INTRODUCTION

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The kinetochore is a three-layer structure located at the centromere of chromosomes. It is required for separation of sister chromatids in eucaryotes during M phase. Recently, accumulated evidence has also shown that, in addition to the ability to `mechanically' move the chromosomes, the kinetochore may also function as a sensor for the mitotic checkpoints to guarantee precise segregation of genetic materials to daughter cells (reviewed in references 1, 10, 21, and 25).

Dissecting its protein components is one of the approaches to understanding the molecular basis of the mammalian kinetochore functions. Since the identification of CENP-A (for centromere protein A), CENP-B, and CENP-C (9), novel centromere-associated proteins, including CENP-D, CENP-E, dynein, and mitosin (also named CENP-F), have also been characterized at the molecular level (reviewed in reference 22). Most of them have been shown by electron microscopy to reside in distinct regions of the kinetochore. For instance, CENP-B, a centromeric DNA-binding protein, associates with human centromeric DNA beneath the inner plate of the kinetochore (4, 18); CENP-A and CENP-C are components of the inner kinetochore plate (26, 33); mitosin/CENP-F is localized to the coronal surface of the outer kinetochore plate (24, 39); and the two motor proteins, dynein and CENP-E, are concentrated in the fibrous corona (5, 35). Recently, the family of centromere proteins has been further expanded by discoveries of the human homologues of yeast Mad2 (15) and Skp1 (3) and the murine homologue of yeast Bub1 (31). These evolutionarily conserved proteins transiently associate with centromeres and are involved in control of the mitotic checkpoints. Clearly, proteins involved in the structure and function of kinetochores must interact in a certain way in mature kinetochores. This issue, however, is presently not clear.

The structural characteristics of centromere proteins have been extensively studied. CENP-A, a histone H3 homologue of 17 kDa, targets the centromere via a histone H3-related domain (30). CENP-B (80 kDa) has been shown to utilize its N-terminal 125 amino acid residues to interact with the centromere (23, 38). The target of this centromere-binding domain is a 17-bp alphoid DNA (18). In addition to this, CENP-B also forms homodimers through a dimerization domain of 59 amino acid residues at its C terminus (13, 38). The centromere localization domain of CENP-C (140 kDa) lies in the homologous region with Mif2, a protein of budding yeast required for correct segregation of chromosomes (14, 36); this region overlaps with a potential DNA-binding domain (29, 36). CENP-E (312 kDa), a kinesin-like protein which is required for metaphase chromosome alignment (27, 34, 37), possesses two microtubule-binding domains (16); its centromere localization domain, however, has not been documented in detail.

Five functional regions have been identified in mitosin, a 350-kDa kinetochore protein. The region between amino acid residues 2961 and 3001 binds to the retinoblastoma protein (Rb) in vitro (39). Residues 2930 through 2958 contain a strong bipartite nuclear localization signal (NLS) (7, 40). Potentially, mitosin can also form homodimers through a C-terminal domain within the region from residue 2488 to 2925 (40). A polypeptide containing residues 2488 through 3113 is able to target the centromere when expressed in monkey kidney CV1 cells (40). Finally, the C-terminal portion from residue 2094 to 3113 has been shown to be capable of spindle pole localization during M phase (41).

In this study, we tried to address the detailed structural requirements for mitosin-kinetochore interaction and to analyze the biochemistry of such an interaction qualitatively. We defined the structural elements important for centromere localization of mitosin in detail. We analyzed the dynamics of mitosin-kinetochore interaction by an in vivo competition experiment. In addition, we showed that mitosin might contribute to nuclear organization, possibly through association with chromatin. The minimal polypeptide of mitosin sufficient for centromere localization can be used as a probe to isolate a gene(s) coding for its downstream target(s) on kinetochores. Connections between mitosin and other kinetochore components can therefore be further explored.

