Removal of centrosomal PP1 by NIMA kinase unlocks the MPF feedback loop to promote mitotic commitment in S. pombe.

BACKGROUND: Activation of the Cdk1/cyclin B complex, also known as mitosis-promoting factor (MPF), drives commitment to mitosis. Interphase MPF is inhibited through phosphorylation of Cdk1 by Wee1-related kinases. Because Cdc25 phosphatases remove this phosphate, Cdc25 activity is an essential part of the switch that drives cells into mitosis. The generation of a critical "trigger" of active MPF promotes a positive feedback loop that employs Polo kinase to boost Cdc25 activity and inhibit Wee1, thereby ensuring that mitotic commitment is a bistable switch. Mutations in the spindle pole body (SPB) component Cut12 suppress otherwise lethal deficiencies in Cdc25. RESULTS: Cut12 harbors a bipartite protein phosphatase 1 (PP1) docking domain. Mutation of either element alone suppressed the temperature-dependent lethality of cdc25.22, whereas simultaneous ablation of both allowed cells to divide in the complete absence of Cdc25. Late G2 phase phosphorylation between the two elements by MPF and the NIMA kinase Fin1 blocked PP1(Dis2) recruitment, thereby promoting recruitment of Polo to Cut12 and the SPB and elevating global Polo kinase activity throughout the cell. CONCLUSIONS: PP1 recruitment to Cut12 sets a threshold for Polo's feedback-loop activity that locks the cell in interphase until Cdc25 pushes MPF activity through this barrier to initiate mitosis. We propose that events on the SPB (and, by inference, the centrosome) integrate inputs from diverse signaling networks to generate a coherent decision to divide that is appropriate for the particular environmental context of each cell. PP1 recruitment sets one or more critical thresholds for single or multiple local events within this switch.

Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle.

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.

Septins from the phytopathogenic fungus Ustilago maydis are required for proper morphogenesis but dispensable for virulence.

BACKGROUND: Septins are a highly conserved family of GTP-binding proteins involved in multiple cellular functions, including cell division and morphogenesis. Studies of septins in fungal cells underpin a clear correlation between septin-based structures and fungal morphology, providing clues to understand the molecular frame behind the varied morphologies found in fungal world. METHODOLOGY/PRINCIPAL FINDINGS: Ustilago maydis genome has the ability to encode four septins. Here, using loss-of-function as well as GFP-tagged alleles of these septin genes, we investigated the roles of septins in the morphogenesis of this basidiomycete fungus. We described that septins in U. maydis could assemble into at least three different structures coexisting in the same cell: bud neck collars, band-like structures at the growing tip, and long septin fibers that run from pole to pole near the cell cortex. We also found that in the absence of septins, U. maydis cells lost their elongated shape, became wider at the central region and ended up losing their polarity, pointing to an important role of septins in the morphogenesis of this fungus. These morphological defects were alleviated in the presence of an osmotic stabilizer suggesting that absence of septins affected the proper formation of the cell wall, which was coherent with a higher sensitivity of septin defective cells to drugs that affect cell wall construction as well as exocytosis. As U. maydis is a phytopathogen, we analyzed the role of septins in virulence and found that in spite of the described morphological defects, septin mutants were virulent in corn plants. CONCLUSIONS/SIGNIFICANCE: Our results indicated a major role of septins in morphogenesis in U. maydis. However, in contrast to studies in other fungal pathogens, in which septins were reported to be necessary during the infection process, we found a minor role of septins during corn infection by U. maydis.

Cdk5 kinase regulates the association between adaptor protein Bem1 and GEF Cdc24 in the fungus Ustilago maydis.

Cyclin-dependent kinases from the Cdk5/Pho85 family are thought to play important roles in morphogenesis in species as diverse as yeast and humans. In the phytopathogenic fungus Ustilago maydis Cdk5 has a major role in the maintenance of cell polarity and virulence. This role seems to be related to the ability of the guanine-nucleotide exchange factor (GEF) Cdc24 to localize at the cell tips. However, the elements behind the Cdk5-dependent stabilization of Cdc24 at the cell poles are not well understood. Here we investigate the role of the adaptor protein Bem1 in polarity maintenance in U. maydis. We found that Bem1 and Cdc24 physically interact and colocalize at cell tips and that Cdk5 regulates this interaction. Our data suggest a method by which Cdk5 could regulate polar growth in this phytopathogenic fungus.

