The AAA-ATPase p97 in mitosis and fertilization

Late mitotic events are chiefly controlled by proteolysis of key regulatory proteins via the ubiquitin-proteasome pathway. In this pathway ubiquitin ligases modify substrates by attachment of ubiquitin (“ubiquitylation”), which usually results in their subsequent degradation by the 26S proteasome. The crucial ubiquitin ligase involved in late mitosis is the anaphase-promoting complex or cyclosome (APC/C). Among the many substrates of the APC/C is the anaphase inhibitor securin, whose destruction leads to activation of separase, which in turn triggers sister chromatid separation by proteolytic cleavage of cohesin. The APC/C also targets cyclin B1, an activating subunit of Cdk1 kinase, whose inactivation is a prerequisite for mitotic exit. The unstable APC/C substrates are often found in association with stable partner proteins. How single subunits of multi-protein complexes are selectively extracted and eventually degraded is largely unknown, but there is increasing evidence that additional factors assist to extract ubiquitin-carrying subunits from stable binding partners. One such factor is vertebrate p97 (Cdc48 in yeast), an abundant and highly conserved member of the AAA-ATPase family. It is involved in such diverse processes as transcriptional regulation, membrane fusion, and ER-associated protein degradation (ERAD). The unifying scheme in these seemingly unrelated functions is that p97 is able to “extract” preferentially ubiquitylated proteins from their environment. Roles of p97 in mitosis have recently emerged: p97 was reported to be required for spindle disassembly and for nuclear envelope reformation during mitotic exit in Xenopus. Furthermore, a genetic interaction between p97, separase and securin, as well as a requirement of p97 for separase stability, were discovered in fission yeast. Given these hints and the importance of ubiquitylation in both mitosis and p97 pathways, this study intended to elucidate additional mitotic roles of p97 in vertebrates. Towards this end, tools to interfere with p97 function in Xenopus egg extracts were developed. These included immunodepletion of the p97 adaptors Npl4, Ufd1 and p47 and addition of recombinant dominant-negative p97-mutants. ERAD, which could be established here for the first time in Xenopus egg extracts, was greatly impaired in the absence of p97 function. However, many aspects of mitosis were found to be unaffected. Importantly, p97’s proposed role in spindle disassembly was clearly falsified within this thesis. Furthermore, p97 was shown to be dispensable for activity and stability of vertebrate separase. Disassembly of the mitotic checkpoint complex, which prevents premature APC/C activation by sequestering its activator Cdc20, did also not require functional p97 despite its dependence on ubiquitylation of Cdc20. However, a novel function of p97 at fertilization was discovered. p97 was found to interact with nucleoplasmin, a histone-binding chaperone that catalyzes the exchange of sperm-specific basic proteins (SBPs) to histones. Indeed, interference with p97 function delayed sperm decondensation in Xenopus egg extracts, thereby confirming a novel role of this AAA-ATPase in sperm chromatin remodelling. In another project the role of securin in human cells was investigated. Human cells lacking securin had been reported to suffer from massive chromosome missegregation, which was in sharp contrast to the mild phenotype of securin knockout mice. In collaboration with the group of M. Speicher it could be demonstrated that chromosome losses in securin-/- cells are transient and give way to a stable segregation pattern after just a few passages. This was despite persisting biochemical defects such as reduced level and activity of separase. These data demonstrate that securin is dispensable for chromosomal stability in human cells

Securin Is Not Required for Chromosomal Stability in Human Cells

Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin(−/−) cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation

Non‐proteolytic ubiquitylation counteracts the APC/C‐inhibitory function of XErp1

Mature Xenopus oocytes are arrested in meiosis by the activity of XErp1/Emi2, an inhibitor of the ubiquitin-ligase anaphase-promoting complex/cyclosome (APC/C). On fertilization, XErp1 is degraded, resulting in APC/C activation and the consequent degradation of cell-cycle regulators and exit from meiosis. In this study, we show that a modest increase in the activity of the ubiquitin-conjugating enzyme UbcX overrides the meiotic arrest in an APC/C-dependent reaction. Intriguingly, XErp1 remains stable in these conditions. We found that UbcX causes the ubiquitylation of XErp1, followed by its dissociation from the APC/C. Our data support the idea that ubiquitylation regulates the APC/C-inhibitory activity of XErp1

