Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.     

Genome sequence of Shigella flexneri serotype 5a strain M90T Sm

Bacteria of the genus Shigella are a major cause of death worldwide (L. von Seidlein et al., PLoS Med. 3:e353, 2006). We sequenced the genome of Shigella flexneri strain M90T Sm (serotype 5a) and compared it to the published genome sequence of S. flexneri strain 8401 (serotype 5b).     

Do Low-Mercury Terrestrial Resources Subsidize Low-Mercury Growth of Stream Fish? Differences between Species along a Productivity Gradient

Low productivity in aquatic ecosystems is associated with reduced individual growth of fish and increased concentrations of methylmercury (MeHg) in fish and their prey. However, many stream-dwelling fish species can use terrestrially-derived food resources, potentially subsidizing growth at low-productivity sites, and, because terrestrial resources have lower MeHg concentrations than aquatic resources, preventing an increase in diet-borne MeHg accumulation. We used a large-scale field study to evaluate relationships among terrestrial subsidy use, growth, and MeHg concentrations in two stream-dwelling fish species across an in-stream productivity gradient. We sampled young-of-the-year brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar), potential competitors with similar foraging habits, from 20 study sites in streams in New Hampshire and Massachusetts that encompassed a wide range of aquatic prey biomass. Stable isotope analysis showed that brook trout used more terrestrial resources than Atlantic salmon. Over their first growing season, Atlantic salmon tended to grow larger than brook trout at sites with high aquatic prey biomass, but brook grew two-fold larger than Atlantic salmon at sites with low aquatic prey biomass. The MeHg concentrations of brook trout and Atlantic salmon were similar at sites with high aquatic prey biomass and the MeHg concentrations of both species increased at sites with low prey biomass and high MeHg in aquatic prey. However, brook trout had three-fold lower MeHg concentrations than Atlantic salmon at low-productivity, high-MeHg sites. These results suggest that differential use of terrestrial resource subsidies reversed the growth asymmetry between potential competitors across a productivity gradient and, for one species, moderated the effect of low in-stream productivity on MeHg accumulation.     

Molecular components of the mitotic spindle.

Mitotic spindles constitute the machinery responsible for equidistribution of the genetic material into each daughter cell during cell division. They are transient and hence quite labile structures, changing their morphology even while performing their function. Biochemical, immunological and genetic analyses of mitotic cells have allowed us to identify a variety of molecules that are recruited to form the spindle at the onset of mitosis. Evaluation of the roles of these molecules in both the formation and in the dynamics of spindle microtubules should be important for understanding the molecular basis of mitosis and its regulation. We have recently identified a novel mitosis-specific microtubule-associated protein (MAP) using a monoclonal antibody probe raised against the mitotic spindles isolated from cultured mammalian cells. This 95/105 kDa antigen represents a unique component of the spindle distinct from any of the other MAPs reported so far. Antibody microinjection resulted in mitotic inhibition in a stage-specific and dose-dependent manner, indicating that the protein is an essential spindle component.     

An integrated platform of genomic assays reveals small-molecule bioactivities

Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.     

Chemical–Genetic Profiling of Imidazo[1,2-a]pyridines and -Pyrimidines Reveals Target Pathways Conserved between Yeast and Human Cells

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships.     

Nest distribution shaping within-stream variation in Atlantic salmon juvenile abundance and competition over small spatial scales

1. Spatial heterogeneity in population density is predicted to have important effects on population characteristics, such as competition intensity and carrying capacity. Patchy breeding distributions will tend to increase spatial heterogeneity in population density, whereas dispersal from breeding patches will tend to decrease it. The potential for dispersal to homogenize densities is likely to differ both among organisms (e.g. plants vs. mobile animals) and throughout ontogeny (e.g. larvae vs. adults). However, for mobile organisms, experimental studies of the importance of breeding distributions from the wild are largely lacking. 2. In the present study, experimental manipulations replicated over eight natural streams and 2 years enabled us to test for effects of the distribution of Atlantic salmon eggs over spatial scales which are relevant to local interactions among individuals. Artificial nests were placed along 250 m study reaches at one of two levels of nest dispersion - patchy (two nests per stream) and dispersed (10 nests per stream) - while holding total egg density (eggs m(-2) stream area) constant. 3. Nest dispersion had significant effects on the spatial distribution of the resulting juveniles in their first summer. Patchy nest distributions resulted in a highly right-skewed frequency distribution of local under-yearling densities (among 25 m sampling sections), as sample sections adjacent to the nest sites had relatively high densities. In contrast, dispersed nest distributions yielded approximately normal density distributions. Sections with high relative densities in the patchy nest distribution treatments also had relatively small juvenile body sizes, and patchy egg distribution appeared to produce a higher redistribution of individuals from the first to the second juvenile growth season than the dispersed distribution. 4. Because patchy breeding distribution combined with limited early dispersal can create spatial variation in density over scales directly relevant for individual interactions, this will be one important component in determining mean levels of early juvenile competition and its spatial variation within populations. Assuming random or ideal-free distribution of individuals may therefore underestimate the mean level of density experienced by juveniles over surprisingly small spatial scales (orders of magnitude smaller than total spatial extent of populations), even for mobile organisms.