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Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclindependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 trimethylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a ‘dNTP supply and demand’ model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

PMCID: PMC5609853, Clathrin facilitates vesicle formation during endocytosis and sorting in the trans-Golgi network (TGN)/endosomal system. Unlike in mammals, yeast clathrin function requires both the clathrin heavy (CHC) and clathrin light (CLC) chain, since Chc1 does not form stable trimers without Clc1. To further delineate clathrin subunit functions, we constructed a chimeric CHC protein (Chc-YR), which fused the N-terminus of yeast CHC (1–1312) to the rat CHC residues 1318–1675, including the CHC trimerization region. The novel CHC-YR allele encoded a stable protein that fractionated as a trimer. CHC-YR also complemented chc1Δ slow growth and clathrin TGN/endosomal sorting defects. In strains depleted for Clc1 (either clc1Δ or chc1Δ clc1Δ), CHC-YR, but not CHC1, suppressed TGN/endosomal sorting and growth phenotypes. Chc-YR-GFP (green fluorescent protein) localized to the TGN and cortical patches on the plasma membrane, like Chc1 and Clc1. However, Clc1-GFP was primarily cytoplasmic in chc1Δ cells harboring pCHC-YR, indicating that Chc-YR does not bind yeast CLC. Still, some partial phenotypes persisted in cells with Chc-YR, which are likely due either to loss of CLC recruitment or chimeric HC lattice instability. Ultimately, these studies have created a tool to examine non-trimerization roles for the clathrin LC., This work was supported primarily by the National Institute of Health grants to SKL (R01-GM055796), DRB (F32-GM084677) and an institutional training grant supporting VAS (T32-HL07188). Further support was provided by the American Cancer Society fellowship to JC (FL Summer Research Fellowship), American Heart Association Postdoctoral fellowship to JRC (6-6253Y), and a European Molecular Biology Organization Short Term Fellowship to ND (EMBO145-2012).

In metazoans the AP-2 complex has a well-defined role in clathrin-mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co- immunoprecipitation between the fission yeast AP-2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4-64 uptake. In an otherwise wild-type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP-2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP-2 in endocytosis has been conserved throughout evolution., Financial support from the Ministerio de Economía y Competitividad (Spain)/European Union FEDER program (grants BFU2010-15085 and BFU2013-48582-C2-2-P) and Junta de Castilla y León (grant SA073U14) made this work possible. NdL, MAC and SMR were supported by FPU fellowships from the Spanish Ministry of Education; MH and FY were supported by JAE-PREDOC CSIC and USAL fellowships,respectively.

Resumen del póster presentado al Young Life Scientists’ Symposium: "Cell cycle: the challenge to coordinate genome stability and morphogenesis", celebrado en Salamanca (España) el 4 de diciembre del 2015., Intracellular protein transport requires the generation and traffic of coated vesicles that originate in, and fuse to organelle membranes. The correct timing and directionality of this transport is guaranteed by guanosine tri-phosphate hydrolases (GTPases) nd phosphoinositides, which contribute to organelle identity. In metazoa, Arf6p participates in clathrin-mediated and clathrin-independemt endocytosis, and has been suggested to participate in traffic through endosomes. On the contrary, no direct evidence of the involvement of this GTPase in endocytosis has been found in unciellular eukaryotes. we have found that in fission yeast arf6Δ mutants interact genetically with mutants in clc1+ and end4+, and that the absence of Arf6p produces a defect in the behavior of the endocytic patches. Additionally, apl3Δ (defective in the clathrin adaptor AP2) and arf6Δ mutants are defective in cell wall synthesis, sterol distribution at the plasma membrane, and polarity. All phenotypes are severly aggravated in double apl3Δ arf6Δ mutant. The severity of these phenotypes correlates with the defect in the dynamics of the endocytic patches. These results show that the participation of Arf6p in endocytosis has been conserved along evolution, and suggest that Arf6p might play AP2-independent roles in vesicle trafficking and morphogenesis. As a tool for the future analysis of these roles, several fluorescent phosphoinositide-binding probes have been constructed. Also GFP-tagged phosphoinositide kinases and phosphatases are under construction.

