Deciphering protein function during mitosis in PtK cells using RNAi
© Stout et al; licensee BioMed Central Ltd. 2006
Received: 22 March 2006
Accepted: 23 June 2006
Published: 23 June 2006
Studying mitosis requires a system in which the dramatic movements of chromosomes and spindle microtubules can be visualized. PtK cells, due to their flat morphology and their small number of large chromosomes, allow microscopic visualizations to be readily performed.
By performing RNAi in PtK cells, we can explore the function of many proteins important for spindle assembly and chromosome segregation. Although it is difficult to transfect DNA into PtK cells (efficiency ~ 10%), we have transfected a fluorescent siRNA at nearly 100% efficiency. Using a cDNA expression library, we then isolated a complete PtK MCAK (P-MCAK) cDNA. P-MCAK shares 81% identity to Human-MCAK (H-MCAK) protein and 66% identity to H-MCAK DNA. Knockdown of P-MCAK by RNAi caused defects in chromosome congression and defective spindle organization. Live imaging revealed that chromosomes had defects in congression and segregation, similar to what we found after microinjection of inhibitory anti-MCAK antibodies. Because it is laborious to isolate full-length clones, we explored using RT-PCR with degenerate primers to yield cDNA fragments from PtK cells from which to design siRNAs. We isolated a cDNA fragment of the mitotic kinesin Eg5 from PtK cells. This fragment is 93% identical to H-Eg5 protein and 87% identical to H-Eg5 DNA. A conserved 21 bp siRNA was used for RNAi in both HeLa and PtK cells in which Eg5 knockdown resulted in an increased mitotic index and cells with monopolar spindles. In addition, we used RT-PCR to isolate fragments of 5 additional genes, whose sequence identity ranged from 76 to 90% with human, mouse, or rat genes, suggesting that this strategy is feasible to apply to any gene of interest.
This approach will allow us to effectively probe mitotic defects from protein knockdowns by combining genomic information from other organisms with the tractable morphology of PtK cells.
The mitotic spindle consists of a dynamic array of microtubules and their associated proteins [1, 2]. Dynamic microtubules are critical both for spindle assembly and for chromosome movement by capturing chromosomes at the kinetochore, the specialized site where microtubules contact mitotic chromosomes [3, 4]. The mitotic spindle is comprised of three classes of microtubules whose dynamics appear to be differentially regulated . Within the spindle, the non-kinetochore or spindle microtubules turn over rapidly; in contrast, the bundles of microtubules in the kinetochore fibers are overall more stable, but locally highly dynamic [6–8]. The astral microtubules are more dynamic relative to interphase microtubules, but it is not known how their stability compares to those of the spindle .
Microtubule polymerization dynamics are of fundamental importance for the intracellular functions of the microtubule cytoskeleton and are highly regulated. In general, microtubules in cells turn over much more rapidly than microtubules assembled from pure tubulin in vitro, due to cellular factors that contribute to increased microtubule turnover, including Op18 [11–15], Tog [16–23], and the microtubule depolymerizing kinesins, MCAK and Kif2A [24–28]. In particular, the essential role that MCAK plays to regulate microtubules in the spindle and at the kinetochore has been the recent focus of much attention.
MCAK is a member of the Kinesin-13 family , whose members depolymerize microtubules rather than translocate along them [30–32]. MCAK was originally identified as a protein that localizes to centromeres in mitosis , and was shown to be critical for spindle assembly in Xenopus egg extracts . MCAK directly destabilizes microtubules by binding to either microtubule end and inducing a conformational change in the microtubule that leads to depolymerization [25, 34]. Furthermore, MCAK regulates microtubule dynamics in the cell both during interphase and mitosis . More recently, it has been shown to be a member of the microtubule plus-end tip tracking proteins, but the functional significance of this activity is not known [35, 36]. The exact role of MCAK in chromosome movement and segregation has been a subject of debate, specifically, whether it is needed solely for chromosome congression before anaphase or whether it also functions directly in chromosome segregation at anaphase [37–40].
