During mitosis, kinetochores need to attach to microtubules emanating from spindle poles. Several protein complexes have been shown to mediate the kinetochore-microtubule interaction. However, with the continually growing number of newly identified kinetochore proteins, it is unclear whether all major components of the kinetochore-microtubule interface have been identified. We therefore performed a high-throughput RNAi screen to identify additional factors involved in kinetochore-microtubule attachment, and identified RAMA1 as a novel regulator of this process. Depletion of RAMA1 results in severe chromosome alignment defects and a checkpoint-dependent mitotic arrest. We show that this is due to reduced kinetochore-microtubule attachments. RAMA1 localizes to the spindle and to outer kinetochores throughout all phases of mitosis and is recruited to kinetochores by the core kinetochore-microtubule attachment factor Hec1. Interestingly, unlike Hec1, the association of RAMA1 with kinetochores is highly dynamic, suggesting that it is not a structural component of the kinetochore. Consistent with this, all other kinetochore proteins tested do not require RAMA1 for their kinetochore localization. Taken together, these results identify RAMA1 as a novel kinetochore protein and suggest that RAMA1 may have a direct role in mediating kinetochore-microtubule interactions.
During mitotic entry, the relatively stable interphase microtubule array breaks down and is replaced by a highly dynamic mitotic spindle. Spindle microtubules nucleated at the centrosomes go through repeated cycles of growth and shrinkage until they are captured by a kinetochore in a process termed `search-and-capture' (Mimori-Kiyosue and Tsukita, 2003). In addition, microtubules are nucleated in the vicinity of chromatin and at kinetochores, and these microtubules are integrated with centrosomally derived microtubules to rapidly form stable kinetochore-microtubule bundles (Walczak and Heald, 2008). In human cells, each kinetochore can interact with ∼25 microtubules in an `end-on' fashion, in which the plus-ends of the microtubules are embedded in the outer plate of the kinetochore (Cheeseman and Desai, 2008; Maiato et al., 2004; Rieder, 1982). Initially, kinetochores are thought to interact with the lattice of microtubules and this promotes dynein-dependent transport to the spindle pole (or Kar3-dependent in yeast) (Rieder and Alexander, 1990; Tanaka et al., 2007; Tanaka et al., 2005; Yang et al., 2007). These lateral microtubule interactions are then converted to end-on microtubule attachments through an ill-defined mechanism.
Many proteins have been implicated in the initial interaction with microtubules, as well as in the stable end-on attachment. In the current view, the NDC80 complex plays a central role in stable kinetochore-microtubule attachment (Cheeseman et al., 2006; Ciferri et al., 2007; Ciferri et al., 2008; DeLuca et al., 2006; McCleland et al., 2003; Wigge and Kilmartin, 2001). The NDC80 complex is a four-subunit complex (Spc24, Spc25, Nuf2 and Hec1 in humans) of which the Hec1 and Nuf2 subunits possess microtubule-binding activity (Cheeseman et al., 2006; Guimaraes et al., 2008; Miller et al., 2008; Wei et al., 2007). Interestingly, although individual Hec1 molecules have relatively low microtubule-binding affinity, this affinity is strongly increased when Hec1 is in complex with other NDC80 subunits, KNL-1 and the Mis12 kinetochore complex (Cheeseman et al., 2006).
