Report |
Address correspondence to Jennifer DeLuca, Department of Biology, 607 Fordham Hall, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Tel.: (919) 962-2354. Fax: (919) 962-1625. E-mail: jgdeluca{at}email.unc.edu
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Abstract |
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Key Words: hNuf2; microtubules; kinetochores; mitosis; siRNA
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Introduction |
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Identification of proteins that dynamically couple vertebrate kinetochores to microtubule plus ends is a critical issue that remains to be resolved in order to understand how chromosomes are accurately aligned on the spindle in prometaphase and then segregated to the poles in anaphase. The kinetochore-bound microtubule motors identified thus far are not sufficient because depletion of either CENP-E or dynein/dynactin from kinetochores suppresses, but does not block, kinetochore microtubule formation or chromosome movement in mammalian tissue cells (Howell et al., 2001; McEwen et al., 2001). Because the spindle checkpoint proteins were initially discovered in yeast and are conserved in humans, we suspected that key proteins at the kinetochoremicrotubule interface might also be conserved. In budding yeast, a molecular complex of Nuf2p, Ndc80p, Spc24p, and Spc25p has been suggested to be essential for kinetochore microtubule formation (He et al., 2001; Wigge and Kilmartin, 2001). Human homologues of Nuf2 (hNuf2) and Ndc80 (HEC) have been identified and shown to localize to mitotic kinetochores (Chen et al., 1997; Nabetani et al., 2001; Wigge and Kilmartin, 2001). Because the fission yeast homologue of Nuf2 also plays a critical role in attaching chromosomes to spindles (Nabetani et al., 2001), and the Caenorhabditis elegans homologue, HIM-10, is essential for proper chromosome segregation (Howe et al., 2001), the vertebrate homologue of Nuf2 appeared to be an excellent candidate for a protein with key functions at the kinetochoremicrotubule interface in human cells.
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Results and discussion |
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To further examine the pathway of mitotic exit by cell death resulting from depletion of hNuf2, we determined the DNA content by flow cytometry (Fig. 2 E). 48 h after hNuf2 siRNA transfection, a significant population of the cells had accumulated with a 4n DNA content, and by 72 h, the large population of cells containing 4n DNA had shifted to a state in which they contained a variable amount of sub-4n DNA. For comparison, HeLa cells treated with vinblastine to depolymerize microtubules remained blocked in mitosis with a 4n DNA content, even after 48 h (Fig. 2 F). These results suggest that the hNuf2 siRNAtransfected cells persist in mitosis for some time and subsequently undergo cell death. To test this directly, we incubated cells with a Trypan blue solution 72 h after transfection for 10 min, and confirmed that cells transfected with hNuf2 siRNA had undergone cell death, as only these cells were permeable to the dye (Fig. 2 G). Furthermore, we stained cells at 48 and 72 h after hNuf2 siRNA transfection with DAPI to directly image cellular DNA (Fig. 2 H) and observed the DNA to be very dense, opaque, and present in globules of varying sizes and shapes, characteristic of cells undergoing cell death (Mills et al., 1999; Zhang and Xu, 2002). We conclude from these data that depletion of hNuf2 from HeLa cells results in a prolonged mitotic block followed by cell death. This aberrant exit from mitosis has characteristics of both apoptosis and mitotic catastrophe (also known as mitotic cell death) (Nabha et al., 2002). Although researchers are beginning to understand the differences in these processes, there remains much controversy regarding distinctions between their biochemical pathways. It will be interesting for both cancer and cell biology fields to determine the specific suicidal mechanisms that are executed in hNuf2 depletioninduced cell death.
