Spindle Pole Body Duplication Research Articles

An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony.

Aurora kinases compose a family of conserved Ser/Thr protein kinases playing essential roles in eukaryotic cell division. To date, Aurora homologues remain uncharacterized in the protozoan phylum Apicomplexa. In malaria parasites, the characterization of Aurora kinases may help understand the cell cycle control during erythrocytic schizogony where asynchronous nuclear divisions occur. In this study, we revisited the kinome of Plasmodium falciparum and identified three Aurora-related kinases, Pfark-1, -2, -3. Among these, Pfark-1 is highly conserved in malaria parasites and also appears to be conserved across Apicomplexa. By tagging the endogenous Pfark-1 gene with the green fluorescent protein (GFP) in live parasites, we show that the Pfark-1–GFP protein forms paired dots associated with only a subset of nuclei within individual schizonts. Immunofluorescence analysis using an anti-α-tubulin antibody strongly suggests a recruitment of Pfark-1 at duplicated spindle pole bodies at the entry of the M phase of the cell cycle. Unsuccessful attempts at disrupting the Pfark-1 gene with a knockout construct further indicate that Pfark-1 is required for parasite growth in red blood cells. Our study provides new insights into the cell cycle control of malaria parasites and reports the importance of Aurora kinases as potential targets for new antimalarials.

Changes in the Nuclear Envelope Environment Affect Spindle Pole Body Duplication in Saccharomyces cerevisiae

The Saccharomyces cerevisiae nuclear membrane is part of a complex nuclear envelope environment also containing chromatin, integral and peripheral membrane proteins, and large structures such as nuclear pore complexes (NPCs) and the spindle pole body. To study how properties of the nuclear membrane affect nuclear envelope processes, we altered the nuclear membrane by deleting the SPO7 gene. We found that spo7Δ cells were sickened by the mutation of genes coding for spindle pole body components and that spo7Δ was synthetically lethal with mutations in the SUN domain gene MPS3. Mps3p is required for spindle pole body duplication and for a variety of other nuclear envelope processes. In spo7Δ cells, the spindle pole body defect of mps3 mutants was exacerbated, suggesting that nuclear membrane composition affects spindle pole body function. The synthetic lethality between spo7Δ and mps3 mutants was suppressed by deletion of specific nucleoporin genes. In fact, these gene deletions bypassed the requirement for Mps3p entirely, suggesting that under certain conditions spindle pole body duplication can occur via an Mps3p-independent pathway. These data point to an antagonistic relationship between nuclear pore complexes and the spindle pole body. We propose a model whereby nuclear pore complexes either compete with the spindle pole body for insertion into the nuclear membrane or affect spindle pole body duplication by altering the nuclear envelope environment.

N-terminal regions of Mps1 kinase determine functional bifurcation

Mps1 is a conserved kinase that in budding yeast functions in duplication of the spindle pole body (SPB), spindle checkpoint activation, and kinetochore biorientation. The identity of Mps1 targets and the subdomains that convey specificity remain largely unexplored. Using a novel combination of systematic deletion analysis and chemical biology, we identified two regions within the N terminus of Mps1 that are essential for either SPB duplication or kinetochore biorientation. Suppression analysis of the MPS1 mutants defective in SPB duplication and biochemical enrichment of Mps1 identified the essential SPB components Spc29 and the yeast centrin Cdc31 as Mps1 targets in SPB duplication. Our data suggest that phosphorylation of Spc29 by Mps1 in G1/S recruits the Mps2-Bbp1 complex to the newly formed SPB to facilitate its insertion into the nuclear envelope. Mps1 phosphorylation of Cdc31 at the conserved T110 residue controls substrate binding to Kar1 protein. These findings explain the multiple SPB duplication defects of mps1 mutants on a molecular level.

