AMS and EBSD maps of rock salt - lamprophyre dyke contact zone, Loule diapir, Algarve basin, Portugal

A rock salt-lamprophyre dyke contact zone (sub-vertical, NE-SW strike) was investigated for its petrographic, mechanic and physical properties by means of anisotropy of magnetic susceptibility (AMS) and rock magnetic properties, coupled with quantitative microstructural analysis and thermal mathematical modelling. The quantitative microstructural analysis of halite texture and solid inclusions revealed good spatial correlation with AMS and halite fabrics. The fabrics of both lamprophyre and rock salt record the magmatic intrusion, "plastic" flow and regional deformation (characterized by a NW-SE trending steep foliation). AMS and microstructural analysis revealed two deformation fabrics in the rock salt: (1) the deformation fabrics in rock salt on the NW side of the dyke are associated with high temperature and high fluid activity attributed to the dyke emplacement; (2) On the opposite side of the dyke, the emplacement-related fabric is reworked by localized tectonic deformation. The paleomagnetic results suggest significant rotation of the whole dyke, probably during the diapir ascent and/or the regional Tertiary to Quaternary deformation.

Identificación y evaluación de la creación de valor mediante la implantación y desarrollo de políticas de responsabilidad social corporativa estratégica

La Responsabilidad Social Corporativa (RSC) sigue constituyendo en la actualidad un área de estudio de elevado interés tanto para la comunidad académica como para los negocios en general. A pesar del gran número de investigaciones realizadas en las pasadas décadas sobre los distintos aspectos que la caracterizan, y la definición generalizada de políticas relacionadas en las compañías más importantes, existen todavía algunos asuntos clave sobre los que se plantean interrogantes fundamentales. La complejidad asociada al constructo RSC y su carácter intrínsecamente dinámico explican en parte esta afirmación. En su aplicación práctica, las dudas sobre la RSC se enfocan hoy en día hacia su implantación con carácter permanente en el día a día de las organizaciones, la relevancia estratégica de las principales iniciativas, o la posibilidad de obtención de beneficios a medio y largo plazo. Se observa de esta forma la traslación de los debates principales hacia las consecuencias más estratégicas de dichas políticas, influenciados por prestigiosos estudios académicos en los que se caracteriza la denominada RSC Estratégica (RSCE), y por las principales organizaciones de certificación de memorias anuales de RSC y sostenibilidad. En este contexto se sitúa el objeto principal de esta investigación, consistente en el diseño de un modelo de implantación de RSCE que permita no sólo identificar los factores más importantes a tener en consideración para su éxito, sino para caracterizar las potenciales formas de creación de valor que pueden surgir de la aplicación del mismo. Se argumenta la elección del tema por considerarse que los asuntos asociados a la RSC no están lo suficientemente explorados desde la visión estratégica más actual, y por constituir la creación de valor el objetivo más crítico dentro de los procesos directivos de planificación estratégica. De esta forma, se utilizan dos metodologías para destacar qué factores son esenciales en la implantación de la RSCE, con qué fines las compañías aplican esas políticas, y qué resultados obtienen como consecuencia: análisis comparativo de casos de estudio y análisis estadístico cuantitativo. Los casos de estudio analizan en profundidad políticas globales de RSCE bajo diferentes puntos de vista, para derivar conclusiones sobre los factores que facilitan u obstaculizan su implantación permanente en las organizaciones. Su desarrollo se estructura en torno a un marco conceptual de referencia obtenido a través de la revisión bibliográfica específica, y se complementa con la información primaria y secundaria de investigación. Por su parte, el análisis cuantitativo se desarrolla mediante tres técnicas exploratorias: estadística descriptiva, regresión múltiple y análisis de componentes principales. Su aplicación combinada va a posibilitar el contraste de aspectos destacados en los análisis de casos, así como la configuración final del modelo de implantación, y la expresión numérica de la creación de valor a través de la RSCE en función de las dimensiones estratégicas consideradas. En consecuencia, los resultados de la tesis se estructuran alrededor de tres preguntas de investigación: ¿cómo se están produciendo y qué caracterización presentan los beneficios que resultan como consecuencia de la implantación de la RSCE en los procesos de planificación estratégica de las compañías?, ¿qué factores esenciales y característicos de la RSCE pueden resultar críticos en los procesos de implantación y futuro desarrollo?, y ¿qué importancia puede tener en el medio y largo plazo el poder de decisión de compra de los consumidores y usuarios finales en la implantación y desarrollo de políticas de RSCE? ABSTRACT Corporate Social Responsibility (CSR) remains a study area of high interest today to both the academic community and businesses in general. Despite the large number of investigations of various aspects of CSR in past decades, and its generalized consideration by the world’s most important companies, there are still some key issues and fundamental questions to resolve. The complexity associated with the CSR construct and its inherently dynamic character, partly explains this statement. In its practical application, doubts about CSR arise today about its permanent implementation in normal business activities, the strategic relevance of related policies, and the possibility of making profits in the medium and long term. It is observed in this way the translation of the main debates towards the more strategic consequences of these policies, influenced by prestigious academic studies that characterize the so-called Strategic CSR (SCSR), and by leading certification agencies of CSR and sustainability reports. In this context, the main purpose of this investigation is to design a model of SCSR for implementation that allows one to not only identify the most important factors to consider for SCSR success, but also to characterize potential forms of value creation that can arise from its application. The selection of this research approach is justified because it is believed that important issues that are associated with CSR have not been sufficiently explored from the aspect of the strategic vision in the current context, and because value creation constitutes the most critical objective within the strategic planning steering processes. Thus, two methods are used to highlight which factors are essential in SCSR implementation processes, the end to which companies apply these policies, and the kind of results that they expect. These methods are: comparative analysis of case studies and quantitative statistical analysis. The case studies discuss in depth SCSR global policies under different perspectives to draw conclusions about the factors that facilitate or hinder permanent implantation in organizations. Their development is structured around a conceptual framework that is obtained by review of specific literature, and is complemented by primary and secondary research information. On the other hand, quantitative analysis is developed by means of three exploratory techniques: descriptive statistics, multiple regression and principal component analysis. Their combined application facilitates a contrast of highlighted aspects in analyzing cases, the final configuration of the implementation model, and the numerical expression of value creation by SCSR as a consequence of the strategic dimensions considered by companies. Finally, the results of the thesis are structured around three research questions: what are the benefits that result from the implementation of SCSR policies in companies’ strategic planning processes?, which essential SCSR factors are potentially critical in the implementation and future development of companies’ processes?, and how decisive in the medium and long term will be the purchase decision power of consumers to the success of SCSR policies?