To test how depletion of hNuf2 blocks chromosome alignment into a metaphase plate, we used immunofluorescence microscopy. Fig. 3 A (top) shows the typical distribution of spindle microtubules and hNuf2 localization for a control cell. The formation of bundles of kinetochore microtubules and their subsequent pulling forces make the spindle short and oblate, on average 11.3 µm from pole to pole (±1.2 µm; n = 8). In hNuf2-depleted cells (Fig. 3 A, bottom), robust microtubule arrays extended from the poles and penetrated the chromosomes, but these spindles were 60% longer (17.0 ± 2.2 µm; n = 18) than control spindles, consistent with the absence of pulling forces from kinetochore fibers, which were not detectable (Fig. 3 A). Furthermore, in cells stained for CREST to clearly mark the kinetochores, we could detect no kinetochore fibers (Fig. 3 B). We next measured the distance between sister kinetochores to determine if hNuf2 kinetochores were stretched, which would suggest tension produced by attached microtubules. The average interkinetochore distance in hNuf2-depleted cells was 0.88 µm (±0.19 µm; n = 152 kinetochores), compared with the control metaphase distance of 1.56 µm (±0.36 µm; n = 138 kinetochores). Cells treated with vinblastine to depolymerize all microtubules had an average interkinetochore distance of 0.88 µm (±0.14 µm; n = 258 kinetochores). These results suggest that kinetochores in hNuf2-depleted cells are not under tension due to microtubule forces. It is known that kinetochore microtubules are differentially stable to cooling at 4°C in comparison to nonkinetochore microtubules (Rieder, 1981). When we cooled cells for 10 min before fixation, kinetochore fibers were abundant in control cells (Fig. 3 C, top), and the spindle length was reduced to 4.8 µm (±1.1 µm; n = 9). However, in hNuf2 siRNAtransfected cells, only a few microtubules were observed (Fig. 3 C, bottom), and in cells with detectable microtubules, the spindles remained elongated with an average spindle length of 13.6 ± 1.7 µm (n = 17). Those cells that did exhibit a low level of hNuf2 staining at kinetochores contained more prominent fluorescence bundles of kinetochore microtubules in the cold-treated preparations (unpublished data). Thus, the formation of stable kinetochore microtubules depends critically on hNuf2.
Consistent with our time-lapse data, we observed by DAPI staining that although most hNuf2 siRNAtreated mitotic cells were unable to properly align their chromosomes (90%), 10% of the treated cells that entered mitosis did achieve a metaphase alignment of chromosomes. The kinetochores of these cells contained hNuf2, although the levels were decreased to an average of 28% of control metaphase levels (unpublished data). Our time-lapse data indicate that such cells also block in mitosis and subsequently undergo cell death, suggesting that reduced levels of hNuf2 at kinetochores can inhibit chromosome segregation and induce mitotic cell death, but permit sufficient kinetochore fiber formation for metaphase chromosome alignment.
In both budding and fission yeast, depletion of Nuf2 blocks microtubule attachment to chromosomes and also inactivates the spindle checkpoint, a result that suggests that Nuf2 depletion produces major disruption of the assembly of many proteins at kinetochores rather than a specific effect on kinetochoremicrotubule attachment (Janke et al., 2001; Nabetani et al., 2001). In contrast, we found for HeLa cells that depletion of hNuf2 not only prevents kinetochore microtubule formation, but it also blocks cells in mitosis, indicating that spindle checkpoint activity is not disrupted by depletion of hNuf2. To test this prediction, we examined spindle checkpoint proteins at kinetochores in hNuf2-depleted cells, and as shown in Fig. 4 , in hNuf2 siRNA transfected cells, both Mad2 and BubR1 were detectable at kinetochores. Mad2 and BubR1 appeared diminished compared to unattached kinetochores in prometaphase control cells (data not shown), however, both proteins showed strong localization to all kinetochores in nocodozole-treated hNuf2 siRNA transfected cells, indicating that hNuf2 depletion does not prevent their binding to kinetochores (data not shown).
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Our results presented here highlight a specific role of hNuf2 for stable kinetochoremicrotubule attachment and predict that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends. hNuf2 is an -helical coiled-coil protein without an ATP binding or motor domain (Osborne et al., 1994; Nabetani et al., 2001). Whether it can bind microtubules directly or in concert with members of an Ndc80 complex or components of other complexes remain important unanswered questions. hNuf2 is a potentially attractive target for stopping the proliferation of tumor or cancer cells, because hNuf2 depletion appears selective for blocking cells in mitosis and producing subsequent cell death.
