Spindle Pole Body Duplication Research Articles

Translational control of MPS1 links protein synthesis with the initiation of cell division and spindle pole body duplication in Saccharomyces cerevisiae.

Protein synthesis underpins cell growth and controls when cells commit to a new round of cell division at a point in late G1 of the cell cycle called Start. Passage through Start also coincides with the duplication of the microtubule-organizing centers, the yeast spindle pole bodies, which will form the two poles of the mitotic spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the spindle pole body in Saccharomyces cerevisiae. Here, we show that the MPS1 transcript has a short upstream open reading frame that represses the synthesis of Mps1p. Mutating the MPS1 uORF makes the cells smaller, accelerates the appearance of Mps1p in late G1, and promotes completion of Start. Monitoring the spindle pole body in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their spindle pole body earlier at a smaller cell size. The accelerated Start of MPS1 uORF mutants depends on the G1 cyclin Cln3p and the transcriptional repressor Whi5p but not on the Cln1,2p G1 cyclins. These results identify growth inputs in mechanisms that control duplication of the microtubule-organizing center and implicate these processes in the coupling of cell growth with division.

Loss of RanGAP1 drives chromosome instability and rapid tumorigenesis of osteosarcoma.

Chromothripsis is a catastrophic event of chromosomal instability that involves intensive fragmentation and rearrangements within localized chromosomal regions. However, its cause remains unclear. Here, we show that reduction and inactivation of Ran GTPase-activating protein 1 (RanGAP1) commonly occur in human osteosarcoma, which is associated with a high rate of chromothripsis. In rapidly expanding mouse osteoprogenitors, RanGAP1 deficiency causes chromothripsis in chr1q, instant inactivation of Rb1 and degradation of p53, consequent failure in DNA damage repair, and ultrafast osteosarcoma tumorigenesis. During mitosis, RanGAP1 anchors to the kinetochore, where it recruits PP1-γ to counteract the activity of the spindle-assembly checkpoint (SAC) and prevents TOP2A degradation, thus safeguarding chromatid decatenation. Loss of RanGAP1 causes SAC hyperactivation and chromatid decatenation failure. These findings demonstrate that RanGAP1 maintains mitotic chromosome integrity and that RanGAP1 loss drives tumorigenesis through its direct effects on SAC and decatenation and secondary effects on DNA damage surveillance.

Human SFI1 and Centrin form a complex critical for centriole architecture and ciliogenesis.

Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.

Absolute quantitation of the yeast Whi5 transcriptional repressor during G1 phase growth

To establish and maintain an homeostatic size optimized to their growth conditions, cells set and sense a critical size threshold for commitment to division in late G1 phase, known as Start in yeast. Start depends on the expression of over two hundred genes required for budding, DNA replication spindle pole body duplication, and other functions, collectively termed the G1/S regulon. The timing of Start is determined in part by two antagonistic proteins: an activator of Start transcription, Swi4, and its repressor, Whi5.

The N-terminus of Sfi1 and yeast centrin Cdc31 provide the assembly site for a new spindle pole body.

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G1 the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G1. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.

A mutation in budding yeast BRR6 affecting nuclear envelope insertion of the spindle pole body.

BRR6 and BRL1 are two paralogs that encode transmembrane proteins of the nuclear envelope (NE) involved in membrane fluidity and nuclear pore complex biogenesis in organisms that undergo a closed mitosis. We show that mutation of a conserved cysteine in the intralumenal domain of Saccharomyces cerevisiae Brr6p results in a novel temperature sensitive allele, brr6-Y100H, that arrests growth due to defects in spindle formation. Analysis of brr6-Y100H cells by electron tomography and Brr6p localization by super-resolution imaging supports the idea that Brr6p is involved in insertion of the newly duplicated spindle pole body into the NE.

The role of gene dosage in budding yeast centrosome scaling and spontaneous diploidization.

Ploidy is the number of whole sets of chromosomes in a species. Ploidy is typically a stable cellular feature that is critical for survival. Polyploidization is a route recognized to increase gene dosage, improve fitness under stressful conditions and promote evolutionary diversity. However, the mechanism of regulation and maintenance of ploidy is not well characterized. Here, we examine the spontaneous diploidization associated with mutations in components of the Saccharomyces cerevisiae centrosome, known as the spindle pole body (SPB). Although SPB mutants are associated with defects in spindle formation, we show that two copies of the mutant in a haploid yeast favors diploidization in some cases, leading us to speculate that the increased gene dosage in diploids ‘rescues’ SPB duplication defects, allowing cells to successfully propagate with a stable diploid karyotype. This copy number-based rescue is linked to SPB scaling: certain SPB subcomplexes do not scale or only minimally scale with ploidy. We hypothesize that lesions in structures with incompatible allometries such as the centrosome may drive changes such as whole genome duplication, which have shaped the evolutionary landscape of many eukaryotes.