	MATERIALS AND METHODS

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Plasmid constructs. Construction of most deletion mutants was based on pTN, which is able to express a FLAG-tagged mitosin mutant containing amino acids 2094 to 3113 under control of the tetracycline-responsive system (11, 41). All mutants expressed were thus tagged with a FLAG epitope at their N termini. Schematic diagrams of all constructs are shown in Fig. 1, 4A, and 5A. Sequencing was always performed to confirm faithful ligation at junctions during plasmid construction whenever analysis by restriction cleavage was not possible.

Transfection. Chinese hamster ovary (CHO) cells were maintained in Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 10% calf serum (Sijiqin Company, Hangzhou, China) in an atmosphere containing 5% CO₂. For stable expression, CHO cells were transfected and selected with G418 as described previously (41). G418-resistant colonies were then cultured as a whole in DMEM containing 0.2 mg of G418/ml. For transient expression, cells were assayed 48 h after transfection. To prevent unscheduled expression, all the transfected cells were maintained in DMEM containing tetracycline (1 µg/ml). For centromere localization, both transient and ectopic experiments generated similar results.

IIF studies. For indirect immunofluorescence (IIF) staining, transfected cells were grown on coverslips overnight in the absence of tetracycline to induce expression before fixation in methanol for 15 min at 20°C. For preparation of metaphase chromosome spread, transfected cells were split onto glass coverslips to about 40% confluency, synchronized first in the presence of both thymidine (2 mM) and tetracycline (1 µg/ml) for 8 h, then cultured overnight in fresh DMEM containing nocodazole (0.4 µg/ml) but no tetracycline. The chromosome spread was then prepared as previously described (8). Samples on the coverslips were fixed in cold methanol. Immunostaining was performed with anti-FLAG M2 antibody (IBI) and fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (Sino-American Biotechnology Company, Shanghai, China) as described previously (39). Nuclear DNA was stained by DAPI (4',6-diamidino-2-phenylindole). Due to the difficulty of controlling the expression levels of different mutants in different individual cells, it was not possible to precisely measure the intensities of centromere-specific signals. Our results were therefore based on both extensive observation of 10 to 20 mitotic cells expressing each mutant and careful comparisons with records for other related mutants. Multiple representative images were recorded on Kodak Gold III (ASA400) or Lucky Pan SHD400 films with an Olympus BX50 fluorescence microscope. Prints were digitized using a Umax Vista-S6 scanner. Images for publication were organized by using Photoshop software and directly exported from a Tektronix Phaser 450 image printer.

	RESULTS

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Identification of the structural elements important for mitosin-kinetochore interaction. To further understand the structural basis of control of the centromere association of mitosin, we performed detailed deletion analysis to define its centromere localization domain. In a previous study, my colleagues and I showed that a mutant containing amino acid residues 2488 to 3113 localizes to the centromere region (40). Attempts to further define the domain, however, were precluded due to strong cytoplasmic IF in mitotic cells expressing shorter mutants; the centromere-specific signals appeared to be overwhelmed by the background generated by free mitosin. Furthermore, mitotic cells expressing mutants were rare in many cases. We thus considered that if we could reduce the expression levels, subcellular targets of the mutants might be highlighted. Moreover, a lower expression level might also reduce the extent of cell cycle block caused by overexpression of mitosin (39). We therefore utilized the tetracycline-responsive system (11) to express FLAG-tagged mitosin mutants. The CHO cell line was used as the host cell line due to its relatively low chromosome number (2N = 20). Expression levels were indeed reduced when interphase CHO cells expressing identical mutants under control of either the cytomegalovirus promoter or the tetracycline-responsive promoter were compared by IIF microscopy (data not shown). Similar examination showed that the average levels of most mutants expressed in CHO cells were compatible; only mitosin-pTCP showed a relatively low expression level (data not shown). For IIF study, we prepared chromosome spreads by using nocodazole-arrested cells to achieve clearer results. To avoid a possible effect of different subcellular localization on centromere targeting, we introduced an NLS into mutants in which the intrinsic NLS had been deleted.

