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. 2016 Jan;6(1):150230.
doi: 10.1098/rsob.150230.

Auxin/AID Versus Conventional Knockouts: Distinguishing the Roles of CENP-T/W in Mitotic Kinetochore Assembly and Stability

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Free PMC article

Auxin/AID Versus Conventional Knockouts: Distinguishing the Roles of CENP-T/W in Mitotic Kinetochore Assembly and Stability

Laura Wood et al. Open Biol. .
Free PMC article

Abstract

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1-3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).

Keywords: AID system; CENP-T/W; SILAC; chromosomes; kinetochore; mass spectrometry.

Figures

Figure 1.
Figure 1.
Schematic of the conditional knockout strategy and its adaption for the AID-CENP-T cell line. (a) Doxycycline regulated shut-down of CENP-T or CENP-W gene expression. (b) Combining the doxycycline regulated shut-down of gene expression with the auxin-inducible degron (AID) system. (c) A schematic of the auxin-induced degradation system applied in eukaryotes. The SCF E3 ubiquitin ligase consists of 4 units: Rbx1, Cul-1, Skp1 and F-box containing protein, TIR1. Auxin hormone (such as IAA) binding to the ectopically expressed TIR1 receptor promotes contacts with target proteins fused to an AID tag. The SCF-TIR1 E3 ligase can then polyubiquitinate the AID tag, promoting degradation of the substrate by the 26S proteasome.
Figure 2.
Figure 2.
Rapid degradation of CENP-T at the protein level using the AID system. (a) Immunoblot detection of CENP-T in wild-type (WT), CENP-TON/OFF conditional KO or AID-CENP-T:CENP-TON/OFF cell extracts. Cell lines were treated with doxycycline for up to 3 days to turn off the expression of the CENP-T transgene. AID-CENP-T cell lines were further treated with auxin and cell lysates collected for the time points indicated. Cells (5 × 105) were loaded per well and anti-tubulin blots used as a loading control. (b) AID-CENP-T localizes to kinetochores in doxycycline-treated AID-CENP-T:CENP-TON/OFF cells. GFP-CENP-A was expressed in wild-type, CENP-TON or AID-CENP-T:CENP-TOFF cell lines and CENP-T detected using antibodies against GgCENP-T. DAPI depicts DNA staining. Scale bar: 5 µm. (c) Growth curves of WT, CENP-T conditional KO and the AID-CENP-T cell lines in the presence or absence of doxycycline. Doxycycline was introduced at time 0 and viable cells counted every 24 h using trypan blue staining. At 96 h, auxin was added to all cell populations and cells counted as described above. (d) AID-CENP-T is lost from kinetochores of doxycycline-treated AID-CENP-T:CENP-TOFF cells following auxin addition. After GFP-CENP-A transfection AID-CENP-T:CENP-TOFF cells were cultured with ethanol (+EtOH) or auxin (+Auxin) for 1 h. DAPI depicts DNA staining. Scale bar: 5 µm. (e) Fluorescence intensity measurements of GgCENP-T staining co-localizing with GFP-CENP-A loci. Approximately 20% of normal CENP-T levels remain at centromeric regions upon 1 h of auxin treatment. The graph displays the mean percentage ± s.e.m.
Figure 3.
Figure 3.
Acute degradation of CENP-T leads to prometaphase arrest and cell death. (a) AID-CENP-T:CENP-TOFF cells were treated with ethanol (minus) or auxin (plus) for the indicated times. Graph reports the mean percentage ± s.e.m. of mitotic and interphase cell number (two repeats; n = ∼500 per experiment). (b) Fluorescence intensity of the PE-annexin V signal using flow cytometry. A graph displaying the mean percentage ± s.e.m. of apoptotic cells with high PE-annexin V signals from three repeats is shown. (c) Panels 1, 2 and 3 depict untreated WT, CENP-TON and AID-CENP-T:CENP-TOFF cell lines, respectively. Panels 4 and 5 show AID-CENP-T:CENP-TOFF treated with ethanol (+EtOH) or auxin (+auxin) for the indicated times. This was followed by fixation and staining with DAPI. The inset shows a zoomed in view as defined by the white squares. Scale bar: 20 µm. (d) Images showing phenotypes scored in panel (e). Hypercondensed: chromosomes that have a severe hypercondensed appearance. Prometaphase 1: typical prometaphase seen in a control cell. Prometaphase 2 and 3: prometaphase morphologies commonly observed in auxin-treated cell populations. Scale bar: 5 µm. (e) Scoring of mitotic phenotypes seen after auxin addition. After the indicated time period, cells were processed for immunofluorescence and stained with DAPI (blue) and anti-tubulin antibodies (red), and categorized into mitotic phases and phenotypes highlighted in (d). The means as a percentage ± s.e.m. are depicted for two repeats (n = ∼100 per experiment). Cells were categorized into mitotic phases and phenotypes highlighted in (d).
Figure 4.
Figure 4.