In addition to the NDC80 complex, several other proteins that localize to kinetochores have microtubule-binding activity, including both motor and non-motor microtubule-associated proteins (MAPs) and many of these have been shown to be involved in chromosome alignment and kinetochore-microtubule attachment (Cheeseman and Desai, 2008; Maiato et al., 2004). These proteins can roughly be divided into three subgroups. First, several microtubule-binding proteins are recruited specifically to unattached kinetochores, including dynein and the microtubule plus-end tracking protein CLIP-170. On the basis of their transient recruitment to kinetochores, these proteins have been implicated in the initial interaction of kinetochores with microtubules (Draviam et al., 2006; Green et al., 2005; Tanenbaum et al., 2006; Yang et al., 2007). A second group of proteins appears to bind to kinetochores only when they are attached to microtubules. This group includes the plus-end tracking proteins APC and EB1 (Draviam et al., 2006; Fodde et al., 2001; Green et al., 2005; Kaplan et al., 2001; Tirnauer et al., 2002), the Ska1-Ska2 complex (Hanisch et al., 2006) and the yeast Dam1 complex (Cheeseman et al., 2001), although a clear human counterpart of the latter complex has not yet been identified. This group of proteins is probably involved in the maintenance of the dynamic kinetochore-microtubule interaction. Finally, several microtubule-binding proteins have been shown to localize to kinetochores throughout mitosis, including the microtubule plus-end tracking proteins CLASP1/2 (Maiato et al., 2003) and the microtubule motor Cenp-E (Yen et al., 1991). Whereas CLASPs have a major role in regulating kinetochore-microtubule dynamics (Maiato et al., 2003; Maiato et al., 2005), Cenp-E has been suggested to be a microtubule capture factor (Putkey et al., 2002), as well as a motor driving chromosome congression (Kapoor et al., 2006).
It is clear from these studies that kinetochore-microtubule interactions are very complex, probably involving many different regulators that act during different phases of the kinetochore-microtubule interaction. Furthermore, the list of proteins that localizes to kinetochores is continuously expanding, suggesting that major attachment factors might still remain undiscovered. We therefore set out to identify additional proteins required for kinetochore-microtubule attachment. Using an RNAi library targeting 180 MAPs, we identified RAMA1 as a novel regulator of kinetochore-microtubule attachment. RAMA1 localizes to kinetochores and the spindle throughout mitosis, and loss of RAMA1 results in clear defects in kinetochore-microtubule attachments. Interestingly, we find that RAMA1 is recruited to kinetochores by the NDC80 complex, suggesting that this complex not only plays an important role in kinetochore-microtubule attachments through direct binding of microtubules, but also acts indirectly by recruiting additional attachment factors to the kinetochore.
Identification of RAMA1 as a novel regulator of mitosis
To search for novel proteins that could link kinetochores to spindle microtubules, we generated an siRNA library targeting 180 proteins that were previously shown to associate with the mitotic spindle (Sauer et al., 2005) (supplementary material Table S1). Each protein was targeted by a pool consisting of four siRNAs that were chemically modified to reduce the chance of off-target effects (Jackson et al., 2006). In addition, we have previously established a high-throughput platform to screen for proteins required for mitosis (M.E.T. and R.H.M., unpublished) and we used this approach to systematically test these 180 proteins for an essential role in mitosis. Using this setup, we identified eight proteins for which the knockdown resulted in a mitotic index above 15% in HeLa cells (Fig. 1A). The strongest accumulation of mitotic cells was observed after knockdown of an uncharacterized protein called C13Orf3/RAMA1 (hereafter referred to as RAMA1) and we therefore decided to investigate this protein further.
To ensure that the observed effects were not due to off-target effects, the four siRNAs targeting RAMA1 were tested individually. Indeed, all four independent siRNAs resulted in a potent accumulation of mitotic cells (Fig. 1B), demonstrating that RAMA1 is indeed required for proper mitotic progression. In addition, depletion of RAMA1 also resulted in a significant increase in the mitotic index in a second cell line (U2OS) (Fig. 1C), indicating that the role of RAMA1 in mitosis is not cell-type specific. As a final proof that RAMA1 is required for mitosis, we performed RNAi rescue experiments. For this, RAMA1 was cloned from a U2OS cDNA library and N-terminally tagged with GFP. Furthermore, two silent point mutations were introduced in the cDNA to render the cDNA resistant to siRNA-mediated depletion. Control cells and cells depleted of RAMA1 were then transfected with either GFP or GFP-RAMA1 and the percentage of cells that entered anaphase was determined as a measure of mitotic progression. In control cells, 28±5% of mitotic cells were in anaphase or telophase and this was reduced to only 3±1% in RAMA1-depleted cells, consistent with a potent block in mitotic progression after loss of RAMA1. However, when RAMA1-depleted cells were transfected with GFP-RAMA1, the percentage of cells that had entered anaphase or telophase was substantially increased, demonstrating that the observed mitotic defects were indeed due to loss of RAMA1 (14±3%) (Fig. 1D). Taken together, these results show that RAMA1 is essential for mitotic progression.
