Spindle Pole Body Research Articles

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.

Yeast centrosome components form a noncanonical LINC complex at the nuclear envelope insertion site.

Bipolar spindle formation in yeast requires insertion of centrosomes (known as spindle pole bodies [SPBs]) into fenestrated regions of the nuclear envelope (NE). Using structured illumination microscopy and bimolecular fluorescence complementation, we map protein distribution at SPB fenestrae and interrogate protein-protein interactions with high spatial resolution. We find that the Sad1-UNC-84 (SUN) protein Mps3 forms a ring-like structure around the SPB, similar to toroids seen for components of the SPB insertion network (SPIN). Mps3 and the SPIN component Mps2 (a Klarsicht-ANC-1-Syne-1 domain [KASH]-like protein) form a novel noncanonical linker of nucleoskeleton and cytoskeleton (LINC) complex that is connected in both luminal and extraluminal domains at the site of SPB insertion. The LINC complex also controls the distribution of a soluble SPIN component Bbp1. Taken together, our work shows that Mps3 is a fifth SPIN component and suggests both direct and indirect roles for the LINC complex in NE remodeling.

Spindle pole body movement is affected by glucose and ammonium chloride in fission yeast

The complexity of chromatin dynamics is orchestrated by several active processes. In fission yeast, the centromeres are clustered around the spindle pole body (SPB) and oscillate in a microtubule- and adenosine triphosphate (ATP)-dependent manner. However, whether and how SPB oscillation are affected by different environmental conditions remain poorly understood. In this study, we quantitated movements of the SPB component, which colocalizes with the centromere in fission yeast. We found that SPB movement was significantly reduced at low glucose concentrations. Movement of the SPB was also affected by the presence of ammonium chloride. Power spectral analysis revealed that periodic movement of the SPB is disrupted by low glucose concentrations. Measurement of ATP levels in living cells by quantitative single-cell imaging suggests that ATP levels are not the only determinant of SPB movement. Our results provide novel insight into how SPB movement is regulated by cellular energy status and additional factors such as the medium nutritional composition.

Quantifying Tubulin Concentration and Microtubule Number Throughout the Fission Yeast Cell Cycle.

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αβ-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αβ-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.

Expression of TorsinA in a heterologous yeast system reveals interactions with lumenal domains of LINC and nuclear pore complex components.

DYT1 dystonia is caused by an in-frame deletion of a glutamic acid codon in the gene encoding the AAA+ ATPase TorsinA (TorA). TorA localizes within the lumen of the nuclear envelope/endoplasmic reticulum and binds to a membrane-spanning cofactor, lamina associated polypeptide 1 (LAP1) or lumenal domain like LAP1 (LULL1), to form an ATPase; the substrate(s) of TorA remains ill-defined. Here we use budding yeast, which lack Torsins, to interrogate TorA function. We show that TorA accumulates at nuclear envelope-embedded spindle pole bodies (SPBs) in a way that requires its oligomerization and the SUN (Sad1 and UNc-84)-domain protein, Mps3. We further show that TorA physically interacts with human SUN1/2 within this system, supporting the physiological relevance of these interactions. Consistent with the idea that TorA acts on a SPB substrate, its binding to SPBs is modulated by the ATPase-stimulating activity of LAP1. TorA and TorA-ΔE reduce the fitness of cells expressing mps3 alleles, whereas TorA alone inhibits growth of cells lacking Pom152, a component of the nuclear pore complex. This genetic specificity is mirrored biochemically as TorA, but not TorA-ΔE, binds Pom152. Thus, TorA–nucleoporin interactions might be abrogated by TorA-ΔE, suggesting new experimental avenues to interrogate the molecular basis behind nuclear envelope herniations seen in mammalian cells lacking TorA function.

Meiosis-specific cohesin component, Rec8, promotes the localization of Mps3 SUN domain protein on the nuclear envelope.

Proteins in the nuclear envelope (NE) play a role in the dynamics and functions of the nucleus and of chromosomes during mitosis and meiosis. Mps3, a yeast NE protein with a conserved SUN domain, predominantly localizes on a yeast centrosome equivalent, spindle pole body (SPB), in mitotic cells. During meiosis, Mps3, together with SPB, forms a distinct multiple ensemble on NE. How meiosis-specific NE localization of Mps3 is regulated remains largely unknown. In this study, we found that a meiosis-specific component of the protein complex essential for sister chromatid cohesion, Rec8, binds to Mps3 during meiosis and controls Mps3 localization and proper dynamics on NE. Ectopic expression of Rec8 in mitotic yeast cells induced the formation of Mps3 patches/foci on NE. This required the cohesin regulator, WAPL ortholog, Rad61/Wpl1, suggesting that a meiosis-specific cohesin complex with Rec8 controls NE localization of Mps3. We also observed that two domains of the nucleoplasmic region of Mps3 are essential for NE localization of Mps3 in mitotic as well as meiotic cells. We speculate that the interaction of Mps3 with the meiosis-specific cohesin in the nucleoplasm is a key determinant for NE localization/function of Mps3.

