In JoVE (1)

Other Publications (80)

Articles by Christian Landry in JoVE

Other articles by Christian Landry on PubMed

Alternative Life Histories Shape Brain Gene Expression Profiles in Males of the Same Population

Proceedings. Biological Sciences / The Royal Society. Aug, 2005  |  Pubmed ID: 16087419

Atlantic salmon (Salmo salar) undergo spectacular marine migrations before homing to spawn in natal rivers. However, males that grow fastest early in life can adopt an alternative 'sneaker' tactic by maturing earlier at greatly reduced size without leaving freshwater. While the ultimate evolutionary causes have been well studied, virtually nothing is known about the molecular bases of this developmental plasticity. We investigate the nature and extent of coordinated molecular changes that accompany such a fundamental transformation by comparing the brain transcription profiles of wild mature sneaker males to age-matched immature males (future large anadromous males) and immature females. Of the ca. 3000 genes surveyed, 15% are differentially expressed in the brains of the two male types. These genes are involved in a wide range of processes, including growth, reproduction and neural plasticity. Interestingly, despite the potential for wide variation in gene expression profiles among individuals sampled in nature, consistent patterns of gene expression were found for individuals of the same reproductive tactic. Notably, gene expression patterns in immature males were different both from immature females and sneakers, indicating that delayed maturation and sea migration by immature males, the 'default' life cycle, may actually result from an active inhibition of development into a sneaker.

Compensatory Cis-trans Evolution and the Dysregulation of Gene Expression in Interspecific Hybrids of Drosophila

Genetics. Dec, 2005  |  Pubmed ID: 16143608

Hybrids between species are often characterized by novel gene-expression patterns. A recent study on allele-specific gene expression in hybrids between species of Drosophila revealed cases in which cis- and trans-regulatory elements within species had coevolved in such a way that changes in cis-regulatory elements are compensated by changes in trans-regulatory elements. We hypothesized that such coevolution should often lead to gene misexpression in the hybrid. To test this hypothesis, we estimated allele-specific expression and overall expression levels for 31 genes in D. melanogaster, D. simulans, and their F1 hybrid. We found that 13 genes with cis-trans compensatory evolution are in fact misexpressed in the hybrid. These represent candidate genes whose dysregulation might be the consequence of coevolution of cis- and trans-regulatory elements within species. Using a mathematical model for the regulation of gene expression, we explored the conditions under which cis-trans compensatory evolution can lead to misexpression in interspecific hybrids.

Large-scale Genetic Variation of the Symbiosis-required Megaplasmid PSymA Revealed by Comparative Genomic Analysis of Sinorhizobium Meliloti Natural Strains

BMC Genomics. 2005  |  Pubmed ID: 16283928

Sinorhizobium meliloti is a soil bacterium that forms nitrogen-fixing nodules on the roots of leguminous plants such as alfalfa (Medicago sativa). This species occupies different ecological niches, being present as a free-living soil bacterium and as a symbiont of plant root nodules. The genome of the type strain Rm 1021 contains one chromosome and two megaplasmids for a total genome size of 6 Mb. We applied comparative genomic hybridisation (CGH) on an oligonucleotide microarrays to estimate genetic variation at the genomic level in four natural strains, two isolated from Italian agricultural soil and two from desert soil in the Aral Sea region.

Genome-wide Scan Reveals That Genetic Variation for Transcriptional Plasticity in Yeast is Biased Towards Multi-copy and Dispensable Genes

Gene. Feb, 2006  |  Pubmed ID: 16427747

One of the most important aspects of the evolution of development and physiology is the interplay between gene expression and the environment, by which traits become altered in response to environmental triggers. This feature is known as phenotypic plasticity. When different genotypes show different levels of plasticity for a trait, then they show genotype-by-environment interaction, or GEI. It is now clear that gene expression plays an important role in organismic-level phenotypic plasticity, but we know very little about whether gene expression itself is subject to genetic variation for phenotypic plasticity (GEI). Given that gene regulation is likely to have evolved to respond to environmental changes, it is of central importance to understand how environmental and genetic variation interact to produce variation in gene expression. Here we investigate genetic variation for phenotypic plasticity in the yeast transcriptome for the whole genome. Six strains of Saccharomyces cerevisiae were grown in four different environments representing a continuum of rich and poor natural conditions. Using DNA-microarray data and an ANOVA analysis with a stringent criterion of significance, we found significant genetic variation for transcriptional plasticity (GEI) among strains for approximately 5% of the genes in the genome. There are about twice as many genes that show genetic variation for phenotypic plasticity as show genetic variation in transcription level independent of the environment. We also found that genes with genetic variation for plasticity were less likely to be essential and were significantly biased towards genes that have paralogs.

Ecological and Evolutionary Genomics of Saccharomyces Cerevisiae

Molecular Ecology. Mar, 2006  |  Pubmed ID: 16499686

Saccharomyces cerevisiae, the budding yeast, is the most thoroughly studied eukaryote at the cellular, molecular, and genetic levels. Yet, until recently, we knew very little about its ecology or population and evolutionary genetics. In recent years, it has been recognized that S. cerevisiae occupies numerous habitats and that populations harbour important genetic variation. There is therefore an increasing interest in understanding the evolutionary forces acting on the yeast genome. Several researchers have used the tools of functional genomics to study natural isolates of this unicellular fungus. Here, we review some of these studies, and show not only that budding yeast is a prime model system to address fundamental molecular and cellular biology questions, but also that it is becoming a powerful model species for ecological and evolutionary genomics studies as well.

Systems-level Analysis and Evolution of the Phototransduction Network in Drosophila

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2007  |  Pubmed ID: 17360639

Networks of interacting genes are responsible for generating life's complexity and for mediating how organisms respond to their environment. Thus, a basic understanding of genetic variation in gene networks in natural populations is important for elucidating how changes at the genetic level map to higher levels of biological organization. Here, using the well-characterized phototransduction network in Drosophila, we analyze variation in gene expression within and between two closely related species, Drosophila melanogaster and Drosophila simulans, under different environmental conditions. Gene expression levels in the pathway are largely conserved between these two sibling species. For most genes in the network, differences in level of gene expression between species are correlated with degree of polymorphism within species. However, one gene encoding the light-induced ion channel TRPL (transient receptor potential-like) shows an excess of expression divergence relative to polymorphism, suggesting a possible role for natural selection in shaping this expression difference between species. Finally, this difference in TRPL expression likely has significant functional consequences, because it is known that a high level of rhabdomeral TRPL leads to increased sensitivity to dim background light and an increased response to a wider range of light intensities. These results provide a preliminary quantification of variation and divergence of gene expression between species in a known gene network and provide a foundation for a system-level understanding of functional and evolutionary change.

Genetic Properties Influencing the Evolvability of Gene Expression

Science (New York, N.Y.). Jul, 2007  |  Pubmed ID: 17525304

Identifying the properties of gene networks that influence their evolution is a fundamental research goal. However, modes of evolution cannot be inferred solely from the distribution of natural variation, because selection interacts with demography and mutation rates to shape polymorphism and divergence. We estimated the effects of naturally occurring mutations on gene expression while minimizing the effect of natural selection. We demonstrate that sensitivity of gene expression to mutations increases with both increasing trans-mutational target size and the presence of a TATA box. Genes with greater sensitivity to mutations are also more sensitive to systematic environmental perturbations and stochastic noise. These results provide a mechanistic basis for gene expression evolvability that can serve as a foundation for realistic models of regulatory evolution.

Indel Arrays: an Affordable Alternative for Genotyping

The Plant Journal : for Cell and Molecular Biology. Aug, 2007  |  Pubmed ID: 17645438

Natural variation and induced mutations are important resources for gene discovery and the elucidation of genetic circuits. Mapping such polymorphisms requires rapid and cost-efficient methods for genome-wide genotyping. Here we report the development of a microarray-based method that assesses 240 unique markers in a single hybridization experiment at a cost of less than US$50 in materials per line. Our genotyping array is built with 70-mer oligonucleotide elements representing insertion/deletion (indel) polymorphisms between the Arabidopsis thaliana accessions Columbia-0 (Col) and Landsberg erecta (Ler). These indel polymorphisms are recognized with great precision by comparative genomic hybridization, eliminating the need for array replicates and complex statistical analysis. Markers are present genome-wide, with an average spacing of approximately 500 kb. PCR primer information is provided for all array indels, allowing rapid single-locus inquiries. Multi-well chips allow groups of 16 lines to be genotyped in a single experiment. We demonstrate the utility of the array for accurately mapping recessive mutations, RIL populations and mixed genetic backgrounds from accessions other than Col and Ler. Given the ease of use of shotgun sequencing to generate partial genomic sequences of unsequenced species, this approach is readily transferable to non-model organisms.

