Generation of temperature sensitive mutations with error-prone PCR in a gene encoding a component of the spindle pole body in fission yeast.

Temperature-sensitive (ts) mutants provide powerful tools for investigation of cellular functions of essential genes. We report here asimple procedure to generate ts mutations using error-prone PCR within pcp1 that encodes aspindle pole body (SPB) component in Schizosaccharomyces pombe. This manipulation is not restricted to pcp1, and can be suited to any essential genes involved in other processes.

The exocyst subunit Sec3 is regulated by a protein quality control pathway.

Exocytosis involves fusion of secretory vesicles with the plasma membrane, thereby delivering membrane proteins to the cell surface and releasing material into the extracellular space. The tethering of the secretory vesicles before membrane fusion is mediated by the exocyst, an essential phylogenetically conserved octameric protein complex. Exocyst biogenesis is regulated by several processes, but the mechanisms by which the exocyst is degraded are unknown. Here, to unravel the components of the exocyst degradation pathway, we screened for extragenic suppressors of a temperature-sensitive fission yeast strain mutated in the exocyst subunit Sec3 (sec3-913). One of the suppressing DNAs encoded a truncated dominant-negative variant of the 26S proteasome subunit, Rpt2, indicating that exocyst degradation is controlled by the ubiquitin-proteasome system. The temperature-dependent growth defect of the sec3-913 strain was gene dosage-dependent and suppressed by blocking the proteasome, Hsp70-type molecular chaperones, the Pib1 E3 ubiquitin-protein ligase, and the deubiquitylating enzyme Ubp3. Moreover, defects in cell septation, exocytosis, and endocytosis in sec3 mutant strains were similarly alleviated by mutation of components in this pathway. We also found that, particularly under stress conditions, wild-type Sec3 degradation is regulated by Pib1 and the 26S proteasome. In conclusion, our results suggest that a cytosolic protein quality control pathway monitors folding and proteasome-dependent turnover of an exocyst subunit and, thereby, controls exocytosis in fission yeast.

Fission yeast sec3 bridges the exocyst complex to the actin cytoskeleton.

The exocyst complex tethers post-Golgi secretory vesicles to the plasma membrane prior to docking and fusion. In this study, we identify Sec3, the missing component of the Schizosaccharomyces pombe exocyst complex (SpSec3). SpSec3 shares many properties with its orthologs, and its mutants are rescued by human Sec3/EXOC1. Although involved in exocytosis, SpSec3 does not appear to mark the site of exocyst complex assembly at the plasma membrane. It does, however, mark the sites of actin cytoskeleton recruitment and controls the organization of all three yeast actin structures: the actin cables, endocytic actin patches and actomyosin ring. Specifically, SpSec3 physically interacts with For3 and sec3 mutants have no actin cables as a result of a failure to polarize this nucleating formin. SpSec3 also interacts with actin patch components and sec3 mutants have depolarized actin patches of reduced endocytic capacity. Finally, the constriction and disassembly of the cytokinetic actomyosin ring is compromised in these sec3 mutant cells. We propose that a role of SpSec3 is to spatially couple actin machineries and their independently polarized regulators. As a consequence of its dual role in secretion and actin organization, Sec3 appears as a major co-ordinator of cell morphology in fission yeast.

Crystal structure and Hirshfeld analysis of trans-di-iodido-bis-[(methyl-sulfan-yl)benzene-κS]platinum(II).

The title complex, [PtI2(C7H8I2)2], represents a further example of a square-planar PtII-di-thio-ether complex. It crystallizes in the monoclinic space group P21/c. Additional Hirshfeld analyses indicate a C-H⋯π inter-action along the [010] axis to be the most important packing factor.

Silver-based coordination polymers assembled by dithioether ligands: potential antibacterial materials despite received ideas.

We report on the first examples on the antibacterial activity towards Gram-negative and Gram-positive bacteria of 2D silver-based coordination polymers obtained by self-assembly with acetylenic dithioether ligands. Their structure imparts a good stability that allows a sustainable release of Ag+ in the media.

[µ-Bis(diphenyl-phosphan-yl)methane]-tricarbon-yl(µ-p-toluene-sulfonyl-meth-yl isocyanato)(triphenyl-phosphane)ironplatinum(Fe-Pt).