Non-proteolytic ubiquitylation counteracts the APC/C-inhibitory function of XErp1

Mature Xenopus oocytes are arrested in meiosis by the activity of XErp1/Emi2, an inhibitor of the ubiquitin-ligase anaphase-promoting complex/cyclosome (APC/C). On fertilization, XErp1 is degraded, resulting in APC/C activation and the consequent degradation of cell-cycle regulators and exit from meiosis. In this study, we show that a modest increase in the activity of the ubiquitin-conjugating enzyme UbcX overrides the meiotic arrest in an APC/C-dependent reaction. Intriguingly, XErp1 remains stable in these conditions. We found that UbcX causes the ubiquitylation of XErp1, followed by its dissociation from the APC/C. Our data support the idea that ubiquitylation regulates the APC/C-inhibitory activity of XErp1

Assessment of Chromosomal Stability in Interphase Nuclei of Parental HCT116 Cells and Chromosomally Stable <i>hSecurin^−/−</i> Cells Using Centromere-Specific Probes for Chromosomes 7, 8, 11, and 17

<div><p>(A and B) Representative interphase FISH images of parental HCT116 (<i>hSecurin^+/+</i>) (A) and <i>hSecurin^−/−</i> (B) cell nuclei after hybridization of a four-color probe set consisting of centromere probes for chromosomes 7 (Cy5.5; purple), 8 (FITC; green), 11 (Cy5; blue), and 17 (Cy3; yellow). In each nucleus, two signals are visible for each probe.</p> <p>(C and D) Graphic summary of chromosome gains and losses in parental HCT116 (C) and <i>hSecurin^−/−</i> (D) cells. The percentage of signals per nucleus for chromosomes 7, 8, 11, and 17 was determined from 300 cells of each genotype (100 cells each in three separate experiments).</p></div

hSecurin^−/− Cells Regain Chromosomal Stability Quickly after <i>hSecurin</i> Knockout by Homologous Recombination: Summary of M-FISH Analysis of <i>hSecurin^−/−</i> Cells at Different Passages

<div><p>(A–D) Graphic summary of M-FISH data from <i>hSecurin^−/−</i> cells at passages 2 (A), 3 (B), 8 (C), and 12 (D). At each passage point 20 or 30 metaphase spreads were painted by M-FISH and analyzed for alterations of chromosome structure and number. Loss of a single copy of a given chromosome is marked in red, loss of both copies is marked in crimson, and gain of a single chromosome is marked in green. Rows indicate the analyzed metaphase spreads (m1 to m30 or m20); columns indicate the chromosome number (1–22 and X).</p> <p>(E) Graphic representation of the percentages of metaphase spreads with chromosomal copy number aberrations at different passages for the series of experiments shown in (A–D) (blue line) and for a repeat experiment (purple line).</p> <p>(F) M-FISH karyotype of a passage 12 <i>hSecurin^−/−</i> cell, showing that the karyotype is identical to that of the parent cell line HCT116 (for details, see text).</p></div

Chromosomally Stable <i>hSecurin</i>^−/− Cells of Passage 12 and Higher Show Reduction in Both the Level and the Activity of Separase

<div><p>(A) Quantitation of full-length separase and the N-terminal cleavage product in both <i>hSecurin</i>^+/+ and <i>hSecurin</i>^−/− cells. Lysates from nocodazole-arrested cells were analyzed by immunoblotting with an antibody against the N-terminus of separase. The chromosomally stable <i>hSecurin</i>^−/− cells show reduced levels of both the full-length and the cleaved N-terminal form of separase. β-tubulin was used as a loading control.</p> <p>(B) Separase was immunoprecipitated from nocodazole-arrested <i>hSecurin</i>^+/+ and <i>hSecurin</i>^−/− cells, activated by incubation in <i>Xenopus</i> anaphase extracts, and incubated with ³⁵S-hScc1 for 0, 20, or 90 min before analysis by SDS-PAGE and autoradiography. For these experiments we used four times as many <i>hSecurin</i>^−/− cells as <i>hSecurin</i>^+/+ cells. Note the absence of detectable Scc1 cleavage fragments in the <i>hSecurin</i>^−/− samples.</p> <p>(C) Separase used for the activity assay in (B) was analyzed by Western blotting before (−) and after (+) exposure to <i>Xenopus</i> anaphase extracts. The <i>hSecurin</i>^+/+ cells clearly demonstrate an increase in self-cleavage of separase upon activation in the extract. Occurrence of auto-cleavage in <i>hSecurin</i>^−/− cells even before incubation in <i>Xenopus</i> extract suggests deregulation of separase, at least under the given conditions of this in vitro experiment.</p></div