The MST1 and hMOB1 Tumor Suppressors Control Human Centrosome Duplication by Regulating NDR Kinase Phosphorylation

Human MST/hSAV/LATS/hMOB tumor suppressor cascades are regulators of cell death and proliferation; however, little is known about other functions of MST/hMOB signaling. Mob1p, one of two MOB proteins in yeast, appears to play a role in spindle pole body duplication (the equivalent of mammalian centrosome duplication). We therefore investigated the role of human MOB proteins in centrosome duplication. We also addressed the regulation of human centrosome duplication by mammalian serine/threonine Ste20-like (MST) kinases, considering that MOB proteins can function together with Ste20-like kinases in eukaryotes. By studying the six human MOB proteins and five MST kinases, we found that MST1/hMOB1 signaling controls centrosome duplication. Overexpression of hMOB1 caused centrosome overduplication, whereas RNAi depletion of hMOB1 or MST1 impaired centriole duplication. Significantly, we delineated an hMOB1/MST1/NDR1 signaling pathway regulating centrosome duplication. More specifically, analysis of shRNA-resistant hMOB1 and NDR1 mutants revealed that a functional NDR/hMOB1 complex is critical for MST1 to phosphorylate NDR on the hydrophobic motif that in turn is required for human centrosome duplication. Furthermore, shRNA-resistant MST1 variants revealed that MST1 kinase activity is crucial for centrosome duplication whereas MST1 binding to the hSAV and RASSF1A tumor suppressor proteins is dispensable. Finally, by studying the PLK4/HsSAS-6/CP110 centriole assembly machinery, we also observed that normal daughter centriole formation depends on intact MST1/hMOB1/NDR signaling, although HsSAS-6 centriolar localization is not affected. Our observations propose a novel pathway in control of human centriole duplication after recruitment of HsSAS-6 to centrioles.

The SESA network links duplication of the yeast centrosome with the protein translation machinery

The yeast spindle pole body (SPB), the functional equivalent of mammalian centrosome, duplicates in G1/S phase of the cell cycle and then becomes inserted into the nuclear envelope. Here we describe a link between SPB duplication and targeted translation control. When insertion of the newly formed SPB into the nuclear envelope fails, the SESA network comprising the GYF domain protein Smy2, the translation inhibitor Eap1, the mRNA-binding protein Scp160 and the Asc1 protein, specifically inhibits initiation of translation of POM34 mRNA that encodes an integral membrane protein of the nuclear pore complex, while having no impact on other mRNAs. In response to SESA, POM34 mRNA accumulates in the cytoplasm and is not targeted to the ER for cotranslational translocation of the protein. Reduced level of Pom34 is sufficient to restore viability of mutants with defects in SPB duplication. We suggest that the SESA network provides a mechanism by which cells can regulate the translation of specific mRNAs. This regulation is used to coordinate competing events in the nuclear envelope.

Budding Yeast Centrosome Duplication Requires Stabilization of Spc29 via Mps1-mediated Phosphorylation

Protein phosphorylation plays an important role in the regulation of centrosome duplication. In budding yeast, numerous lines of evidence suggest a requirement for multiple phosphorylation events on individual components of the centrosome to ensure their proper assembly and function. Here, we report the first example of a single phosphorylation event on a component of the yeast centrosome, or spindle pole body (SPB), that is required for SPB duplication and cell viability. This phosphorylation event is on the essential SPB component Spc29 at a conserved Thr residue, Thr(240). Mutation of Thr(240) to Ala is lethal at normal gene dosage, but an increased copy number of this mutant allele results in a conditional phenotype. Phosphorylation of Thr(240) was found to promote the stability of the protein in vivo and is catalyzed in vitro by the Mps1 kinase. Furthermore, the stability of newly synthesized Spc29 is reduced in a mutant strain with reduced Mps1 kinase activity. These results demonstrate the first evidence for a single phosphorylation event on an SPB component that is absolutely required for SPB duplication and suggest that the Mps1 kinase is responsible for this protein-stabilizing phosphorylation.

Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating

During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.