The title compound, [FePt(C(9)H(9)NO(2)S)(C(18)H(15)P)(C(25)H(22)P(2))(CO)(3)], represents a rare example of an isonitrile-bridged heterobimetallic complex (here Pt and Fe) and is an inter-esting precursor for the preparation of heterodinuclear µ-amino-carbyne complexes, since the basic imine-type N atom of the µ(2)-C=N-R ligand readily undergoes addition with various electrophiles to afford iminium-like salts. In the crystal, the almost symmetrically bridging µ(2)-C=N-R ligand (neglecting the different atomic radii of Fe and Pt) is strongly bent towards the Fe(CO)(3) fragment, with a C=N-R angle of only 121.1 (4)°.

Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain.

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.

Identification of a conserved F-box protein 6 interactor essential for endocytosis and cytokinesis in fission yeast.

The F-box domain is a degenerated motif consisting of approximately 40 amino acid residues that specifically bind Skp1, a core component of the SCF (Skp1-Cdc53/Cullin 1-F-box protein) ubiquitin ligase. Recent work, mainly performed in budding yeast, indicates that certain F-box proteins form non-SCF complexes together with Skp1 in the absence of cullins and play various roles in cell cycle and signalling pathways. However, it is not established whether these non-SCF complexes are unique to budding yeast or common in other eukaryotes. In the present paper, using TAP (tandem affinity purification) coupled to MudPIT (Multidimensional Protein Identification Technology) analysis, we have identified a novel conserved protein, Sip1, in fission yeast, as an interacting partner of an essential F-box protein Pof6. Sip1 is a large HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor)-repeats containing protein (217 kDa) and forms a complex with Pof6 and Skp1. This complex does not contain cullins, indicating that it is a novel non-SCF complex. Like Pof6 and Skp1, Sip1 is essential for cell viability and temperature-sensitive sip1 mutants display cell division arrest as binucleate cells with septa. Sip1 localizes to the nucleus and dynamic cytoplasmic dots, which are shown in the present study to be endocytic vesicles. Consistent with this, sip1 mutants are defective in endocytosis. Furthermore, towards the end of cytokinesis, constriction of the actomyosin ring and dissociation of type II myosin and septum materials are substantially delayed in the absence of functional Sip1. These results indicate that the conserved Sip1 protein comprises a novel non-SCF F-box complex that plays an essential role in endocytosis, cytokinesis and cell division.

The dynamin related protein Dnm1 fragments mitochondria in a microtubule-dependent manner during the fission yeast cell cycle.

Mitochondria are dynamic organelles that undergo cycles of fission and fusion. In the fission yeast, Schizosaccharomyces pombe, mitochondria align with microtubules and mitochondrial integrity is dependent upon an intact microtubule cytoskeleton. Here we show that mitochondria re-organize during the cell cycle and that this process is both dynamin- and microtubule-dependent. Microtubule depolymerization results in mitochondrial fragmentation but only when the dynamin-related protein Dnm1 is present. Mitochondrial fusion is, on the other hand, microtubule-independent. dnm1Delta cells, besides showing extensively fused mitochondria, are specifically resistant to anti-microtubule drugs. Dnm1-YFP localizes to foci at sites of mitochondrial severing which occupy the interface between adjacent nucleoids, suggesting the existence of defined mitochondrial "territories," each of which contains a nucleoid. Such territories are lost in dnm1Delta in which nucleoids become aggregated. Mitochondrial ends exhibit motile behavior, extending towards and retracting from the cell poles, independently of the cytoskeleton. We conclude that: (a) mitochondria are organized by microtubules in fission yeast but are not moved by them; (b) Dnm1 mediates mitochondrial fission during interphasic growth and at cell division; (c) the interaction between microtubules and mitochondria, either directly or indirectly via Dnm1, not only modifies the disposition of mitochondria it also modifies the behavior of microtubules. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Crystal structure of {µ2-1,2-bis-[(4-methyl-phenyl-sulfan-yl]-3-oxoprop-1-ene-1,3-di-yl-1:2κ2 C 3:C 1}dicarbon-yl-1κ2 C-[µ2-methyl-enebis(di-phenyl-phos-phane)-1:2κ2 P:P'](tri-phenyl-phosphane-2κP)iron-platinum(Fe-Pt), [(OC)2Fe(µ-dppm){µ-C(=O)C(4-MeC6H4SCH2)=CCH2SC6H4Me-4}Pt(PPh3)].