Resumen del póster presentado al Biochemical Society Meeting: "Organelle Crosstalk in Membrane Dynamics and Cell Signalling", celebrado en Edimburgo (UK) del 26 al 29 de octubre de 2015., The term exomer refers to a Saccharomyces cerevisiae Golgi protein complex that is required for the delivery of certain proteins to the plasma membrane. The bestcharacterized cargo is the chitin synthase Chs3p. Schizosaccharomyces pombe Cfr1p and Bch1p are proteins similar to some of the exomer components. In this yeast, there is no homologue to Chs3p and βglucans are the main cell wall components. Cfr1p and Bch1p co-immunoprecipitate and co-localize. Cfr1 co-localizes with Sec72 but not with Anp1p. Additionally, 30% of Cfr1p co-fractionates with prevacuolar and vacuole markers. We have found co-immunoprecipitation with AP-1 and AP-2 components, and genetic interaction with mutants in the AP-1, AP-2, and GGA clathrin adaptors. No relationship with AP-3, COPI and COPII components has been found. In the absence of apm1+, the βglucan synthase Bgs1p accumulates in structures that might correspond to early endosomes. We have found similar results for other glucan synthases. In double cfr1Δ apm1Δ mutants this abnormal accumulation is enhanced. These aggregates are partially resolved by latrunculin in the apm1Δ but not in the cfr1Δ apm1Δ mutant. These results show that the fission yeast exomer plays a pervasive role in the traffic between the Golgi and the endosomal and prevacuolar compartments.

Resumen del póster presentado al Young Life Scientists’ Symposium: "Cell cycle: the challenge to coordinate genome stability and morphogenesis", celebrado en Salamanca (España) el 4 de diciembre del 2015., In the budding yeast Saccharomyces cerevisiae a protein complex termed exomer has been shown to be required for the delivery of certain transmembrane proteins to the cell surface. The exomer is composed od Chs5p and four ChAPs (Chs5 and Arf-bindinig proteins). Chs5p is reuired for the localization of the complex at the Trans-Golgi-Network (TGN). The best characterized cargo is the chitin synthase Chs3p. In Schizosaccharomyces pombe there are no homologues to Chs3p and glucans are the main components of the cell wall. In this fission yeast Cfr1p is similar to Chs5p, and Bch1p is the only protein similar tl the ChAPs. Cfr1p and Bch1p co-immunoprecipitate, co-localize, and are interdependent for their localization. Cfr1 co-localizes with Sec72 but not with Anp1p. Additionally, 30% of Cfr1p co-fractionates with prevacuolar compartment (PVC) and vacuole markers. We have found co-immunoprecipitation between Cfr1p and AP-1 and AP-2 components, and genetic interaction with mutants in the AP-1, AP-2, and GGA clathrin adaptors. No relationship with AP-3, COPI and COPII components has been found. In cfr1Δ mutants, co-migration of the β(1,3)glucan synthases Bgs1p and Bgs4p accumulates in structures that might correspond to early endosomes, and in the double cfr1Δ apm 1Δ mutants this abnormal accumulation is enhanced. While these aggregates are partially resolved by latrunculin in the apm1Δ mutant, they are not resolved in the cfr1Δ apm 1Δ mutant. Finally, in the cfr1Δ, bch1Δ, and cfr1Δ bch1Δ strains, the vacuolar carboxypeptidase Y is missorted to the cell surface. These results show that the fission yeast exomer plays a pervasive role in the traffic between the Golgi and the endosomal and prevacuolar compartments.

Memoria presentada por la licenciada Dña. Nagore Tamara De León Marcos para optar al grado de Doctor en Bioquímica por la Universidad de Salamanca, ha sido realizada en el Centro Mixto Instituto de Biología Funcional y Genómica, CSIC/Universidad de Salamanca (Departamento de Micro-biología y Genética)., La pared celular es un elemento morfogenético esencial que determina la forma final de las células y que las protege contra la lisis. En S. pombe ésta está constituida por ¿ y ß-glucano y manoproteínas y tanto la síntesis como remodelación de su estructura requiere de diferentes enzimas estrictamente reguladas. En S. pombe existe poca información de cómo se lleva a cabo la incorporación del material de membrana y sobre la regulación de las enzimas implicadas en la síntesis y remodelación de la pared celular por los mecanismos de transporte vesicular. Para abordar el estudio de cómo el tráfico vesicular mediado por clatrina afecta a la morfogénesis de S. pombe y en particular cuál es su papel en la regulación de la síntesis de la pared celular se ha analizado el papel tanto de la clatrina, mediante el análisis de diferentes mutantes de la cadena ligera de la clatrina, como el del adaptador AP-2, que interviene en el proceso de endocitosis mediada por clatrina. Se ha demostrado que la deleción de la cadena ligera de la clatrina resulta letal para las células de S. pombe y que esta letalidad se rescata al incubar las células en un medio suplementado con sorbitol. En este caso aunque las células pueden sobrevivir poseen graves defectos morfológicos, en crecimiento, en tráfico vesicular, en desarrollo sexual, etc., Se ha podido comprobar que la ausencia de Clc1p afecta drásticamente a la estabilidad de Chc1p hecho que hace que, a diferencia de otros organismos, la supervivencia de S. pombe sea más dependiente de la presencia clatrina. Además se ha demostrado que la letalidad causada por la ausencia de Clc1p se debe principalmente a defectos graves en la síntesis de la pared celular que afectan directamente a la síntesis del glucano. Los resultados obtenidos muestran que una reducción en la cantidad de clatrina causa un leve impacto en el transporte vesicular en general y en otros procesos y elementos biológicos, pero afecta gravemente a la secreción de enzimas de síntesis/remodelación de la pared celular, como las ß(1,3)glucán sintasa y endoglucanasas. En cuanto al complejo adaptador AP-2 se ha comprobado, que a diferencia de lo que se conoce hasta el momento en otros organismos unicelulares, éste forma un complejo con la clatrina y se ha demostrado que tiene un papel en la endocitosis general de S. pombe. Así mismo se ha descubierto que AP-2 puede estar interviniendo en la síntesis de la pared celular ya que su ausencia afecta a la actividad ß-glucán sintasa y hace que S. pombe sea hiper-sensible a compuestos que afectan a la integridad de la pared celular., Esta Tesis Doctoral ha sido realizada gracias a la financiación de una beca predoctoral del Programa de Formación del Profesorado Universitario (2009/2013) del Ministerio de Educación del Gobierno de España, proyectos del Plan Nacional de I+D (MICINN/Programa FEDER) (BFU2007-61866 y BFU2010-15085), el proyecto de la Junta de Castilla y León GR231 y la Fundación Ramón Areces.

