ABSTRACT
Kinetochores assemble on chromosomes in mitosis to allow microtubules to attach and bring about accurate chromosome segregation. The kinases Cyclin B-Cdk1 and Aurora B are crucial for the formation of stable kinetochores. However, the activity of these two kinases appears to decline dramatically at centromeres during anaphase onset, precisely when microtubule attachments are required to move chromosomes toward opposite poles of the dividing cell. We find that, although Aurora B leaves centromeres at anaphase, a gradient of Aurora B activity centered on the central spindle is still able to phosphorylate kinetochore substrates such as Dsn1 to modulate kinetochore stability in anaphase and to regulate kinetochore disassembly as cells enter telophase. We provide a model to explain how Aurora B co-operates with Cyclin B-Cdk1 to maintain kinetochore function in anaphase.
Subject(s)
Anaphase , Aurora Kinase B/metabolism , Chromosome Segregation , Kinetochores/enzymology , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cyclin B/genetics , Cyclin B/metabolism , Female , HeLa Cells , Humans , Phosphorylation , Protein Binding , Time FactorsABSTRACT
Micronuclei are a hallmark of cancer and several other human disorders. Recently, micronuclei were implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here, we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reassembly to prevent micronuclei formation. Using high-resolution live-cell imaging of human cancer and non-cancer cells, we uncover that anaphase lagging chromosomes are more frequent than previously anticipated, yet they rarely form micronuclei. Micronuclei formation from anaphase lagging chromosomes is prevented by a midzone-based Aurora B phosphorylation gradient that stabilizes kinetochore-microtubule attachments and assists spindle forces required for anaphase error correction while delaying nuclear envelope reassembly on lagging chromosomes, independently of microtubule density. We propose that a midzone-based Aurora B phosphorylation gradient actively monitors and corrects frequent chromosome segregation errors to prevent micronuclei formation during human cell division.
Subject(s)
Anaphase , Aurora Kinase B/metabolism , Chromosome Segregation , Kinetochores/enzymology , Micronuclei, Chromosome-Defective , Nuclear Envelope/enzymology , Spindle Apparatus/enzymology , HeLa Cells , Humans , Mechanotransduction, Cellular , Nuclear Envelope/genetics , Phosphorylation , Spindle Apparatus/genetics , Time FactorsABSTRACT
Local phosphatase regulation is needed at kinetochores to silence the mitotic checkpoint (a.k.a. spindle assembly checkpoint [SAC]). A key event in this regard is the dephosphorylation of MELT repeats on KNL1, which removes SAC proteins from the kinetochore, including the BUB complex. We show here that PP1 and PP2A-B56 phosphatases are primarily required to remove Polo-like kinase 1 (PLK1) from the BUB complex, which can otherwise maintain MELT phosphorylation in an autocatalytic manner. This appears to be their principal role in the SAC because both phosphatases become redundant if PLK1 is inhibited or BUB-PLK1 interaction is prevented. Surprisingly, MELT dephosphorylation can occur normally under these conditions even when the levels or activities of PP1 and PP2A are strongly inhibited at kinetochores. Therefore, these data imply that kinetochore phosphatase regulation is critical for the SAC, but primarily to restrain and extinguish autonomous PLK1 activity. This is likely a conserved feature of the metazoan SAC, since the relevant PLK1 and PP2A-B56 binding motifs have coevolved in the same region on MADBUB homologues.
