RESUMEN
The polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. The budding yeast Saccharomyces cerevisiae divides asymmetrically, like many other cells, to generate two distinct progeny cells. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We screened a collection of essential genes to analyze the effects of core cellular processes in asymmetric cell division based on Ace2 localization. This screen identified mutations that affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model, we blocked cells from progressing through mitosis and found that prolonged metaphase delay is sufficient to disrupt Ace2 asymmetry after release, and that Ace2 asymmetry is restored after cytokinesis. We also demonstrate that members of the evolutionarily conserved facilitates chromatin transcription (FACT) chromatin-reorganizing complex are required for both asymmetric and cell cycle-regulated localization of Ace2, and for localization of the RAM network components.
Asunto(s)
División Celular Asimétrica , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Ensamble y Desensamble de Cromatina , Citocinesis , Proteínas de Unión al ADN/genética , Mitosis , Mutación , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library. RESULTS: We present a new recombination-based method, CATS - Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining. CONCLUSIONS: The method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Proteínas Fluorescentes Verdes/genética , Péptidos/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Sistemas CRISPR-Cas , Edición Génica , Proteínas Fluorescentes Verdes/metabolismo , Ensayos Analíticos de Alto Rendimiento , Péptidos/metabolismo , Plásmidos/genética , ARN Guía de Kinetoplastida/genética , Saccharomyces cerevisiae/genética , Coloración y EtiquetadoRESUMEN
To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations - a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells.
Asunto(s)
Mapas de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Unión Proteica , Transporte de ProteínasRESUMEN
Knowledge of how genetic effects arising from parental care influence the evolution of offspring traits comes almost exclusively from studies of maternal care. However, males provide care in some taxa, and often this care differs from females in quality or quantity. If variation in paternal care is genetically based then, like maternal care and maternal effects, paternal effects may have important consequences for the evolution of offspring traits via indirect genetic effects (IGEs). IGEs and direct-indirect genetic covariances associated with parental care can contribute substantially to total heritability and influence predictions about how traits respond to selection. It is unknown, however, if the magnitude and sign of parental effects arising from fathers are the same as those arising from mothers. We used a reciprocal cross-fostering experiment to quantify environmental and genetic effects of paternal care on offspring performance in the burying beetle, Nicrophorus vespilloides. We found that IGEs were substantial and direct-indirect genetic covariances were negative. Combined, these patterns led to low total heritabilities for offspring performance traits. Thus, under paternal care, offspring performance traits are unlikely to evolve in response to selection, and variation in these traits will be maintained in the population despite potentially strong selection on these traits. These patterns are similar to those generated by maternal care, indicating that the genetic effects of care on offspring performance are independent of the caregiver's sex.