Ecological Annotation of Genes and Genomes Through Ecological Genomics

Molecular Ecology. Nov, 2007  |  Pubmed ID: 17883391

Ecological genomics is a research field that aims to determine how a genome or a population of genomes interacts with its environment across ecological and evolutionary timescales. This matter was the central theme of the symposium on Ecological Genomics that took place at the First meeting of the Canadian Society for Ecology and Evolution, held at the University of Toronto in May 2007. Through their research on a diverse array of organisms, the various speakers illustrated how ecology and evolution benefit from genomics, and indirectly how genomics can benefit from evolutionary ecology.

Cascading Transcriptional Effects of a Naturally Occurring Frameshift Mutation in Saccharomyces Cerevisiae

Molecular Ecology. Jun, 2008  |  Pubmed ID: 18422925

Gene-expression variation in natural populations is widespread, and its phenotypic effects can be acted upon by natural selection. Only a few naturally segregating genetic differences associated with expression variation have been identified at the molecular level. We have identified a single nucleotide insertion in a vineyard isolate of Saccharomyces cerevisiae that has cascading effects through the gene-expression network. This allele is responsible for about 45% (103/230) of the genes that show differential gene expression among the homozygous diploid progeny produced by a vineyard isolate. Using isogenic laboratory strains, we confirm that this allele causes dramatic differences in gene-expression levels of key genes involved in amino acid biosynthesis. The mutation is a frameshift mutation in a mononucleotide run of eight consecutive T's in the coding region of the gene SSY1, which encodes a key component of a plasma-membrane sensor of extracellular amino acids. The potentially high rate of replication slippage of this mononucleotide repeat, combined with its relatively mild effects on growth rate in heterozygous genotypes, is sufficient to account for the persistence of this phenotype at low frequencies in natural populations.

An in Vivo Map of the Yeast Protein Interactome

Science (New York, N.Y.). Jun, 2008  |  Pubmed ID: 18467557

Protein interactions regulate the systems-level behavior of cells; thus, deciphering the structure and dynamics of protein interaction networks in their cellular context is a central goal in biology. We have performed a genome-wide in vivo screen for protein-protein interactions in Saccharomyces cerevisiae by means of a protein-fragment complementation assay (PCA). We identified 2770 interactions among 1124 endogenously expressed proteins. Comparison with previous studies confirmed known interactions, but most were not known, revealing a previously unexplored subspace of the yeast protein interactome. The PCA detected structural and topological relationships between proteins, providing an 8-nanometer-resolution map of dynamically interacting complexes in vivo and extended networks that provide insights into fundamental cellular processes, including cell polarization and autophagy, pathways that are evolutionarily conserved and central to both development and human health.

A Genome-wide View of the Spectrum of Spontaneous Mutations in Yeast

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2008  |  Pubmed ID: 18583475

The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.

Chromatin- and Transcription-related Factors Repress Transcription from Within Coding Regions Throughout the Saccharomyces Cerevisiae Genome

PLoS Biology. Nov, 2008  |  Pubmed ID: 18998772

Previous studies in Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.

Systems Biology Spins off a New Model for the Study of Canalization

Trends in Ecology & Evolution. Feb, 2009  |  Pubmed ID: 19100652

The tools of systems biology make possible the study of phenotypic robustness at the molecular level. A recent investigation by Ansel et al. shows that alleles with different capabilities to buffer noise in gene expression - variation in protein abundance among genetically identical cells - segregate in populations of budding yeasts. These findings show that there is raw material for robust gene expression to evolve by natural selection, enabling the study of canalization of gene networks as it evolves in nature.

How Perfect Can Protein Interactomes Be?

Science Signaling. 2009  |  Pubmed ID: 19261595

Any engineered device should certainly not contain nonfunctional components, for this would be a waste of energy and money. In contrast, evolutionary theory tells us that biological systems need not be optimized and may very well accumulate nonfunctional elements. Mutational and demographic processes contribute to the cluttering of eukaryotic genomes and transcriptional networks with "junk" DNA and spurious DNA binding sites. Here, we question whether such a notion should be applied to protein interactomes-that is, whether these protein interactomes are expected to contain a fraction of nonselected, nonfunctional protein-protein interactions (PPIs), which we term "noisy." We propose a simple relationship between the fraction of noisy interactions expected in a given organism and three parameters: (i) the number of mutations needed to create and destroy interactions, (ii) the size of the proteome, and (iii) the fitness cost of noisy interactions. All three parameters suggest that noisy PPIs are expected to exist. Their existence could help to explain why PPIs determined from large-scale studies often lack functional relationships between interacting proteins, why PPIs are poorly conserved across organisms, and why the PPI space appears to be immensely large. Finally, we propose experimental strategies to estimate the fraction of evolutionary noise in PPI networks.

Weak Functional Constraints on Phosphoproteomes

Trends in Genetics : TIG. May, 2009  |  Pubmed ID: 19349092

Owing to their crucial roles in regulating protein function, phosphorylation sites (phosphosites) are expected to be evolutionarily conserved. However, mixed results regarding this prediction have been reported. We resolve these contrasting conclusions to show that phosphosites are, on average, more conserved than non-phosphorylated equivalent residues when their enrichment in disordered regions of proteins is taken into account. Phosphosites of known function are dramatically more conserved than those with no characterized function, indicating that the apparent rapid evolution of phosphoproteomes results from a large fraction of phosphosites being non-functional. Our findings highlight the need to use evolutionary information to identify functional regulatory features such as post-translational modifications of eukaryotic proteomes.

Cytokine Signaling Mediates UV-induced Nociceptive Sensitization in Drosophila Larvae

Current Biology : CB. May, 2009  |  Pubmed ID: 19375319

Heightened nociceptive (pain) sensitivity is an adaptive response to tissue damage and serves to protect the site of injury. Multiple mediators of nociceptive sensitization have been identified in vertebrates, but the complexity of the vertebrate nervous system and tissue-repair responses has hindered identification of the precise roles of these factors.

Key Considerations for Measuring Allelic Expression on a Genomic Scale Using High-throughput Sequencing

Molecular Ecology. Mar, 2010  |  Pubmed ID: 20331781

Differences in gene expression are thought to be an important source of phenotypic diversity, so dissecting the genetic components of natural variation in gene expression is important for understanding the evolutionary mechanisms that lead to adaptation. Gene expression is a complex trait that, in diploid organisms, results from transcription of both maternal and paternal alleles. Directly measuring allelic expression rather than total gene expression offers greater insight into regulatory variation. The recent emergence of high-throughput sequencing offers an unprecedented opportunity to study allelic transcription at a genomic scale for virtually any species. By sequencing transcript pools derived from heterozygous individuals, estimates of allelic expression can be directly obtained. The statistical power of this approach is influenced by the number of transcripts sequenced and the ability to unambiguously assign individual sequence fragments to specific alleles on the basis of transcribed nucleotide polymorphisms. Here, using mathematical modelling and computer simulations, we determine the minimum sequencing depth required to accurately measure relative allelic expression and detect allelic imbalance via high-throughput sequencing under a variety of conditions. We conclude that, within a species, a minimum of 500-1000 sequencing reads per gene are needed to test for allelic imbalance, and consequently, at least five to 10 millions reads are required for studying a genome expressing 10 000 genes. Finally, using 454 sequencing, we illustrate an application of allelic expression by testing for cis-regulatory divergence between closely related Drosophila species.

Cell Signaling. Signaling Through Cooperation

Science (New York, N.Y.). May, 2010  |  Pubmed ID: 20489011

Moving from Transcriptional to Phospho-evolution: Generalizing Regulatory Evolution?