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Determination of structural elements important for centromere localization of mitosin. (A) Diagrams of representative mutants used in this study. A diagram showing both structural characteristics of the full-length mitosin and restriction sites on its cDNA used for plasmid construction is displayed on top. Names of the constructs are printed on the left for each mutant. Numbers represent positions of amino acid residues. PKKKRKV indicates an insertion of the NLS of SV40 large T antigen. All the listed mutants were nuclear proteins in interphase as expected (data not shown). Their abilities for centromere localization (+, positive; , negative) are summarized on the right. Dashed lines indicate common boundaries shared by multiple mutants. The essential core region is located between residues 2792 and 2887. (B) Sequence of the core region. Leucines in each putative leucine zipper are underlined. Arrowheads indicate the two cysteine residues on which we performed mutagenesis studies (Fig. 4).

View larger version (106K): [in this window] [in a new window]   FIG. 2.   IIF images of representative mitosin mutants. CHO cells transfected with mitosin mutants were plated on coverslips, synchronized to prometaphase, and subjected to preparation of in situ chromosome spreads. Samples were then immunostained with anti-FLAG M2 monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G. Chromosomes were visualized by DAPI staining. All the left panels are IIF images of different mutants. All the right panels are images of chromosomes. The middle panels consist of superimposed images. The names of plasmids used for transfection are listed on the left. Arrowheads indicate typical centromere IF of positive mutants. One typical mitotic cell is shown for each mutant. The insets in panels 16 and 17 are amplified images included to highlight the weak centromere-specific IF. Scale bar, 10 µm.

View larger version (89K): [in this window] [in a new window]   FIG. 3.   Subcellular localization of mitosin-pTCB-N and mitosin-pTCB in the cell cycle. CHO cells transfected with pTCB-N and pTCB were grown, respectively, on coverslips overnight in the absence of tetracycline and fixed in cold methanol. Mitosin mutants were stained with anti-FLAG M2 monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (panels 1, 3, 5, and 7). Chromosomes were visualized by DAPI (panels 2, 4, 6, and 8). Both mutants tended to form aggregates in the majority of cells throughout the cell cycle. To highlight the centromere-specific localization of mitosin-pTCB-N, only mitotic cells with few aggregates are shown. (A) Diagrams of constructs used in this assay. Numbers indicate positions of amino acid residues. (B) Mitosin-pTCB-N forms strong centromere dots during M phase. Arrowheads indicate positions of centromere-specific IF. (C) Localization of mitosin-pTCB to centromeres is hardly detectable throughout M phase. A typical cell is shown at interphase (panels 1 and 2), at prophase (panels 3 and 4), at metaphase (panels 5 and 6), and at telophase (panels 7 and 8). Scale bar, 20 µm.

Binding of mitosin to kinetochores is hindered after NEBD. To explore the dynamics of the mitosin-kinetochore interaction, we studied the ability of a competitor, mitosin-pTCB, to target to centromeres in unsynchronized cells. As a cytoplasmic protein, mitosin-pTCB would be sequestered outside the nucleus until nuclear envelope breakdown (NEBD). Then it would compete with endogenous mitosin for sites on kinetochores. Mitosin-pTCB-N and mitosin-pTCB differed by only the presence of an artificial NLS in the former (Fig. 3A). Mitosin-pTCB-N was therefore a nuclear protein in interphase (Fig. 3B, panels 1 to 2), while mitosin-pTCB was cytoplasmic (Fig. 3C, panels 1 to 2). Although both mutants formed huge aggregates in cells at different stages of the cell cycle, their centromere localization properties were not significantly affected, similar to other mutants discussed previously. Similar to the wild-type mitosin, mitosin-pTCB-N redistributed to the centromeres in prophase (Fig. 3B, panels 3 to 4). In contrast to this, mitosin-pTCB stayed in the cytoplasm in prophase; no detectable IF was observed in the nucleus in prophase (Fig. 3C, panels 3 to 4). In addition to that at prophase, strong centromere localization of mitosin-pTCB-N persisted in cells at metaphase (Fig. 3B, panels 5 to 6) and anaphase (panels 7 to 8). Mitosin-pTCB, however, was still hardly observed at centromeres, even after NEBD, e.g., in cells at metaphase (Fig. 3C, panels 5 to 6) and anaphase (panels 7 to 8). Nevertheless, mitosin-pTCB was capable of centromere localization: when it was exposed to kinetochores for a prolonged period, e.g., in mitotic cells arrested by nocodazole, positive signals at centromeres were observed (data not shown). These data indicated that mitosin molecules available after NEBD could no longer be incorporated into kinetochores efficiently.

