Biology subjects

Kasimer, D.

Publications and source records attributed to Kasimer, D..

3 recordsLinked to original sources

bioRxiv2019

Cell cycle regulation of mitochondrial protein import revealed by genome-scale pooled bimolecular fluorescence complementation screening

A central focus of systems biology is the functional mapping of protein-protein interactions under physiological conditions. Here we describe MaGiCaL-BiFC, a lentivirus-based bimolecular fluorescence protein-fragment complementation approach for the high-throughput, genome-scale identification of protein-protein interactions in mammalian cells. After developing and validating this methodology using known protein-protein interaction pairs, we constructed genome-scale pooled BiFC libraries using the human ORFeome cDNA collection. These pooled libraries, containing [~] 12,000 unique human cDNAs, were used to screen for candidate interaction partners of the mitochondrial transmembrane protein TOMM22. Following infection of cells with the TOMM22 bait and the pooled cDNA libraries, cells harboring candidate TOMM22 interacting proteins were isolated from the cell pool via fluorescence activated cell sorting, and identified via microarray analysis. This approach identified several known interaction partners of TOMM22, as well as novel physical and functional partners that link the mitochondrial network to proteins involved in diverse cellular processes. Notably, protein kinase CK2 was identified as a novel physical interaction partner of human TOMM22. We found that this association occurs preferentially during mitosis and involves direct phosphorylation of TOMM22, an event that may lead to attenuation of mitochondrial protein import. Together, this data contributes to the growing body of evidence suggesting eloquent coordination between cell cycle progression and mitochondrial physiology. Importantly, through high-throughput screening and focused validation, our study demonstrates the power of the MaGiCaL-BiFC approach to uncover novel functional protein-protein interactions, including those involving proteins with membrane-spanning domains, or of a transient nature, all within their native cellular environment.

systems biology↗

bioRxiv2018

The humungous fungus of Michigan three decades on

In the late 1980s, a genetic individual of the fungus Armillaria gallica that extended over at least 37 hectares of forest floor and encompassed hundreds of tree root systems was discovered on the Upper Peninsula of Michigan. Based on observed growth rates, the individual was estimated to be at least 1500 years old with a mass of more than 105 kg. Nearly three decades on, we returned to the site of individual for new sampling. We report here that the same genetic individual of A. gallica is still alive on its original site, but we estimated that it is older and larger than originally estimated, at least 2,500 years and 4 x 105 kg, respectively. We also show that mutation has occurred within the somatic cells of the individual, reflecting its historical pattern of growth from a single point. The overall rate of mutation, however, was extremely low. The large individual of A. gallica has been remarkably resistant to genomic change as it has persisted in place.

evolutionary biology↗

bioRxiv2018

Persistence of Resident and Transplanted Genotypes of the Undomesticated Yeast, Saccharomyces paradoxus in Forest Soil

One might expect yeasts in soil to be highly dispersed via water or insects, forming ephemeral, genetically heterogeneous populations subject to competition and environmental stochasticity. Here, we report years-long persistence of genotypes of the yeast Sacchaormyces paradoxus in space and time. Within 1 km2 in a mixed hardwood forest on scales from centimeters to tens of meters, we detect persistence over 3 years of native genotypes, identified by SNPs genome-wide, of the wild yeast, Saccharomyces paradoxus around Quercus rubra and Q. alba. Yeasts were recovered by enrichment in ethanol-containing medium, which measures only presence or absence, not abundance. Additional transplantation experiments employed strains marked with spontaneous defects in the URA3 gene, which also confer resistance to 5-Fluoroorotic acid (5FOA). Plating soil suspensions from transplant sites on 5FOA medium permitted one-step quantification of yeast colony-forming units, with no interference from other unmarked yeasts or microorganisms. After an initial steep decrease in abundance, the yeast densities fluctuated over time, increasing in association with rainfall and decreasing in association with drought. After 18 months, the transplanted yeasts remain in place on the nine sites. In vitro transplantation experiments into non-sterile soil in petri dishes showed similar patterns of persistence and response to moisture and drought. To determine whether S. cerevisiae, not previously recovered from soils regionally, can persist in our cold-climate sites, we transplanted marked S. cerevisiae alone and in mixture with S. paradoxus in fall, 2017. Five months on, S. cerevisiae persist to the same extent as S. paradoxus.

ecology↗