Trends in Genetics : TIG. Nov, 2010  |  Pubmed ID: 20817339

Much of biological diversity is thought to arise from changes in regulatory networks. Although the role of transcriptional regulation has been well established, the contribution to evolution of changes at other levels of regulation has yet to be addressed. Using examples from the literature and recent studies on the evolution of protein phosphorylation, we argue that protein regulatory networks also play a prime role in generating diversity within and between species. Because there are several analogies between the regulation of protein functions by kinases and the regulation of gene expression by transcription factors, the principles that guide transcriptional regulatory evolution can also be explored in kinase-substrate networks. These comparisons will allow us to generalize existing models of evolution across the complex layers of the cell's regulatory links.

A Toolkit of Protein-fragment Complementation Assays for Studying and Dissecting Large-scale and Dynamic Protein-protein Interactions in Living Cells

Methods in Enzymology. 2010  |  Pubmed ID: 20946817

Protein-fragment complementation assays (PCAs) are a family of assays for detecting protein-protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism or in vitro. PCAs can be used to detect PPI between proteins of any molecular weight and expressed at their endogenous levels. Proteins are expressed in their appropriate cellular compartments and can undergo any posttranslational modification or degradation that, barring effects of the PCA fragment fusion, they would normally undergo. Applications of PCAs in yeast have been limited until recently, simply because appropriate expression plasmids or cassettes had not been developed. However, we have now developed and reported on several PCAs in Saccharomyces cerevisiae that cover the gamut of applications one could envision for studying any aspect of PPIs. Here, we present detailed protocols for large-scale analysis of PPIs with the survival-selection dihydrofolate reductase (DHFR) reporter PCA and a new PCA based on a yeast cytosine deaminase reporter that allows for both survival and death selection. This PCA should prove a powerful way to dissect PPIs. We then present a method to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs and finally, two luciferase reporter PCAs that have proved useful for studies of dynamics of PPIs.

Molecular Characterization of the Evolution of Phagosomes

Molecular Systems Biology. Oct, 2010  |  Pubmed ID: 20959821

Amoeba use phagocytosis to internalize bacteria as a source of nutrients, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in vertebrates, initiate a sustained immune response. By using a large-scale approach to identify and compare the proteome and phosphoproteome of phagosomes isolated from distant organisms, and by comparative analysis over 39 taxa, we identified an 'ancient' core of phagosomal proteins around which the immune functions of this organelle have likely organized. Our data indicate that a larger proportion of the phagosome proteome, compared with the whole cell proteome, has been acquired through gene duplication at a period coinciding with the emergence of innate and adaptive immunity. Our study also characterizes in detail the acquisition of novel proteins and the significant remodeling of the phagosome phosphoproteome that contributed to modify the core constituents of this organelle in evolution. Our work thus provides the first thorough analysis of the changes that enabled the transformation of the phagosome from a phagotrophic compartment into an organelle fully competent for antigen presentation.

Gene Network Architecture As a Canvas for the Interpretation of Ecological Genomics Investigations

Molecular Ecology. Dec, 2010  |  Pubmed ID: 21091660

New technologies promise to revolutionize the field of molecular ecology. This technological progress comes with its own set of challenges. Among the most important ones is the analysis and interpretation of the data in a way that tells us about the molecular causes of the phenotype of interest and its consequences. In this issue, Whitehead et al. (2010) reveal part of the mechanistic basis of evolved pollution tolerance by studying the developmental and transcriptional response of tolerant and sensitive fish embryos to polychlorinated biphenyls (PCBs), a pollutant commonly found in coastal waters of the United States. By integrating their gene expression profiling data with phenotypic data on individuals along with what is known about pathways by which this pollutant acts in zebrafish and mammals, they are able to suggest detailed mechanisms that have evolved to allow a fish population to adapt to a very damaging pollutant and develop normally.

Chromatin Regulators Shape the Genotype-phenotype Map

Molecular Systems Biology. Nov, 2010  |  Pubmed ID: 21119628

Phosphorylation Network Rewiring by Gene Duplication

Molecular Systems Biology. 2011  |  Pubmed ID: 21734643

Elucidating how complex regulatory networks have assembled during evolution requires a detailed understanding of the evolutionary dynamics that follow gene duplication events, including changes in post-translational modifications. We compared the phosphorylation profiles of paralogous proteins in the budding yeast Saccharomyces cerevisiae to that of a species that diverged from the budding yeast before the duplication of those genes. We found that 100 million years of post-duplication divergence are sufficient for the majority of phosphorylation sites to be lost or gained in one paralog or the other, with a strong bias toward losses. However, some losses may be partly compensated for by the evolution of other phosphosites, as paralogous proteins tend to preserve similar numbers of phosphosites over time. We also found that up to 50% of kinase-substrate relationships may have been rewired during this period. Our results suggest that after gene duplication, proteins tend to subfunctionalize at the level of post-translational regulation and that even when phosphosites are preserved, there is a turnover of the kinases that phosphorylate them.

Cell Biology. A Cellular Roadmap for the Plant Kingdom

Science (New York, N.Y.). Jul, 2011  |  Pubmed ID: 21798921

Protein-fragment Complementation Assays for Large-scale Analysis, Functional Dissection and Dynamic Studies of Protein-protein Interactions in Living Cells

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21870242

Protein-fragment Complementation Assays (PCAs) are a family of assays for detecting protein-protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism, or in vitro. PCAs can be used to detect PPI between proteins of any molecular weight and expressed at their endogenous levels. Proteins are expressed in their appropriate cellular compartments and can undergo any posttranslational modification or degradation that, barring effects of the PCA fragment fusion, they would normally undergo. Assays can be performed in any cell type or model organism that can be transformed or transfected with gene expression DNA constructs. Here we focus on recent applications of PCA in the budding yeast, Saccharomyces cerevisiae, that cover the gamut of applications one could envision for studying any aspect of PPIs. We present detailed protocols for large-scale analysis of PPIs with the survival-selection dihydrofolate reductase (DHFR), reporter PCA, and a new PCA based on a yeast cytosine deaminase reporter that allows for both survival and death selection. This PCA should prove a powerful way to dissect PPIs. We then present methods to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs.

Characterization of Spindle Checkpoint Kinase Mps1 Reveals Domain with Functional and Structural Similarities to Tetratricopeptide Repeat Motifs of Bub1 and BubR1 Checkpoint Kinases

The Journal of Biological Chemistry. Feb, 2012  |  Pubmed ID: 22187426

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

Compositional Differences Between Size Classes of Dissolved Organic Matter from Freshwater and Seawater Revealed by an HPLC-FTIR System

Environmental Science & Technology. Feb, 2012  |  Pubmed ID: 22216934

The molecular complexity of dissolved organic matter (DOM) hinders its characterization. New approaches are thus needed for a better understanding of DOM reactivity and fate in aquatic systems. In this study, high-performance liquid chromatography (HPLC), using size-exclusion separation, was coupled with Fourier transform infrared spectroscopy (FTIR). A solvent-elimination interface was used to deposit DOM fractions onto a germanium disk that were then analyzed by FTIR. Samples included ultrafiltered DOM (UDOM) and fulvic acids from the St. Lawrence Estuary and its tributaries. Results showed significant compositional changes with molecular size and origin, especially in UDOM. Larger fractions of UDOM contained more carbohydrates, amides, aromatics/alkenes and aliphatics, while smaller fractions contained more carboxylate and OH groups. Small marine molecules (500-900 Da) were also enriched in sulfate groups that appeared bound to UDOM. Large marine molecules were the most amide-rich fractions. Fulvic acids were enriched in carboxylate and OH groups, showed little changes in composition, and appeared similar to small terrigenous (riverine) UDOM even in marine water. This work shows that an HPLC-FTIR system is a powerful and complementary tool in the characterization of DOM. The compositional changes observed may explain the reported contrasting reactivity and fate of DOM having different size and origin.

Where Do Phosphosites Come from and Where Do They Go After Gene Duplication?

International Journal of Evolutionary Biology. 2012  |  Pubmed ID: 22779031

Gene duplication followed by divergence is an important mechanism that leads to molecular innovation. Divergence of paralogous genes can be achieved at functional and regulatory levels. Whereas regulatory divergence at the transcriptional level is well documented, little is known about divergence of posttranslational modifications (PTMs). Protein phosphorylation, one of the most important PTMs, has recently been shown to be an important determinant of the retention of paralogous genes. Here we test whether gains and losses of phosphorylated amino acids after gene duplication may specifically modify the regulation of these duplicated proteins. We show that when phosphosites are lost in one paralog, transitions from phosphorylated serines and threonines are significantly biased toward negatively charged amino acids, which can mimic their phosphorylated status in a constitutive manner. Our analyses support the hypothesis that divergence between paralogs can be generated by a loss of the posttranslational regulatory control on a function rather than by the complete loss of the function itself. Surprisingly, these favoured transitions cannot be reached by single mutational steps, which suggests that the function of a phosphosite needs to be completely abolished before it is restored through substitution by these phosphomimetic residues. We conclude by discussing how gene duplication could facilitate the transitions between phosphorylated and phosphomimetic amino acids.

