S. cerevsiae has served as a proving ground for new genomics technologies, application of these technologies and the development of the bioinformatics tools required to analyze these datasets. In addition, focused biological investigations by a large research community has resulted detailed understandings of how individual genes contribute to basic cellular functions. We propose to harness the genetic power of yeast to unify existing knowledge with the functions of the actin cytoskeleton, a major integrator of cellular systems. Actin filaments are constructed of a single protein but diversity in actin function is mediated by a large battery of accessory proteins. Investigations on the yeast actin cytoskeleton have led to the development of powerful tools and reagents to study how interactions with the actin cytoskeleton contribute to its regulation and utilization by cellular systems, but many of these tools and reagents have not been utilized to their fullest extent to realize a complete picture of the yeast actin cytoskeleton or the cellular systems it affects. Notable among these reagents is a large collection of mutants in the single essential actin gene ACT1 that were specifically designed to disrupt protein-protein interactions. We propose to use these reagents and the power of yeast genetics and genomics to uncover a complete or at least nearly complete genetic description of those genes that impact, or whose functions are impacted by, the actin cytoskeleton. Complex haploinsufficiency screens will be performed with a null allele of actin against the entire ordered array of yeast gene knock-outs to try and uncover genes sensitive to reductions in actin stoichiometry. This sub- network of genes will then be retested against the large collection of mutant actin alleles to determine which of the genetic interactions can be attributed to reductions in subsets of actin functions. To complete the """"""""actin interactome"""""""", this analysis will be complemented by synthetic lethal screening with the set of viable actin alanine scan mutants. All interactions will be correlated back to the loss or reduction in specific subsets of actin functions as defined by the actin mutants. To bring further coherence to the data, genes of the actin interactome will also be examined for genetic interactions with the genes for all known actin-binding proteins. Analysis of this highly integrated data set will be used to arrange both the actin alleles and their interacting genes into groups that we hypothesize will reflect shared defects such as the loss of binding between actin and specific actin binding proteins. Bayesian data integration with other datasets will be used for function prediction and to uncover novel relationships between gene sets and cluster analysis will be used to correlate these patterns back to the structure of actin. Visualization of the network will be facilitated by the development of a three-dimensional version of the network display tool Osprey that will connect nodes of the network back to the structure of actin. All data and data analysis resulting from this proposal will be maintained in a free access, Web-based format within the Toronto GRID database, the SGD, and PIXIE.Princeton.edu. ? ? ?
| DiPrima, Stephanie; Haarer, Brian; Viggiano, Susan et al. (2014) Linking genetics to structural biology: complex heterozygosity screening with actin alanine scan alleles identifies functionally related surfaces on yeast actin. G3 (Bethesda) 4:1491-501 |
| Haarer, Brian; Mi-Mi, Lei; Cho, Jessica et al. (2013) Actin dosage lethality screening in yeast mediated by selective ploidy ablation reveals links to urmylation/wobble codon recognition and chromosome stability. G3 (Bethesda) 3:553-61 |
| Haarer, Brian; Aggeli, Dimitra; Viggiano, Susan et al. (2011) Novel interactions between actin and the proteasome revealed by complex haploinsufficiency. PLoS Genet 7:e1002288 |
| Viggiano, Susan; Haarer, Brian; Amberg, David C (2010) Correction/completion of the yeast actin, alanine scan alleles. Genetics 185:391-4 |