Montecarlo method applied to comparison of tenders in construction projects.

La comparación de las diferentes ofertas presentadas en la licitación de un proyecto,con el sistema de contratación tradicional de medición abierta y precio unitario cerrado, requiere herramientas de análisis que sean capaces de discriminar propuestas que teniendo un importe global parecido pueden presentar un impacto económico muy diferente durante la ejecución. Una de las situaciones que no se detecta fácilmente con los métodos tradicionales es el comportamiento del coste real frente a las variaciones de las cantidades realmente ejecutadas en obra respecto de las estimadas en el proyecto. Este texto propone abordar esta situación mediante un sistema de análisis cuantitativo del riesgo como el método de Montecarlo. Este procedimiento, como es sabido, consiste en permitir que los datos de entrada que definen el problema varíen unas funciones de probabilidad definidas, generar un gran número de casos de prueba y tratar los resultados estadísticamente para obtener los valores finales más probables,con los parámetros necesarios para medir la fiabilidad de la estimación. Se presenta un modelo para la comparación de ofertas, desarrollado de manera que puede aplicarse en casos reales aplicando a los datos conocidos unas condiciones de variación que sean fáciles de establecer por los profesionales que realizan estas tareas. ABSTRACT: The comparison of the different bids in the tender for a project, with the traditional contract system based on unit rates open to and re-measurement, requires analysis tools that are able to discriminate proposals having a similar overall economic impact, but that might show a very different behaviour during the execution of the works. One situation not easily detected by traditional methods is the reaction of the actual cost to the changes in the exact quantity of works finally executed respect of the work estimated in the project. This paper intends to address this situation through the Monte Carlo method, a system of quantitative risk analysis. This procedure, as is known, is allows the input data defining the problem to vary some within well defined probability functions, generating a large number of test cases, the results being statistically treated to obtain the most probable final values, with the rest of the parameters needed to measure the reliability of the estimate. We present a model for the comparison of bids, designed in a way that it can be applied in real cases, based on data and assumptions that are easy to understand and set up by professionals who wish to perform these tasks.

