In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.     

Motor-mediated Cortical versus Astral Microtubule Organization in Lipid-monolayered Droplets

The correct spatial organization of microtubules is of crucial importance for determining the internal architecture of eukaryotic cells. Microtubules are arranged in space by a multitude of biochemical activities and by spatial constraints imposed by the cell boundary. The principles underlying the establishment of distinct intracellular architectures are only poorly understood. Here, we studied the effect of spatial confinement on the self-organization of purified motors and microtubules that are encapsulated in lipid-monolayered droplets in oil, varying in diameter from 5–100 μm, which covers the size range of typical cell bodies. We found that droplet size alone had a major organizing influence. The presence of a microtubule-crosslinking motor protein decreased the number of accessible types of microtubule organizations. Depending on the degree of spatial confinement, the presence of the motor caused either the formation of a cortical array of bent microtubule bundles or the generation of single microtubule asters in the droplets. These are two of the most prominent forms of microtubule arrangements in plant and metazoan cells. Our results provide insights into the combined organizing influence of spatial constraints and cross-linking motor activities determining distinct microtubule architectures in a minimal biomimetic system. In the future, this simple lipid-monolayered droplet system characterized here can be expanded readily to include further biochemical activities or used as the starting point for the investigation of motor-mediated microtubule organization inside liposomes surrounded by a deformable lipid bilayer.     

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1''s phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.     

Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at 0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.     

Kar9p Is a Novel Cortical Protein Required for Cytoplasmic Microtubule Orientation in Yeast

kar9 was originally identified as a bilateral karyogamy mutant, in which the two zygotic nuclei remained widely separated and the cytoplasmic microtubules were misoriented (Kurihara, L.J., C.T. Beh, M. Latterich, R. Schekman, and M.D. Rose. 1994. J. Cell Biol. 126:911–923.). We now report a general defect in nuclear migration and microtubule orientation in kar9 mutants. KAR9 encodes a novel 74-kD protein that is not essential for life. The kar9 mitotic defect was similar to mutations in dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5. kar9Δ dhc1Δ, kar9Δ jnm1Δ, and kar9Δ act5Δ double mutants were synthetically lethal, suggesting that these genes function in partially redundant pathways to carry out nuclear migration. A functional GFP-Kar9p fusion protein localized to a single dot at the tip of the shmoo projection. In mitotic cells, GFP-Kar9p localized to a cortical dot with both mother–daughter asymmetry and cell cycle dependence. In small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud. In telophase and G1 unbudded cells, no localization was observed. By indirect immunofluorescence, cytoplasmic microtubules intersected the GFP-Kar9p dot. Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent. We propose that Kar9p is a component of a cortical adaptor complex that orients cytoplasmic microtubules.     

Phosphorylation Regulates Kinase and Microtubule Binding Activities of the Budding Yeast Chromosomal Passenger Complex in Vitro

The chromosomal passenger complex (CPC) is a key regulator of mitosis in eukaryotes. It comprises four essential and conserved proteins known in mammals/yeasts as Aurora B/Ipl1, INCENP/Sli15, Survivin/Bir1, and Borealin/Nbl1. These subunits act together in a highly controlled fashion. Regulation of Aurora B/Ipl1 kinase activity and localization is critical for CPC function. Although regulation of CPC localization and kinase activity in vivo has been investigated elsewhere, studies on the complete, four-subunit CPC and its basic biochemical properties are only beginning. Here we describe the biochemical characterization of purified and complete Saccharomyces cerevisiae four-subunit CPC. We determined the affinity of the CPC for microtubules and demonstrated that the binding of CPC to microtubules is primarily electrostatic in nature and depends on the acidic C-terminal tail (E-hook) of tubulin. Moreover, phosphorylation of INCENP/Sli15 on its microtubule binding region also negatively regulates CPC affinity for microtubules. Furthermore, we show that phosphorylation of INCENP/Sli15 is required for activation of the kinase Aurora B/Ipl1 and can occur in trans. Although phosphorylation of INCENP/Sli15 is essential for activation, we determined that a version of the CPC lacking the INCENP/Sli15 microtubule binding region (residues Glu-91 to Ile-631) is able to form an intact complex that retains microtubule binding activity. Thus, we conclude that this INCENP/Sli15 linker domain plays a largely regulatory function and is not essential for complex formation or microtubule binding.     

Chromosome Separation and Exit from Mitosis in Budding Yeast: Dependence on Growth Revealed by cAMP-Mediated Inhibition

Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.     

Budding Yeast Swe1 Is Involved in the Control of Mitotic Spindle Elongation and Is Regulated by Cdc14 Phosphatase during Mitosis

Cyclin-dependent kinase (Cdk1) activity is required for mitotic entry, and this event is restrained by an inhibitory phosphorylation of the catalytic subunit Cdc28 on a conserved tyrosine (Tyr¹⁹). This modification is brought about by the protein kinase Swe1 that inhibits Cdk1 activation thus blocking mitotic entry. Swe1 levels are regulated during the cell cycle, and they decrease during G₂/M concomitantly to Cdk1 activation, which drives entry into mitosis. However, after mitotic entry, a pool of Swe1 persists, and we collected evidence that it is involved in controlling mitotic spindle elongation. We also describe that the protein phosphatase Cdc14 is implicated in Swe1 regulation; in fact, we observed that Swe1 dephosphorylation in vivo depends on Cdc14 that, in turn, is able to control its subcellular localization. In addition we show that the lack of Swe1 causes premature mitotic spindle elongation and that high levels of Swe1 block mitotic spindle elongation, indicating that Swe1 inhibits this process. Importantly, these effects are not dependent upon the role of in Cdk1 inhibition. These data fit into a model in which Cdc14 binds and inhibits Swe1 to allow timely mitotic spindle elongation.     