Spindle pole body duplication defective yeast cells are more prone to membrane damage

ABSTRACTCorrect separation of chromosomes during mitosis is essential for preventing genetic instability and aneuploidy. Such separation is dependent on correct duplication of the nuclear-associated microtubular organizing center, i.e., spindle pole body (SPB), in fungi. MonoPolar Spindle 2 (MPS2) is an essential gene, encoding a membrane protein required for the insertion of SPB into the nuclear envelope. We recently reported that the SESA complex, which is composed of Smy2, Eap1, Scp160, and Asc1, suppresses the essential role of MPS2 (Sezen et al. 2009, Genes & Development 23:1559–1570), i.e., in SESA-active cells Mps2 becomes nonessential. We also proposed that the SESA network facilitates this insertion by altering the membrane lipid composition (Sezen 2015, FEMS Yeast Research 15:fov089). In addition, we are interested in the antifungal properties of essential oils and previously reported that membrane integrity of yeast cells is impaired upon exposure to turpentine, thyme, oregano, and orange peel essential oils (Konuk and Ergüden 2017, BioCell 41:13–18). Due to our continuing interest in the SESA system and the mechanisms by which essential oils affect yeast cells, we aimed to investigate the effects of essential oils on yeast cell membranes. Herein, we show that mps2∆ 2µm-SMY2 and mps2∆ pom34∆ cells, in which the SESA complex is active and SPB duplication is defective, are more prone to membrane damage upon treatment with essential oils.

Structure and function of Spc42 coiled-coils in yeast centrosome assembly and duplication.

Centrosomes and spindle pole bodies (SPBs) are membraneless organelles whose duplication and assembly is necessary for bipolar mitotic spindle formation. The structural organization and functional roles of major proteins in these organelles can provide critical insights into cell division control. Spc42, a phosphoregulated protein with an N-terminal dimeric coiled-coil (DCC), assembles into a hexameric array at the budding yeast SPB core, where it functions as a scaffold for SPB assembly. Here, we present in vitro and in vivo data to elucidate the structural arrangement and biological roles of Spc42 elements. Crystal structures reveal details of two additional coiled-coils in Spc42: a central trimeric coiled-coil and a C-terminal antiparallel DCC. Contributions of the three Spc42 coiled-coils and adjacent undetermined regions to the formation of an ∼145 Å hexameric lattice in an in vitro lipid monolayer assay and to SPB duplication and assembly in vivo reveal structural and functional redundancy in Spc42 assembly. We propose an updated model that incorporates the inherent symmetry of these Spc42 elements into a lattice, and thereby establishes the observed sixfold symmetry. The implications of this model for the organization of the central SPB core layer are discussed.

Dhh1 is a member of the SESA network.

The correct separation of chromosomes during mitosis is necessary to prevent genetic instability and aneuploidy, which are responsible for cancer and other diseases, and it depends on proper centrosome duplication. In a recent study, we found that Smy2 can suppress the essential role of Mps2 in the insertion of yeast centrosome into the nuclear membrane by interacting with Eap1, Scp160, and Asc1 and designated this network as SESA (Smy2, Eap1, Scp160, Asc1). Detailed analysis showed that the SESA network is part of a mechanism which regulates translation of POM34 mRNA. Thus, SESA is a system that suppresses spindle pole body duplication defects by repressing the translation of POM34 mRNA. In this study, we performed a genome-wide screening in order to identify new members of the SESA network and confirmed Dhh1 as a putative member. Dhh1 is a cytoplasmic DEAD-box helicase known to regulate translation. Therefore, we hypothesized that Dhh1 is responsible for the highly selective inhibition of POM34 mRNA by SESA.

A role for Saccharomyces cerevisiae Centrin (Cdc31) in mitochondrial function and biogenesis.

Centrins belong to a family of proteins containing calcium-binding EF-hand motifs that perform well-established roles in centrosome and spindle pole body (SPB) duplication. Yeast encodes a single Centrin protein (Cdc31) that binds components in the SPB. However, further studies revealed a role for Centrins in mRNA export, and interactions with contractile filaments and photoreceptors. In addition, human Centrin-2 can bind the DNA-lesion recognition factor XPC, and improve the efficiency of nucleotide excision repair. Similarly, we reported that yeast Cdc31 binds Rad4, a functional counterpart of the XPC DNA repair protein. We also found that Cdc31 is involved in the ubiquitin/proteasome system, and mutations interfere with intracellular protein turnover. In this report, we describe new findings that indicate a role for Cdc31 in the energy metabolism pathway. Cdc31 and cdc31 mutant proteins showed distinct interactions with proteins in energy metabolism, and mutants showed sensitivity to oxidative stress and poor growth on non-fermentable carbon. Significant alteration in mitochondrial morphology was also detected. Although it is unclear how Cdc31 contributes to so many unrelated mechanisms, we propose that by controlling SPB duplication Centrin proteins might link the cellular responses to DNA damage, oxidative load and proteotoxic stresses to growth control.

