Cell migration and division in amoeboid-like fission yeast

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.Yeast cells are non-motile and are encased in a cell wall that supports high internal turgor pressure. The cell wall is also essential for cellular morphogenesis and cell division. Here, we report unexpected morphogenetic changes in a Schizosaccharomyces pombe mutant defective in cell wall biogenesis. These cells form dynamic cytoplasmic protrusions caused by internal turgor pressure and also exhibit amoeboid-like cell migration resulting from repeated protrusive cycles. The cytokinetic ring responsible for cell division in wild-type yeast often fails in these cells; however, they were still able to divide using a ring-independent alternative mechanism relying on extrusion of the cell body through a hole in the cell wall. This mechanism of cell division may resemble an ancestral mode of division in the absence of cytokinetic machinery. Our findings highlight how a single gene change can lead to the emergence of different modes of cell growth, migration and division.This work was supported by grants to R.R.D. from the Spanish Ministerio de Ciencia e Inovación BFU2010-21310 and P09-CTS-4697 (Proyecto de Excelencia) from La Junta de Andalucia. I.F.-P. was supported by the Spanish Ministerio de Educación (Juan de la Cierva Program). M.B. was supported by the Spanish Ministerio de Educación (FPI Fellowship, BES-2008-004018). J.Z. was supported by grant P09-CTS-4697.Peer Reviewe

The ceramide synthase subunit lac1 regulates cell growth and size in fission yeast

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.Junta de Andalucía P18-FRJ1132Universidad de Sevilla VIPPIT-2020-I.5Japan Society for the Promotion of Science JP19H02922, JP21K19088Ministerio de Ciencia, Innovación y Universidades BFU2017-89700-

Effects of the microtubule nucleator Mto1 on chromosomal movement, DNA repair, and sister chromatid cohesion in fission yeast.

Although the function of microtubules (MTs) in chromosomal segregation during mitosis is well characterized, much less is known about the role of MTs in chromosomal functions during interphase. In the fission yeast Schizosaccharomyces pombe, dynamic cytoplasmic MT bundles move chromosomes in an oscillatory manner during interphase via linkages through the nuclear envelope (NE) at the spindle pole body (SPB) and other sites. Mto1 is a cytoplasmic factor that mediates the nucleation and attachment of cytoplasmic MTs to the nucleus. Here, we test the function of these cytoplasmic MTs and Mto1 on DNA repair and recombination during interphase. We find that mto1Δ cells exhibit defects in DNA repair and homologous recombination (HR) and abnormal DNA repair factory dynamics. In these cells, sister chromatids are not properly paired, and binding of Rad21 cohesin subunit along chromosomal arms is reduced. Our findings suggest a model in which cytoplasmic MTs and Mto1 facilitate efficient DNA repair and HR by promoting dynamic chromosomal organization and cohesion in the nucleus.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness BFU2011-15216-E, P09-CTS-4697, and PGC2018-099849-B-100 to R.R.D.; National Institutes of Health (NIH) R01, GM067690, and GM115185 to F.C.; and NIH grants R01-GM085145 and R35-GM126910 to S.J

The transcription factor rbf1 is the master regulator for b-mating type controlled pathogenic development in Ustilago maydis

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development.We would like to thank R. Kahmann and the Max-Planck Institute for terrestrial Microbiology, Marburg, for generous support.Peer Reviewe

Caracterizacion de factores especificos del programa de virulencia en Ustilago maydis