Full-Length Synaptonemal Complex Grows Continuously during Meiotic Prophase in Budding Yeast

The synaptonemal complex (SC) links two meiotic prophase chromosomal events: homolog pairing and crossover recombination. SC formation involves the multimeric assembly of coiled-coil proteins (Zip1 in budding yeast) at the interface of aligned homologous chromosomes. However, SC assembly is indifferent to homology and thus is normally regulated such that it occurs only subsequent to homology recognition. Assembled SC structurally interfaces with and influences the level and distribution of interhomolog crossover recombination events. Despite its involvement in dynamic chromosome behaviors such as homolog pairing and recombination, the extent to which SC, once installed, acts as an irreversible tether or maintains the capacity to remodel is not clear. Experiments presented here reveal insight into the dynamics of the full-length SC in budding yeast meiotic cells. We demonstrate that Zip1 continually incorporates into previously assembled synaptonemal complex during meiotic prophase. Moreover, post-synapsis Zip1 incorporation is sufficient to rescue the sporulation defect triggered by SCs built with a mutant version of Zip1, Zip1-4LA. Post-synapsis Zip1 incorporation occurs initially with a non-uniform spatial distribution, predominantly associated with Zip3, a component of the synapsis initiation complex that is presumed to mark a subset of crossover sites. A non-uniform dynamic architecture of the SC is observed independently of (i) synapsis initiation components, (ii) the Pch2 and Pph3 proteins that have been linked to Zip1 regulation, and (iii) the presence of a homolog. Finally, the rate of SC assembly and SC central region size increase in proportion to Zip1 copy number; this and other observations suggest that Zip1 does not exit the SC structure to the same extent that it enters. Our observations suggest that, after full-length assembly, SC central region exhibits little global turnover but maintains differential assembly dynamics at sites whose distribution is patterned by a recombination landscape.     

Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin,a Ubiquitous Actin Monomer-sequestering Protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role.     

Characterization of a Mutation in Yeast Causing Nonrandom Chromosome Loss during Mitosis

Diploid strains of the yeast Saccharomyces cerevisiae homozygous for a recessive chromosome loss mutation (chl) exhibit a high degree of mitotic instability. Cells become monosomic for chromosome III at a frequency of approximately one percent of all cell divisions. Chromosome loss at this high frequency is also found for chromosome I, and at lesser frequencies for chromosomes VIII and XVI. In contrast, little or no chromosome loss is found for six other linkage groups tested (II, V, VI, VII, XI and XVII). The chl mutation also induces a ten-fold increase in both intergenic and intragenic mitotic recombination on all ten linkage groups tested. The chl mutation does not cause an increase in spontaneous mutations, nor are mutant strains sensitive to UV or γ irradiation. The effects of chl during meiosis are observed primarily in reduced spore viability. A decrease in chromosome III linkage relationships is also found.     

The CAF-1 and Hir Histone Chaperones Associate with Sites of Meiotic Double-Strand Breaks in Budding Yeast

In the meiotic prophase, programmed DNA double-strand breaks (DSB) are introduced along chromosomes to promote homolog pairing and recombination. Although meiotic DSBs usually occur in nucleosome-depleted, accessible regions of chromatin, their repair by homologous recombination takes place in a nucleosomal environment. Nucleosomes may represent an obstacle for the recombination machinery and their timely eviction and reincorporation into chromatin may influence the outcome of recombination, for instance by stabilizing recombination intermediates. Here we show in budding yeast that nucleosomes flanking a meiotic DSB are transiently lost during recombination, and that specific histone H3 chaperones, CAF-1 and Hir, are mobilized at meiotic DSBs. However, the absence of these chaperones has no effect on meiotic recombination, suggesting that timely histone reincorporation following their eviction has no influence on the recombination outcome, or that redundant pathways are activated. This study is the first example of the involvement of histone H3 chaperones at naturally occurring, developmentally programmed DNA double-strand breaks.     

The Ecm11-Gmc2 Complex Promotes Synaptonemal Complex Formation through Assembly of Transverse Filaments in Budding Yeast

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation.     

The Anaphase-promoting Complex Promotes Actomyosin-Ring Disassembly during Cytokinesis in Yeast

The anaphase-promoting complex (APC) is a ubiquitin ligase that controls progression through mitosis by targeting specific proteins for degradation. It is unclear whether the APC also contributes to the control of cytokinesis, the process that divides the cell after mitosis. We addressed this question in the yeast Saccharomyces cerevisiae by studying the effects of APC mutations on the actomyosin ring, a structure containing actin, myosin, and several other proteins that forms at the division site and is important for cytokinesis. In wild-type cells, actomyosin-ring constituents are removed progressively from the ring during contraction and disassembled completely thereafter. In cells lacking the APC activator Cdh1, the actomyosin ring contracts at a normal rate, but ring constituents are not disassembled normally during or after contraction. After cytokinesis in mutant cells, aggregates of ring proteins remain at the division site and at additional foci in other parts of the cell. A key target of APC^Cdh1 is the ring component Iqg1, the destruction of which contributes to actomyosin-ring disassembly. Deletion of CDH1 also exacerbates actomyosin-ring disassembly defects in cells with mutations in the myosin light-chain Mlc2, suggesting that Mlc2 and the APC employ independent mechanisms to promote ring disassembly during cytokinesis.