The Genotype-phenotype Maps of Systems Biology and Quantitative Genetics: Distinct and Complementary

Advances in Experimental Medicine and Biology. 2012  |  Pubmed ID: 22821467

The processes by which genetic variation in complex traits is generated and maintained in populations has for a long time been treated in abstract and statistical terms. As a consequence, quantitative genetics has provided limited insights into our understanding of the molecular bases of quantitative trait variation. With the developing technological and conceptual tools of systems biology, cellular and molecular processes are being described in greater detail. While we have a good description of how signaling and other molecular networks are organized in the cell, we still do not know how genetic variation affects these pathways, because systems and molecular biology usually ignore the type and extent of genetic variation found in natural populations. Here we discuss the quantitative genetics and systems biology approaches for the study of complex trait architecture and discuss why these two disciplines would synergize with each other to answer questions that neither of the two could answer alone.

Protein Abundance is Key to Distinguish Promiscuous from Functional Phosphorylation Based on Evolutionary Information

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. Sep, 2012  |  Pubmed ID: 22889910

In eukaryotic cells, protein phosphorylation is an important and widespread mechanism used to regulate protein function. Yet, of the thousands of phosphosites identified to date, only a few hundred at best have a characterized function. It was recently shown that these functional sites are significantly more conserved than phosphosites of unknown function, stressing the importance of considering evolutionary conservation in assessing the global functional landscape of phosphosites. This leads us to review studies that examined the impact of phosphorylation on evolutionary conservation. While all these studies have shown that conservation is greater among phosphorylated sites compared with non-phosphorylated ones, the magnitude of this difference varies greatly. Further, not all studies have considered key factors that may influence the rate of phosphosite evolution. Such key factors are their localization in ordered or disordered regions, their stoichiometry or the abundance of their corresponding protein. Here we take into account all of these factors simultaneously, which reveals remarkable evolutionary patterns. First, while it is well established that protein conservation increases with abundance, we show that phosphosites partly follow an opposite trend. More precisely, Saccharomyces cerevisiae phosphosites present among abundant proteins are 1.5 times more likely to diverge in the closely related species Saccharomyces bayanus when compared with phosphosites present in the 5 per cent least abundant proteins. Second, we show that conservation is coupled to stoichiometry, whereby sites frequently phosphorylated are more conserved than those rarely phosphorylated. Finally, we provide a model of functional and noisy or 'accidental' phosphorylation that explains these observations.

What is Needed for Next-generation Ecological and Evolutionary Genomics?

Trends in Ecology & Evolution. Dec, 2012  |  Pubmed ID: 22902072

Ecological and evolutionary genomics (EEG) aims to link gene functions and genomic features to phenotypes and ecological factors. Although the rapid development of technologies allows central questions to be addressed at an unprecedented level of molecular detail, they do not alleviate one of the major challenges of EEG, which is that a large fraction of genes remains without any annotation. Here, we propose two solutions to this challenge. The first solution is in the form of a database that regroups associations between genes, organismal attributes and abiotic and biotic conditions. This database would result in an ecological annotation of genes by allowing cross-referencing across studies and taxa. Our second solution is to use new functional techniques to characterize genes implicated in the response to ecological challenges.

Proteomic Characterization of Phagosomal Membrane Microdomains During Phagolysosome Biogenesis and Evolution

Molecular & Cellular Proteomics : MCP. Nov, 2012  |  Pubmed ID: 22915823

After their formation at the cell surface, phagosomes become fully functional through a complex maturation process involving sequential interactions with various intracellular organelles. In the last decade, series of data indicated that some of the phagosome functional properties occur in specialized membrane microdomains. The molecules associated with membrane microdomains, as well as the organization of these structures during phagolysosome biogenesis are largely unknown. In this study, we combined proteomics and bioinformatics analyses to characterize the dynamic association of proteins to maturing phagosomes. Our data indicate that groups of proteins shuffle from detergent-soluble to detergent-resistant membrane microdomains during maturation, supporting a model in which the modulation of the phagosome functional properties involves an important reorganization of the phagosome proteome by the coordinated spatial segregation of proteins.

Evidence for the Robustness of Protein Complexes to Inter-species Hybridization

PLoS Genetics. 2012  |  Pubmed ID: 23300466

Despite the tremendous efforts devoted to the identification of genetic incompatibilities underlying hybrid sterility and inviability, little is known about the effect of inter-species hybridization at the protein interactome level. Here, we develop a screening platform for the comparison of protein-protein interactions (PPIs) among closely related species and their hybrids. We examine in vivo the architecture of protein complexes in two yeast species (Saccharomyces cerevisiae and Saccharomyces kudriavzevii) that diverged 5-20 million years ago and in their F1 hybrids. We focus on 24 proteins of two large complexes: the RNA polymerase II and the nuclear pore complex (NPC), which show contrasting patterns of molecular evolution. We found that, with the exception of one PPI in the NPC sub-complex, PPIs were highly conserved between species, regardless of protein divergence. Unexpectedly, we found that the architecture of the complexes in F1 hybrids could not be distinguished from that of the parental species. Our results suggest that the conservation of PPIs in hybrids likely results from the slow evolution taking place on the very few protein residues involved in the interaction or that protein complexes are inherently robust and may accommodate protein divergence up to the level that is observed among closely related species.

Transcriptional Divergence Plays a Role in the Rewiring of Protein Interaction Networks After Gene Duplication

Journal of Proteomics. Apr, 2013  |  Pubmed ID: 23063722

Gene duplication plays a key role in the evolution of protein-protein interaction (PPI) networks. After a gene duplication event, paralogous proteins may diverge through the gain and loss of PPIs. This divergence can be explained by two non-exclusive mechanisms. First, mutations may accumulate in the coding sequences of the paralogs and affect their protein sequences, which can modify, for instance, their binding interfaces and thus their interaction specificity. Second, mutations may accumulate in the non-coding region of the genes and affect their regulatory sequences. The resulting changes in expression profiles can lead to paralogous proteins being differentially expressed and occurring in the cell with different sets of potential interaction partners. These changes could also alter splicing regulation and lead to the inclusion or exclusion of alternative exons. The evolutionary role of these regulatory mechanisms remains largely unexplored. We use bioinformatics analyses of existing PPI data and proteome-wide PPI screening to show that the divergence of transcriptional regulation between paralogs plays a significant role in determining their PPI specificity. Because many gene duplication events are followed by rapid changes in transcriptional regulation, our results suggest that PPI networks may be rewired by gene duplication, without the need for protein to diverge in their binding specificities. This article is part of a Special Issue entitled: From protein structures to clinical applications.

QPCA: a Scalable Assay to Measure the Perturbation of Protein-protein Interactions in Living Cells

Molecular BioSystems. Jan, 2013  |  Pubmed ID: 23099892

One of the most important challenges in systems biology is to understand how cells respond to genetic and environmental perturbations. Here we show that the yeast DHFR-PCA, coupled with high-resolution growth profiling (DHFR-qPCA), is a straightforward assay to study the modulation of protein-protein interactions (PPIs) in vivo as a response to genetic, metabolic and drug perturbations. Using the canonical Protein Kinase A (PKA) pathway as a test system, we show that changes in PKA activity can be measured in living cells as a modulation of the interaction between its regulatory (Bcy1) and catalytic (Tpk1 and Tpk2) subunits in response to changes in carbon metabolism, caffeine and methyl methanesulfonate (MMS) treatments and to modifications in the dosage of its enzymatic regulators, the phosphodiesterases. Our results show that the DHFR-qPCA is easily implementable and amenable to high-throughput. The DHFR-qPCA will pave the way to the study of the effects of drug, genetic and environmental perturbations on in vivo PPI networks, thus allowing the exploration of new spaces of the eukaryotic interactome.