CENP-T loss leads to microtubule attachment defects. GFP-CENP-A was transfected into (a) wild-type, (b) CENP-TON/OFF conditional knockouts and (c,d) AID-CENP-T:CENP-TON/OFF cells. Attachment to PLL-coated coverslips was followed by fixation and staining using antibodies against tubulin. DAPI depicts DNA staining. Scale bar: 5 µm. AID-CENP-T:CENP-TOFF cells were either cultured with (c) ethanol (+EtOH) or (d) auxin (+auxin) for 6.5 h. (e) (1) Light microscopy image (DAPI; left) of a wild-type metaphase cell subsequently processed for thin-section transmission electron microscopy (TEM; right). White arrows depict a metaphase aligned chromosome, with lower panels displaying magnified electron micrographs of the chromosome of interest. In these images red rimmed arrows highlight inner and outer kinetochore plates, while black arrows signify microtubules. (2) Mild and (3) severe phenotypes associated with rapid AID-CENP-T degradation. AID-CENP-T cells were treated with auxin for 6.5 h before immediate processing for electron microscopy. Cells analysed by light microscopy (DAPI; left) and TEM (right) are depicted. White arrows mark the chromosome of interest. Magnified EM images show sites of potential lateral microtubule interactions. Black arrows depict microtubules, while white arrows show possible interactions with chromosomes. Scale bars as indicated.
Figure 5.
Figure 5.
AID-CENP-T protein levels are not reversible in mitosis. (a) Schematic of indicated lengths of nocodazole and auxin treatments before chromosome isolation procedures. Coomassie stained gel showing the chromosome isolates from wild-type (WT), CENP-TON and AID-CENP-T:CENP-TOFF cell lines. Chromosomes isolated from the AID-CENP-T cell line after ethanol (+EtOH) or auxin treatment (+Aux) for the indicated time periods are also shown. Corresponding immunoblots of chromosome samples using anti-GgCENP-T, anti-GgINCENP, anti-GgTopo IIalpha, anti-GgNdc80 and anti-Histone H3 antibodies are depicted. (b) Immunoblot detection of CENP-T and tubulin in whole-cell extracts. Doxycycline was added at time 0 and cells harvested every 24 h for 3 days. AID-CENP-T:CENP-TOFF cells were further treated with auxin and cell lysates taken 15 min, 30 min and 1 h after addition. This was followed by additional wash steps to remove the auxin and cell lysates taken as indicated. Cells (2.5 × 105) were loaded per well. (c) Schematic of nocodazole and auxin treatments to test whether auxin-induced degradation of AID-CENP-T was reversible in mitosis. (d) Western blot analysis of whole-cell extracts from asynchronous and synchronised mitotic cell populations (as depicted in 5c). Blots were probed with antibodies against GgCENP-T, GgCyclinB2 and tubulin. (e) Coomassie stained gel of chromosomes isolated under the indicated conditions. Immunoblots of corresponding chromosome isolations using anti-GgCENP-T and anti-Histone H3 antibodies. The indicated mitotic index (%) of the individual cell populations at the time of chromosome isolation procedures is depicted.
Figure 6.
Figure 6.
Identification of CENP-T-dependent chromosomal proteins using proteomics analysis. (a) Schematic of the SILAC/proteomics experimental strategy. Wild-type cells are grown in heavy SILAC medium, while the AID-CENP-T:CENP-TOFF cells are cultured in the presence of light amino acids. AID-CENP-T cells are maintained in doxycycline to ‘turn off’ the expression of the inducible transgene and ensure that AID-tagged CENP-T becomes the dominant form of the protein. Both cell types are blocked in mitosis with nocodazole (+Noc) for 12 h. Ten hours into the nocodazole block auxin is added (+Aux) to the AID-CENP-T:CENP-TOFF cells to promote the degradation of the AID-tagged protein. The CENP-T and CENP-W KO cell line is cultured in the presence of light amino acids and doxycycline (+Dox) added for 30 h before nocodazole addition. Equal numbers of mitotic cells from the two populations are mixed and the chromosomes isolated. After chromosome isolation, mass spectrometry is used to determine SILAC ratios for individual proteins. (b) Barplots of mean log2 H/L ratios from AID-CENP-T:CENP-TOFF, CENP-TOFF and CENP-WOFF SILAC experiments. The red line indicates a greater than 1.5-fold-change in the abundance of a protein. All assemblies listed have previously been identified as centromeric, kinetochore components or proteins closely associated with kinetochore-fibres (K-fibres). Groupings have been made based on the current literature of kinetochore subcomplex formation.
Figure 7.
Figure 7.
Comparison of significantly depleted chromosomal proteins after conditional shut-down or auxin-induced degradation of CENP-T. Volcano plots represent the average log2 fold-change between two biological replicates versus –log10 (p-values) calculated using the limma package in R [–27]. Proteins that have an adjusted p < 0.05 and a H/L SILAC ratio representative of a greater than 1.5-fold-change are found within the significant areas indicated by the blue boxes. Key kinetochore proteins are colour coded based on known subcomplex formation.

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