Leader of the SAC: molecular mechanisms of Mps1/TTK regulation in mitosis.

Discovered in 1991 in a screen for genes involved in spindle pole body duplication, the monopolar spindle 1 (Mps1) kinase has since claimed a central role in processes that ensure error-free chromosome segregation. As a result, Mps1 kinase activity has become an attractive candidate for pharmaceutical companies in the search for compounds that target essential cellular processes to eliminate, for example, tumour cells or pathogens. Research in recent decades has offered many insights into the molecular function of Mps1 and its regulation. In this review, we integrate the latest knowledge regarding the regulation of Mps1 activity and its spatio-temporal distribution, highlight gaps in our understanding of these processes and propose future research avenues to address them.

The budding yeast RSC complex maintains ploidy by promoting spindle pole body insertion.

Ploidy is tightly regulated in eukaryotic cells and is critical for cell function and survival. Cells coordinate multiple pathways to ensure replicated DNA is segregated accurately to prevent abnormal changes in chromosome number. In this study, we characterize an unanticipated role for the Saccharomyces cerevisiae "remodels the structure of chromatin" (RSC) complex in ploidy maintenance. We show that deletion of any of six nonessential RSC genes causes a rapid transition from haploid to diploid DNA content because of nondisjunction events. Diploidization is accompanied by diagnostic changes in cell morphology and is stably maintained without further ploidy increases. We find that RSC promotes chromosome segregation by facilitating spindle pole body (SPB) duplication. More specifically, RSC plays a role in distributing two SPB insertion factors, Nbp1 and Ndc1, to the new SPB. Thus, we provide insight into a role for a SWI/SNF family complex in SPB duplication and ploidy maintenance.

The half-bridge component Kar1 promotes centrosome separation and duplication during budding yeast meiosis.

The budding yeast centrosome, often called the spindle pole body (SPB), nucleates microtubules for chromosome segregation during cell division. An appendage, called the half bridge, attaches to one side of the SPB and regulates SPB duplication and separation. Like DNA, the SPB is duplicated only once per cell cycle. During meiosis, however, after one round of DNA replication, two rounds of SPB duplication and separation are coupled with homologue segregation in meiosis I and sister-chromatid segregation in meiosis II. How SPB duplication and separation are regulated during meiosis remains to be elucidated, and whether regulation in meiosis differs from that in mitosis is unclear. Here we show that overproduction of the half-bridge component Kar1 leads to premature SPB separation during meiosis. Furthermore, excessive Kar1 induces SPB overduplication to form supernumerary SPBs, leading to chromosome missegregation and erroneous ascospore formation. Kar1-­mediated SPB duplication bypasses the requirement of dephosphorylation of Sfi1, another half-bridge component previously identified as a licensing factor. Our results therefore reveal an unexpected role of Kar1 in licensing meiotic SPB duplication and suggest a unique mechanism of SPB regulation during budding yeast meiosis.

Duplication and Nuclear Envelope Insertion of the Yeast Microtubule Organizing Centre, the Spindle Pole Body.

The main microtubule organizing centre in the unicellular model organisms Saccharomyces cerevisiae and Schizosaccharomyces pompe is the spindle pole body (SPB). The SPB is a multilayer structure, which duplicates exactly once per cell cycle. Unlike higher eukaryotic cells, both yeast model organisms undergo mitosis without breakdown of the nuclear envelope (NE), a so-called closed mitosis. Therefore, in order to simultaneously nucleate nuclear and cytoplasmic MTs, it is vital to embed the SPB into the NE at least during mitosis, similarly to the nuclear pore complex (NPC). This review aims to embrace the current knowledge of the SPB duplication cycle with special emphasis on the critical step of the insertion of the new SPB into the NE.

Novel phosphorylation states of the yeast spindle pole body.

ABSTRACTPhosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of enriched Saccharomyces cerevisiae SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a cdc4-1 temperature-sensitive mutation, with particular focus on phosphorylation events on the γ-tubulin small complex (γ-TuSC). The cdc4-1 arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally, in vivo analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites that influence microtubule formation.

Characterization of spindle pole body duplication reveals a regulatory role for nuclear pore complexes.