RAMA1 is a novel kinetochore and spindle protein
Next, we expressed GFP-RAMA1 in asynchronous growing cells to determine its localization throughout the cell cycle. In interphase, RAMA1 was observed diffusely in the cytoplasm, but also specifically at the centrosome (Interphase, Fig. 2A). In prophase, when separated centrosomes had initiated the assembly of microtubule asters, RAMA1 colocalized with the two forming microtubule asters (Fig. 2A, Prophase). Strikingly, after nuclear-envelope breakdown (NEB) RAMA1 was not only observed on the spindle, but also accumulated strongly at foci on the chromosomes (Prometaphase, Fig. 2A). These foci were observed throughout prometaphase and metaphase (Prometaphase and Metaphase, Fig. 2A) and were also present in anaphase at similar levels (Anaphase, Fig. 2A). However, in late telophase, when chromosomes started to decondense and the nuclear envelope reformed, the dot-like staining on chromosomes disappeared (Telophase, Fig. 2A). This type of staining is highly reminiscent of kinetochore localization and, indeed, RAMA1 foci were always observed in close proximity of CREST staining, a marker for centromeres (Fig. 2B). Interestingly, RAMA1 staining did not overlap with CREST, but localized substantially more towards the outside (Fig. 2B), suggesting that it is a component of the outer kinetochore. We therefore conclude that RAMA1 is a constitutive component of the kinetochore and spindle. We also attempted to detect endogenous RAMA1 at kinetochores, but unfortunately our antibodies were unable to reliably detect the endogenous protein.
RAMA1 is recruited to kinetochores by the NDC80 complex
To confirm that RAMA1 is a component of the outer kinetochore, cells expressing GFP-RAMA1 were co-stained for CREST and Hec1, a known outer-kinetochore component. Indeed, GFP-RAMA1 largely overlapped with Hec1 staining (Fig. 3A), confirming that RAMA1, like Hec1, is a component of the outer kinetochore. Furthermore, like Hec1, RAMA1 recruitment to kinetochores did not depend on microtubule attachment because RAMA1 could clearly be observed at kinetochores in cells treated with nocodazole (lower panel, Fig. 3B).
Next, we tested whether Hec1 was required to recruit RAMA1 to kinetochores. Hec1 was depleted as described previously (Tanenbaum et al., 2008), which resulted in a very strong defect in chromosome alignment (Fig. 3C). Strikingly, RAMA1 was completely lost from kinetochores after depletion of Hec1, but still localized to the spindle (Fig. 3C,D). Similarly, knockdown of Nuf2, another component of the NDC80 complex, also prevented RAMA1 recruitment to kinetochores (Fig. 3C,D). We also attempted to co-immunoprecipitate RAMA1 with components of the NDC80 complex. However, we were unable to detect an interaction, suggesting that the interaction is either indirect or very unstable (as described below). Taken together, these results show that RAMA1 is a component of the outer kinetochore whose recruitment to kinetochores depends on the NDC80 complex.