Cysteine²⁸⁶⁴ is essential for the activity of the core region. In addition to the two leucine zipper motifs in the core region, another notable feature is the two cysteine residues located at positions 2801 and 2864 (Fig. 1B). The first cysteine lies in the first leucine zipper motif, and the second one is close to the second motif. To examine if these cysteines were essential for the activity of the core region, we mutated the codon of each cysteine into serine based on construct pTCB-N. The resulting plasmids, pTCBC1-N and pTCBC2-N, were capable of expressing polypeptides identical to mitosin-pTCB-N except for the point mutations Cys²⁸⁰¹Ser and Cys²⁸⁶⁴Ser, respectively (Fig. 4A). Proper expression of these mutants was confirmed by immunoblotting (data not shown). We found that both mutants formed amorphous aggregates throughout the cell cycle (Fig. 4B and data not shown) like their parental mutant, mitosin-pTCB-N (Fig. 3B). Nevertheless, the point mutation at Cys²⁸⁰¹ did not affect the centromere localization of the mutant (Fig. 4B, panels 1 to 3). The mutation at Cys²⁸⁶⁴, however, completely abolished localization of the mutant to centromeres (Fig. 4B, panels 4 to 6). Cys²⁸⁶⁴ was therefore a critical residue for the activity of the core region. According to these results, Cys²⁸⁰¹ and Cys²⁸⁶⁴ are unlikely to form a disulfide bridge with each other.

View larger version (97K): [in this window] [in a new window]   FIG. 4.   Cysteine2864 is an essential amino acid for the centromere localization of mitosin. (A) Diagrams showing the point mutants used in this study. Both the plasmids were constructed based on pTCB-N (Fig. 1A). Restriction sites used for construction are shown. The positions of point mutations are also indicated. Numbers indicate positions of amino acid residues. (B) IIF images of the point mutants. CHO cells were transfected with pTCBC1-N or pTCBC2-N. Chromosome spreads and IIF staining were performed as described in the legend to Fig. 2. Similar to their parental mutant, mitosin-pTCB-N, both point mutants tended to aggregate in cells. Left panels, IIF images; right panels, chromosomes visualized by DAPI staining; middle panels, superimposed images. Panels 1 to 3 show a typical mitotic cell expressing mitosin-pTCBC1-N; strong centromere localization was observed despite the existence of aggregates. Panels 4 to 6 show a mitotic cell expressing mitosin-pTCBC2-N; no centromere localization was observed. Differences in the sizes of chromosomes are due to different extents of expansion in hypotonic buffer during sample preparation. Scale bar, 10 µm.