Ecología de la dispersión de plantas en los bosques secos del suroccidente Ecuatoriano

La importancia del proceso de dispersión de semillas en la estructura y dinámica de los ecosistemas es ampliamente reconocida. Sin embargo, para los bosques tropicales estacionalmente secos los estudios relacionados con este proceso son aún escasos y dispersos en comparación con los bosques tropicales lluviosos. En este trabajo se estudió la importancia de los síndromes de dispersión de semillas en la estructuración de comunidades, mediante el análisis de los patrones de dispersión de semillas en el espacio y tiempo para comunidades de leñosas en los bosques secos del suroccidente Ecuatoriano. Esta área forma parte de la región Tumbesina, una de las áreas de endemismo más importantes del mundo, pero también uno de los hotspots más amenazados. El clima se caracteriza por una estación seca que va de mayo a noviembre y una estación lluviosa que se extiende desde diciembre a abril. Para toda esta zona se estima una temperatura promedio anual entre 20° y 26°C y una precipitación promedio anual entre 300 y 700 mm. El trabajo de campo se desarrolló entre febrero de 2009 y septiembre de 2012. El primer paso fue la recopilación de información sobre las especies leñosas nativas de los bosques secos del suroccidente de Ecuador, que permitiera asignar a cada especie a un síndrome de dispersión para determinar el espectro de síndromes de dispersión de semillas. Luego, utilizando la información disponible de 109 parcelas establecidas previamente a lo largo de cuatro cantones de la provincia de Loja que conservan bosques secos en buen estado, se analizó la relación entre el síndrome de dispersión y condiciones ambientales. La relación de los síndromes de dispersión con los patrones espaciales de las especies y con los patrones de la lluvia y banco de semillas se estudió dentro de una parcela permanente de 9 ha, en la Reserva Ecológica Arenillas. Dentro de esta parcela se estableció un transecto de aproximadamente 3,4 km, que se recorrió mensualmente para colectar excretas de cérvidos y analizar el rol de este grupo como dispersor de semillas. Una gran variedad de plantas en los bosques secos tropicales del suroccidente de Ecuador requirió la asistencia de animales para la dispersión de semillas. Sin embargo, un análisis del espectro de dispersión considerando no solo la riqueza, sino también la abundancia relativa de especies, permitió determinar que a pesar de la alta variedad de especies zoócoras, la mayor parte de la comunidad correspondía a individuos anemócoros, que no proveen ninguna recompensa para la dispersión por animales. Este patrón puede deberse a la abundancia relativa de hábitats adecuados para especies con diferente síndrome de dispersión. Las condiciones ambientales afectaron la estructura del espectro de dispersión en la comunidad de bosque seco neotropical estudiada. El análisis de la importancia relativa del síndrome de dispersión y de la heterogeneidad espacial en la formación de patrones espaciales de árboles adultos permitió determinar que la heterogeneidad ambiental ejercía un efecto adicional (y en algunos el único) en la formación de patrones agregados de la mayoría de especies estudiadas. Los resultados señalaron diferencias en los patrones espaciales de las especies dependiendo del síndrome de dispersión, pero también una gran variación en los patrones espaciales incluso entre especies del mismo síndrome de dispersión. El análisis simultáneo de los patrones de la lluvia de semillas y banco de semillas de una comunidad de leñosas y su relación con la vegetación establecida indicaron que la lluvia de semillas era temporalmente variable en número de especies y abundancia de semillas, y dependía del síndrome de dispersión. El síndrome de dispersión también influyó en la formación de bancos de semillas, siendo las especies con capacidad de dispersión limitada (autócoras) las de mayor riqueza de especies y abundancia de semillas. Los cérvidos también se consideraron como un elemento clave en el proceso de dispersión de semillas. Al menos ocho especies leñosas fueron dispersadas legítimamente vía endozoócora. La mayoría de las especies dispersadas presentaron diásporas sin adaptaciones obvias para la dispersión, por lo que la ingestión de semillas por cérvidos se constituye en una vía potencial para la dispersión de sus semillas a largas distancias y, con ello, mejora la posibilidad de colonizar nuevos sitios y mantener el flujo genético. Los resultados de este estudio aportan nuevas evidencias para el entendimiento de la importancia de los procesos de dispersión de semillas en la estructura de los bosques secos neotropicales. Uno de los principales hallazgos a partir de estos cuatro capítulos es que los patrones espaciales de las especies, así como las estrategias que utilizan para dispersarse y hacer frente a las condiciones adversas (es decir, lluvia o banco de semillas) llevan consigo un efecto del síndrome de dispersión, y que la intensidad ese efecto depende a la vez de las condiciones ambientales del lugar. ABSTRACT The importance of seed dispersal process in the estructuring and ecosystem dynamic is widely recongnized. However, for seasonally tropical dry forest studies related to this process are still scarce and scattered compared to tropical rain forests. The present research deals with the importance of seed dispersal syndromes as a driver in the community structure, focusing its attention to temporal and spatial patterns of seed dispersal in woody communities of seasonally dry forest at Southwestern Ecuador. This area is part of the Tumbesian region, one of the most important areas of endemism, but also one of the most threatened areas around the world. Climate is characterized by a dry season from May to November, and a rainy season from December to April. For the whole area an average temperature between 20 ° and 26 ° C, and an average annual rainfall between 300 and 700 mm are estimated. Fieldwork was carried out between February 2009 and September 2012. During a first step information about native woody species of dry forests of southwestern Ecuador was gathered, enabling to assign a dispersal syndrome to each species to determine the seed dispersal spectrum. In a second step, available information from 109 established plots along four municipalities in Loja province, which hold the highest and best conserved dry forest remanants, was analyzed to establish the relationship between dispersal syndromes and environmental conditions. The relationships between dispersal syndromes and species spatial patterns; and between dispersal syndromes and seed rain and seed bank patterns, were studied within a permanent plot of 9 ha, in the Arenillas Ecological Reserve. Within this plot one transect of approximately 3.4 km was set to collect monthly deer droppings, which were used to latter analyze the rol of this group as seed dispersers. The results showed that a large variety of plants in tropical dry forest of Southwestern Ecuador require animal assistance to dispers their seeds. However, an analysis of seed dispersal spectrum considering not only species richness, but also the relative abundance of species, allowed to determine that despite the high variety of zoochorous species, most individuals in the community corresponds to anemochoruos species. This shift may be due to the relative abundance of habitats that are suitable for species with different dispersal syndromes. Moreover, quantitative data analysis showed that environmental conditions affect the structure of seed dispersal spectrum in the studied community. The analysis of relative importance of dispersal syndrome, and the environmental heterogeneity on formation of adult trees spatial patterns, indicated that environmental heterogeneity exert an additional (or was the only) effect limiting the distribution of most species in this forest. The findings showed differences in spatial patterns related to dispersal syndrome, but also showed a large variation in spatial patterns even among species sharing the same dispersal syndrome. Simultaneous analysis of seed rain and seed bank patterns of a woody community, and their relationship with established vegetation, suggested that seed rain is temporally variable in species number and seeds abundance, and that variation is related to the dispersal syndrome. Dispersal syndrome also influenced on the formation of seed banks, being species with limited dispersal abilities (autochorous) the ones with highest species richness and seed abundance. Deer were found as a key element in the seed dispersal process. At least to eight woody species were dispersed legitimately by ingestion. Diaspores of most dispersed species had no obvious adaptations to seed dispersal, therefore, seed ingestion by deer represents a potential pathway for long-distance dispersal, and hence, improves the chances to colonizing new sites and to maintain gene flow. Overall, these results provide new evidence for understanding the importance of seed dispersal processes in the structure of Neotropical dry forests. One of the major findings from these four chapters is that spatial patterns of species, and the strategies used to disperse their seeds and to deal with the adverse conditions (i.e. seed rain or seed bank) are related with dispersal syndromes, and the intensity of that relation depends in turn, on environmental conditions.