Intrinsic and Cyclin-dependent Kinase-dependent Control of Spindle Pole Body Duplication in Budding Yeast

Centrosome duplication must be tightly controlled so that duplication occurs only once each cell cycle. Accumulation of multiple centrosomes can result in the assembly of a multipolar spindle and lead to chromosome mis-segregation and genomic instability. In metazoans, a centrosome-intrinsic mechanism prevents reduplication until centriole disengagement. Mitotic cyclin/cyclin-dependent kinases (CDKs) prevent reduplication of the budding yeast centrosome, called a spindle pole body (SPB), in late S-phase and G2/M, but the mechanism remains unclear. How SPB reduplication is prevented early in the cell cycle is also not understood. Here we show that, similar to metazoans, an SPB-intrinsic mechanism prevents reduplication early in the cell cycle. We also show that mitotic cyclins can inhibit SPB duplication when expressed before satellite assembly in early G1, but not later in G1, after the satellite had assembled. Moreover, electron microscopy revealed that SPBs do not assemble a satellite in cells expressing Clb2 in early G1. Finally, we demonstrate that Clb2 must localize to the cytoplasm in order to inhibit SPB duplication, suggesting the possibility for direct CDK inhibition of satellite components. These two mechanisms, intrinsic and extrinsic control by CDK, evoke two-step system that prevents SPB reduplication throughout the cell cycle.

Mps1 Phosphorylates Borealin to Control Aurora B Activity and Chromosome Alignment

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

Activation of the Cdc42p GTPase by cyclin-dependent protein kinases in budding yeast

Cyclin-dependent kinases (CDKs) trigger essential cell cycle processes including critical events in G1 phase that culminate in bud emergence, spindle pole body duplication, and DNA replication. Localized activation of the Rho-type GTPase Cdc42p is crucial for establishment of cell polarity during G1, but CDK targets that link the Cdc42p module with cell growth and cell cycle commitment have remained largely elusive. Here, we identify the GTPase-activating protein (GAP) Rga2p as an important substrate related to the cell polarity function of G1 CDKs. Overexpression of RGA2 in the absence of functional Pho85p or Cdc28p CDK complexes is toxic, due to an inability to polarize growth. Mutation of CDK consensus sites in Rga2p that are phosphorylated both in vivo and in vitro by Pho85p and Cdc28p CDKs results in a loss of G1 phase-specific phosphorylation. A failure to phosphorylate Rga2p leads to defects in localization and impaired polarized growth, in a manner dependent on Rga2p GAP function. Taken together, our data suggest that CDK-dependent phosphorylation restrains Rga2p activity to ensure appropriate activation of Cdc42p during cell polarity establishment. Inhibition of GAPs by CDK phosphorylation may be a general mechanism to promote proper G1-phase progression.

An Sfi1p-Like Centrin-Binding Protein Mediates Centrin-Based Ca 2+ -Dependent Contractility in Paramecium tetraurelia

The previous characterization and structural analyses of Sfi1p, a Saccharomyces cerevisiae centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca2+-dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using Paramecium tetraurelia, which harbors a large Ca2+ -dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived from the high-molecular-mass protein to probe the Paramecium genome sequence, we characterized the PtCenBP1 gene, which encodes a 460-kDa protein. PtCenBP1p displays six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX11F/LX2WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant PtCenBP1 allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca2+-dependent contractile processes.

Novelsfi1Alleles Uncover Additional Functions for Sfi1p in Bipolar Spindle Assembly and Function

A variety of spindle and kinetochore defects have been shown to induce a mitotic delay through activation of the spindle checkpoint. With the aim of identifying novel mitotic defects we carried out a mad1 synthetic lethal screen in budding yeast. In this screen, four novel alleles of sfi1 were isolated. SFI1 is an essential gene, previously identified through its interaction with centrin/CDC31 and shown to be required for spindle pole body (SPB) duplication. The new mutations were all found in the C-terminal domain of Sfi1p, which has no known function, but it is well conserved among budding yeasts. Analysis of the novel sfi1 mutants, through a combination of light and electron microscopy, revealed duplicated SPBs <0.3 microm apart. Importantly, these SPBs have completed duplication, but they are not separated, suggesting a possible defect in splitting of the bridge. We discuss possible roles for Sfi1p in this step in bipolar spindle assembly.