Resumen del póster presentado a la EMBO Conference on Fission Yeast: Pombe 2013; 7th International Fission Yeast Meeting celebrada en Londres (UK) del 24 al 29 de junio de 2013., One of the most attractive areas of Biology is the knowledge of how organisms generate and maintain their shape. In fungi, the cell wall is an essential morphogenetic element, since it is the last organelle responsible for the maintenance of the cellular shape. Our objective is to study how the biosynthesis of β-glucan, an essential component of the cell wall, is regulated by the mechanisms of vesicular transport. In this work we have addressed the study of the regulation of the synthesis of this polymer by the Cfr1/Csr1p complex. Cfr1p and Csr1p share similarity with the exomer proteins from S. cerevisiae. The term exomer refers to a set of proteins that form a vesicle coat required for the transport of certain proteins from the trans-Golgi network to the plasma membrane. The exomer is formed by a scaffold protein, Chs5p, to which Chs6p and its homologues (Bch1p, Bud7p and Bch2p, termed generically “ChAPs”) anchor. The ChAPs are responsible for cargo specificity. S. pombe Cfr1p shares similarity with Chs5p and Csr1p is the only protein similar to the ChAPs. Previous studies have shown that Cfr1p localizes at the Golgi and is required for cell fusion during mating, a function that shares with Chs5p. We have cloned csr1+ and have found that Cfr1p and Csr1p form a complex at the Golgi and that the localization of each protein in this organelle depends on each other. Additionally, cfr1Δ and csr1Δ mutants are sensitive to compounds that interfere with cell wall synthesis, interact genetically with mutants in the β(1,3)glucan synthase, and have a reduced level of β-glucan in their cell wall. Our goal is to study the role that these proteins might play in vesicular transport, and to determine how they regulate cell wall synthesis and morphogenesis.

Resumen del póster presentado a la EMBO Conference on Fission Yeast: Pombe 2013; 7th International Fission Yeast Meeting celebrada en Londres (UK) del 24 al 29 de junio de 2013., The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization, while in other organisms as Saccharomyces cerevisiae and Dictyostelium discoideum clc1Δ mutants are viable in all the conditions tested. Why this difference is? When grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodeling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis and perhaps, this is one of the main reasons why clc1 gen deletion is lethal in S. pombe.

Comunicación presentada a la "8ª Reunión de la Red Española de Levaduras" celebrada en El Escorial del 14 al 16 de diciembre de 2011.

10 páginas, 5 figuras.-- El pdf del artículo es la versión pre-print., In this paper we show that in Schizosaccharomyces pombe, mating-specific cell adhesion is dependent on the exocyst subunit Sec8p, but independent of the exocyst subunit Exo70p. In the absence of Exo70p, the forespore membrane does not develop properly and the leading edge protein Meu14p is abnormally distributed. Additionally, the spindle pole body is aberrant in a significant number of exo70Δ asci. In both the sec8-1 and the exo70Δ mutants, the development of the spore cell wall is impaired. These results show that different steps of sexual development are differentially regulated by the exocyst and suggest the existence of exocyst subcomplexes with distinct roles in mating., This work has been supported by grants BFU2007-61866 from the CICYTand GR231 from the Junta de Castilla y León, Spain. M.R.S. and N.d.L. were supported by fellowships from the Iranian and Spanish Ministry of Science, respectively.