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura:20-04-2007The dimorphic fungus Ustilago maydis is the causative agent of corn smut desease. The process of plant invasion involves regulated growth and highly organized morphological changes. Haploid cells (sporidia) of this fungus are unicellular, and grow saprophytically by budding. The pathogenic form, the filamentous dikaryon, is established after fusion of two sporidia that have to harbor different alleles of the a and b mating type loci of U. maydis. The a locus controls the cell fusion via a pheromone-receptor based system. Upon pheromone stimulation cells arrest budding growth and start the formation of conjugation tubes that are cell cycle arrested in the G2 phase. These mating filaments are highly polaryzed structures that undergo directed tip growth towards the pheromone source, followed by cell fusion and the formation of dikaryotic hyphae. The subsequent steps in filament formation and pathogenic development are controlled by the multiallelic b-locus that encodes two distinct homeodomain transcription factors, bE and bW. A heterodimeric complex of the two proteins is formed when they are derived from different alleles. The dikaryon formed after the fusion of compatible sporidia is arrested in the G2 phase of the cell cycle. On the plant surface, filaments differentiate appressoria and penetrate the cuticule. Finally, spores are generated and dispersed by air. Spores will germinate and upon germination, meiosis takes place and pairs of compatible haploid cells are generated. In this work we have identified two new factors involved in pathogenic development. Pcl7 is a cyclin of Cdk5 implicated in morphogenesis during pathogenic development. Δpcl7 cells have defects on polarization during the formation of the conjugative tube and the infective filament. Biz1 (b-dependent zinc finger protein), is a transcriptional regulator that plays a pivotal role during the infection process. Δbiz1 cells show a severe reduction in appressoria formation and plant penetration. In biz1 mutants, when the hypha penetrates, its pathogenic development is arrested directly after plant penetration. pcl7 is upregulated by the induction of the amating type locus and both biz1 and pcl7 are induced via the bmating type locus. High levels of biz1 or pcl7 expression induce highly polarized growth and a G2 cell cycle arrest supporting roles in morphogenesis and cell cycle regulation

Control de la integridad del genoma en condiciones de estrés

Resumen del trabajo presentado al XXXVII Congreso de la Sociedad Española de Genética, celebrado en Torremolinos (Málaga) del 29 de septiembre al 2 de octubre de 2009.Ante cambios ambientales como un choque de temperatura, la presencia de iones o radicales libres en concentracones tóxicas, o la deprivación de nutrientes, las células eucariotas activan rutas de respuesta a estrés que las protegen de dichas agresiones. En general, una vez que las rutas de estrés son activadas, la señal es transducida y amplificada por medio de proteínas kinasas, para concluir en una respuesta transcripcional y/o en la producción de determinados metabolitos celulares que van a permitir la adaptación y supervivencia celular en la nueva situación que indujo la respuesta. La progresión por el ciclo celular está condicionada por la activación de las rutas de estrés, de modo que, por ejemplo, ante un estrés térmico las células retrasan la transición G2-M hasta que se han adaptado a las nuevas condiciones. En la levadura de fisión Schizosaccharomyces pombe, existen dos rutas principales de respuesta a estrés conocidas como SAPK (stress activated protein kinase pathway) y CIP (cell integrity pathway). Ambas rutas son activadas ante un gran número de condiciones adversas. La progresión por el ciclo celular se puede comprometer si las células son tratadas con drogas que despolimerizan los microtúbulos (MTs). Los MTs son polímeros lineales de alfa y beta tubulina que conforman el huso mitótico, que es la estructura celular encargada de la segregación equitativa de los cromosomas durante la mitosis. Cuando el citoesqueleto de MTs se altera durante mitosis, las células no capturan adecuadamente los cromosomas y esto resulta en la generación de aneuploidias y, en el caso de determinados tumores, en un aumento de la malignidad de los mismos. En nuestro laboratorio realizamos la observación de que células de S. pombe sometidas a diferentes tipos de estrés sobrevivían mucho mejor a la despolimerización de los microtúbulos, sugiriendo que la célula en condiciones de estrés, además de producir una respuesta transcripcional o metabólica para adaptarse al mismo, posee mecanismos que vigilan/protegen la integridad de su genoma en estas circunstancias.Peer reviewe

The XMAP215 Ortholog Alp14 Promotes Microtubule Nucleation in Fission Yeast

The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity in vitro, it is not yet clear whether these proteins participate in MT nucleation in vivo. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, the XMAP215 ortholog Alp14 is critical for efficient MT nucleation in vivo. In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but not plus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor in vivo. We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT.This work was supported by NIH grantsR01GM069670 andR01GM115185 (to F.C.) and by anMEC/Fulbright postdoctoral award (FMECD-2011/6545641100), the Andalucía Talent Hub Program/Marie Skłodowska-Curie actions (COFUND; grant agreement no. 291780), and funding from the research program of Universidad Pablo de Olavide (to I.F.-P.)

