HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL) supplemented with 10% fetal-bovine serum (FBS; Equitech-Bio) at 37°C and 5% CO₂.

The following antibodies were used in immunofluorescence microscopy and immunoblotting. A rabbit ASURA polyclonal antibody was generated as described previously [⁴⁵] and used at a dilution of 1:1000. The other primary antibodies were anti-CENP-F rabbit polyclonal (1:2000, Novus Biologicals), anti-Hec1 mouse monoclonal (1:1000, Affinity Bioreagents), anti-CREST (1:1000, Cortex Biochem), and anti-α-tubulin mouse monoclonal (1:500, Calbiochem). Secondary antibodies for immunoblot analyses are alkaline phosphatase anti-mouse IgG (1:2000, Vector Laboratories) and alkaline phosphatase anti-rabbit IgG (1:2000, Vector Laboratories). For immunofluorescence analyses, secondary antibodies were Alexa Fluor 488 anti-mouse monoclonal (1:500, Molecular Probes), Alexa Fluor 488 anti-rabbit monoclonal (1:500, Molecular Probes), and anti-human IgG (1:200, Sigma).

HeLa cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions at a final concentration of 100 nM with ASURA-siRNA (PHB2 siRNA-1 in [⁴⁵]) or Hec1 siRNA (5'-AAGTTCAAAAGCTGGATGATC-3') [²⁷]. Cells transfected with Lipofectamine alone were used as a control. Cells were collected 48 hr post-transfection for use in analysis.

Cells (siRNA or mock transfected) grown in 24-well plates were collected and lysed in 2× Laemmli Sample Buffer (Santa Cruz Biotechnology) with an equal amount of PBS buffer. Protein extracts were fractionated on 10% polyacrylamide gels and then transferred onto PVDF membrane. The immunoblots were blocked with 1% BSA-TBST (0.1% Tween 20, 25 mM Tris-HCl pH 7.4, 137 mM NaCl, 25 mM KCl) and labeled with the primary and secondary antibodies. The immunoreactive protein bands were detected by NBT/BCIP solution (Roche) diluted in AP buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 1 mM MgCl₂).

HeLa cells grown on coverslips were transfected with target siRNA, fixed with 4% PFA (para-formaldehyde) diluted in PBS (phosphate buffered saline, pH 7.4) for 15 min at 37°C, and stained with Hoechst 33342 (Sigma) for mitotic index calculation. For proteins localization analyses, cells were fixed either with 4% PFA containing 0.5% Triton X-100 diluted in PBS for 15 min (for Hec1 and CREST staining) or 100% ice-cold methanol for 10 min at −20°C (for CENP-F and CREST staining). Alternatively, cells were arrested at metaphase by adding colcemid (final concentration 0.1 µg/ml) in the culture medium for 3 hr at 37°C and were collected for metaphase-chromosome spreads as described earlier [²⁶]. Cells were blocked with 1% BSA in PBS at room temperature for 30 min. Then, primary and secondary antibody interactions were carried out for 1 hr at room temperature, respectively. Samples were then mounted in Vectorshield mounting medium (Vector Laboratories) and examined under an Axioplan II imaging fluorescence microscope (Carl Zeiss) equipped with a CCD camera (MicroMax, Roper Scientific) driven by the IP Lab software.

HeLa cells transfected as above for 48 hr were later fixed in 3% glutaraldehyde and 0.2% tannic acid diluted in PBS buffer for 1 hr at room temperature. Post-fixation was in 2% OsO₄ for 20 min. The cells were dehydrated through an increasing ethanol series and infiltrated with epoxy resin (Quetol 812). The resin was polymerized at 37°C for 12 hr, 45°C for 12 hr and 60°C for 48 hr. Cells of interest embedded in the resin were chosen under an optical microscope and trimmed to ~1.0 mm². Samples were cut into 70–80 nm thick serial sections with an ultramicrotome equipped with a diamond knife (Ultracut E, Reichart-Jung). The sections were stained with uranyl acetate and lead citrate for examination with a transmission electron microscope (JEM-1200EX, JOEL).