In order to study the exact role of MCAK and other Kinesin-13 family members in the regulation of cellular microtubule dynamics, it is essential to use a cell type in which the dynamics of microtubules during mitosis can be readily visualized. One popular cell type is the marsupial PtK2 cell, from the kidney of a normal adult male Potorous tridactylis, which has a large flat morphology and a small number of large chromosomes (for example see [41, 42]). However, functional analysis has been limited to the microinjection of inhibitory antibodies or application of small molecule inhibitors due to the lack of genomic information for RNAi knockdown. In addition, these cells generally transfect poorly, which hinders such studies. Here we report the identification of the PtK MCAK gene and the optimization of the techniques to use siRNA-mediated knockdown to deplete endogenous MCAK and compare those effects to those of antibody inhibition. In addition, we use RT-PCR to identify several other partial gene sequences and show the effects of knockdown of additional PtK genes to demonstrate the applicability of our approach.
Results and discussion
P-MCAK is homologous to Human and Xenopus MCAK
Knockdown of P-MCAK causes defects in spindle morphology and chromosome alignment
Loss of MCAK causes defects in chromosome movement
Previous studies with injection of a centromere dominant-negative form of MCAK  or expression of motorless MCAK  resulted in an increased number of lagging chromosomes, which are chromosomes that remain in the midzone during anaphase and often into telophase. This is in contrast to our current studies in which knockdown of MCAK by RNAi did not result in lagging chromosomes during anaphase upon analysis of fixed samples (data not shown). This might be because the low frequency of anaphase cells in fixed samples in both control and MCAK RNAi cells hindered our analysis of this defect. Because lagging chromosomes are often caught at the midzone beyond anaphase, we also scored telophase cells for lagging chromosomes. Though our results did show a decrease in the percentage of telophase cells after MCAK RNAi (Fig. 2H), we still did not see an increase in lagging chromosomes (data not shown). Another possibility is that during early anaphase, some chromosomes lag behind the mass of segregating chromosomes, but then recover quickly enough so that these "stragglers"  eventually get partitioned to the two daughter cells. This phenotype would be difficult to discern in fixed analysis. To test this possibility, we imaged control and MCAK RNAi cells by time-lapse phase contrast microscopy.
Analysis of the morphological defects and mitotic progression of mitosis in live images of RNAi transfected cells vs. antibody-injected cells.
% cells (n)
% cells (n)
% cells (n)
% cells (n)
Chromosomes lingering at poles
Multiple attempts to congress
Not all chromosomes congress
Loose alignment with high oscillations
Total % cells with alignment defects
% cells (n)
% cells (n)
% cells (n)
% cells (n)
Total % cells with segregation defects
Avg. ± SEM (n)
Avg. ± SEM (n)
Avg. ± SEM (n)
Avg. ± SEM (n)
Spindle length (μm)
15.3 ± 0.5 (14)
12.9 ± 0.5* (12)
14.5 ± 0.6 (16)
12.9 ± 0.4* (17)
Timing of Mitotic Progression
Avg. ± SEM (n)
Avg. ± SEM (n)
Avg. ± SEM (n)
Avg. ± SEM (n)
Time between injection and NEB (min)
5.6 ± 2.2 (17)
2.8 ± 0.8 (18)
Time between NEB and LCC (min)
17.4 ± 1.3 (14)
23.3 ± 7.0 (12)
16.1 ± 1.7 (15)
32.3 ± 3.4* (17)
Time between LCC and Ana A (min)
13.7 ± 2.7 (13)
12.6 ± 1.8 (11)
16.1 ± 1.7 (14)
17.4 ± 2.2 (18)
Total time between NEB and Ana A (min)
30.1 ± 2.7 (17)
32.6 ± 5.9 (14)
33.3 ± 1.7 (17)
47.7 ± 4.1* (18)
In addition to chromosome congression and segregation defects, we determined that MCAK RNAi cells also displayed an overall shorter spindle length (Table 1). This result may at first seem counterintuitive. Because we have shown that depletion of MCAK results in increased astral microtubule length, it could be expected that its depletion would also result in longer spindle microtubules increasing the overall spindle length. However, the increased length of the astral microtubules seen upon the depletion of MCAK may instead sufficiently reduce the overall tubulin pool available so that normal spindle length cannot be maintained in the dividing cell. Alternatively, it has been shown previously that treatment of cells with low concentrations of paclitaxel, which suppress microtubule dynamics without causing a significant change in microtubule polymer, also result in shorter spindles . The authors interpreted this data to indicate that paclitaxel suppressed plus-end dynamics of kinetochore microtubules without affecting the depolymerization at poles due to flux. Inhibition of MCAK may similarly be suppressing plus-end dynamics of kinetochore microtubules. Consistent with this idea, we reported previously that injection of the MCAK centromere dominant-negative also caused shorter spindles .