RAMA1 is a highly dynamic component of the outer kinetochore
The outer most components of the kinetochore, like CLIP-170 and CLASP, interact with kinetochores very dynamically (Pereira et al., 2006; Tanenbaum et al., 2006), whereas structural components of the kinetochore are expected to bind to kinetochores much more stably. Indeed, the NDC80 complex was shown to have an extremely low turnover at kinetochores (Hori et al., 2003). We therefore measured the turnover of RAMA1 at kinetochores by recording fluorescence recovery after photobleaching (FRAP). Surprisingly, RAMA1 recovered very rapidly on metaphase kinetochores after photobleaching, with a half-life of ∼15 seconds (Fig. 3E,F). These results indicate that RAMA1 is not a structural component of the kinetochore. Furthermore, the very dynamic association of RAMA1 with kinetochores, in contrast to the stable association of Hec1 and Nuf2, suggests that these proteins do not form a stable complex. Rather, the NDC80 complex might form a scaffold onto which RAMA1 is recruited, directly or indirectly.
RAMA1 is not required for recruitment of other attachment factors to kinetochores
Because RAMA1 is a kinetochore protein and the kinetochore is structured in a highly hierarchical fashion, we also tested whether depletion of RAMA1 resulted in loss of other kinetochore components, which, in turn, could result in the observed defects in mitotic progression. Transfection of HeLa cells with RAMA1 siRNA resulted in a strong reduction of RAMA1 mRNA levels (Fig. 4A), demonstrating the effectiveness of knockdown. Similarly, GFP-RAMA1 was sufficiently depleted by RAMA1 siRNA (supplementary material Fig. S1). Since the levels of many proteins vary at kinetochores depending on the number of microtubules bound to the kinetochores, we treated cells with nocodazole to allow for a better comparison. First, we found that Cenp-E was recruited normally to kinetochores of RAMA1-depleted cells as compared to control cells (Fig. 4B,C). Similarly, CLIP-170 still localizes to kinetochores after RAMA1 RNAi (Fig. 4D,E). Furthermore, although Hec1 was required to localize RAMA1 to kinetochores, loss of RAMA1 did not lead to a substantial recution in Hec1 levels at kinetochores (Fig. 4F,G), indicating that Hec1 is upstream of RAMA1 in kinetochore assembly. Finally, we found that Cenp-A, the dynactin subunit p150glued and the spindle checkpoint protein BubR1 were all recruited to kinetochores normally in RAMA1-depleted cells (data not shown). Although we cannot exclude the possibility that other kinetochore components are mis-localized, these results strongly suggest that the general kinetochore structure and several well-known attachment factors do not require RAMA1 for their kinetochore localization. Together with the highly dynamic binding of RAMA1 to kinetochores, these results suggest that RAMA1 does not have a structural role at kinetochores.
RAMA1 is required for chromosome alignment
Loss of RAMA1 results in a very strong accumulation of cells in mitosis and a concomitant decrease in the fraction of anaphase cells (see Fig. 1). To better understand the role of RAMA1 in mitosis, we analyzed the loss-of-function phenotype in more detail. A large fraction of RAMA1-depleted cells (68%) showed severe chromosome alignment defects (more than five misaligned chromosomes), even after 1 hour of treatment with the proteasome inhibitor MG132 to block progression to anaphase, whereas an additional 18% displayed a metaphase plate with one to five misaligned chromosomes (Fig. 5A,B). By contrast, in cells transfected with GAPDH siRNA, we did not observe any cells that had severe alignment defects and only 4% of cells had a metaphase plate with one to five misaligned chromosomes after 1 hour of MG132 treatment (Fig. 5A,B).
To analyze the dynamics of chromosome alignment in more detail, we used time-lapse analysis to follow RAMA1-depleted and GAPDH-depleted HeLa cells stably expressing YFP-H2B (Fig. 5C). In GAPDH-transfected cells >90% of cells had formed a metaphase plate within 30 minutes after NEB, and cells entered anaphase 18±4 minutes after full chromosome alignment (Fig. 5D). By contrast, RAMA1-depleted cells showed a strong delay before reaching full alignment, with 45% of cells never reaching anaphase for the duration of the film (10 hours) (Fig. 5C,D). Furthermore, the time from full alignment to anaphase onset was significantly delayed and we often observed cells that reached metaphase alignment in which chromosomes fell out of the metaphase plate (Fig. 5D). The delay in mitosis observed in RAMA1-depleted cells was dependent on the spindle checkpoint, since co-depletion of the essential checkpoint component BubR1 completely overcame the mitotic delay (Fig. 5E). Together, these results show that loss of RAMA1 inhibits efficient chromosome alignment and, in addition, suggest that chromosomes that have aligned at the metaphase plate have reduced kinetochore-microtubule attachments and therefore often fall out of the plate. Furthermore, the results show that these defects induce a checkpoint-dependent delay in mitosis.