Subject(s)
Cell Cycle Proteins/metabolism , Kinetochores/enzymology , Protein Phosphatase 1/metabolism , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Spindle Apparatus/enzymology , Cell Cycle Proteins/genetics , HeLa Cells , Humans , Phosphorylation/genetics , Protein Phosphatase 1/genetics , Protein Phosphatase 2/genetics , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Spindle Apparatus/genetics , Polo-Like Kinase 1ABSTRACT
Kinetochores are instrumental for accurate chromosome segregation by binding to microtubules in order to move chromosomes and by delaying anaphase onset through the spindle assembly checkpoint (SAC). Dynamic phosphorylation of kinetochore components is key to control these activities and is tightly regulated by temporal and spatial recruitment of kinases and phosphoprotein phosphatases (PPPs). Here we focus on PP1, PP2A-B56 and PP2A-B55, three PPPs that are important regulators of mitosis. Despite the fact that these PPPs share a very similar active site, they target unique ser/thr phosphorylation sites to control kinetochore function. Specificity is in part achieved by PPPs binding to short linear motifs (SLiMs) that guide their substrate specificity. SLiMs bind to conserved pockets on PPPs and are degenerate in nature, giving rise to a range of binding affinities. These SLiMs control the assembly of numerous substrate specifying complexes and their position and binding strength allow PPPs to target specific phosphorylation sites. In addition, the activity of PPPs is regulated by mitotic kinases and inhibitors, either directly at the activity level or through affecting PPP-SLiM interactions. Here, we discuss recent progress in understanding the regulation of PPP specificity and activity and how this controls kinetochore biology.
Subject(s)
Kinetochores/enzymology , Mitosis , Phosphoprotein Phosphatases/physiology , Animals , Chromosome Segregation , Humans , Microtubules/enzymology , Phosphorylation , Spindle Apparatus/enzymology , Substrate SpecificityABSTRACT
Faithful chromosome segregation in mitosis and meiosis requires that chromosomes properly attach to spindle microtubules. Initial kinetochore-microtubule attachments are often incorrect and rely on error correction mechanisms to release improper attachments, allowing the formation of new attachments. Aurora B kinase and, in mammalian germ cells, Aurora C kinase function as the enzymatic component of the Chromosomal Passenger Complex (CPC), which localizes to the inner centromere/kinetochore and phosphorylates kinetochore proteins for microtubule release during error correction. In this review, we discuss recent findings of the molecular pathways that regulate the chromosomal localization of Aurora B and C kinases in human cell lines, mice, fission yeast, and budding yeast. We also discuss differences in the importance of localization pathways between mitosis and meiosis.
Subject(s)
Aurora Kinase B/physiology , Aurora Kinase C/physiology , Meiosis , Mitosis , Animals , Cell Line , Humans , Kinetochores/enzymology , Mice , Microtubules/enzymology , YeastsABSTRACT
The fidelity of chromosome segregation during mitosis is intimately linked to the function of kinetochores, which are large protein complexes assembled at sites of centromeric heterochromatin on mitotic chromosomes. These key "orchestrators" of mitosis physically connect chromosomes to spindle microtubules and transduce forces through these connections to congress chromosomes and silence the spindle assembly checkpoint. Kinetochore-microtubule attachments are highly regulated to ensure that incorrect attachments are not prematurely stabilized, but instead released and corrected. The kinase activity of the centromeric protein Aurora B is required for kinetochore-microtubule destabilization during mitosis, but how the kinase acts on outer kinetochore substrates to selectively destabilize immature and erroneous attachments remains debated. Here, we review recent literature that sheds light on how Aurora B kinase is recruited to both centromeres and kinetochores and discuss possible mechanisms for how kinase interactions with substrates at distinct regions of mitotic chromosomes are regulated.