Our EM analyses are based on those of DeLuca et al. [¹³] with some alterations. For control, cells that apparently aligned at metaphase plate were chosen for analysis. ASURA and Hec1 RNAi cells were chosen based on their phenotypes of poor chromosome alignment. All kinetochores observed were included in the analyses regardless of their appearance. For individual cells, only a few sections, containing chromosome-rich regions, which were often close to the center of the cells, were examined. As the boundary between individual chromosomes is not obvious, and sister kinetochore appearances can sometimes show differences depending on kinetochore fiber attachment, kinetochores were analyzed individually rather than as a kinetochore pair of a chromosome. Furthermore, as kinetochore morphology varies even between adjacent serial sections, which has also been reported for Indian muntjac chromosomes [⁵³], we analyzed several adjacent serial sections to classify individual kinetochores. Kinetochore is classified as trilaminar once the canonical layered structure is visible in any of the adjacent serial sections for an individual kinetochore, even if the structure is rather fuzzy in other sections.

To test how ASURA functions in mitosis, we transfected HeLa cells with a 21-nucleotide duplex homologous to a portion of the ASURA sequence. As a result, ASURA expression levels were strongly reduced 48 hr after transfection (26% of control) as shown in Figure 1A. Further observation revealed a significant increase in mitotic index to about 3-fold that of the control (4.7±0.3%) in ASURA RNAi cultures (13.4±1.9%) (Fig. 1B). We previously showed that ASURA RNAi affected the kinetochore localization of several kinetochore proteins, including Hec1 [⁴⁵]. Thus, we checked the effects of Hec1 knockdown as well. The mitotic index of Hec1 RNAi (12.9±2.3%) was similar to that of ASURA RNAi (Fig. 1B). The advantage of employing Hec1 RNAi is that, among the kinetochore proteins that we have tested, Hec1 has been studied the most regarding its function at the kinetochore. DeLuca et al. [¹³] showed by using EM that Hec1 and Nuf2 localized at the kinetochore outer layer. As the structural effect of Nuf2 RNAi (Hec1 is depleted at the same time) is well studied, Hec1 was used as a model protein throughout this study.

We next assessed the role of ASURA on mitotic progression. Cultures subjected to ASURA siRNA treatment displayed a high percentage of prometaphase cells (82.0±2.5%), more than double the control (36.9±3.1%) (Fig. 1C). There were only a few cells with chromosomes aligned in the metaphase plate (Fig. 1C, D), with most of the mitotic cells showing non-alignment or misalignment.

When we examined for ASURA localization profile, immunofluorescence showed that ASURA is predominantly localized at the cytoplasm (Fig. 1E), as revealed by expression of GFP-ASURA [⁴⁵]. Unlike Hec1, which localized to the kinetochore during mitotic phase, there was no obvious signal of ASURA localization specifically to the kinetochore or centromeric region throughout the cell cycle, indicating that ASURA is not a kinetochore component. Next, we investigated ASURA effect on kinetochore proteins localization. In mock transfected cultures, Hec1 (Figs. 1E, 2B) and CENP-F (Fig. 2A) localized normally at the kinetochore. With partial depletion of ASURA, kinetochore localization of CENP-F (Fig. 2A, C) was abolished, whereas Hec1 intensity decreased to 50% of the control (Fig. 2B, C), consistent with our previous report [⁴⁵]. Hec1 expression level was unaltered in the absence of ASURA (Fig. 1A), indicating that this is not an off-target effect.

We confirmed premature sister chromatid separation (Fig. 2B) after knockdown of ASURA [⁴⁵]. Interestingly, we found that even in Hec1 knockdown cultures, sister chromatids were separated in about 30% of the cells, which has not been reported elsewhere (Fig. 2B). The exact reason for this separation remains obscure (refer to discussion), although this is observed only in cells with Hec1 intensity lower than 5% of the normal (Fig. 3). The possibility that the loss of sister chromatid cohesion in ASURA partial depletion was due to the lower levels of Hec1 was not apparent from our data. In particular, when Hec1 intensity at the kinetochore is about 50% of the control, a level similar to that of ASURA disruption, sister chromatids were rarely separated.