The J-domain cochaperone Rsp1 interacts with Mto1 to organize noncentrosomal microtubule assembly.

Microtubule biogenesis initiates at various intracellular sites, including the centrosome, the Golgi apparatus, the nuclear envelope, and preexisting microtubules. Similarly, in the fission yeast Schizosaccharomyces pombe, interphase microtubules are nucleated at the spindle pole body (SPB), the nuclear envelope, and preexisting microtubules, depending on Mto1 activity. Despite the essential role of Mto1 in promoting microtubule nucleation, how distribution of Mto1 in different sites is regulated has remained elusive. Here, we show that the J-domain cochaperone Rsp1 interacts with Mto1 and specifies the localization of Mto1 to non-SPB nucleation sites. The absence of Rsp1 abolishes the localization of Mto1 to non-SPB nucleation sites, with concomitant enrichment of Mto1 to the SPB and the nuclear envelope. In contrast, Rsp1 overexpression impairs the localization of Mto1 to all microtubule organization sites. These findings delineate a previously uncharacterized mechanism in which Rsp1-Mto1 interaction orchestrates non-SPB microtubule formation.

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.

Spindle pole body component 25 regulates stemness of prostate cancer cells.

Spindle pole body component 25 (SPC25) is a component of NDC80 complex that controls spindle assembly checkpoint in the microtubule-binding domain of kinetochores. We recently showed that SPC25 is required for prostate cancer (PrC) cell proliferation and cell cycle progression, and here we investigated whether SPC25 may be a Cancer stem cell (CSC) marker in PrC. We found that the levels of SPC25 were higher in PrC samples than paired normal prostate tissue. The overall survival of PrC patients with high SPC25 was poorer than those with low SPC25. PrC cell lines were transduced with two vectors carrying a luciferase reporter and a mCherry fluorescent reporter under a cytomegalovirus promoter and a nuclear green fluorescent protein reporter under the control of a SPC25 promoter, respectively, to allow differentiating SPC25+ from SPC25- PrC cells by flow cytometry. Compared to SPC25- cells, SPC25+ cells formed significantly more tumor spheres in culture, appeared to be more resistant towards docetaxel-induced cell apoptosis, and generated larger tumors with higher frequency after serial adoptive transplantation. Thus, our data suggest that SPC25 may be highly expressed in the CSC-like cells in PrC and could be a promising target for effective treatment of PrC.

Budding Yeast BFA1 Has Multiple Positive Roles in Directing Late Mitotic Events.

The proper regulation of cell cycle transitions is paramount to the maintenance of cellular genome integrity. In Saccharomyces cerevisiae, the mitotic exit network (MEN) is a Ras-like signaling cascade that effects the transition from M phase to G1 during the cell division cycle in budding yeast. MEN activation is tightly regulated. It occurs during anaphase and is coupled to mitotic spindle position by the spindle position checkpoint (SPoC). Bfa1 is a key component of the SPoC and functions as part of a two-component GAP complex along with Bub2. The GAP activity of Bfa1-Bub2 keeps the MEN GTPase Tem1 inactive in cells with mispositioned spindles, thereby preventing inappropriate mitotic exit and preserving genome integrity. Interestingly, a GAP-independent role for Bfa1 in mitotic exit regulation has been previously identified. However the nature of this Bub2-independent role and its biological significance are not understood. Here we show that Bfa1 also activates the MEN by promoting the localization of Tem1 primarily to the daughter spindle pole body (dSPB). We demonstrate that the overexpression of BFA1 is lethal due to defects in Tem1 localization, which is required for its activity. In addition, our studies demonstrate a Tem1-independent role for Bfa1 in promoting proper cytokinesis. Cells lacking TEM1, in which the essential mitotic exit function is bypassed, exhibit cytokinesis defects. These defects are suppressed by the overexpression of BFA1. We conclude that Bfa1 functions to both inhibit and activate late mitotic events.

Nuclear envelope expansion in budding yeast is independent of cell growth and does not determine nuclear volume.

Most cells exhibit a constant ratio between nuclear and cell volume. The mechanism dictating this constant ratio and the nuclear component(s) that scale with cell size are not known. To address this, we examined the consequences to the size and shape of the budding yeast nucleus when cell expansion is inhibited by down-regulating components of the secretory pathway. We find that under conditions where cell size increase is restrained, the nucleus becomes bilobed, with the bulk of the DNA in one lobe and the nucleolus in the other. The formation of bilobed nuclei is dependent on fatty acid and phospholipid synthesis, suggesting that it is associated with nuclear membrane expansion. Bilobed nuclei appeared predominantly after spindle pole body separation, suggesting that nuclear envelope expansion follows cell-cycle cues rather than cell size. Importantly, cells with bilobed nuclei had the same nuclear:cell volume ratio as cells with round nuclei. Therefore, the bilobed nucleus could be a consequence of continued NE expansion as cells traverse the cell cycle without an accompanying increase in nuclear volume due to the inhibition of cell growth. Our data suggest that nuclear volume is not determined by nuclear envelope availability but by one or more nucleoplasmic factors.