Mitosin colocalizes with nuclear DNA in interphase. The nuclear matrix has been shown to serve as sites of chromatin organization in the nucleus (2, 6). Mitosin/CENP-F is a nuclear matrix protein in interphase (17, 24). We frequently noticed that, in interphase, mitosin tended to closely colocalize with DAPI-stained nuclear DNA. Such a colocalization was not highlighted in wild-type mitosin and some deletion mutants (e.g., mitosin-pTN) due to the relatively homogeneous distributions of both mitosin and DNA (Fig. 5A and 5B, panels 1 to 2) (39-41). Nevertheless, in nuclei swollen by hypotonic buffer, colocalization of wild-type mitosin with nuclear DNA in CV1 cells was observed by IIF microscopy (data not shown). In addition, colocalization was obvious with certain mutants, such as mitosin-pTC (41), mitosin-pTNE (Fig. 5A and 5B, panels 3 to 4), and mitosin-pTZ (Fig. 5A and 5B, panels 7 to 8). In cells expressing these mutants, brightly stained DNA foci were frequently superimposed with strong IF of mitosin mutants. It appeared that these mutants somehow altered the chromatin organization in the nucleus because the distinct DNA foci were usually not observed in mock-transfected cells (Fig. 5B and data not shown). On the other hand, all the tested mutants with truncations to residue 2949 and further removed from the C terminus (i.e., mitosin-pTND-N and -pTCB-N) completely lost such a colocalization (Fig. 5A, 5B, panels 5 to 6, and 3B). These results implied that mitosin might interact with chromatin proteins or DNA. As summarized in Fig. 5A, a putative chromatin-binding domain of mitosin was speculated to lie between residues 2902 and 3037. 

View larger version (67K): [in this window] [in a new window]   FIG. 5.   Mitosin colocalizes with nuclear DNA in interphase. (A) Diagrams of representative mitosin mutants used in this study. Restriction sites used for constructing the listed plasmids are shown at the top. The ability of each mutant to colocalize with DNA is summarized on the right. A putative chromatin-binding domain is speculated to exist between residues 2902 and 3037 and is indicated between dashed lines. (B) IF images of the mutants. CHO cells transfected with pTN, pTNE, pTND-N, and pTZ were fixed on coverslips and subjected to IIF microscopy as described in the legend to Fig. 3. All the listed mutants were nuclear proteins in interphase as expected. Panels 1, 3, 5, and 7, IF images of different mutants; panels 2, 4, 6, and 8, nuclear DNA stained by DAPI. Arrowheads indicate typical areas where mitosin mutants colocalize with condensed DNA. Typical cells at interphase expressing mitosin-pTN (panels 1 and 2), mitosin-pTNE (panels 3 and 4), mitosin-pTND-N (panels 5 and 6), and mitosin-pTZ (panels 7 and 8) are displayed. Scale bar, 20 µm.

Deletion of the core region results in distribution of mitosin along chromosomes. In prophase, mitosin/CENP-F dissociates from the nuclear matrix and redistributes to kinetochores (17, 24, 39). Therefore, activation of the centromere colocalization property should be accompanied by loss of its nuclear matrix-associating as well as the putative chromatin-binding activities. To test if there might be any direct connections among these events, we deleted the core region from mitosin-pTN to form mitosin-pTNC, which lacked residues 2804 to 2901 of mitosin-pTN (Fig. 6A). Mitosin-pTNC did not aggregate in interphase cells; it was distributed in the nucleus in a pattern similar to that of mitosin-pTZ (data not shown). To our surprise, mitosin-pTNC appeared to fail to completely dissociate from chromatin in M phase since bright foci of various sizes were observed along virtually all chromosomes (Fig. 6B). The number of foci on chromosomes varied from cell to cell, as shown in Fig. 6B, panels 1 to 3 and 4 to 6. We also noticed that some of the foci only partially associated with chromosomes (panel 2). In some cases, especially when the foci on chromosomes were dense, immunofluorescence could be observed at centromere regions of some, but not all, chromosomes (data not shown). Detailed study showed that such centromere colocalization was nonspecific, since, in many cases, IF at centromeres was not observed (panels 1 to 3). These results suggest that the core region might down-regulate the putative chromatin-binding activity of mitosin in prophase.