Subject(s)
Aurora Kinase B/metabolism , Centromere/enzymology , Chromosome Segregation , Kinetochores/enzymology , Microtubules/enzymology , Mitosis , Spindle Apparatus/enzymology , Animals , HumansABSTRACT
Aurora B kinase is essential for faithful chromosome segregation during mitosis. During (pro)metaphase, Aurora B is concentrated at the inner centromere by the kinases Haspin and Bub1. However, how Haspin and Bub1 collaborate to control Aurora B activity at centromeres remains unclear. Here, we show that either Haspin or Bub1 activity is sufficient to recruit Aurora B to a distinct chromosomal locus. Moreover, we identified a small, Bub1 kinase-dependent Aurora B pool that supported faithful chromosome segregation in otherwise unchallenged cells. Joined inhibition of Haspin and Bub1 activities fully abolished Aurora B accumulation at centromeres. While this impaired the correction of erroneous KT-MT attachments, it did not compromise the mitotic checkpoint, nor the phosphorylation of the Aurora B kinetochore substrates Hec1, Dsn1, and Knl1. This suggests that Aurora B substrates at the kinetochore are not phosphorylated by centromere-localized pools of Aurora B, and calls for a reevaluation of the current spatial models for how tension affects Aurora B-dependent kinetochore phosphorylation.
Subject(s)
Aurora Kinase B/metabolism , Chromosome Segregation , Intracellular Signaling Peptides and Proteins/metabolism , Kinetochores/enzymology , Microtubules/enzymology , Mitosis , Protein Serine-Threonine Kinases/metabolism , Aurora Kinase B/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , HCT116 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kinesins/genetics , Kinesins/metabolism , M Phase Cell Cycle Checkpoints , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/genetics , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Signal Transduction , Time FactorsABSTRACT
Aurora B kinase has a critical role in regulating attachments between kinetochores and spindle microtubules during mitosis. Early in mitosis, kinase activity at kinetochores is high to promote attachment turnover, and in later mitosis, activity decreases to ensure attachment stabilization. Aurora B localizes prominently to inner centromeres, and a population of the kinase is also detected at kinetochores. How Aurora B is recruited to and evicted from these regions to regulate kinetochore-microtubule attachments remains unclear. Here, we identified and investigated discrete populations of Aurora B at the centromere/kinetochore region. An inner centromere pool is recruited by Haspin phosphorylation of histone H3, and a kinetochore-proximal outer centromere pool is recruited by Bub1 phosphorylation of histone H2A. Finally, a third pool resides ~20 nm outside of the inner kinetochore protein CENP-C in early mitosis and does not require either the Bub1/pH2A/Sgo1 or Haspin/pH3 pathway for localization or activity. Our results suggest that distinct molecular pathways are responsible for Aurora B recruitment to centromeres and kinetochores.
Subject(s)
Aurora Kinase B/metabolism , Centromere/enzymology , Kinetochores/enzymology , Mitosis , Aurora Kinase B/genetics , Binding Sites , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centromere/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , HeLa Cells , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Transport , Signal Transduction , Time FactorsABSTRACT
The discovery of 20 unconventional kinetochore proteins in Trypanosoma brucei has opened a new and interesting area of evolutionary research to study a biological process previously thought to be highly conserved in all eukaryotes. In addition, the discovery of novel proteins involved in a critical cellular process provides an opportunity to exploit differences between kinetoplastid and human kinetochore proteins to develop therapeutics for diseases caused by kinetoplastid parasites. Consequently, we identified two of the unconventional kinetochore proteins as key targets (the highly related kinases KKT10 and KKT19). Recombinant T. brucei KKT19 (TbKKT19) protein was produced, a peptide substrate phosphorylated by TbKKT19 identified (KKLRRTLSVA), Michaelis constants for KKLRRTLSVA and ATP were determined (179 µM and 102 µM respectively) and a robust high-throughput compatible biochemical assay developed. This biochemical assay was validated pharmacologically with inhibition by staurosporine and hypothemycin (IC50 values of 288 nM and 65 nM respectively). Surprisingly, a subsequent high-throughput screen of a kinase-relevant compound library (6,624 compounds) yielded few hits (8 hits; final hit rate 0.12%). The low hit rate observed was unusual for a kinase target, particularly when screened against a compound library enriched with kinase hinge binding scaffolds. In an attempt to understand the low hit rate a TbKKT19 homology model, based on human cdc2-like kinase 1 (CLK1), was generated. Analysis of the TbKKT19 sequence and structure revealed no obvious features that could explain the low hit rates. Further work will therefore be necessary to explore this unique kinetochore kinase as well as to assess whether the few hits identified can be developed into tool molecules or new drugs.