Cell migration and division in amoeboid-like fission yeast

Summary Yeast cells are non-motile and are encased in a cell wall that supports high internal turgor pressure. The cell wall is also essential for cellular morphogenesis and cell division. Here, we report unexpected morphogenetic changes in a Schizosaccharomyces pombe mutant defective in cell wall biogenesis. These cells form dynamic cytoplasmic protrusions caused by internal turgor pressure and also exhibit amoeboid-like cell migration resulting from repeated protrusive cycles. The cytokinetic ring responsible for cell division in wild-type yeast often fails in these cells; however, they were still able to divide using a ring-independent alternative mechanism relying on extrusion of the cell body through a hole in the cell wall. This mechanism of cell division may resemble an ancestral mode of division in the absence of cytokinetic machinery. Our findings highlight how a single gene change can lead to the emergence of different modes of cell growth, migration and division

Biz1, a Zinc Finger Protein Required for Plant Invasion by Ustilago maydis, Regulates the Levels of a Mitotic Cyclin

Plant invasion by pathogenic fungi involves regulated growth and highly organized fungal morphological changes. For instance, when the smut fungus Ustilago maydis infects maize (Zea mays), its dikaryotic infective filament is cell cycle arrested, and appressoria are differentiated prior to plant penetration. Once the filament enters the plant, the cell cycle block is released and fungal cells begin proliferation, suggesting a tight interaction between plant invasion and the cell cycle and morphogenesis control systems. We describe a novel factor, Biz1 (b-dependent zinc finger protein), which has two Cys(2)His(2) zinc finger domains and nuclear localization, suggesting a transcriptional regulatory function. The deletion of biz1 shows no detectable phenotypic alterations during axenic growth. However, mutant cells show a severe reduction in appressoria formation and plant penetration, and those hyphae that invade the plant arrest their pathogenic development directly after plant penetration. biz1 is induced via the b-mating–type locus, the key control instance for pathogenic development. The gene is expressed at high levels throughout pathogenic development, which induces a G2 cell cycle arrest that is a direct consequence of the downregulation of the mitotic cyclin Clb1. Our data support a model in which Biz1 is involved in cell cycle arrest preceding plant penetration as well as in the induction of appressoria

Polar Growth in the Infectious Hyphae of the Phytopathogen Ustilago maydis Depends on a Virulence-Specific Cyclin[W]

The maize smut fungus Ustilago maydis switches from yeast to hyphal growth to infect maize (Zea mays) plants. This switching is promoted by mating of compatible cells and seems to be required for plant penetration. Although many genes distinctively expressed during this dimorphic switch have been identified and shown to be essential for the infection process, none seems to be explicitly required for polar growth control. Here, we report the characterization of pcl12, encoding a cyclin that interacts specifically with Cdk5, an essential cyclin-dependent kinase with regulatory roles in morphogenesis in U. maydis. Pcl12 fulfills the requirements to be a virulence-specific regulator of polar growth in U. maydis. First, pcl12 expression is induced during the pathogenic development. Secondly, Pcl12 is sufficient to induce hyperpolarized growth in U. maydis cells, as haploid cells overexpressing pcl12 in axenic conditions produce filaments that were morphologically indistinguishable from those produced during the infection process. Finally, cells defective in pcl12 showed impaired polar growth during the formation of the b-dependent filament, the induction of the conjugation tubes, or the formation of a promycelium in spore germination. However, in spite of this pivotal role during morphogenesis, pcl12 mutants were virulent. We discuss the implications of these results for the role of polar growth during the infection process