Analysis of Defective Sister Chromatid Separation in <i>hSecurin</i>^−/− Cells

<div><p><i>hSecurin</i>^+/+ cells and <i>hSecurin</i>^−/− cells were stained with DAPI as a counterstain, a cyclin B1 antibody (green/FITC), which stains cells in the early mitosis but not in anaphase, and a CREST antibody (yellow/Cy3) to visualize kinetochores. In each image are telophase cells showing a complete separation of sister chromatids.</p> <p>(A–D) Analysis of several cells of the HCT116 parent cell line (<i>hSecurin</i>^+/+). The sequence of images illustrates the DAPI (A), FITC (B), and Cy3 (C) channels, while (D) shows the merged FITC and Cy3 images. One cell is stained with the cyclin B1 antibody; the majority of cells are in prophase (characteristic “double-dot” pattern of paired centromeres). There are two telophase cells.</p> <p>(E–G) Two telophase <i>hSecurin</i>^+/+ cells with complete separation of sister chromatids. (E) DAPI; (F) Cy3; (G) merged DAPI and Cy3 image.</p> <p>(H–K) Cells from the <i>hSecurin</i>^−/− cell line. The cell in the upper-right corner is stained with the cyclin B1 antibody; at the bottom is a normal telophase cell with complete separation of sister chromatids. (H) DAPI; (I) FITC; (J) Cy3; (K) merged FITC and Cy3 image.</p> <p>(L–N) Prophase and telophase <i>hSecurin</i>^−/− cells. The telophase cell demonstrates a complete separation of sister chromatids. (L) DAPI; (M) Cy3; (N) merged DAPI and Cy3 image.</p> <p>(O) Quantitation of the chromatid separation defect in <i>hSecurin</i>^+/+ and <i>hSecurin</i>^−/− cells. The percentage of anaphase cells with unsegregated sister chromatids at the metaphase plate was determined from 75 cells of each genotype (25 cells each in three separate experiments; the error bars indicate standard deviation).</p></div

Verification that the Chromosomally Stable Cells Indeed Lack Part of <i>hSecurin</i> by Analyses of Genomic DNA from Parental HCT116 Cells (+/+) and Chromosomally Stable <i>hSecurin^−/−</i> Cells (−/−)

<div><p>(A) Transcript structure of the <i>hSecurin</i> gene with its six exons. The lengths of introns and exons are drawn to scale based on the NCBI 35 assembly of the human genome (<a href="http://www.ensembl.org" target="_blank">http://www.ensembl.org</a>). Exons 2 and 3, with the locations of the respective primer pairs, are depicted enlarged.</p> <p>(B) As a control, PCR analysis was done with primers located in exons 8 and 9 of the p53 gene and resulted in the expected amplification product for both cell lines (lanes 2 and 3). In contrast, PCR with primers PTTG-R6 and PTTG-R1, located in the second and third exon of the <i>hSecurin</i> gene (arrows above exons 2 and 3 in [A]), yielded an amplification product only for the parental HCT116 cells (+/+; lane 5) and not for the chromosomally stable <i>hSecurin^−/−</i> cells (−/−; lane 6). Lane 1 shows the 100-bp ladder as a size marker, and lanes 4 and 7 are negative controls for the respective primer pairs.</p> <p>(C) PCR analyses with primers SecP1l, located in exon 2, and SecP2r, located in intron 3–4 (arrows below exons 2 and 3 in [A]), resulted in amplification products with different sizes (lanes 2 and 3), reflecting the deletion of exon 3. Lane 1 shows the 100-bp ladder; lane 4 is the negative control.</p></div