Subject(s)
Peptides/antagonists & inhibitors , Phosphotransferases/antagonists & inhibitors , Trypanosoma brucei brucei/drug effects , Trypanosomiasis, African/diet therapy , Animals , Drug Discovery , High-Throughput Screening Assays , Humans , Kinetochores/drug effects , Kinetochores/enzymology , Peptides/chemistry , Phosphotransferases/chemistry , Phosphotransferases/genetics , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Staurosporine/pharmacology , Trypanosoma brucei brucei/enzymology , Trypanosomiasis, African/parasitology , Zearalenone/analogs & derivatives , Zearalenone/pharmacologyABSTRACT
Candida albicans, an ascomycete, has an ability to switch to diverse morphological forms. While C. albicans is predominatly diploid, it can tolerate aneuploidy as a survival strategy under stress. Aurora kinase B homolog Ipl1 is a critical ploidy regulator that controls microtubule dynamics and chromosome segregation in Saccharomyces cerevisiae. In this study, we show that Ipl1 in C. albicans has a longer activation loop than that of the well-studied ascomycete S. cerevisiae. Ipl1 localizes to the kinetochores during the G1/S phase and associates with the spindle during mitosis. Ipl1 regulates cell morphogenesis and is required for cell viability. Ipl1 monitors microtubule dynamics which is mediated by separation of spindle pole bodies. While Ipl1 is dispensable for maintaining structural integrity and clustering of kinetochores in C. albicans, it is required for the maintenance of bilobed distribution of clustered kinetochores along the mitotic spindle. Depletion of Ipl1 results in erroneous kinetochore-microtubule attachments leading to aneuploidy due to which the organism can survive better in the presence of fluconazole. Taking together, we suggest that Ipl1 spatiotemporally ensures bilobed kinetochore distribution to facilitate bipolar spindle assembly crucial for ploidy maintenance in C. albicans.
Subject(s)
Aurora Kinases/metabolism , Candida albicans/enzymology , Candida albicans/genetics , Chromosome Segregation , Fungal Proteins/metabolism , Kinetochores/enzymology , Aurora Kinases/genetics , Fungal Proteins/genetics , Mitosis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Spindle Apparatus/enzymology , Spindle Apparatus/geneticsABSTRACT
PP2A-B56 is a serine/threonine phosphatase complex that regulates several major mitotic processes, including sister chromatid cohesion, kinetochore-microtubule attachment and the spindle assembly checkpoint. We show here that these key functions are divided between different B56 isoforms that localise to either the centromere or kinetochore. The centromeric isoforms rely on a specific interaction with Sgo2, whereas the kinetochore isoforms bind preferentially to BubR1 and other proteins containing an LxxIxE motif. In addition to these selective binding partners, Sgo1 helps to anchor PP2A-B56 at both locations: it collaborates with BubR1 to maintain B56 at the kinetochore and it helps to preserve the Sgo2/B56 complex at the centromere. A series of chimaeras were generated to map the critical region in B56 down to a small C-terminal loop that regulates the key interactions and defines B56 localisation. Together, this study describes how different PP2A-B56 complexes utilise isoform-specific interactions to control distinct processes during mitosis.