To determine if the defects in MCAK RNAi cells were similar to those we have described earlier with fixed analysis of cells after antibody microinjection, we imaged cells by time-lapse phase contrast microscopy after antibody injection . Prophase cells were injected with either control IgG or anti-MCAK antibodies and then followed through mitosis by phase-contrast microscopy. Similar to the MCAK RNAi cells, the MCAK antibody injected cells had defects during chromosome congression (Figure 3, Table 1, and Videos 3 and 4). Chromosomes lingered at spindle poles, had difficulty in congression and most significantly had increased oscillations at the metaphase plate, which resulted in a loose metaphase plate and perhaps the higher incidence of straggling chromosomes at anaphase. Similar to MCAK RNAi cells, anti-MCAK antibody injected cells also had shorter spindles. One difference between antibody-injected and RNAi cells is that although MCAK RNAi caused a slight increase in lagging chromosomes, the antibody-injected cells had a higher incidence of lagging chromosomes. However, the overall percentage of cells with one or more segregation defects was only slightly higher in MCAK antibody injected cells than in MCAK RNAi cells.
It is not clear why the segregation defects translate to fewer lagging chromosomes in MCAK RNAi knockdown cells versus MCAK antibody inhibited cells. One possibility is that cell-to-cell variability with RNAi knockdown and the lower numbers of cells in our live analysis contributed to the lower overall percentage of lagging chromosomes in MCAK RNAi cells. Unfortunately we could not fix and stain live cells imaged after MCAK RNAi to determine the extent of knockdown in the imaged cell because MCAK staining is quite diffuse in the cytoplasm and in the nucleus at early G1. However, the failure to see lagging chromosomes in fixed analysis of both anaphase and telophase cells where we had analyzed many more cells suggests that these results are not simply due to inefficient knockdown in the imaged cells. It could be that antibody inhibition blocks MCAK activity in ways that RNAi does not. Antibody inhibition may impede MCAK function locally in the cell, such as at the centromere, and this contributes to the increase in lagging chromosomes in ways that global knockdown by RNAi would not, or it could be that our antibodies are interfering with the function of another protein that possibly interacts with MCAK producing a cumulative effect. We think both of these possibilities are unlikely because the antibody-antigen complexes are found in the cytoplasm, not at the centromere , and because we have injected 3 different antibodies, including a newly developed one to the P-MCAK protein, as well as a dominant-negative version of MCAK, and all give similar phenotypes. Another possibility is that RNAi knockdown depletes the protein over a longer time period than antibody injection, which may allow a compensatory mechanism to be activated. In the antibody injection studies, antibodies are injected in a short window of time prior to nuclear envelope breakdown at a time when the microtubules are extremely dynamic, so the effects of the MCAK antibody inhibition are immediate and dramatic, and the cell has no time to invoke any compensatory mechanism. In support of this idea, during the optimization of our knockdown conditions in which the knockdown of MCAK was less efficient (~70%), we did see more lagging chromosomes at anaphase in our fixed analysis (data not shown). It could be that the lower percentage knockdown of MCAK in these initial transfections did not initiate a second compensatory mechanism and therefore more closely resembled the defects associated with MCAK antibody inhibition.