Roles of Protein Phosphatase Type 1 in Schizosaccharomyces pombe

Las proteína fosfatasas están consideradas reguladores globales en muchosprocesos biológicos. Se clasifican en distintas familias según su especificidad desustrato, sus mecanismos de catálisis y sus relaciones evolutivas. Los holoenzimasde proteína fosfatasas de tipo 1 (PP1) están compuestos por un pequeño númerode subunidades catalíticas y un amplio número de subunidades reguladoras. Elgenoma de S. pombe codifica dos subunidades catalíticas muy relacionadas, Dis2y Sds21. Hemos fusionado la proteína verde fluorescente «mejorada» (EGFP) alextremo amino-terminal de los genes endógenos de dis2+ y sds21+. Hemos descritoque Dis2 y Sds21 se localizan en distintos compartimentos y estructuras celulares,como los centromeros-kinetocoros, núcleo, un anillo en el ecuador de células endivisión, vesículas endocíticas y extremos celulares. Cada una de estaslocalizaciones sugiere diferentes funciones para las subunidades catalíticas de PP1que interaccionan con distintas proteínas reguladoras. Esto convierte a PP1 en unenzima multifuncional que actúa como un regulador global en muchos procesoscelulares

Protein phosphatases are considered to be global regulators in many biologicalprocesses. They are divided in families on the basis of substrate specificity,mechanisms of catalysis and evolutionary relations. Protein Phosphatase Type 1(PP1) holoenzymes are composed of a small number of catalytic subunits and anarray of regulatory, targeting, subunits. The S. pombe genome encodes two highlyrelated catalytic subunits, Dis2 and Sds21. We fused enhanced green fluorescence protein (EGFP) coding sequences to the aminus-termini of endogenous dis2+ andsds21+ genes. We have described that Dis2 and Sds21 localize in differentcell compartments and structures as centromeres- kinetochores, nucleoli, a ringat the cell equator in dividing cells, endocytic vesicles and the cell tips. Each ofthese locations suggests different functions of a single catalytic PP1 subunitmediated by its interaction with different targeting proteins. This converts PP1into a multifunctional enzyme that acts as a global regulator in many cellularprocesses

Schizosaccharomyces pombe protein phosphatase 1 in mitosis, endocytosis and a partnership with Wsh3/Tea4 to control polarised growth.

PP1 holoenzymes are composed of a small number of catalytic subunits and an array of regulatory, targeting, subunits. The Schizosaccharomyces pombe genome encodes two highly related catalytic subunits, Dis2 and Sds21. The gene for either protein can be individually deleted, however, simultaneous deletion of both is lethal. We fused enhanced green fluorescent protein (EGFP) coding sequences to the 5' end of the endogenous sds21(+) and dis2(+) genes. Dis2.NEGFP accumulated in nuclei, associated with centromeres, foci at cell tips and endocytic vesicles. This actin-dependent endocytosis occurred between nuclei and growing tips and was polarised towards growing tips. When dis2(+) was present, Sds21.NEGFP was predominantly a nuclear protein, greatly enriched in the nucleolus. When dis2(+) was deleted, Sds21.NEGFP levels increased and Sds21.NEGFP was then clearly detected at centromeres, endocytic vesicles and cell tips. Dis2.NEGFP was recruited to cell tips by the formin binding, stress pathway scaffold Wsh3 (also known as Tea4). Wsh3/Tea4 modulates polarised tip growth in unperturbed cell cycles and governs polarised growth following osmotic stress. Mutating the PP1 recruiting RVXF motif in Wsh3/Tea4 blocked PP1 binding, altered cell cycle regulated growth to induce branching, induced branching from existing tips in response to stress, and blocked the induction of actin filaments that would otherwise arise from Wsh3/Tea4 overproduction.

Sustained cell polarity and virulence in the phytopathogenic fungus Ustilago maydis depends on an essential cyclin-dependent kinase from the Cdk5/Pho85 family.

Cyclin-dependent kinases from the Cdk5/Pho85 family are thought to play important roles in morphogenesis in organisms as diverse as yeast and humans. Here we used the corn smut fungus Ustilago maydis to address the role of Cdk5/Pho85 kinases in the morphogenesis and virulence of dimorphic phytopathogens. We found that Cdk5 is essential for growth in U. maydis. A temperature-sensitive cdk5 mutant caused cell wall and morphology defects at the restrictive temperature. Actin patches labeled with a fimbrin-GFP fusion protein were delocalized and a GFP-Myo5 fusion was directed towards the growing cell pole and rapidly dissociated from the tip. These defects were found to be due to an impairment in the maintenance of cell polarity. Our results indicated that Cdk5 is required for the activity of Rac1, probably at the level of the localization of its GEF, Cdc24. Cdk5 was required for full virulence, probably because mutant cells are unable to sustain the dramatic polar growth required for the formation of the infective structures. These results support a major role for morphogenesis in the virulence program of dimorphic fungi.