Subject(s)
Centromere/enzymology , Kinetochores/enzymology , Mitosis , Multiprotein Complexes/metabolism , Protein Isoforms/metabolism , Protein Phosphatase 2/metabolism , Cell Cycle Proteins/metabolism , HeLa Cells , Humans , Protein Binding , Protein Multimerization , Protein Serine-Threonine Kinases/metabolismABSTRACT
Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cell Nucleus/enzymology , Cyclin B1/metabolism , Kinetochores/enzymology , M Phase Cell Cycle Checkpoints , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/genetics , Cell Nucleus/genetics , Cyclin B1/genetics , HEK293 Cells , HeLa Cells , Humans , Phosphorylation , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Retinal Pigment Epithelium/enzymology , Signal Transduction , Time FactorsABSTRACT
Cyclin B-dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cyclin B1/metabolism , Kinetochores/enzymology , M Phase Cell Cycle Checkpoints , Signal Transduction , Spindle Apparatus/enzymology , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/genetics , Cyclin B1/genetics , HEK293 Cells , HeLa Cells , Humans , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Spindle Apparatus/genetics , Time FactorsABSTRACT
During cell division, duplicated genetic material is separated into two distinct daughter cells. This process is essential for initial tissue formation during development and to maintain tissue integrity throughout an organism's lifetime. To ensure the efficacy and efficiency of this process, the cell employs a variety of regulatory and signaling proteins that function as mitotic regulators and checkpoint proteins. One vital mitotic regulator is polo-like kinase 1 (PLK1), a highly conserved member of the polo-like kinase family. Unique from its paralogues, it functions specifically during mitosis as a regulator of cell division. PLK1 is spatially and temporally enriched at three distinct subcellular locales; the mitotic centrosomes, kinetochores, and the cytokinetic midbody. These localization patterns allow PLK1 to phosphorylate specific downstream targets to regulate mitosis. In this review, we will explore how polo-like kinases were originally discovered and diverged into the five paralogues (PLK1-5) in mammals. We will then focus specifically on the most conserved, PLK1, where we will discuss what is known about how its activity is modulated, its role during the cell cycle, and new, innovative tools that have been developed to examine its function and interactions in cells.
Subject(s)
Cell Cycle Proteins/metabolism , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Centrosome/enzymology , Cytokinesis/physiology , Humans , Kinetochores/enzymology , Signal Transduction/physiology , Polo-Like Kinase 1ABSTRACT
Current methods to disrupt the microtubule cytoskeleton do not easily provide rapid, local control with standard cell manipulation reagents. Here, we develop a new microtubule-disruption tool based on katanin p60 severing activity and demonstrate proof-of-principle by targeting it to kinetochores in Drosophila melanogaster S2 cells. Specifically, we show that human katanin p60 can remove microtubule polymer mass in S2 cells and an increase in misaligned chromosomes when globally overexpressed. When katanin p60 was targeted to the kinetochores via Mis12, we were able to recapitulate the misalignment only when using a phosphorylation-resistant mutant katanin p60. Our results demonstrate that targeting an active version of katanin p60 to the kinetochore can reduce the fidelity of achieving full chromosome alignment in metaphase and could serve as a microtubule disruption tool for the future.
Subject(s)
Katanin , Microtubules , Animals , Cell Line , Drosophila melanogaster , Humans , Katanin/genetics , Katanin/metabolism , Kinetochores/enzymology , Metaphase/physiology , Microtubules/enzymology , Microtubules/geneticsABSTRACT
In budding yeast meiosis, homologous chromosomes become linked by chiasmata and then move back and forth on the spindle until they are bioriented, with the kinetochores of the partners attached to microtubules from opposite spindle poles. Certain mutations in the conserved kinase, Mps1, result in catastrophic meiotic segregation errors but mild mitotic defects. We tested whether Dam1, a known substrate of Mps1, was necessary for its critical meiotic role. We found that kinetochore-microtubule attachments are established even when Dam1 is not phosphorylated by Mps1, but that Mps1 phosphorylation of Dam1 sustains those connections. But the meiotic defects when Dam1 is not phosphorylated are not nearly as catastrophic as when Mps1 is inactivated. The results demonstrate that one meiotic role of Mps1 is to stabilize connections that have been established between kinetochores and microtubles by phosphorylating Dam1.
