ABSTRACT
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.
Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Spindle Pole Bodies/metabolism , Amino Acid Sequence , Anaphase , Binding Sites , Cell Cycle Proteins/genetics , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Mutant Proteins/metabolism , Mutation/genetics , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Kinases/metabolism , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Spindle Pole Bodies/ultrastructure , Structure-Activity RelationshipABSTRACT
Owing to the local enrichment of factors that influence its dynamics and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells, post-Golgi vesicles ride on long actin cables to the bud tip. The proteins Boi1 and Boi2 (Boi1/2) participate in tethering and docking these vesicles to the plasma membrane. Here, we show in Saccharomyces cerevisiae that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation, reduces the directed movement of the vesicles to the tip and shortens the vesicles' tethering time at the cortex. Transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2, through interactions with Bud6 and Bni1, induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusion with the membrane.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Actins/metabolism , Adaptor Proteins, Signal Transducing , Cell Polarity , Exocytosis , Microfilament Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
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.
Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Protein Multimerization , Saccharomyces cerevisiae Proteins/metabolism , Tubulin/metabolism , Microtubule-Associated Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Domains , Protein Stability , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
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.
ABSTRACT
The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is â¼35 to 40 nm, leading to an estimated minimal rootletin length of â¼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator.
Subject(s)
Centrioles/metabolism , Centrosome/metabolism , Cytoskeletal Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Proteins/metabolism , Amino Acid Motifs , Centrioles/chemistry , Centrioles/genetics , Centrosome/chemistry , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , HeLa Cells , Humans , Interphase , Microscopy , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Protein Binding , Proteins/chemistry , Proteins/genetics , tRNA MethyltransferasesABSTRACT
The paralogous Brr6 and Brl1 are conserved integral membrane proteins of the nuclear envelope (NE) with an unclear role in nuclear pore complex (NPC) biogenesis. Here, we analyzed double-degron mutants of Brr6/Brl1 to understand this function. Depletion of Brr6 and Brl1 caused defects in NPC biogenesis, whereas the already assembled NPCs remained unaffected. This NPC biogenesis defect was not accompanied by a change in lipid composition. However, Brl1 interacted with Ndc1 and Nup188 by immunoprecipitation, and with transmembrane and outer and inner ring NPC components by split yellow fluorescent protein analysis, indicating a direct role in NPC biogenesis. Consistently, we found that Brr6 and Brl1 associated with a subpopulation of NPCs and emerging NPC assembly sites. Moreover, BRL1 overexpression affected NE morphology without a change in lipid composition and completely suppressed the nuclear pore biogenesis defect of nup116Δ and gle2Δ cells. We propose that Brr6 and Brl1 transiently associate with NPC assembly sites where they promote NPC biogenesis.
Subject(s)
Membrane Lipids/metabolism , Membrane Proteins/metabolism , Mutation , Nuclear Pore/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Membrane Lipids/genetics , Membrane Proteins/genetics , Nuclear Pore/genetics , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Nuclear Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast Saccharomyces cerevisiae, position of pre-anaphase nucleus is not strongly biased toward bud neck in Ogataea polymorpha and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in S. cerevisiae. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the γ-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of γ-tubulin receptor to SPBs determines the timing of spindle orientation in O. polymorpha.
Subject(s)
Cell Cycle Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomycetales/metabolism , Spindle Pole Bodies/metabolism , Microscopy, Electron , Saccharomycetales/ultrastructureABSTRACT
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.
Subject(s)
Cell Cycle , Nuclear Envelope/metabolism , Nuclear Pore/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Spindle Pole Bodies/metabolism , Calmodulin-Binding Proteins , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Genotype , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mutation , Nuclear Envelope/genetics , Nuclear Envelope/ultrastructure , Nuclear Pore/genetics , Nuclear Pore/ultrastructure , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phenotype , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Spindle Pole Bodies/genetics , Spindle Pole Bodies/ultrastructure , Time Factors , Tubulin/genetics , Tubulin/metabolismABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Schizosaccharomyces/metabolism , Spindle Pole Bodies/physiology , Centrosome/metabolism , Humans , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces pombe Proteins/metabolism , Spindle Apparatus/metabolism , Trimethoprim, Sulfamethoxazole Drug Combination/metabolismABSTRACT
The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1's function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31-Cdc31 interactions between Sfi1-Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.
Subject(s)
Cell Cycle Proteins/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Spindle Pole Bodies/metabolism , Calcium-Binding Proteins/metabolism , Nuclear Proteins/genetics , Protein Binding , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The protein arginine methyltransferase 6 (PRMT6) is a coregulator of gene expression and executes its repressing as well as activating function by asymmetric dimethylation of histone H3 at R2 (H3 R2me2a). Given that elevated expression levels of PRMT6 have been reported in various cancer types, we explore here its role in cell proliferation and senescence. We find that knockdown of PRMT6 results in proliferation defects of transformed as well as non-transformed cells, causes G1-phase arrest and induces senescence. This phenotype is accompanied by transcriptional upregulation of important cell cycle regulators, most prominently the cyclin-dependent kinase (CDK) inhibitor gene p21 (p21(CIP1/WAF1), CDKN1A) and p16 (p16(INK4A), CDKN2A). Chromatin immuno-precipitation analysis reveals that the p21 gene is a direct target of PRMT6 and the corresponding histone mark H3 R2me2a. Using a cell model of oncogene-induced senescence (OIS), in which p21 is an essential activator of the senescent phenotype, we show that PRMT6 expression declines upon induction of senescence and conversely p21 gene expression increases. Moreover, overexpression of PRMT6 leads to reduced levels of OIS. These findings indicate that the transcriptional repressor activity of PRMT6 facilitates cell proliferation and blocks senescence by regulation of tumor suppressor genes and that this might contribute to the oncogenic capacity of PRMT6.