We also measured the timing of mitotic progression in both the RNAi cells and in the antibody-injected cells (Table 1). Previously we had shown that injection of the dominant-negative MCAK, GFP-CEN, causes a delay in prometaphase . The knockdown of MCAK by RNAi in our live analysis did not result in a significant increase in the time between nuclear envelope breakdown and anaphase A onset, however MCAK antibody microinjection did cause a prometaphase delay. These differences may also be explained by the timing of the experiments in that the antibodies are injected just prior to nuclear envelope breakdown. In addition, it is important to note that while the live analysis did not show a major defect in timing, there were several individual live imaged cells that did show a prometaphase delay, and the fixed analysis of cells clearly showed an increase in the percentage of cells in prometaphase, suggesting that with a larger population of cells, such as our fixed analysis, the delay can be detected.
Overall, from our analysis of MCAK RNAi knockdown phenotypes, we conclude that RNAi in PtK2 cells is an effective tool to study the roles of mitotic proteins. Our results with P-MCAK RNAi corroborate previous work showing loss of MCAK leads to aberrant spindle formation as well as aberrant kinetochore-microtubule attachments that lead to improper chromosome congression and missegregation of chromosomes during anaphase. From these studies we also conclude that RNAi and antibody injection studies do give similar overall results, but more subtle details may vary between the methods because of the dramatic difference in the time-course of the inhibition. We feel that these two approaches should therefore be considered complementary rather than strictly redundant.
Partial cDNA sequences can be used to develop SiRNAs
Identification of additional PtK2 partial clones
Homologous genes across mammalian species share a high degree of identity at the DNA level.
Percent identity between paired alignments
# identical 21 base pair stretches in paired alignments
# nucleotides compared
Accession Numbers for DNA sequences used to compare to PtK2 RTPCR fragments
Here we have developed a method to use genomic information from other organisms with a sequenced genome to knockdown protein levels in PtK cells. This will allow us to systematically explore the roles of mitotic proteins taking advantage of the excellent morphology and ease with which live analysis of cellular dynamics can be performed in PtK cells. While our interest is in understanding mitosis and PtK cells provide a model system to research this process, the approach we outlined here should be adaptable to any cell type regardless of the existence of a sequenced genome. In addition, our data showing that the efficiency of transfection between DNA and siRNAs can be significantly different suggests that other cell types that have been experimentally intractable to transfection techniques may be able to be utilized for gene silencing by siRNAs.
Cloning of P-MCAK and P-Eg5
A cDNA expression screen of a PtK1 cDNA library was carried out utilizing conventional methods . An antibody raised against the N-terminus of X-MCAK  was used as a probe in the screen. We screened 900,000 plaques and isolated a single full-length clone encoding P-MCAK [GenBank: DQ444242]. To clone a portion of the catalytic domain of P-Eg5, we generated degenerate oligonucleotides by aligning human, mouse, and rat sequences encoding Eg5 and used these in RT-PCR reactions (Qiagen one-step RT PCR kit) using total RNA isolated from PtK2 cells (Qiagen RNAeasy) as a template. The Eg5 fragments were then subcloned into pEGFPC1 (Clontech) plasmid and fully sequenced on both strands. Sequence analysis was performed using Sequencher (Gene Codes Corporation), and the percent identity of fragments was calculated using DNA Strider.