Loss of RAMA1 results in defects in kinetochore-microtubule attachment and tension
The results of the time-lapse analysis suggest that RAMA1-depleted cells have reduced kinetochore-microtubule attachments. To test this directly, we quantified the amount of cold-stable microtubules in RAMA1-depleted cells, as kinetochore microtubules are more stable at 4°C than unattached microtubules (Rieder, 1981). Indeed, RAMA1-depleted cells showed a significant reduction in the amount of cold-stable microtubules, although this effect was less severe than in cells depleted for Hec1 (Fig. 6A,B). To determine the defects in attachment in more detail, we stained cells for p150glued or CLIP-170, both markers for unattached kinetochores (King et al., 2000; Tanenbaum et al., 2006). In control cells, p150glued- and CLIP-170-positive kinetochores were observed in all prometaphase cells as expected, whereas they were mostly absent from metaphase cells (Fig. 6C,D; and data not shown). Misaligned chromosomes in RAMA1-depleted cells displayed very high p150glued and CLIP-170 staining at kinetochores, demonstrating that they were indeed unattached (Fig. 6C,D; and data not shown). These results confirm the suggestion that the chromosomes that failed to align at the metaphase plate lack kinetochore-microtubule attachments.
We next investigated chromosomes within the metaphase plate, because the results of the time-lapse analysis suggested that kinetochores of fully aligned chromosomes in RAMA1-depleted cells contain reduced kinetochore-microtubule attachments. Indeed, whereas only 6% of controls cells that appeared to be in metaphase showed more than two kinetochores that were positive for p150glued, this was the case in 38% of RAMA1-depleted cells (Fig. 6C,D). Taken together, these results show that chromosome alignment defects in RAMA1-depleted cells are due to lack of stable microtubule attachments and show, consistent with time-lapse analysis, that the cells that are able to align all chromosomes at the metaphase plate remain in a metaphase-like state due to a few unattached kinetochores.
Because RAMA1-depleted cells show increased numbers of unattached kinetochores, not only on misaligned chromosomes, but also on kinetochores of chromosomes that had aligned at the metaphase plate, we assessed whether these attachment defects also resulted in decreased inter-kinetochore tension. To determine the tension per kinetochore pair, we measured the distance between sister kinetochores (as determined by Hec1 staining) in cells blocked in mitosis for 1 hour with the proteasome inhibitor MG132 (Fig. 6E). In control cells, the average inter-kinetochore distance was 1.21±0.18 μm (n=382 kinetochore pairs in ten cells), whereas in RAMA1-depleted cells that inter-kinetochore distance was significantly reduced to 1.10±0.18 μm (P<0.001, Student's t-test) (n=456 kinetochore pairs in ten cells). Importantly, for these experiments only cells were chosen that showed full chromosome alignment. Consistent with the p150glued staining, the decrease in average inter-kinetochore distance in RAMA1-depleted cells was largely due to a relatively small amount of kinetochore pairs in each cell that were under very low tension. Whereas in control cells 2.3% of kinetochore pairs per cell displayed an inter-kinetochore tension of less than 0.86 μm (twice the standard deviation below average), this was the case for an average of 11.6% of kinetochore pairs in each RAMA1-depleted cell (Fig. 6F). Taken together, these data show that RAMA1 is necessary for the formation of stable kinetochore-microtubule attachments and for the generation of inter-kinetochore tension.