The title compound, [FePt(C19H18OS2)(C18H15P)(C25H22P2)(CO)2], 1, [(OC)2Fe(µ-dppm)(µ-C(=O)C(CH2SC6H4Me-4)=CCH2SC6H4Me-4)Pt(PPh3)], represents the first example of a diphosphane-bridged heterobimetallic Fe-Pt dimetalla-cyclo-pentenone complex resulting from a bimetallic activation of metal-coordinated carbonyl ligand with an inter-nal alkyne, namely 1,4-bis-(p-tolyl-thio)-but-2-yne. The bridging µ2-C(=O)C(CH2SC6H4Me-4)=CCH2SC6H4Me-4 unit (stemming from a carbon-carbon coupling reaction between CO and the triple bond of the alkyne di-thio-ether) forms a five-membered dimetalla-cyclo-pentenone ring, in which the C=C bond is π-coordinated to the Fe center. The latter is connected to the Pt center through a short metal-metal bond of 2.5697 (6) Å.

Mutations in a Single Signaling Pathway Allow Cell Growth in Heavy Water.

Life is completely dependent on water. To analyze the role of water as a solvent in biology, we replaced water with heavy water (D2O) and investigated the biological effects by a wide range of techniques, using Schizosaccharomyces pombe as model organism. We show that high concentrations of D2O lead to altered glucose metabolism and growth retardation. After prolonged incubation in D2O, cells displayed gross morphological changes, thickened cell walls, and aberrant cytoskeletal organization. By transcriptomics and genetic screens, we show that the solvent replacement activates two signaling pathways: (1) the heat-shock response pathway and (2) the cell integrity pathway. Although the heat-shock response system upregulates various chaperones and other stress-relieving enzymes, we find that the activation of this pathway does not offer any fitness advantage to the cells under the solvent-replaced conditions. However, limiting the D2O-triggered activation of the cell integrity pathway allows cell growth when H2O is completely replaced with D2O. The isolated D2O-tolerant strains may aid biological production of deuterated biomolecules.

The dynamin-related protein Vps1 regulates vacuole fission, fusion and tubulation in the fission yeast, Schizosaccharomyces pombe.

Fission yeast cells lacking the dynamin-related protein (DRP) Vps1 had smaller vacuoles with reduced capacity for both fusion and fission in response to hypotonic and hypertonic conditions respectively. vps1Delta cells showed normal vacuolar protein sorting, actin organisation and endocytosis. Over-expression of vps1 transformed vacuoles from spherical to tubular. Tubule formation was enhanced in fission conditions and required the Rab protein Ypt7. Vacuole tubulation by Vps1 was more extensive in the absence of a second DRP, Dnm1. Both dnm1Delta and the double mutant vps1Delta dnm1Delta showed vacuole fission defects similar to that of vps1Delta. Over-expression of vps1 in dnm1Delta, or of dnm1 in vps1Delta failed to rescue this phenotype. Over-expression of dnm1 in wild-type cells, on the other hand, induced vacuole fission. Our results are consistent with a model of vacuole fission in which Vps1 creates a tubule of an appropriate diameter for subsequent scission by Dnm1.

Crystal structure of dicarbon-yl[µ2-methyl-enebis(di-phenyl-phosphane)-κ2 P:P'][µ2-2-(2,4,5-tri-methyl-phen-yl)-3-oxoprop-1-ene-1,3-di-yl](tri-phenyl-phosphane-κP)ironplatinum(Fe-Pt)-di-chloro-methane-toluene (1/1/2), [(OC)2Fe(µ-dppm)(µ-C(=O)C(2,4,5-C6H2Me3)=CH)Pt(PPh3)].

The title compound, [FePt(C12H12O)(C18H15P)(C25H22P2)(CO)2]·2C7H8·CH2Cl2 or [(OC)2Fe(µ-dppm)(µ-C(=O)C(2,4,5-C6H2Me3)=CH)Pt(PPh3)], represents an example of a diphosphane-bridged heterobimetallic dimetalla-cyclo-pentenone complex resulting from a bimetallic activation of 1-ethynyl-2,4,5-tri-methyl-benzene and a metal-coordinated carbonyl ligand. The bridging µ2-C(=O)C(2,4,5-C6H2Me3)=CH unit (stemming from a carbon-carbon coupling reaction between CO and the terminal alkyne) forms a five-membered dimetalla-cyclo-pentenone ring, in which the C=C bond is π-coordinated to the Fe centre. The latter is connected to the Pt centre through a short metal-metal bond of 2.5770 (5) Å. In the crystal, the complex is solvated by one di-chloro-methane and two toluene mol-ecules.