Fashion in Actions on Social Media – Spanish SME Fashion Brands Case Studies

Fashion is one of the most vibrant sectors in Europe and important contributors to the European Union (EU) economy. In particular, Small and Medium Enterprises (SMEs) play a major part in European fashion industry (EU 2012). Just like fashion, where people¿s style has inherently meant to be shared as it is foremost a representation of one¿s self-image, social media allow the reflection of ones' personality and emotions. Although fashion practitioners have embraced social media in their marketing activities, it is still relatively few known at an academic level about the specificities of fashion industry when approaching social media marketing (SMM) strategies. This study sets out to explore fashion companies' SMM strategy and its activities. From an exploratory approach, we present case studies of two Spanish SME fashion companies, anonymously named hereafter as Company A and Company B, to deepen our understanding on how fashion brands implement their SMM strategy. Company A offers high-end fashion products while Company B produces medium fashion products. We analyzed the case studies using qualitative (interviews to companies' executives) and a mix of qualitative and quantitative (content analysis of companies' social media platform) methods. Public posts data of both companies' Facebook brand pages were used to perform the content analysis. Our findings through case studies of the two companies reveal that branding-oriented strategic objectives are the main drivers of their SMM implementations. There are significant differences between both companies. The main strategic action employed by Company A is engaging customers to participate into brand's offline social gathering events by inviting them through social media platform, while Company B focuses its effort on posting product promotion related contents and engaging influencers such as fashion bloggers. Our results are expected to serve as a basis of further investigations on how SMM strategy and strategic actions implemented by fashion brands may influence marketing outcomes.