Are Long-lived Trees Poised for Evolutionary Change? Single Locus Effects in the Evolution of Gene Expression Networks in Spruce

Molecular Ecology. May, 2013  |  Pubmed ID: 23294325

Genetic variation in gene expression traits contributes to phenotypic diversity and may facilitate adaptation following environmental change. This is especially important in long-lived organisms where adaptation to rapid changes in the environment must rely on standing variation within populations. However, the extent of expression variation in most wild species remains to be investigated. We address this question by measuring the segregation of expression levels in white spruce [Picea glauca (Moench), Voss] in a transcriptome-wide manner and examining the underlying evolutionary and biological processes. We applied a novel approach for the genetic analysis of expression variation by measuring its segregation in haploid meiotic seed tissue. We identified over 800 transcripts whose abundances are most likely controlled by variants in single loci. Cosegregation analysis of allelic expression levels was used to construct regulatory associations between genes and define regulatory networks. The majority (67%) of segregating transcripts were under linkage. Regulatory associations were typically among small groups of genes (2-3 transcripts), indicating that most segregating expression levels can evolve independently from one another. One notable exception was a large putative trans effect that altered the expression of 180 genes that includes key regulators of protein metabolism, highlighting a regulatory cascade affected by variation in a single locus in this conserved metabolic pathway. Overall, segregating expression variation was associated with stress response- and duplicated genes, whose evolution may be linked to functional innovations. These observations indicate that expression variation might be important in facilitating diversity of molecular responses to environmental stresses in wild trees.

Integrative Avenues for Exploring the Dynamics and Evolution of Protein Interaction Networks

Current Opinion in Biotechnology. Aug, 2013  |  Pubmed ID: 23571097

Over the past decade, the study of protein interaction networks (PINs) has shed light on the organizing principles of living cells. However, PINs have been mostly mapped in one single condition. We outline three of the most promising avenues of investigation in this field, namely the study of first, how PINs are rewired by mutations and environmental perturbations; secondly, how inter-species interactions affect PIN achitectures; thirdly, what mechanisms and forces drive PIN evolution. These investigations will unravel the dynamics and condition dependence of PINs and will thus lead to a better functional annotation of network architecture. One major challenge to reach these goals is the integration of PINs with other cellular regulatory networks in the context of complex cellular phenotypes.

A Systematic Approach for the Genetic Dissection of Protein Complexes in Living Cells

Cell Reports. Jun, 2013  |  Pubmed ID: 23746448

Cells contain many important protein complexes involved in performing and regulating structural, metabolic, and signaling functions. One major challenge in cell biology is to elucidate the organization and mechanisms of robustness of these complexes in vivo. We developed a systematic approach to study structural dependencies within complexes in living cells by deleting subunits and measuring pairwise interactions among other components. We used our methodology to perturb two conserved eukaryotic complexes: the retromer and the nuclear pore complex. Our results identify subunits that are critical for the assembly of these complexes, reveal their structural architecture, and uncover mechanisms by which protein interactions are modulated. Our results also show that paralogous proteins play a key role in the robustness of protein complexes and shape their assembly landscape. Our approach paves the way for studying the response of protein interactomes to mutations and enhances our understanding of genotype-phenotype maps.

Extracting Insight from Noisy Cellular Networks

Cell. Nov, 2013  |  Pubmed ID: 24267884

Network biologists attempt to extract meaningful relationships among genes or their products from very noisy data. We argue that what we categorize as noisy data may sometimes reflect noisy biology and therefore may shield a hidden meaning about how networks evolve and how matter is organized in the cell. We present practical solutions, based on existing evolutionary and biophysical concepts, through which our understanding of cell biology can be enormously enriched.

Exploring the Northern Limit of the Distribution of Saccharomyces Cerevisiae and Saccharomyces Paradoxus in North America

FEMS Yeast Research. Mar, 2014  |  Pubmed ID: 24119009

We examined the northern limit of Saccharomyces cerevisiae and Saccharomyces paradoxus in northeast America. We collected 876 natural samples at 29 sites and applied enrichment methods for the isolation of mesophilic yeasts. We uncovered a large diversity of yeasts, in some cases, associated with specific substrates. Sequencing of the ITS1, 5.8S and ITS2 loci allowed to assign 226 yeast strains at the species level, including 41 S. paradoxus strains. Our intensive sampling suggests that if present, S. cerevisiae is rare at these northern latitudes. Our sampling efforts spread across several months of the year revealed that successful sampling increases throughout the summer and diminishes significantly at the beginning of the fall. The data obtained on the ecological context of yeasts corroborate what was previously reported on Pichiaceae, Saccharomycodaceae, Debaryomycetaceae and Phaffomycetaceae yeast families. We identified 24 yeast isolates that could not be assigned to any known species and that may be of taxonomic, medical, or biotechnological importance. Our study reports new data on the taxonomic diversity of yeasts and new resources for studying the evolution and ecology of S. paradoxus.

Modulation of the Yeast Protein Interactome in Response to DNA Damage

Journal of Proteomics. Apr, 2014  |  Pubmed ID: 24262151

Cells deploy diverse mechanisms to physiologically adapt to potentially detrimental perturbations. These mechanisms include changes in the organization of protein-protein interaction networks (PINs). Most PINs characterized to date are portrayed in a single environmental condition and are thus likely to miss important connections among biological processes. In this report, we show that the yeast DHFR-PCA on high-density arrays allows to detects modulations of protein-protein interactions (PPIs) in different conditions by testing more than 1000 PPIs in standard and in a drug-inducing DNA damage conditions. We identify 156 PPIs that show significant modulation in response to DNA damage. We provide evidence that modulated PPIs involve essential genes (NOP7, EXO84 and LAS17) playing critical roles in response to DNA damage. Additionally, we show that a significant proportion of PPI changes are likely explained by changes in protein localization and, to a lesser extent, protein abundance. The protein interaction modules affected by changing PPIs support the role of mRNA stability and translation, protein degradation and ubiquitylation and the regulation of the actin cytoskeleton in response to DNA damage. Overall, we provide a valuable tool and dataset for the study of the rewiring of PINs in response to environmental perturbations.

Recent Advances in Ecological Genomics: from Phenotypic Plasticity to Convergent and Adaptive Evolution and Speciation

Advances in Experimental Medicine and Biology. 2014  |  Pubmed ID: 24277292

Biological diversity emerges from the interaction between genomes and their environment. Recent conceptual and technological developments allow dissecting these interactions over short and long time-scales. The 16 contributions to this book by leaders in the field cover major recent progresses in the field of Ecological Genomics. Altogether, they illustrate the interplay between the life-history and genomic architecture of organisms, how the interaction of the environment and the genome is shaping phenotypic variation through phenotypic plasticity, how the process of adaptation may be constrained and fueled by internal and external features of organisms and finally, how species formation is the result of intricate interactions between genomes and the ecological conditions. These contributions also show how fundamental questions in biology transcend the boundaries of kingdoms, species and environments and illustrate how integrative approaches are powerful means to answer the most important and challenging questions in ecology and evolution.

Molecular Mechanisms of Paralogous Compensation and the Robustness of Cellular Networks

Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution. Nov, 2014  |  Pubmed ID: 24376223

Robustness is the ability of a system to maintain its function despite environmental or genetic perturbation. Genetic robustness is a key emerging property of living systems and is achieved notably by the presence of partially redundant parts that result from gene duplication. Functional overlap between paralogs allows them to compensate for each other's loss, as commonly revealed by aggravating genetic interactions. However, the molecular mechanisms linking the genotype (loss of function of a gene) to the phenotype (genetic buffering by a paralog) are still poorly understood and the molecular aspects of this compensation are rarely addressed in studies of gene duplicates. Here, we review molecular mechanisms of functional compensation between paralogous genes, many of which from studies that were not meant to study this phenomenon. We propose a standardized terminology and, depending on whether or not the molecular behavior of the intact gene is modified in response to the deletion of its paralog, we classify mechanisms of compensation into passive and active events. We further describe three non-exclusive mechanisms of active paralogous compensation for which there is evidence in the literature: changes in abundance, in localization, and in protein interactions. This review will serve as a framework for the genetic and molecular analysis of paralogous compensation, one of the universal features of genetic systems.