View larger version (62K): [in this window] [in a new window]   FIG. 6.   Mitosin-pTNC is distributed along chromosomes in M phase. Chromosome spreads prepared from CHO cells expressing mitosin-pTNC were subjected to IIF staining as described in the legend to Fig. 2. (A) Diagrams for mitosin-pTNC. Numbers indicate positions of amino acids. (B) IF images. Left panels, IIF images of transfected cells; right panels, chromosome images stained by DAPI; middle panels, superimposed images. Panels 1 to 3 show a mitotic cell bearing large foci along chromosomes; arrows indicate centromeres without IF label. Panels 4 to 6 show a mitotic cell bearing dense IF dots along chromosomes. Scale bar, 10 µm.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

The structural basis for mitosin-kinetochore interaction. By performing deletion analysis, we found that two major structural elements, the internal repeats from residues 2094 through 2487 and the core region located between residues 2792 and 2887, were critical for strong mitosin-kinetochore interaction. Their contributions to the centromere localization property, however, were clearly different. Although independent localization to centromeres was not detected, the core region was indeed essential for the centromere localization of mitosin: without this region (e.g., in mitosin-pTNC and -pTZ), or even when the region was partially deleted (e.g., in mitosin-pTSE and -pTNF-N) or point mutated (e.g., in mitosin-pTCBC2-N), the corresponding mutants never exhibited centromere localization. Without the repeat region, in contrast, some mutants containing the core region (e.g., mitosin-pTG and -pTC) were still able to manifest centromere localization in spite of their weak affinities. Clearly, some sequences in the vicinity of the core region could partially compensate for the function of the internal repeats. In spite of this, clearly strong centromere localization was observed only in mutants containing both the internal repeats and the core region (e.g., mitosin-pTN, -pTS, -pTCB-N, and -pTKB-N). In addition, we found that both the repeat units were required for maximal mitosin-kinetochore interaction. How the internal repeats coordinate with the core region to achieve strong centromere localization is still an intriguing mystery. Our data suggest that the internal repeats (and other minor elements adjacent to the core region), instead of binding to kinetochores directly, might facilitate and/or stabilize the core region-kinetochore interaction.

The dynamics of mitosin-kinetochore interaction. With the study of a cytoplasmic mutant, mitosin-pTCB, we explored the kinetics of mitosin-kinetochore interaction qualitatively. We found that bound mitosin did not exchange its binding sites easily with free forms after NEBD. Significant exchange could be observed only when mitosis was blocked to allow prolonged time of competition. This result implied that the assembly of mitosin into kinetochores was completed prior to NEBD. After this point, free mitosin could no longer be recruited into kinetochores effectively. One of the possible explanations is that, after NEBD, access to the bound mitosin or mitosin-binding sites is denied by another component(s) of kinetochores assembled after mitosin. It has long been suspected that functional kinetochores are assembled in multiple stages. In cells at interphase, precursors of kinetochores, or prekinetochores, are found as discrete foci which contain certain centromere proteins (e.g., CENP-A, CENP-B, and CENP-C) colocalized with alphoid satellite DNA (12, 19, 20). In contrast, mitosin is one of the components assembled onto kinetochores in prophase (39).

The core region may regulate the putative chromatin-binding activity of mitosin. Several lines of evidence suggest that mitosin might bind to chromatin proteins or DNA in interphase. First, several mitosin mutants colocalized with brightly stained DNA foci in cells at interphase. Among cells expressing mitosin-pTZ, for instance, those bright DNA foci were observed only in transfected cells; mitosin-pTZ appeared to have reorganized chromatin distribution in these cells (Fig. 5B). This phenomenon was also observed in cells expressing mitosin-pTNE (Fig. 5B), mitosin-pTC (41), and several other mutants (data not shown). Second, our data suggested that there might be a chromatin-binding domain between residues 2902 and 3037. This region covers the previously identified in vitro Rb-binding domain and the intrinsic NLS (39, 40). Third, mitosin-pTNC exhibited both the colocalization with nuclear DNA in interphase and localization along chromosomes in mitotic cells. Last, during interphase, wild-type mitosin also colocalized with nuclear DNA in cells treated with hypotonic buffer (data not shown). The nuclear matrix is a complex structure implicated in multiple functions including chromatin organization (reviewed in references 2 and 6). The present results further imply that, as a nuclear matrix protein in interphase (17), mitosin/CENP-F might provide sites of attachment for proper chromatin organization. Detailed studies at the electron microscopic level are required to further test these speculations.