Chromatin assembly in a yeast whole-cell extract

A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.

Copy Number Variations in DISC1 and DISC1-Interacting Partners in Major Mental Illness

Acknowledgements We would like to thank all of the patients, relatives and control individuals who participated in the study. We are indebted to the late Prof. Walter Muir, Chair of Developmental Psychiatry and Honorary Consultant in Learning Disability Psychiatry, University of Edinburgh, who initiated these studies and whose work was dedicated to the welfare of the patients who generously participated. We are also grateful to Mrs. Pat Malloy for her assistance with DNA collection and MAQ assays screening of the Scottish samples. The Scottish sample collection was supported by a grant from the Chief Scientist Office (CSO), part of the Scottish Government Health and Social Care Directorates. This research was funded by grants from the CSO to B.S.P. (grant CZB/4/610), The Academy of Medical Sciences/Wellcome Trust to M.J. (grant R41455) and The RS Macdonald Charitable Trust (grant D21419 together with J.H.), the Swedish Research Council (grants 2003-5158 and 2006-4472), the Medical Faculty, Umeå University, and the County Councils of Västerbotten and Norrbotten, Sweden, as well as by grants from the Fund for Scientific Research Flanders (FWO-F), the Industrial Research Fund (IWT) and the Special Research Fund of the University of Antwerp, Belgium. M.J. is funded by a Wellcome Trust Clinical Research Fellowship for MB PhD graduates (R42811). We acknowledge the contribution of the personnel of the VIB Genetic Service Facility (http://www.vibgeneticservicefacility.be/) for the genetic analysis of the Swedish samples. Research nurses Gunnel Johansson, Lotta Kronberg, Tage Johansson and Lisbeth Bertilsson are thankfully acknowledged for their help and expertise. The Betula Study was funded by the Swedish Research Council (grants 345-2003-3883 and 315-2004-6977). We also acknowledge the contribution by the staff in the Betula project

Sulfate reduction in higher plants: Molecular evidence for a novel 5′-adenylylsulfate reductase

Sulfate-assimilating organisms reduce inorganic sulfate for Cys biosynthesis. There are two leading hypotheses for the mechanism of sulfate reduction in higher plants. In one, adenosine 5′-phosphosulfate (APS) (5′-adenylylsulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione. Alternatively, the mechanism may be similar to that in bacteria in which the enzyme, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase, catalyzes thioredoxin (Trx)-dependent reduction of PAPS. Three classes of cDNA were cloned from Arabidopsis thaliana termed APR1, -2, and -3, that functionally complement a cysH, PAPS reductase mutant strain of Escherichia coli. The coding sequence of the APR clones is homologous with PAPS reductases from microorganisms. In addition, a carboxyl-terminal domain is homologous with members of the Trx superfamily. Further genetic analysis showed that the APR clones can functionally complement a mutant strain of E. coli lacking Trx, and an APS kinase, cysC. mutant. These results suggest that the APR enzyme may be a Trx-independent APS reductase. Cell extracts of E. coli expressing APR showed Trx-independent sulfonucleotide reductase activity with a preference for APS over PAPS as a substrate. APR-mediated APS reduction is dependent on dithiothreitol, has a pH optimum of 8.5, is stimulated by high ionic strength, and is sensitive to inactivation by 5′-adenosinemonophosphate (5′-AMP). 2′-AMP, or 3′-phosphoadenosine-5′-phosphate (PAP), a competitive inhibitor of PAPS reductase, do not affect activity. The APR enzymes may be localized in different cellular compartments as evidenced by the presence of an amino-terminal transit peptide for plastid localization in APR1 and APR3 but not APR2. Southern blot analysis confirmed that the APR clones are members of a small gene family, possibly consisting of three members.

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Δ strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4β,14α-dimethyl-4α-carboxy-cholesta-8,24-dien-3β-ol and 4β-methyl-4α-carboxy-cholesta-8,24-dien-3β-ol, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

Direct Visualization of a Protein Nuclear Architecture

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.

Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2+ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2+; overexpression of atb2+ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

The Kinesin-related Proteins, Kip2p and Kip3p, Function Differently in Nuclear Migration in Yeast

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δ kar9Δ double mutants were viable. Conversely, kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δ dhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.

End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis in Saccharomyces cerevisiae

end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.