PtK2 cells were plated at 15,000 – 20,000 cells/ml, and PtK1 cells were plated at 20,000 – 25,000 cells/ml in D-MEM (Invitrogen) supplemented with penicillin/streptomycin and 10% FBS 24 hrs before transfection onto coverslips in 6-well dishes and grown at 37°C, 5% CO2. Cells were then transfected with a non-fluorescently labelled 21 bp siRNA (Dharmacon) to P-MCAK (UGACUUUCUUUGAGAUCUA [dT] [dT]), or Eg5 (NNCAAGGAUGAAGUCUAUCAA), and either control oligo GFP (GCAAGCUGACCCUGAAGUUCAU) or siCONTROL Non-Targeting siRNA #2 to luciferase using Oligofectamine (Invitrogen) following their protocol with the following exceptions: 200 pmol siRNA/35 mm well was used, siRNA/lipid complexes were formed in D-MEM without penicillin/streptomycin and FBS, complexes were diluted in D-MEM/10% FBS, and 1 ml of siRNA/lipid dilution was added to each well the day of transfection, with an additional 1 ml/well of D-MEM/10% FBS added 24 hr post-transfection. Cells were examined at 48 hrs for Eg5 and 72 hrs for MCAK post-transfection. These time points were chosen after a qualitative analysis of the efficiency of knockdown by phenotypic analysis of spindle morphology. To determine the efficiency of transfection, cells were plated identically as above except that they were transfected with siGlo Risc-free RNA (Dharmacon) and then analyzed at 24–72 hrs post-transfection. No differences in the efficiency of transfection were observed by determining the percentage of cells containing fluorescent RNA. It should be noted that during our optimizations, we found that silencing efficiency decreased with increased number of passages beyond 6–8 weeks after thawing the cells. For HeLa cell transfections of Eg5, cells were plated at 5,000 – 6,000 cells/ml 24 hrs before transfection and then processed identically as described for PtK cells except that cells were passaged and transfected in Opti-MEM (Invitrogen) and processed 24 hrs post transfection. For treatment with monastrol, both HeLa and PtK cells were plated at the same density as for RNAi, grown for either 24 hrs for HeLa cells or 48 hrs for PtK cells, at which time the growth media was replaced with media containing 100 μM monastrol, and the cells incubated for a further 24 hrs. Monastrol treated cells were fixed and stained identically to the Eg5 siRNA transfected cells.
For Eg5 staining, HeLa cells or PtK cells were rinsed in PBS (12 mM Phosphate, 137 mM NaCl, 3 mM KCl, pH 7.4) and then fixed for 5' in -20°C MeOH. All subsequent immunofluorescence-labelling procedures were performed at room temperature. Fixed cells were rinsed with TBS-TX [TBS (20 mM Tris, 150 mM NaCl, pH 7.5) + 0.1% Triton X-100] and blocked in AbDil (2% BSA, 0.1% NaN3 in TBS-TX) for 30 min. All subsequent rinses between antibody incubations were performed using TBS-TX, and all antibodies were diluted in AbDil. Anti-H-Eg5 antibody (1/500 dilution; Cytoskeleton) was incubated for 30' on cells followed by incubation in 1/50 fluorescein-conjugated goat anti-rabbit secondary antibody (Jackson Immunologicals). For staining of microtubules, cells were incubated for 30 min in DM1alpha (Sigma), 1/500 for PtK cells and 1/1000 for HeLa cells, followed by a 30 min incubation in 1/50 Texas Red-conjugated donkey anti-mouse secondary antibody (Jackson Immunological). DNA was visualized using 2 μg/ml Hoechst in TBS-TX for 5 min. Coverslips were mounted onto microscope slides using 0.5% p-phenylenediamine, 20 mM Tris (pH 8.8) in 90% glycerol. For immunofluorescence of MCAK, cells were fixed for 20 min in PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgSO4, pH 7.0) supplemented with 2% formaldehyde. Cells were blocked in AbDil for 30' and then incubated for 30 min in 5 μg/ml anti-XMCAK-CT  followed by a 30 min incubation in 1/50 fluorescein-conjugated goat anti-rabbit secondary antibody (Jackson Immunologicals). For microtubule staining after MCAK knockdown, cells were fixed and processed as described for MCAK staining except that the fix was supplemented with 4% formaldehyde and 0.1% glutaraldehyde. Free aldehydes were quenched in TBS plus trace NaBH4 for 5 min.
Microinjections and microscopy
Microinjections were performed essentially as described previously . Time-lapse analysis was performed 70–87 hrs post-transfection using a Nikon TE-300 inverted microscope and a 40x/0.60 numerical aperture Plan Fluor objective (Nikon). Specimens were maintained at 36–37.5°C by using a 400 ASI airstream incubator (Nevtek). Phase contrast images were collected with a Cool Snap Cf camera (Roper Scientific) at 30-s intervals at 100-ms exposure times, and all cameras, shutters, and filter wheels were controlled by MetaMorph. Micrographs were assembled in Adobe Photoshop for identical contrast enhancement, and montages were prepared using Adobe Illustrator. Movies were prepared in MetaMorph and converted to Quicktime format.