Combining all the data [⁴⁵], ASURA is required for kinetochore localization of Hec1, CENP-F, and also CENP-E. Thus, we suggest that ASURA depletion leads to improper kinetochore assembly.

To confirm the above hypothesis, we adopted electron microscopy for our RNAi analyses. As the defects observed in kinetochore may be derived from the disruption of the maturation process, we first ascertained the kinetochore development in HeLa cells. Figure 4A showed a prophase cell. The nuclear envelope (green arrows) and nucleoli (red arrowheads) are visible. Kinetochores were observed as either a fibrous mass (Fig. 4B, D, blue arrows) or fuzzy ball with a partially constructed kinetochore plate (Fig. 4B, C, red arrows). After nuclear envelope breakdown, the outer plates became clearer and began to interact with MTs. Figure 4E shows a pair of sister kinetochores from a chromosome located near the metaphase plate in a prometaphase cell. The right one is interacting end-on with the robust kinetochore MTs, although the plate structure is less obvious. The kinetochore at the left is interacting side-on with a MT running close-by, and the fibrous corona (blue arrowheads in Fig. 4E) is visible [¹⁴]. During prometaphase, kinetochores facing the poles are favorable in capturing MTs [⁴³]. Once this association is established, kinetochores were transported poleward via the corona [⁴³], forming lateral interactions with any stabilized MT bundles [⁵]. Even if chromosomes fail to achieve bi-orientation at the poles, the chromosomes glide along the kinetochore fibers with the aid of CENP-E from the corona to obtain bi-orientation at the metaphase plate [²¹], which is known as the mono-oriented pathway [⁵]. In the prometaphase, some kinetochores remained as fuzzy balls without internal structure (data not shown).

The final step of the maturation is achieved when the electron-dense outer plate and inner plate are completely formed, enabling the kinetochore to capture robust MTs and achieve bi-orientation (Fig. 4F) by the end of prometaphase. A schematic model showing the development of the kinetochore is presented based on the observations above (Fig. 4G). Kinetochores are classified into three developmental groups, Class 1, 2, and 3.

Mock transfected control (Fig. 5A), ASURA RNAi (Fig. 5B) and Hec1 RNAi (Fig. 5C) cells were analyzed under the aforementioned conditions. Kinetochores were classified by referring to their maturation process (Fig. 4G). The quantitative data are shown in Figure 5M. Normally, Class 3 kinetochores form the majority, more than 75% of the population, as shown in the control. This is rarely the case in the ASURA and Hec1 RNAi cultures, where less than 10% are classified as Class 3. DeLuca et al. [¹³] showed that in Nuf2-Hec1 depletion, more than 50% of the kinetochores observed failed to form a well-recognized outer plate. Liu et al. [²⁵] also revealed that fuzzy balls were frequently observed in the absence of Nuf2. We made a similar observation for Hec1 RNAi, where highly disorganized kinetochores were significantly increased (Fig. 5M, Class 1). As expected, kinetochore outer plates were either undeveloped (Class 1) or poorly-formed (Class 2) with the partial depletion of ASURA. Even when a layered structure was constructed, the outer plates, and sometimes even the inner plates, were often pulled out or stretched (Fig. 5H, I), indicating that without ASURA the kinetochore lacks physical rigidity against the MT pulling forces, although MT attachments were less frequent compared to the authentic trilaminar structure (Fig. 5D). The morphological defects observed in ASURA knockdown cultures were similar to those of Hec1 disruption (Fig. 5J–L). This suggests that the abnormalities observed in kinetochore formation derived from the effect of mislocalization of Hec1 and other outer kinetochore proteins downstream. Altogether, this clearly shows that ASURA is an essential protein for proper kinetochore formation, most probably by targeting kinetochore proteins.