Local Climatic Adaptation in a Widespread Microorganism

Proceedings. Biological Sciences / The Royal Society. Feb, 2014  |  Pubmed ID: 24403328

Exploring the ability of organisms to locally adapt is critical for determining the outcome of rapid climate changes, yet few studies have addressed this question in microorganisms. We investigated the role of a heterogeneous climate on adaptation of North American populations of the wild yeast Saccharomyces paradoxus. We found abundant among-strain variation for fitness components across a range of temperatures, but this variation was only partially explained by climatic variation in the distribution area. Most of fitness variation was explained by the divergence of genetically distinct groups, distributed along a north-south cline, suggesting that these groups have adapted to distinct climatic conditions. Within-group fitness components were correlated with climatic conditions, illustrating that even ubiquitous microorganisms locally adapt and harbour standing genetic variation for climate-related traits. Our results suggest that global climatic changes could lead to adaptation to new conditions within groups, or changes in their geographical distributions.

Functional Divergence and Evolutionary Turnover in Mammalian Phosphoproteomes

PLoS Genetics. Jan, 2014  |  Pubmed ID: 24465218

Protein phosphorylation is a key mechanism to regulate protein functions. However, the contribution of this protein modification to species divergence is still largely unknown. Here, we studied the evolution of mammalian phosphoregulation by comparing the human and mouse phosphoproteomes. We found that 84% of the positions that are phosphorylated in one species or the other are conserved at the residue level. Twenty percent of these conserved sites are phosphorylated in both species. This proportion is 2.5 times more than expected by chance alone, suggesting that purifying selection is preserving phosphoregulation. However, we show that the majority of the sites that are conserved at the residue level are differentially phosphorylated between species. These sites likely result from false-negative identifications due to incomplete experimental coverage, false-positive identifications and non-functional sites. In addition, our results suggest that at least 5% of them are likely to be true differentially phosphorylated sites and may thus contribute to the divergence in phosphorylation networks between mouse and humans and this, despite residue conservation between orthologous proteins. We also showed that evolutionary turnover of phosphosites at adjacent positions (in a distance range of up to 40 amino acids) in human or mouse leads to an over estimation of the divergence in phosphoregulation between these two species. These sites tend to be phosphorylated by the same kinases, supporting the hypothesis that they are functionally redundant. Our results support the hypothesis that the evolutionary turnover of phosphorylation sites contributes to the divergence in phosphorylation profiles while preserving phosphoregulation. Overall, our study provides advanced analyses of mammalian phosphoproteomes and a framework for the study of their contribution to phenotypic evolution.

The Calcineurin Signaling Network Evolves Via Conserved Kinase-phosphatase Modules That Transcend Substrate Identity

Molecular Cell. Aug, 2014  |  Pubmed ID: 24930733

To define a functional network for calcineurin, the conserved Ca(2+)/calmodulin-regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by copurification and dephosphorylation assays. This study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the network by acquiring a calcineurin-recognition motif. Calcineurin substrates in yeast and mammals are distinct due to network rewiring but, surprisingly, are phosphorylated by similar kinases. We postulate that corecognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin-kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase-phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop that establish common regulatory motifs in signaling networks.

Chromosomal Variation Segregates Within Incipient Species and Correlates with Reproductive Isolation

Molecular Ecology. Sep, 2014  |  Pubmed ID: 25039979

Reproductive isolation is a critical step in the process of speciation. Among the most important factors driving reproductive isolation are genetic incompatibilities. Whether these incompatibilities are already present before extrinsic factors prevent gene flow between incipient species remains largely unresolved in natural systems. This question is particularly challenging because it requires that we catch speciating populations in the act before they reach the full-fledged species status. We measured the extent of intrinsic postzygotic isolation within and between phenotypically and genetically divergent lineages of the wild yeast Saccharomyces paradoxus that have partially overlapping geographical distributions. We find that hybrid viability between lineages progressively decreases with genetic divergence. A large proportion of postzygotic inviability within lineages is associated with chromosomal rearrangements, suggesting that chromosomal differences substantially contribute to the early steps of reproductive isolation within lineages before reaching fixation. Our observations show that polymorphic intrinsic factors may segregate within incipient species before they contribute to their full reproductive isolation and highlight the role of chromosomal rearrangements in speciation. We propose different hypotheses based on adaptation, biogeographical events and life history evolution that could explain these observations.

Turnover of Protein Phosphorylation Evolving Under Stabilizing Selection

Frontiers in Genetics. 2014  |  Pubmed ID: 25101120

Most proteins are regulated by posttranslational modifications and changes in these modifications contribute to evolutionary changes as well as to human diseases. Phosphorylation of serines, threonines, and tyrosines are the most common modifications identified to date in eukaryotic proteomes. While the mode of action and the function of most phosphorylation sites remain unknown, functional studies have shown that phosphorylation affects protein stability, localization and ability to interact. Two broad modes of action have been described for protein phosphorylation. The first mode corresponds to the canonical and qualitative view whereby single phosphorylation sites act as molecular switches that either turn on or off specific protein functions through direct or allosteric effects. The second mode is more akin to a rheostat than a switch. In this case, a group of phosphorylation sites in a given protein region contributes collectively to the modification of the protein, irrespective of the precise position of individual sites, through an aggregate property. Here we discuss these two types of regulation and examine how they affect the rate and patterns of protein phosphorylation evolution. We describe how the evolution of clusters of phosphorylation sites can be studied under the framework of complex traits evolution and stabilizing selection.

Detecting Functional Divergence After Gene Duplication Through Evolutionary Changes in Posttranslational Regulatory Sequences

PLoS Computational Biology. Dec, 2014  |  Pubmed ID: 25474245

Gene duplication is an important evolutionary mechanism that can result in functional divergence in paralogs due to neo-functionalization or sub-functionalization. Consistent with functional divergence after gene duplication, recent studies have shown accelerated evolution in retained paralogs. However, little is known in general about the impact of this accelerated evolution on the molecular functions of retained paralogs. For example, do new functions typically involve changes in enzymatic activities, or changes in protein regulation? Here we study the evolution of posttranslational regulation by examining the evolution of important regulatory sequences (short linear motifs) in retained duplicates created by the whole-genome duplication in budding yeast. To do so, we identified short linear motifs whose evolutionary constraint has relaxed after gene duplication with a likelihood-ratio test that can account for heterogeneity in the evolutionary process by using a non-central chi-squared null distribution. We find that short linear motifs are more likely to show changes in evolutionary constraints in retained duplicates compared to single-copy genes. We examine changes in constraints on known regulatory sequences and show that for the Rck1/Rck2, Fkh1/Fkh2, Ace2/Swi5 paralogs, they are associated with previously characterized differences in posttranslational regulation. Finally, we experimentally confirm our prediction that for the Ace2/Swi5 paralogs, Cbk1 regulated localization was lost along the lineage leading to SWI5 after gene duplication. Our analysis suggests that changes in posttranslational regulation mediated by short regulatory motifs systematically contribute to functional divergence after gene duplication.

The Yeast Galactose Network As a Quantitative Model for Cellular Memory

Molecular BioSystems. Jan, 2015  |  Pubmed ID: 25328105

Recent experiments have revealed surprising behavior in the yeast galactose (GAL) pathway, one of the preeminent systems for studying gene regulation. Under certain circumstances, yeast cells display memory of their prior nutrient environments. We distinguish two kinds of cellular memory discovered by quantitative investigations of the GAL network and present a conceptual framework for interpreting new experiments and current ideas on GAL memory. Reinduction memory occurs when cells respond transcriptionally to one environment, shut down the response during several generations in a second environment, then respond faster and with less cell-to-cell variation when returned to the first environment. Persistent memory describes a long-term, arguably stable response in which cells adopt a bimodal or unimodal distribution of induction levels depending on their preceding environment. Deep knowledge of how the yeast GAL pathway responds to different sugar environments has enabled rapid progress in uncovering the mechanisms behind GAL memory, which include cytoplasmic inheritance of inducer proteins and positive feedback loops among regulatory genes. This network of genes, long used to study gene regulation, is now emerging as a model system for cellular memory.