Using a systematic high-throughput siRNA screen, we have identified RAMA1 as a novel regulator of chromosome alignment. We showed that RAMA1 localizes to kinetochores and to the spindle and we found that loss of RAMA1 results in severe chromosome alignment defects. Kinetochores of unaligned chromosomes were unattached, suggesting that defects in chromosome alignment are due to reduced kinetochore-microtubule attachment. Indeed, even chromosomes that were able to congress to the metaphase plate showed decreased kinetochore-microtubule attachments and reduced inter-kinetochore tension, and these chromosomes were often observed to fall out of the metaphase plate. Furthermore, we found that RAMA1 is a highly dynamic component of the kinetochore and that several other key attachment factors still localize to kinetochores in the absence of RAMA1, suggesting that the observed phenotypes are not simply a consequence of mis-localization of other essential kinetochore components. Taken together, these results show that RAMA1 is a novel regulator of kinetochore-microtubule attachments.
Depletion of RAMA1 resulted in severe chromosome alignment defects. Nonetheless, we did find cells that had fully aligned their chromosomes to the metaphase plate in both live and fixed cell populations. It is possible that these cells only had a partial knockdown for RAMA1, or alternatively, parallel attachment pathways could compensate for loss of RAMA1 in these cells. However, many of these cells were also delayed in the metaphase-to-anaphase transition and careful examination revealed that these metaphase-like cells contained an increased frequency of unattached kinetochores and reduced inter-kinetochore tension, explaining why anaphase onset was delayed. These results show that loss of RAMA1 reduces the number of stable kinetochore-microtubule attachments, which, depending on the severity of the effect, results in a delay in anaphase onset, with misaligned chromosomes or cells remaining in a metaphase-like state.
GFP-RAMA1 was not only observed at kinetochores, but also on the mitotic spindle, indicating that RAMA1 has an affinity for microtubules, either directly or indirectly through binding to a MAP. This suggests that RAMA1 could act as a linker protein to physically tether kinetochores to microtubules, similar to the proposal for CLIP-170, Cenp-E and Hec1 (Cheeseman et al., 2006; DeLuca et al., 2006; Draviam et al., 2006; Green et al., 2005; Guimaraes et al., 2008; Miller et al., 2008; Tanenbaum et al., 2006; Wei et al., 2007; Wood et al., 1997). It will be important in the future to determine whether RAMA1 binds microtubules directly or indirectly. If the latter is true, it is possible that RAMA1 links kinetochores to microtubules through proteins associated with the tips of kinetochore-microtubules. Several proteins have been suggested to localize specifically to the microtubule-kinetochore interface, including EB1, APC, the Ska1/Ska2 complex and the Dam1 complex (in yeast). It will be interesting to determine whether RAMA1 could act as the kinetochore anchor for any of these proteins.
Although several proteins have been implicated in the regulation of kinetochore-microtubule attachment, loss of only very few outer kinetochore proteins results in complete loss of attachments. The NDC80 complex has a well-established role in the formation of stable kinetochore-microtubule attachments from yeast to mammals, and loss of the NDC80 complex from kinetochores results in an almost complete loss of kinetochore-microtubule attachments in mammalian cells (DeLuca et al., 2002; McCleland et al., 2003; Wigge and Kilmartin, 2001). However, our data show that loss of the NDC80 complex also displaces RAMA1 from kinetochores. Similarly, Cenp-E no longer localizes to kinetochores after depletion of the NDC80 component Nuf2 (Liu et al., 2007). It is therefore possible that the severity of the NDC80 loss-of-function phenotype could be explained by the fact that multiple attachment pathways are eliminated simultaneously, whereas loss of RAMA1, CLIP-170 or Cenp-E does not perturb the localization of the other kinetochore attachment factors. Future work will hopefully resolve the contribution of each individual component to the formation and maintenance of stable kinetochore-microtubule attachments and to the generation of tension across kinetochore pairs.