Expanded Interactome of the Intrinsically Disordered Protein Dss1.

Dss1 (also known as Sem1) is a conserved, intrinsically disordered protein with a remarkably broad functional diversity. It is a proteasome subunit but also associates with the BRCA2, RPA, Csn12-Thp1, and TREX-2 complexes. Accordingly, Dss1 functions in protein degradation, DNA repair, transcription, and mRNA export. Here in Schizosaccharomyces pombe, we expand its interactome further to include eIF3, the COP9 signalosome, and the mitotic septins. Within its intrinsically disordered ensemble, Dss1 forms a transiently populated C-terminal helix that dynamically interacts with and shields a central binding region. The helix interfered with the interaction to ATP-citrate lyase but was required for septin binding, and in strains lacking Dss1, ATP-citrate lyase solubility was reduced and septin rings were more persistent. Thus, even weak, transient interactions within Dss1 may dynamically rewire its interactome.

The Exocyst Complex in Health and Disease.

Exocytosis involves the fusion of intracellular secretory vesicles with the plasma membrane, thereby delivering integral membrane proteins to the cell surface and releasing material into the extracellular space. Importantly, exocytosis also provides a source of lipid moieties for membrane extension. The tethering of the secretory vesicle before docking and fusion with the plasma membrane is mediated by the exocyst complex, an evolutionary conserved octameric complex of proteins. Recent findings indicate that the exocyst complex also takes part in other intra-cellular processes besides secretion. These various functions seem to converge toward defining a direction of membrane growth in a range of systems from fungi to plants and from neurons to cilia. In this review we summarize the current knowledge of exocyst function in cell polarity, signaling and cell-cell communication and discuss implications for plant and animal health and disease.

A synergistic relationship between three regions of stathmin family proteins is required for the formation of a stable complex with tubulin.

Stathmin is a ubiquitous 17 kDa cytosolic phosphoprotein proposed to play a general role in the integration and relay of intracellular signalling pathways. It is believed to regulate microtubule dynamics by sequestering tubulin in a complex made of two tubulin heterodimers per stathmin molecule (T2S complex). The other proteins of the stathmin family can also bind two tubulin heterodimers through their SLD (stathmin-like domain), but the different tubulin:SLD complexes display varying stabilities. In this study, we analysed the relative influence of three regions of SLDs on the interaction with tubulin and the mechanistic processes that lead to its sequestration. Tubulin-binding properties of fragments and chimaeras of stathmin and RB3(SLD) were studied in vitro by tubulin polymerization, size-exclusion chromatography and surface plasmon resonance assays. Our results show that the N-terminal region of SLDs favours the binding of the first tubulin heterodimer and that the second C-terminal tubulinbinding site confers the specific stability of a given tubulin:SLD complex. Our results highlight the molecular processes by which tubulin co-operatively interacts with the SLDs. This knowledge may contribute to drug development aimed at disturbing microtubules that could be used for the treatment of cancer.

Crystal structure of tricarbon-yl(µ-di-phenyl-phosphido-κ(2) P:P)(methyl-diphenyl-silyl-κSi)bis(tri-phenyl-phosphane-κP)iron(II)platinum(0)(Fe-Pt).

The title compound, [FePt(C12H10P)(C13H13Si)(C18H15P)2(CO)3]·0.5CH2Cl2, represents an example of a phosphido-bridged heterobimetallic silyl complex; these are inter-esting precursors for the coordination and activation of small unsaturated organic mol-ecules. The µ2-PPh2 ligand spans the iron and platinum atoms, which are connected via a metal-metal bond of 2.7738 (4) Å. In contrast to most other complexes of the [(OC)3Fe(SiR 3)(µ-PR 2)PtL 2] family, where the iron-bound SiR 3 group is trans-arranged with respect to the µ2-PPh2 ligand, the SiPh2Me ligand is roughly collinear with the Fe-Pt vector [Si-Fe-Pt = 169.07 (3)°].