The Last Enzyme of the De Novo Purine Synthesis Pathway ATIC Plays a Central Role in Insulin Signalling and the Golgi/endosomes Protein Network

Molecular & Cellular Proteomics : MCP. Feb, 2015  |  Pubmed ID: 25687571

Insulin is internalized with its cognate receptor into the endosomal apparatus rapidly after binding to hepatocytes. We performed a bioinformatic screen of Golgi/endosome (G/E) hepatic protein fractions and found that ATIC, which is a rate limiting enzyme in the de novo purine biosynthesis pathway, and PTPLAD1 are associated with IR internalisation. The IR interactome (IRGEN) connects ATIC to AMPK within the G/E protein network (GEN). 45% of the IRGEN have common heritable variants associated with type 2 diabetes, including ATIC and AMPK. We show that PTPLAD1 and AMPK are rapidly compartmentalised within the plasma membrane (PM) and G/E fractions after insulin stimulation and that ATIC later accumulates in the G/E fraction. Using an in vitro reconstitution system and siRNA mediated partial knockdown of ATIC and PTPLAD1 in HEK293 cells, we show that both ATIC and PTPLAD1 affect IR tyrosine phosphorylation and endocytosis. We further show that insulin stimulation and ATIC knockdown readily increase the level of AMPKThr172 phosphorylation in IR complexes. We observed that IR internalisation was markedly decreased after AMPKα2 knockdown, and treatment with the ATIC substrate AICAR, which is an allosteric activator of AMPK, increased IR endocytosis in cultured cells and in the liver. These results suggest the presence of a signalling mechanism that senses adenylate synthesis, ATP levels and IR activation states and that acts in regulating IR autophosphorylation and endocytosis.

Metabolic Variation in Natural Populations of Wild Yeast

Ecology and Evolution. Feb, 2015  |  Pubmed ID: 25691993

Ecological diversification depends on the extent of genetic variation and on the pattern of covariation with respect to ecological opportunities. We investigated the pattern of utilization of carbon substrates in wild populations of budding yeast Saccharomyces paradoxus. All isolates grew well on a core diet of about 10 substrates, and most were also able to grow on a much larger ancillary diet comprising most of the 190 substrates we tested. There was substantial genetic variation within each population for some substrates. We found geographical variation of substrate use at continental, regional, and local scales. Isolates from Europe and North America could be distinguished on the basis of the pattern of yield across substrates. Two geographical races at the North American sites also differed in the pattern of substrate utilization. Substrate utilization patterns were also geographically correlated at local spatial scales. Pairwise genetic correlations between substrates were predominantly positive, reflecting overall variation in metabolic performance, but there was a consistent negative correlation between categories of substrates in two cases: between the core diet and the ancillary diet, and between pentose and hexose sugars. Such negative correlations in the utilization of substrate from different categories may indicate either intrinsic physiological trade-offs for the uptake and utilization of substrates from different categories, or the accumulation of conditionally neutral mutations. Divergence in substrate use accompanies genetic divergence at all spatial scales in S. paradoxus and may contribute to race formation and speciation.

Found in Translation: Functions and Evolution of a Recently Discovered Alternative Proteome

Current Opinion in Structural Biology. Jun, 2015  |  Pubmed ID: 25795211

A major goal in biology is to map entire proteomes to better understand the biology and evolution of cells. However, our current views of proteomes are conservative and biased against small proteins. Besides serendipitous discoveries of small proteins, it has been largely assumed that eukaryotic mature mRNAs contain a single ORF and that non-coding RNAs are not translated because their ORFs are too short to play a functional role. A flurry of recent studies brought to light an unexplored proteome that is mainly translated from short ORFs in non-coding regions and from alternative ORFs (AltORFs) in reference genes. The detection of these small proteins and the elucidation of their functions remain challenging and open a new dimension of eukaryotic proteomes, including the birth of novel genes and proteins.

Systematic Identification of Signal Integration by Protein Kinase A

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2015  |  Pubmed ID: 25831502

Cellular processes and homeostasis control in eukaryotic cells is achieved by the action of regulatory proteins such as protein kinase A (PKA). Although the outbound signals from PKA directed to processes such as metabolism, growth, and aging have been well charted, what regulates this conserved regulator remains to be systematically identified to understand how it coordinates biological processes. Using a yeast PKA reporter assay, we identified genes that influence PKA activity by measuring protein-protein interactions between the regulatory and the two catalytic subunits of the PKA complex in 3,726 yeast genetic-deletion backgrounds grown on two carbon sources. Overall, nearly 500 genes were found to be connected directly or indirectly to PKA regulation, including 80 core regulators, denoting a wide diversity of signals regulating PKA, within and beyond the described upstream linear pathways. PKA regulators span multiple processes, including the antagonistic autophagy and methionine biosynthesis pathways. Our results converge toward mechanisms of PKA posttranslational regulation by lysine acetylation, which is conserved between yeast and humans and that, we show, regulates protein complex formation in mammals and carbohydrate storage and aging in yeast. Taken together, these results show that the extent of PKA input matches with its output, because this kinase receives information from upstream and downstream processes, and highlight how biological processes are interconnected and coordinated by PKA.

Identification of Candidate Mimicry Proteins Involved in Parasite-driven Phenotypic Changes

Parasites & Vectors. Apr, 2015  |  Pubmed ID: 25888917

Endoparasites with complex life cycles are faced with several biological challenges, as they need to occupy various ecological niches throughout their development. Host phenotypes that increase the parasite's transmission rate to the next host have been extensively described, but few mechanistic explanations have been proposed to describe their proximate causes. In this study we explore the possibility that host phenotypic changes are triggered by the production of mimicry proteins from the parasite by using an ecological model system consisting of the infection of the threespine stickleback (Gasterosteus aculeatus) by the cestode Schistocephalus solidus.

Feedback Regulation Between Autophagy and PKA

Autophagy. 2015  |  Pubmed ID: 26046386

Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

Lachancea Quebecensis Sp. Nov., a Yeast Species Consistently Isolated from Tree Bark in the Canadian Province of Québec

International Journal of Systematic and Evolutionary Microbiology. Oct, 2015  |  Pubmed ID: 26297665

A thorough sampling of maple, oak, birch, and apple tree bark in North America yielded a set of isolates that represent a yeast species not yet formally described. The strains obtained were all isolated from the Canadian province of Québec. These four isolates have identical electrophoretic karyotypes, distinct from other species of the genus Lachancea, and are most closely related to the formally recognized species Lachancea thermotolerans according to the D1/D2 domain of the LSU rDNA gene and 5.8S–ITS region. Previous studies revealed the existence of a population of strains closely related to L. thermotolerans, with unique D1/D2 sequences and the ability to grow on melibiose, which is also true for these isolates. The sequences obtained here (for the D1/D2, and 5.8S–ITS region) are identical among the four strains, and in a phylogenetic analysis of the D1/D2 region, the strains form a distinct clade with the previously described population closely related to L. thermotolerans, composed of isolates from Japan, as well as from the provinces of Ontario and Québec in Canada. On the basis of select physiological and phylogenetic characteristics, a novel ascosporogenous yeast species, Lachancea quebecensis sp. nov., is proposed. The type strain LL11_022T ( = CBS 14138T = CLIB 1763T = UCDFST 15-106T) was isolated from maple tree bark in the Station Duchesnay, QC region of Québec, Canada. The MycoBank number is MB811749.

RNAseq Analysis Highlights Specific Transcriptome Signatures of Yeast and Mycelial Growth Phases in the Dutch Elm Disease Fungus Ophiostoma Novo-ulmi

G3 (Bethesda, Md.). Sep, 2015  |  Pubmed ID: 26384770

Fungal dimorphism is a complex trait and our understanding of the ability of fungi to display different growth morphologies is limited to a small number of model species. Here we study a highly aggressive dimorphic fungus, the ascomycete Ophiostoma novo-ulmi, which is a model in plant pathology and the causal agent of Dutch elm disease. The two growth phases that this fungus displays, i.e., a yeast phase and mycelial phase, are thought to be involved in key steps of disease development. We used RNAseq to investigate the genome-wide gene expression profiles that are associated with yeast and mycelial growth phases in vitro. Our results show a clear molecular distinction between yeast and mycelial phase gene expression profiles. Almost 12% of the gene content is differentially expressed between the two phases, which reveals specific functions related to each growth phase. We compared O. novo-ulmi transcriptome profiles with those of two model dimorphic fungi, Candida albicans and Histoplasma capsulatum. Few orthologs showed similar expression regulation between the two growth phases, which suggests that, globally, the genes associated with these two life forms are poorly conserved. This poor conservation underscores the importance of developing specific tools for emerging model species that are distantly related to the classical ones. Taken together, our results provide insights into transcriptome regulation and molecular specificity in O. novo-ulmi and offer a new perspective for understanding fungal dimorphism.