Materials and Methods
Cell culture, transfection and drug treatments
U2OS and HeLa cells were cultured in DMEM (Gibco) with 6% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. siRNA was transfected using reverse transfection with Hiperfect (Qiagen) according to the manufacturers' guidelines. Gene names and siRNA sequences of the siRNA library are listed in supplementary material Table S1. Additional siRNAs used in this study were: 5′-RAMA1 AAUCCAGGCUCAAUGAUAA-3′, Hec1 and Nuf2 OTP SMART pool (Dharmacon) and BubR1 5′-AGATCCTGGCTAACTGTTC-3′. DNA transfections were performed using Fugene 6 (Roche) according to the manufacturer's guidelines. For FRAP experiments, U2OS cell lines created stably expressing GFP-RAMA1 using zeocin (Invitrogen) selection. STLC and MG132 (Sigma) were dissolved in DMSO and were both used at 5 μM final concentration.
Cloning of GFP-RAMA1
RAMA1 was amplified from a U2OS-derived cDNA library by PCR using Takara LA polymerase (Takara Bio, Shiga, Japan) with the following primers: forward 5′-CAGACCCTATCCGGAGCTTCTGCGGGGAAG-3′ and reverse 5′-TCAGTTTTCTTTGTTGCTGACATCTC-3′ and cloned into the pGEM-T vector. RAMA1 was then digested with SacII-Not1 and cloned into a modified version of pcDNA4-TO vector (Invitrogen), which includes an N-terminal biotinylation tag and a GFP (pTON-bEGFP). The plasmid was then fully sequenced and silent mutations were inserted by site-directed mutagenesis.
Cells were grown on 10 mm glass coverslips and fixed with 3.7% formaldehyde with 1% Triton X-100, washed once with PBS and post-fixed with cold methanol. The following antibodies were used: α-tubulin antibody (Sigma) (1:7500), anti-pHistone H3 (Upstate) (1:1000), Hec1 (Genetex) (1:500), CLIP-170 antibody #2360 (1:1000) (Coquelle et al., 2002), p150glued (BD) (1:500), Cenp-E (a gift from Geert Kops) (1:500), CREST anti-serum (Cortex Biochem) (1:1000) and anti-GFP (custom-made) (1:500). Primary antibodies were incubated overnight at room temperature and secondary antibodies (Alexa Fluor 488, 561 and 647, Molecular Probes) were incubated for 1 hour at room temperature. DAPI was added to all samples before mounting in Vectashield mounting fluid (Vector Labs). Images were acquired either on a Zeiss LSM510 META confocal microscope (Carl Zeiss) with a Plan Apochromat 63× 1.4NA objective with 1 μm intervals between Z-planes or on a DeltaVision RT system (Applied Precision) with a 60× 1.42NA PlanApoN objective (Olympus) using SoftWorx software. Images acquired on the DeltaVision are maximum projections of deconvolved images, unless stated otherwise. Statistic analysis of the interkinetochore distance was performed using SPSS software version 11.
Automated analysis of mitotic index
Cells were grown in 96-well plates (Viewplate-96, Perkin Elmer) in 100 μl of culture medium. Cells were fixed by addition of 50 μl of a 10% formaldehyde solution to the medium to prevent loss of mitotic cells. Cells were then washed with PBS and post-fixed with cold methanol. Wells were stained with anti-pHistoneH3 antibody and DAPI. Image acquisition was performed using a Cellomics ArrayScan VTI (Thermo Scientific) using a 10× 0.50NA objective and five images were acquired per well, which contained around 1000-2000 cells in total. Image analysis was performed using Cellomics ArrayScan HCS Reader (Thermo Scientific). In short, cells were identified on the basis of DAPI staining and they were scored as `mitotic' if the pHistoneH3 staining reached a preset threshold. All images and automated image quantifications were visually checked.