MPS3 mediates meiotic bouquet formation in Saccharomyces cerevisiae

In meiotic prophase, telomeres associate with the nuclear envelope and accumulate adjacent to the centrosome/spindle pole to form the chromosome bouquet, a well conserved event that in Saccharomyces cerevisiae requires the meiotic telomere protein Ndj1p. Ndj1p interacts with Mps3p, a nuclear envelope SUN domain protein that is required for spindle pole body duplication and for sister chromatid cohesion. Removal of the Ndj1p-interaction domain from MPS3 creates an ndj1 Delta-like separation-of-function allele, and Ndj1p and Mps3p are codependent for stable association with the telomeres. SUN domain proteins are found in the nuclear envelope across phyla and are implicated in mediating interactions between the interior of the nucleus and the cytoskeleton. Our observations indicate a general mechanism for meiotic telomere movements.

Yeast UBL-UBA proteins have partially redundant functions in cell cycle control

BackgroundProteins containing ubiquitin-like (UBL) and ubiquitin associated (UBA) domains have been suggested to shuttle ubiquitinated substrates to the proteasome for degradation. There are three UBL-UBA containing proteins in budding yeast: Ddi1, Dsk2 and Rad23, which have been demonstrated to play regulatory roles in targeting ubiquitinated substrates to the proteasome for degradation. An involvement of these proteins in cell cycle related events has also been reported. We tested whether these three proteins act redundantly in the cell cycle.ResultsHere we show that the UBL-UBA proteins are partially redundant for cell cycle related roles. RAD23 is redundant with DDI1 and DSK2, but DDI1 and DSK2 are not redundant with each other and the triple deletion shows a synthetic effect, suggesting the existence of at least two roles for RAD23 in cell cycle control. The rad23Δddi1Δdsk2Δ triple deletion strain delays both in G2/M-phase and in mid-anaphase at high temperatures with duplicated spindle pole bodies. Cell cycle progression in the triple deletion strain can only be partially rescued by a rad23 allele lacking the c-terminal UBA domain, suggesting that RAD23 requires its c-terminal UBA domain for full function. In addition to their ability to bind ubiquitin and the proteasome, the UBL-UBA proteins also share the ability to homodimerize. Rad23 and Dsk2 dimerization requires their UBL and/or UBA domains whereas Ddi1 dimerization does not. Here we show that Ddi1 homodimerization is necessary for its cell cycle related functions.ConclusionThe three yeast UBL-UBA proteins have partially redundant roles required for progression through mitosis.

Deletion of RNQ1 gene reveals novel functional relationship between divergently transcribed Bik1p/CLIP-170 and Sfi1p in spindle pole body separation

Spindle pole body (SPB; the microtubule organizing center in yeast) duplication is essential to form a bipolar spindle. The duplicated SPBs must then separate and migrate to opposite sides of the nucleus. We identified a novel functional relationship in SPB separation between the microtubule stabilizing protein Bik1p/CLIP-170 and the SPB half-bridge protein Sfi1p. A genetic interaction between BIK1 and SFI1 was discovered in a synthetic lethal screen using a strain deficient in the prion protein gene RNQ1. RNQ1 deletion reduced expression from the divergently transcribed BIK1, allowing us to identify genetic interactors with bik1. The sfi1-1 bik1 synthetic lethality was suppressed by over-expression of CIK1, KAR1, and PPH21. Genetic analysis indicated that the sfi1-1 bik1 synthetic lethality was unlikely related to the function of Bik1p in the dynein pathway or to defects in spindle position. Furthermore, a sfi1-1 Deltakip2 mutant was viable, suggesting that the Bik1p pool at the cytoplasmic microtubule plus-ends may not be required in sfi1-1. Microscopic examination indicated the sfi1-1 mutant was delayed in SPB duplication, SPB separation, or spindle elongation and the sfi-1 Deltabik1 double mutant arrested with duplicated but unseparated SPBs. These results suggest that Bik1p has a previously uncharacterized function in the separation of duplicated SPBs.