The spindle pole body (SPB) of budding yeast duplicates once per cell cycle. In G1, the satellite, an SPB precursor, assembles next to the mother SPB (mSPB) on the cytoplasmic side of the nuclear envelope (NE). How the growing satellite subsequently inserts into the NE is an open question. To address this, we have uncoupled satellite growth from NE insertion. We show that the bridge structure that separates the mSPB from the satellite is a distance holder that prevents deleterious fusion of both structures. Binding of the γ-tubulin receptor Spc110 to the central plaque from within the nucleus is important for NE insertion of the new SPB. Moreover, we provide evidence that a nuclear pore complex associates with the duplicating SPB and helps to insert the SPB into the NE. After SPB insertion, membrane-associated proteins including the conserved Ndc1 encircle the SPB and retain it within the NE. Thus, uncoupling SPB growth from NE insertion unmasks functions of the duplication machinery.

Molecular model of fission yeast centrosome assembly determined by superresolution imaging.

Microtubule-organizing centers (MTOCs), known as centrosomes in animals and spindle pole bodies (SPBs) in fungi, are important for the faithful distribution of chromosomes between daughter cells during mitosis as well as for other cellular functions. The cytoplasmic duplication cycle and regulation of the Schizosaccharomyces pombe SPB is analogous to centrosomes, making it an ideal model to study MTOC assembly. Here, we use superresolution structured illumination microscopy with single-particle averaging to localize 14 S. pombe SPB components and regulators, determining both the relationship of proteins to each other within the SPB and how each protein is assembled into a new structure during SPB duplication. These data enabled us to build the first comprehensive molecular model of the S. pombe SPB, resulting in structural and functional insights not ascertained through investigations of individual subunits, including functional similarities between Ppc89 and the budding yeast SPB scaffold Spc42, distribution of Sad1 to a ring-like structure and multiple modes of Mto1 recruitment.

A ternary membrane protein complex anchors the spindle pole body in the nuclear envelope in budding yeast

In budding yeast (Saccharomyces cerevisiae) the multilayered spindle pole body (SPB) is embedded in the nuclear envelope (NE) at fusion sites of the inner and outer nuclear membrane. The SPB is built from 18 different proteins, including the three integral membrane proteins Mps3, Ndc1, and Mps2. These membrane proteins play an essential role in the insertion of the new SPB into the NE. How the huge core structure of the SPB is anchored in the NE has not been investigated thoroughly until now. The present model suggests that the NE protein Mps2 interacts via Bbp1 with Spc29, one of the coiled-coil proteins forming the central plaque of the SPB. To test this model, we purified and reconstituted the Mps2-Bbp1 complex from yeast and incorporated the complex into liposomes. We also demonstrated that Mps2-Bbp1 directly interacts with Mps3 and Ndc1. We then purified Spc29 and reconstituted the ternary Mps2-Bbp1-Spc29 complex, proving that Bbp1 can simultaneously interact with Mps2 and Spc29 and in this way link the central plaque of the SPB to the nuclear envelope. Interestingly, Bbp1 induced oligomerization of Spc29, which may represent an early step in SPB duplication. Together, this analysis provides important insights into the interaction network that inserts the new SPB into the NE and indicates that the Mps2-Bbp1 complex is the central unit of the SPB membrane anchor.

Duplication of the Yeast Spindle Pole Body Once per Cell Cycle.

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. Centrosomes and SPBs duplicate exactly once per cell cycle by mechanisms that use the mother structure as a platform for the assembly of the daughter. The conserved Sfi1 and centrin proteins are essential components of the SPB duplication process. Sfi1 is an elongated molecule that has, in its center, 20 to 23 binding sites for the Ca(2+)-binding protein centrin. In the yeastSaccharomyces cerevisiae, all Sfi1 N termini are in contact with the mother SPB whereas the free C termini are distal to it. During S phase and early mitosis, cyclin-dependent kinase 1 (Cdk1) phosphorylation of mainly serine residues in the Sfi1 C termini blocks the initiation of SPB duplication ("off" state). Upon anaphase onset, the phosphatase Cdc14 dephosphorylates Sfi1 ("on" state) to promote antiparallel and shifted incorporation of cytoplasmic Sfi1 molecules into the half-bridge layer, which thereby elongates into the bridge. The Sfi1 C termini of the two Sfi1 layers localize in the bridge center, whereas the N termini of the newly assembled Sfi1 molecules are distal to the mother SPB. These free Sfi1 N termini then assemble the new SPB in G1phase. Recruitment of Sfi1 molecules into the anaphase SPB and bridge formation were also observed inSchizosaccharomyces pombe, suggesting that the Sfi1 bridge cycle is conserved between the two organisms. Thus, restricting SPB duplication to one event per cell cycle requires only an oscillation between Cdk1 kinase and Cdc14 phosphatase activities. This clockwork regulates the "on"/"off" state of the Sfi1-centrin receiver.