length of the central spindle was quantified by measuring the distance between spindle poles in KIF4-deficient and control cells. Figure 4E shows the plots of the distance between the poles versus the cell size in KIF4-deficient cells (open circles) and control cells (closed circles) at anaphase. The central spindle was elongated by about 20% in KIF4-deficient cells compared with control cells. At telophase in KIF4 siRNA-treated cells, in which KIF4 levels were significantly reduced, a much reduced midbody was formed (Figure 4D, panels d and f), and the midbody microtubules were less prominent and less organized (Figure 4D, panel e), compared with the robust midbodies in control cells (Figure 4D, panel b). We failed to observe cells in cytokinesis that were completely negative for KIF4. This could be either because cells lacking KIF4 fail to undergo cytokinesis or because even minute amounts of residual KIF4 become detectable when concentrated into the highly compact midbody structure. Regardless of the explanation, examination of cultures suggests that cytokinesis ultimately failed, as a significant increase in the number of binucleate cells (about 8%) was observed. These observations suggest that KIF4 is required for the formation of an organized central spindle midzone and midbody and for successful cytokinesis. KIF4 deficiency leads to mislocalization of PRC1, MKLP1, CENP-E and chromosomal passenger proteins Since KIF4 binds to PRC1, we next determined whether PRC1 targeting late in mitosis is upstream or downstream of KIF4 function by examining the subcellular localization of PRC1 in KIF4-depleted cells. Instead of the compact midzone/midbody localization of PRC1 with KIF4 seen in control cells (Figure 5A and B, panel b), KIF4 deficiency caused PRC1 to be dispersed along microtubules throughout the central spindle during anaphase (Figure 5A, panel d). In KIF4-deficient cells during cytokinesis, PRC1 was not concentrated in the midbody but was dispersed all along the microtubule bundles (Figure 5B, panel d). These results suggest that PRC1 can bind to central spindle microtubules without KIF4, but that its concentration into compact foci at the midline of the central spindle requires the presence of KIF4. Similarly, in KIF4-deficient cells, CENP-E, MKLP1 and the chromosomal passenger proteins, Aurora B, Survivin and INCENP, were all dispersed throughout the diffuse central spindle (Figure 5C–G), similar to PRC1 as shown in Figure 5A. The lack of focus in the central spindle in KIF4-deficient cells was quantified by measuring the width of the region stained by PRC1, CENP-E, MKLP1 and Aurora B antibodies. Figure 5H shows this width in KIF4-deficient cells (open circles) and control cells (closed circles). In KIF4-deficient cells, the value obtained was about 2.5 times larger than that of control cells at anaphase. These results show that KIF4 is not required for this group of midzone-associated proteins to bind to midzone microtubules. However, it is required for those microtubules to form an organized central spindle, and for the midzone-associated proteins to concentrate into compact structures that will ultimately give rise to the midbody. PRC1 is required for KIF4 and CENP-E to relocalize from chromosomes to microtubules during anaphase We next examined the effects of PRC1 deficiency on the subcellular localization of this group of midzone-associated proteins. PRC1 could be effectively suppressed by 100 nM of siRNA (Figure 6A), and immunofluorescence analyses revealed that almost no PRC1 could be detected in PRC1-depleted cells (Figure 6B, panel d). No effect was detected in control cells transfected with single-stranded sense RNA (Figure 6B, panel b). Figure 6.PRC1 deficiency causes loss of the spindle midzone and absence of KIF4 and CENP-E in the central spindle, but localization of MKLP1 and chromosomal passenger proteins in the half-spindle. (A) Immunoblot analysis of HeLa cells treated with various concentrations of PRC1 siRNA (0–100 nM) or single-stranded sense RNA alone (100 nM). Cell extracts were probed with anti-PRC1 antibody and anti-tubulin antibody as an internal control. (B–H) HeLa cells were treated with single-stranded sense PRC1 RNA (panels a and b; control) or with PRC1 siRNA (panels c and d), and PRC1 (B, shown in red), α-tubulin (C, red), CENP-E (D, red), MKLP1 (E, red), Aurora B (F, red), Survivin (G, red), INCENP (H, red), KIF4 (green) and DNA (blue) were stained in cells at anaphase. Scale bar, 5 μm. Download figure Download PowerPoint In PRC1-depleted anaphase cells, KIF4 was absent from the central spindle, but appeared to be localized normally to chromosomes (Figure 6B, panel c). Failure of KIF4 relocation was not unlikely to be simply due to a loss of midzone structure since MKLP1 was localized to a diffuse midzone in PRC1-deficient cells (see below). Because of the technical difficulty of double staining PRC1 together with other midzone-associated proteins, we subsequently exploited this change in the distribution of KIF4 as a monitor of the effectiveness of PRC1 depletion in particular cells (Figure 6B–H, panel c). This approach was validated by immunostaining of these cells with anti-α-tubulin antibody, which clearly demonstrated that an integrated spindle was not formed in anaphase cells judged to be PRC1-deficient as a result of their KIF4 distribution (Figure 6C, panel d). Instead, the half-spindles remained disconnected from one another, as reported previously (Mollinari et al, 2002). CENP-E has been reported to redistribute during anaphase from kinetochores to the central spindle and later to the midbody at telophase (Yen et al, 1991). In PRC1-deficient cells, CENP-E remained on kinetochores or chromosomes as well as diffusely throughout the cells at anaphase (Figure 6D, panel d). Together, these results suggest that KIF4 and CENP-E require PRC1 to exit from chromosomes and bind to central spindle microtubules during anaphase B. MKLP1 and Aurora B localization to half-spindles does not require PRC1 MKLP1 and all three chromosomal passenger proteins were concentrated in equatorial regions of PRC1-depleted anaphase cells (Figure 6E–H). Double staining of Aurora B and tubulin clearly demonstrated that Aurora B was localized near the plus ends of the microtubules in the disconnected half-spindles (Figure 7A). MKLP1 was also localized near the plus ends of the same microtubules (Figure 7B), but did not exactly overlap with Aurora B (Figure 7C). Instead, it occupied an extended region proximal to the plus ends. Figure 7.MKLP1 and Aurora B localize to the half-spindle of PRC1-deficient cells. (A) Staining of tubulin (green), Aurora B (red) and DNA (blue) in PRC1-deficient cells and control cells at anaphase. Scale bar, 5 μm. (B) Staining of tubulin (green), MKLP1 (red) and DNA (blue) in PRC1-deficient cells and control cells at anaphase. Scale bar, 5 μm. (C) Staining of MKLP1 (green), Aurora B (red) and DNA (blue) in PRC1-deficient cells at anaphase. Scale bar, 5 μm. Download figure Download PowerPoint These distributions of MKLP1 and Aurora B in PRC1-depleted cells resemble what is seen for the peripheral microtubules in control cells early in anaphase B (Figure 3C). In those cells, MKLP1 and KIF4 concentrate in foci flanked by Aurora B in the compact central spindle; however at the periphery of the midzone, Aurora B can be seen near the plus ends of single microtubules surrounded by a more extensive domain of MKLP1 (Figure 3B and C). Apparently, MKLP1 only concentrates into foci as the microtubules are incorporated into the compact central spindle. These observations suggest that the phenotype of the PRC1 RNAi may correspond to an intermediate stage in the formation of a normal bipolar anaphase spindle, and that the targeting of Aurora B and MKLP1 on anaphase midzone microtubules does not require PRC1. When anaphase cells were subjected to a brief treatment with colcemid, central spindles persisted but astral microtubules disappeared (Supplementary Figure 2). In these cells, KIF4 and CENP-E remained associated with chromosomes and/or kinetochores, respectively. In contrast, PRC1, MKLP1 and Aurora B were localized in the central spindle remnants. This experiment shows that KIF4 and CENP-E exhibit different requirements for targeting and/or binding to central spindles than do PRC1, MKLP1 and Aurora B. Binding of KIF4 and CENP-E to central spindles may require dynamic microtubules. When a YFP-fused phosphorylation-null PRC1 mutant was overexpressed in HeLa cells, a number of bundled microtubules were detected in cells at mitosis (Supplementary Figure 3A) and interphase, as previously reported (Mollinari et al, 2002). Thi
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