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Materials and methods |
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For lysates, cells were grown in 25-cm2 flasks at a confluency of 106 cells/ml. Cells were removed from the flasks with trypsin, pelleted in a tabletop clinical centrifuge at 5,000 rpm, washed with PBS (140 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4, 1.5 mM Na2HPO4, pH 7.8), and lysed by sonication. Samples were then clarified by centrifugation, and the protein concentration of the supernatants was determined. Samples were run on 10% SDSpolyacrylamide gels, transferred to nitrocellulose, and subjected to immunoblot analysis using hNuf2 antibodies (1:1,000; Wigge and Kilmartin, 2001) and actin antibodies (1:10,000; Sigma-Aldrich) for a loading control.
siRNA
A 21-nucleotide siRNA duplex was synthesized by Dharmacon Research to target the hNuf2 sequence 5'-AAGCATGCCGTGAAACGTATA-3'. Transfections were performed following the protocol provided by Dharmacon Research using the oligofectamine transfection reagent (Invitrogen). For controls, cells were transfected with a Cy3-labeled luciferase GL2 duplex (Dharmacon Research), or cells were mock transfected with oligofectamine alone. Transfection efficiency was determined using the Cy3luciferase GL2 siRNA; 48 h after transfection, cells were fluorescently imaged on an inverted Nikon TE30 using a 10x objective. Cells were counted from a total of four experiments, and, in each case, there were virtually no cells lacking the fluorescent label; thus the transfection efficiency was near 100%.
Immunofluorescence microscopy
Images were obtained using a Nikon 100x/NA 1.4 planapochromat oil immersion lens on a spinning disc confocal fluorescence microscope (Cimini et al., 2001). Mad2 antibodies were prepared as described by Waters et al. (1998) and used at a dilution of 1:100. Tubulin antibodies (DM1; Sigma-Aldrich) were used at a dilution of 1:350. T.J. Yen (Fox Chase Cancer Center, Philadelphia, PA) provided BubR1 and CENP-E antibodies, both used at a dilution of 1:500. CREST serum, provided by B.R. Brinkley (Baylor College of Medicine, Houston, TX), was used at a dilution of 1:10,000.
For determination of cells with cold-stable microtubules, cell media was removed from the culture dishes 48 h after siRNA transfection and replaced with ice-cold media. Cells were then incubated on ice for 10 min to induce microtubule disassembly of all nonkinetochore microtubules and subsequently fixed and processed for immunofluorescence using hNuf2 and anti-tubulin antibodies and DAPI to stain cellular DNA. Cells were imaged as described by Howell et al. (2000).
Phase-contrast microscopy and time-lapse imaging
Cells were transfected with either hNuf2 siRNA or a control 21-nucleotide siRNA duplex and imaged 24, 48, and 72 h after transfection using a Nikon 10x apodized phase objective on a Nikon TE300 microscope with an inverted stand. For live-cell time-lapse imaging, coverslips were mounted in Rose chambers and the chambers were filled with L-15 medium (Sigma-Aldrich) supplemented with 7 mM Hepes, pH 7.2, and 10% FBS. Cells were time-lapse recorded using a Nikon 10x or 40x apodized phase objective on a Nikon inverted TE300 stand. Using a Ludl rotary-encoded scanning stage and a Hamamatsu Orca II camera with Metamorph digital imaging software (Universal Imaging Corp.), cells were imaged every 90 s for 8 h over 10 fields of view.
Flow cytometry and cell death analysis
For flow cytometry, cells were grown in 25-cm2 culture dishes and transfected with either control siRNA or hNuf2 siRNA. After transfection at various times, cells were trypsinized, pelleted, and washed in PBS. Cells were fixed with 1% paraformaldehyde for 20 min, pelleted, and washed in PBS. Cells were resuspended in ice-cold 70% ethanol and stored at 4°C. For analysis, cells were pelleted, washed with PBS, and resuspended in 500 µl propidium iodide (20 µg/ml)/RNase A (30 µg/ml). Cells were analyzed within 3 h on a Becton Dickinson FACScan® interfaced to a Cytomation data acquisition system.
For determination of cell death, mock-transfected and hNuf2 siRNAtransfected cells grown on coverslips were incubated for 10 min with 0.2% Trypan blue 72 h after transfection. Coverslips were mounted onto slides and observed by phase-contrast microscopy using a 20x objective and also by epifluorescence microscopy, as Trypan blue is a fluorescent dye that emits a red fluorescence upon excitation by blue light (Reno et al., 1997).
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health grants GM24364 to E.D. Salmon and GM66588 to J.G. DeLuca.
Submitted: 27 August 2002
Revised: 3 October 2002
Accepted: 4 October 2002
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References |
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