Fission yeast Nod1 is a component of cortical nodes involved in cell size control and division site placement.

Most cells enter mitosis once they have reached a defined size. In the fission yeast Schizosaccharomyces pombe, mitotic entry is orchestrated by a geometry-sensing mechanism that involves the Cdk1/Cdc2-inhibiting Wee1 kinase. The factors upstream of Wee1 gather together in interphase to form a characteristic medial and cortical belt of nodes. Nodes are also considered to be precursors of the cytokinesis contractile actomyosin ring (CAR). Here we describe a new component of the interphase nodes and cytokinesis rings, which we named Nod1. Consistent with its role in cell size control at division, nod1Δ cells were elongated and epistatic with regulators of Wee1. Through biochemical and localisation studies, we placed Nod1 in a complex with the Rho-guanine nucleotide exchange factor Gef2. Nod1 and Gef2 mutually recruited each other in nodes and Nod1 also assembles Gef2 in rings. Like gef2Δ, nod1Δ cells showed a mild displacement of their division plane and this phenotype was severely exacerbated when the parallel Polo kinase pathway was also compromised. We conclude that Nod1 specifies the division site by localising Gef2 to the mitotic cell middle. Previous work showed that Gef2 in turn anchors factors that control the spatio-temporal recruitment of the actin nucleation machinery. It is believed that the actin filaments originated from the nodes pull nodes together into a single contractile ring. Surprisingly however, we found that node proteins could form pre-ring helical filaments in a cdc12-112 mutant in which nucleation of the actin ring is impaired. Furthermore, the deletion of either nod1 or gef2 created an un-expected situation where different ring components were recruited sequentially rather than simultaneously. At later stages of cytokinesis, these various rings appeared inter-fitted rather than merged. This study brings a new slant to the understanding of CAR assembly and function.

Dynamin-dependent biogenesis, cell cycle regulation and mitochondrial association of peroxisomes in fission yeast.

Peroxisomes were visualized for the first time in living fission yeast cells. In small, newly divided cells, the number of peroxisomes was low but increased in parallel with the increase in cell length/volume that accompanies cell cycle progression. In cells grown in oleic acid, both the size and the number of peroxisomes increased. The peroxisomal inventory of cells lacking the dynamin-related proteins Dnm1 or Vps1 was similar to that in wild type. By contrast, cells of the double mutant dnm1Delta vps1Delta contained either no peroxisomes at all or a small number of morphologically aberrant organelles. Peroxisomes exhibited either local Brownian movement or longer-range linear displacements, which continued in the absence of either microtubules or actin filaments. On the contrary, directed peroxisome motility appeared to occur in association with mitochondria and may be an indirect function of intrinsic mitochondrial dynamics. We conclude that peroxisomes are present in fission yeast and that Dnm1 and Vps1 act redundantly in peroxisome biogenesis, which is under cell cycle control. Peroxisome movement is independent of the cytoskeleton but is coupled to mitochondrial dynamics.

Functional analysis of a fungal endophyte stress-activated MAP kinase.

The ability of fungi to sense and respond rapidly to environmental stress is crucial for their survival in the wild. One of the most important pathways involved in this response is the stress-activated MAP (mitogen-activated protein) kinase pathway. We report here on the isolation of the stress-activated MAP kinase, sakA, from the fungal endophyte Epichloë festucae. Complementation of the stress sensitivity and cell cycle defects of an Schizosaccharomyces pombe sty1Delta mutant with sakA confirmed it encodes a functional MAP kinase. Analysis of an E. festucae DeltasakA mutant revealed sakA is essential for growth under conditions of temperature and osmotic stress in culture, and for sensitivity to the fungicide fludioxonil. However, the DeltasakA mutant shows no increased sensitivity to hydrogen peroxide. Given sakA can rescue the sty1Delta mutant from sensitivity to oxidative stress, SakA has the potential to sense and transduce oxidative stress signals. The DeltasakA mutant is also defective in conidia formation, suggesting a role for SakA in asexual development of E. festucae. The detection of elevated hydrogen peroxide production in the DeltasakA mutant suggests there may be a link between MAP kinase and ROS (reactive oxygen species) signalling pathways in E. festucae.