HeLa cells stably expressing YFP-H2B were plated on four- or eight-well glass-bottom dishes (Labtek). Slides were imaged on a Zeiss Axiovert 200M microscope equipped with a Plan-Neofluar 63× 1.25 oil objective in a permanently heated chamber in Leibovitz L15 CO2-independent medium. Images were acquired every 5 minutes using a Photometrics CoolSNAP HQ charged-coupled device (CCD) camera (Scientific Instruments, Tucson, AZ) and an YFP filter cube (Chroma Technology, Rockingham, VT). Z-stacks were acquired with 2 μm interval between Z-stacks. Images were processed using Metamorph software (Universal Imaging, Downington, PA).
U2OS stably expressing GFP-RAMA1 were grown in eight-well glass bottom dishes (Labtek). FRAP analysis was performed on a Zeiss LSM510 META confocal microscope. Two images were acquired before bleaching and these were averaged to give the starting fluorescence. Bleaching was preformed by scanning an area of 3×3 pixels with 100% laser power of the 488 nm laser, with 50 iterations. Images were acquired every 10 seconds after bleaching. Fluorescence intensities of the bleached area were measured over time using Metamorph software, and total cell fluorescence was also measured to correct for bleaching.
HeLa cells lysates were prepared 24 hours after siRNA transfection and whole-cell RNA was purified using the Qiagen RNA Easy kit according to the manufacturer's guidelines. cDNA was synthesized using Superscript II (Invitrogen). 1 μg cDNA was used per reaction and products were amplified in 25 cycles. Real-time PCR primer pairs were designed with Tm of close to 60°C to generate 200–300 bp amplicons, mostly spanning introns. Primers used were RAMA1 forward 5′-CAGATCCCTCTTCACCTACGA-3′ and reverse 5′-TCAACGTTTAAAGGGGGACA-3′ and β-actin forward 5′-GGCATCCTCACCCTGAAGTA-3′ and reverse 5′-GGGGTGTTGAAGGTCTCAAA-3′.
HeLa cells were transfected with siRNA targeting either GAPDH or RAMA1. After 8 hours, cells were transfected with GFP-RAMA. After 24 hours, cells were harvested and lysed using Laemmli buffer [120 mM Tris (pH 6.8), 4% SDS, 20% glycerol]. Equal amounts of protein were seperated on a polyacrylamide gel and subsequently transferred to nitrocellulose membranes. Membranes were probed with the following primary antibodies: anti-GFP (custom made) (1:1000) and anti-actin (Santa Cruz Biotechnology) (1:2500). HRP-coupled secondary antibodies (Dako, Carpinteria, CA) were used in a 1:2500 dilution. The immunopositive bands were visualized using ECL western blotting reagent (GE Healthcare).
Note added in proof
During the final revision of this manuscript three additional studies identified RAMA1 as novel kinetochore component involved in kinetochore-microtubule attachment. In these studies the same protein was called Rama1 (Welburn et al., 2009), C13orf3 (Theis et al., 2009) and Ska3 (Gaitanos et al., 2009).
We thank Anna Akhmanova (Erasmus Medical Centre, Rotterdam, The Netherlands) for the plasmid containing the biotinylation-tag-GFP construct, Niels Galjart (Erasmus Medical Centre, Rotterdam, The Netherlands) for the CLIP-170 antibody, Geert Kops (UMC Utrecht, Utrecht, The Netherlands) for the Cenp-E and GFP antibodies and Jagesh Shah (Harvard Medical School, Boston, MA) for the HeLa YFP-H2B cell line. We would also like to thank Joost Vermaat for help with statistical analysis and Livio Kleij for maintaining the microscopes. Furthermore we would like to thank the members of the Medema, Kops and Lens laboratories for helpful discussion. This work was supported by the Dutch Organization for Scientific Research (NWO-VICI, ZonMw 918.46.616 and NWO-ALW, 81802003) and a `UMC Internationalizering' grant. R.H.M. was funded by the Netherlands Genomics Initiative of the Netherlands Organization for Scientific Research.