Fixed cells were imaged on a Nikon E-600 microscope equipped with 40X/1.0 NA Plan Apo oil, 60X/1.4 NA Plan Apo oil, and 100X/1.3 NA Plan Fluor oil objectives (Nikon). Digital images were collected with either a Micromax 1300 Y cooled CCD camera or a CoolSnap HQ (Roper Scientific, Inc.). All cameras, shutters, and filter wheels were controlled by MetaMorph software (Molecular Devices). Z-series optical sections through each cell were obtained at 0.5 μm steps with the use of MetaMorph software and a stepping motor (Prior Scientific Instrument). Images were then deconvolved (blind iterative deconvolution) using AutoDeblur 9.3 software (AutoQuant Imaging). All micrographs were assembled in Adobe Photoshop CS for contrast enhancement, and montages were prepared using Adobe Illustrator CS.
For determining the amount of protein knockdown by immunoblotting, PtK2 cells were harvested by trypsinization at 72 hrs post-transfection, washed in PBS and then counted. Equal numbers of cells (serially diluted) were run on SDS-PAGE followed by immunoblotting. The blots were simultaneously probed with anti-XMCAK NT2 at 2 μg/ml in AbDil-T (made with 0.1% Tween-20 instead of TX-100) and DM1alpha (1/5000), washed in TBST, followed by incubation in a mixture of 1/20000 of anti-rabbit and 1/20000 anti-mouse horse-radish peroxidase secondary antibodies. The blots were washed and developed using Pierce SuperSignal WestPico ECL reagents. Scanned images of the western were quantified using ImageJ 1.34s v10.2 (NIH).
Quantification and analysis
Mitotic indices were calculated for both control and experimental conditions by scoring mitotic versus interphase cells for 200–300 cells/per coverslip for a total of 600 cells and taking the percentage of mitotic cells per cell total. To calculate the monopolar phenotype of Eg5, 100 mitotic cells between prometaphase and anaphase (when there is normally a bipolar spindle in control cells) were scored as either bipolar or monopolar spindles, and the percentages for both control and experimental cells were calculated. For MCAK RNAi analysis, 100 mitotic cells of both control and MCAK RNAi cells were scored for mitotic cell stage, and for monopolar spindles, for "hairy" (excess microtubules) prometaphase cells, and for lagging chromosomes in anaphase or telophase. The mean and SEM for 3 or more experiments are reported. Significance was considered if p < 0.5 as calculated by a paired t-test using F-test two-sample to determine variance. For analysis of time-lapse movies, the movies were coded and then scored blind by two individuals for presence or absence of the described mitotic defects. Any discrepancies were then scored blind by a third individual. All scores are reported as the percentage of cells scored for each defect. Distance measurements of spindle length were made using MetaMorph software. The spindle length was measured from the time-lapse movie at the frame of anaphase A onset. Measurements were collected in pixel number then converted to micrometers. All calculations for fixed and live analyses were performed in Excel.
We thank Guowei Fang for the suggestion of transfecting siRNAs into PtK cells and for helpful suggestions regarding siRNA transfection protocols and reagents. We thank Gary Gorbsky for the PtK1 cDNA library and Lisa Cameron for the PtK1 cell line. We thank Alexey Khodjakov and Tarun Kapoor for discussion and encouragement, Kevin Bohannon for coding all of the movies, and Nathan Stout for many helpful suggestions on statistical analysis and use of Excel. This work was supported by NIH GM059618 to CEW. This research was supported in part by the Indiana METACyt Initiative of Indiana University, funded in part through a major grant from the Lilly Endowment, Inc. SLK is a fellow of the American Cancer Society, RSR is funded by an American Heart Association predoctoral fellowship, and CEW is a fellow of the Leukemia and Lymphoma Society.
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