Subject(s)
Cell Cycle Proteins/physiology , Chromosome Segregation , Meiosis , Microtubule-Associated Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/physiology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Kinetochores/enzymology , Microtubule-Associated Proteins/genetics , Microtubules/enzymology , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Spindle Apparatus/metabolism , Time-Lapse ImagingABSTRACT
Polo-like kinase 1 (Plk1) is a crucial cell cycle regulator by its specific localization and activity during cell cycle. It has been shown that the phosphorylation and ubiquitylation of Plk1 are required for its own activation and localization. Here, we report that SUMOylation regulates the activity of Plk1 in the lepidopteran insect of Bombyx mori. In the absence of SUMOylation, it causes the lost localization of Plk1 on centrosomes and kinetochores, as well as an uneven distribution in midzone. We further identify that the putative SUMOylation site of Bombyx Plk1 at lysine 466 is required for its localization on centrosomes, and K466 mutation in Plk1 could influence its interaction with Smt3/Ubc9 complex. These findings are also confirmed by Drosophila Polo and human Plk1, which together reveals a conserved role of Plk1 SUMOylation in mammals. Moreover, conjugation of Smt3 to Plk1 SUMOylation mutant promotes its localization on centrosomes and kinetochores, and rescues functional defects of chromosome alignment in cells depleted of endogenous Plk1. Altogether, the present data indicate that the SUMOylation of Plk1 could participate in proper chromosome alignment and segregation during mitosis, and provides a novel layer for the regulation of Plk1 localization and activity throughout cell cycle.
Subject(s)
Bombyx/cytology , Bombyx/enzymology , Cell Cycle Proteins/metabolism , Cell Cycle , Insect Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Sumoylation , Animals , Bombyx/genetics , Cell Cycle Proteins/genetics , Centrosome/enzymology , Chromosome Segregation , Drosophila/metabolism , Kinetochores/enzymology , Mitosis , Mutation , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Polo-Like Kinase 1ABSTRACT
The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans, CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening.
Subject(s)
Autoantigens/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/enzymology , Chromatin/enzymology , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/enzymology , DNA Topoisomerases, Type II/metabolism , Mitosis , Animals , Autoantigens/genetics , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/genetics , Centromere Protein A , Chromatin/genetics , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/genetics , Chromosomes/genetics , DNA Topoisomerases, Type II/genetics , Gene Expression Regulation, Developmental , Kinetochores/enzymology , RNA InterferenceABSTRACT
Filamentous fungi have devastating negative impacts as pathogens and agents of food spoilage but also have critical ecological importance and are utilized for industrial applications. The characteristic multinucleate nature of filamentous fungi is facilitated by limiting if, when and where septation, the fungal equivalent of cytokinesis, occurs. In the model filamentous fungus Aspergillus nidulans septation does not occur immediately after mitosis and is an incomplete process resulting in the formation of a septal pore whose permeability is cell cycle regulated. How mitotic regulators, such as the Aurora kinase, contribute to the often unique biology of filamentous fungi is not well understood. The Aurora B kinase has not previously been investigated in any detail during hyphal growth. Here we demonstrate for the first time that Aurora displays cell cycle dependent locations to the region of forming septa, the septal pore and mature septa as well as the mitotic apparatus. To functionally analyze Aurora, we generated a temperature sensitive allele revealing essential mitotic and spindle assembly checkpoint functions consistent with its location to the kinetochore region and spindle midzone. Our analysis also reveals that cellular and kinetochore Aurora levels increase during a mitotic spindle assembly checkpoint arrest and we propose that this could be important for checkpoint inactivation when spindle formation is prevented. We demonstrate that Aurora accumulation at mature septa following mitotic entry does not require mitotic progression but is dependent upon a timing mechanism. Surprisingly we also find that Aurora inactivation leads to cellular swelling and lysis indicating an unexpected function for Aurora in fungal cell growth. Thus in addition to its conserved mitotic functions our data suggest that Aurora has the capacity to be an important regulator of septal biology and cell growth in filamentous fungi.