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.

Recruitment of the mitotic exit network to yeast centrosomes couples septin displacement to actomyosin constriction

In many eukaryotic organisms cytokinesis is driven by a contractile actomyosin ring (CAR) that guides membrane invagination. What triggers CAR constriction at a precise time of the cell cycle is a fundamental question. In budding yeast CAR is assembled via a septin scaffold at the division site. A Hippo-like kinase cascade, the Mitotic Exit Network (MEN), promotes mitotic exit and cytokinesis, but whether and how these two processes are independently controlled by MEN is poorly understood. Here we show that a critical function of MEN is to promote displacement of the septin ring from the division site, which in turn is essential for CAR constriction. This is independent of MEN control over mitotic exit and involves recruitment of MEN components to the spindle pole body (SPB). Ubiquitination of the SPB scaffold Nud1 inhibits MEN signaling at the end of mitosis and prevents septin ring splitting, thus silencing the cytokinetic machinery.

The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation.

Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72-γ-TuSC interaction by binding to Spc72 and γ-TuSC. Stu2 together with Spc72-γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72-γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.

Pap1 + confers microtubule damage resistance to mut2a, an extragenic suppressor of the rad26:4A allele in S. pombe.

The DNA structure checkpoint protein Rad26ATRIP is also required for an interphase microtubule damage response. This checkpoint delays spindle pole body separation and entry into mitosis following treatment of cells with microtubule poisons. This checkpoint requires cytoplasmic Rad26ATRIP, which is compromised by the rad26:4A allele that inhibits cytoplasmic accumulation of Rad26ATRIP following microtubule damage. The rad26::4a allele also disrupts minichromosome stability and cellular morphology, suggesting that the interphase microtubule damage checkpoint pathway operates in an effort to maintain chromosome stability and proper cell shape. To identify other proteins of the Rad26-dependent interphase microtubule damage response, we used ultra violet (UV) radiation to identify extragenic interaction suppressors of the rad26::4A growth defect on microtubule poisons. One suppressor mutation, which we named mut2a, permitted growth of rad26:4A cells on MBC media and conferred sensitivity to a microtubulin poison upon genetic outcross. In an attempt to clone this interaction suppressor using a genomic library complementation strategy, we instead isolated pap1 + as an extracopy suppressor of the mut2a growth defect. We discuss the mechanism by which pap1 + overexpression may allow growth of mut2a cells in conditions that destabilize microtubules.

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.

Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication.

Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.

Centrosome Remodelling in Evolution.

The centrosome is the major microtubule organizing centre (MTOC) in animal cells. The canonical centrosome is composed of two centrioles surrounded by a pericentriolar matrix (PCM). In contrast, yeasts and amoebozoa have lost centrioles and possess acentriolar centrosomes—called the spindle pole body (SPB) and the nucleus-associated body (NAB), respectively. Despite the difference in their structures, centriolar centrosomes and SPBs not only share components but also common biogenesis regulators. In this review, we focus on the SPB and speculate how its structures evolved from the ancestral centrosome. Phylogenetic distribution of molecular components suggests that yeasts gained specific SPB components upon loss of centrioles but maintained PCM components associated with the structure. It is possible that the PCM structure remained even after centrosome remodelling due to its indispensable function to nucleate microtubules. We propose that the yeast SPB has been formed by a step-wise process; (1) an SPB-like precursor structure appeared on the ancestral centriolar centrosome; (2) it interacted with the PCM and the nuclear envelope; and (3) it replaced the roles of centrioles. Acentriolar centrosomes should continue to be a great model to understand how centrosomes evolved and how centrosome biogenesis is regulated.

Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization.

Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein Sid4 at the spindle pole body (SPB), and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Pololike kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

Asymmetric Segregation of Aged Spindle Pole Bodies During Cell Division: Mechanisms and Relevance Beyond Budding Yeast?

Asymmetric cell division generates cell diversity and contributes to cellular aging and rejuvenation. Here, we review the molecular mechanisms enabling budding yeast to recognize spindle pole bodies (SPB, centrosome equivalent) based on their age, and guide their non-random mitotic segregation: SPB inheritance requires the distinction of old from new SPBs and is regulated by the SPB-inheritance network (SPIN) and the mitotic exit network (MEN). The SPIN marks the pre-existing SPB as old and the MEN recognizes these marks translating them into spindle orientation. We next revisit other molecules and structures that partition depending on their age rather than their abundance at mitosis as, for example, DNA, centrosomes, mitochondria, and histones in yeast and other systems. The recurrence of this differential behavior suggests a functional significance for numerous cell types, which we then discuss. We conclude that non-random segregation may facilitate asymmetric cell fate determination and thereby indirectly aging and rejuvenation. Also see the video abstract here: https://youtu.be/1sQ4rAomnWY.