The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication.

Structural role of Sfi1p–centrin filaments in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p–centrin complexes containing several repeats show Sfi1p as an α helix with centrins wrapped around each repeat and similar centrin–centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p–centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous α helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p–centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.

Cdk1 regulates centrosome separation by restraining proteolysis of microtubule-associated proteins

In yeast, separation of duplicated spindle pole bodies (SPBs) (centrosomes in higher eukaryotes) is an indispensable step in the assembly of mitotic spindle and is triggered by severing of the bridge that connects the sister SPBs. This process requires Cdk1 (Cdc28) activation by Tyrosine 19 dephosphorylation. We show that cells that fail to activate Cdk1 are devoid of spindles due to persistently active APCCdh1, which targets microtubule-associated proteins Cin8, Kip1 and Ase1 for degradation. Tyrosine 19 dephosphorylation of Cdk1 is necessary to specifically prevent proteolysis of these proteins. Interestingly, SPB separation is dependent on the microtubule-bundling activity of Cin8 but not on its motor function. Since ectopic expression of proteolysis-resistant Cin8, Kip1 or Ase1 is sufficient for SPB separation even in the absence of Cdc28-Clb activity, we suggest that stabilization of these mechanical force-generating proteins is the predominant role of Cdc28-Clb in centrosome separation.

Disappearance of the budding yeast Bub2–Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit

Budding yeast spindle position checkpoint is engaged by misoriented spindles and prevents mitotic exit by inhibiting the G protein Tem1 through the GTPase-activating protein (GAP) Bub2/Bfa1. Bub2 and Bfa1 are found on both duplicated spindle pole bodies until anaphase onset, when they disappear from the mother-bound spindle pole under unperturbed conditions. In contrast, when spindles are misoriented they remain symmetrically localized at both SPBs. Thus, symmetric localization of Bub2/Bfa1 might lead to inhibition of Tem1, which is also present at SPBs. Consistent with this hypothesis, we show that a Bub2 version symmetrically localized on both SPBs throughout the cell cycle prevents mitotic exit in mutant backgrounds that partially impair it. This effect is Bfa1 dependent and can be suppressed by high Tem1 levels. Bub2 removal from the mother-bound SPB requires its GAP activity, which in contrast appears to be dispensable for Tem1 inhibition. Moreover, it correlates with the passage of one spindle pole through the bud neck because it needs septin ring formation and bud neck kinases.

The Saccharomyces cerevisiae spindle pole body (SPB) component Nbp1p is required for SPB membrane insertion and interacts with the integral membrane proteins Ndc1p and Mps2p.

The spindle pole body (SPB) in Saccharomyces cerevisiae functions to nucleate and organize spindle microtubules, and it is embedded in the nuclear envelope throughout the yeast life cycle. However, the mechanism of membrane insertion of the SPB has not been elucidated. Ndc1p is an integral membrane protein that localizes to SPBs, and it is required for insertion of the SPB into the nuclear envelope during SPB duplication. To better understand the function of Ndc1p, we performed a dosage suppressor screen using the ndc1-39 temperature-sensitive allele. We identified an essential SPB component, Nbp1p. NBP1 shows genetic interactions with several SPB genes in addition to NDC1, and two-hybrid analysis revealed that Nbp1p binds to Ndc1p. Furthermore, Nbp1p is in the Mps2p-Bbp1p complex in the SPB. Immunoelectron microscopy confirmed that Nbp1p localizes to the SPB, suggesting a function at this location. Consistent with this hypothesis, nbp1-td (a degron allele) cells fail in SPB duplication upon depletion of Nbp1p. Importantly, these cells exhibit a "dead" SPB phenotype, similar to cells mutant in MPS2, NDC1, or BBP1. These results demonstrate that Nbp1p is a SPB component that acts in SPB duplication at the point of SPB insertion into the nuclear envelope.