Evolutionary Rescue by Compensatory Mutations is Constrained by Genomic and Environmental Backgrounds

Molecular Systems Biology. Oct, 2015  |  Pubmed ID: 26459777

Since deleterious mutations may be rescued by secondary mutations during evolution, compensatory evolution could identify genetic solutions leading to therapeutic targets. Here, we tested this hypothesis and examined whether these solutions would be universal or would need to be adapted to one's genetic and environmental make-ups. We performed experimental evolutionary rescue in a yeast disease model for the Wiskott-Aldrich syndrome in two genetic backgrounds and carbon sources. We found that multiple aspects of the evolutionary rescue outcome depend on the genotype, the environment, or a combination thereof. Specifically, the compensatory mutation rate and type, the molecular rescue mechanism, the genetic target, and the associated fitness cost varied across contexts. The course of compensatory evolution is therefore highly contingent on the initial conditions in which the deleterious mutation occurs. In addition, these results reveal biologically favored therapeutic targets for the Wiskott-Aldrich syndrome, including the target of an unrelated clinically approved drug. Our results experimentally illustrate the importance of epistasis and environmental evolutionary constraints that shape the adaptive landscape and evolutionary rate of molecular networks.

The Genomics of Wild Yeast Populations Sheds Light on the Domestication of Man's Best (micro) Friend

Molecular Ecology. Nov, 2015  |  Pubmed ID: 26509691

The domestication of plants, animals and microbes by humans are the longest artificial evolution experiments ever performed. The study of these long-term experiments can teach us about the genomics of adaptation through the identification of the genetic bases underlying the traits favoured by humans. In laboratory evolution, the characterization of the molecular changes that evolved specifically in some lineages is straightforward because the ancestors are readily available, for instance in the freezer. However, in the case of domesticated species, the ancestor is often missing, which leads to the necessity of going back to nature in order to infer the most likely ancestral state. Significant and relatively recent examples of this approach include wolves as the closest wild relative to domestic dogs (Axelsson et al. 2013) and teosinte as the closest relative to maize (reviewed in Hake & Ross-Ibarra 2015). In both cases, the joint analysis of domesticated lineages and their wild cousins has been key in reconstructing the molecular history of their domestication. While the identification of closest wild relatives has been done for many plants and animals, these comparisons represent challenges for micro-organisms. This has been the case for the budding yeast Saccharomyces cerevisiae, whose natural ecological niche is particularly challenging to define. For centuries, this unicellular fungus has been the cellular factory for wine, beer and bread crafting, and currently for bioethanol and drug production. While the recent development of genomics has lead to the identification of many genetic elements associated with important wine characteristics, the historical origin of some of the domesticated wine strains has remained elusive due to the lack of knowledge of their close wild relatives. In this issue of Molecular Ecology, Almeida et al. (2015) identified what is to date the closest known wild population of the wine yeast. This population is found associated with oak trees in Europe, presumably its natural host. Using population genomics analyses, Almeida and colleagues discovered that the initial divergence between natural and domesticated wine yeasts in the Mediterranean region took place around the early days of wine production. Surprisingly, genomic regions that are key to wine production today appeared not to be derived from these natural populations but from genes gained from other yeast species.

Multi-scale Perturbations of Protein Interactomes Reveal Their Mechanisms of Regulation, Robustness and Insights into Genotype-phenotype Maps

Briefings in Functional Genomics. Mar, 2016  |  Pubmed ID: 26476431

Cellular architectures and signaling machineries are organized through protein-protein interactions (PPIs). High-throughput methods to study PPIs in yeast have opened a new perspective on the organization of the cell by allowing the study of whole protein interactomes. Recent investigations have moved from the description of this organization to the analysis of its dynamics by experimenting how protein interaction networks (PINs) are rewired in response to perturbations. Here we review studies that have used the budding yeast as an experimental system to explore these altered networks. Given the large space of possible PPIs and the diversity of potential genetic and environmental perturbations, high-throughput methods are an essential requirement to survey PIN perturbations on a large scale. Network perturbations are typically conceptualized as the removal of entire proteins (nodes), the modification of single PPIs (edges) or changes in growth conditions. These studies have revealed mechanisms of PPI regulation, PIN architectural organization, robustness and sensitivity to perturbations. Despite these major advances, there are still inherent limits to current technologies that lead to a trade-off between the number of perturbations and the number of PPIs that can be considered simultaneously. Nevertheless, as we exemplify here, targeted approaches combined with the existing resources remain extremely powerful to explore the inner organization of cells and their responses to perturbations.

Evidence of Natural Hybridization in Brazilian Wild Lineages of Saccharomyces Cerevisiae

Genome Biology and Evolution. Jan, 2016  |  Pubmed ID: 26782936

The natural biology of Saccharomyces cerevisiae, the best known unicellular model eukaryote, remains poorly documented and understood although recent progress has started to change this situation. Studies carried out recently in the Northern Hemisphere revealed the existence of wild populations associated with oak trees in North America, Asia, and in the Mediterranean region. However, in spite of these advances, the global distribution of natural populations of S. cerevisiae, especially in regions were oaks and other members of the Fagaceae are absent, is not well understood. Here we investigate the occurrence of S. cerevisiae in Brazil, a tropical region where oaks and other Fagaceae are absent. We report a candidate natural habitat of S. cerevisiae in South America and, using whole-genome data, we uncover new lineages that appear to have as closest relatives the wild populations found in North America and Japan. A population structure analysis revealed the penetration of the wine genotype into the wild Brazilian population, a first observation of the impact of domesticated microbe lineages on the genetic structure of wild populations. Unexpectedly, the Brazilian population shows conspicuous evidence of hybridization with an American population of Saccharomyces paradoxus. Introgressions from S. paradoxus were significantly enriched in genes encoding secondary active transmembrane transporters. We hypothesize that hybridization in tropical wild lineages may have facilitated the habitat transition accompanying the colonization of the tropical ecosystem.

Dissection of Expression-quantitative Trait Locus and Allele Specificity Using a Haploid/diploid Plant System - Insights into Compensatory Evolution of Transcriptional Regulation Within Populations

The New Phytologist. Jul, 2016  |  Pubmed ID: 26891783

Regulation of gene expression plays a central role in translating genotypic variation into phenotypic variation. Dissection of the genetic basis of expression variation is key to understanding how expression regulation evolves. Such analyses remain challenging in contexts where organisms are outbreeding, highly heterozygous and long-lived such as in the case of conifer trees. We developed an RNA sequencing (RNA-seq)-based approach for both expression-quantitative trait locus (eQTL) mapping and the detection of cis-acting (allele-specific) vs trans-acting (non-allele-specific) eQTLs. This method can be potentially applied to many conifers. We used haploid and diploid meiotic seed tissues of a single self-fertilized white spruce (Picea glauca) individual to dissect eQTLs according to linkage and allele specificity. The genetic architecture of local eQTLs linked to the expressed genes was particularly complex, consisting of cis-acting, trans-acting and, surprisingly, compensatory cis-trans effects. These compensatory effects influence expression in opposite directions and are neutral when combined in homozygotes. Nearly half of local eQTLs were under compensation, indicating that close linkage between compensatory cis-trans factors is common in spruce. Compensated genes were overrepresented in developmental and cell organization functions. Our haploid-diploid eQTL analysis in spruce revealed that compensatory cis-trans eQTLs segregate within populations and evolve in close genetic linkage.

[Wine Yeast As a Model for the Study of Human Genes and Diseases and Their Personalized Effects]

Medecine Sciences : M/S. Apr, 2016  |  Pubmed ID: 27137687

Transcriptome Sequences Spanning Key Developmental States As a Resource for the Study of the Cestode Schistocephalus Solidus, a Threespine Stickleback Parasite

GigaScience. Jun, 2016  |  Pubmed ID: 27259971

Schistocephalus solidus is a well-established model organism for studying the complex life cycle of cestodes and the mechanisms underlying host-parasite interactions. However, very few large-scale genetic resources for this species are available. We have sequenced and de novo-assembled the transcriptome of S. solidus using tissues from whole worms at three key developmental states - non-infective plerocercoid, infective plerocercoid and adult plerocercoid - to provide a resource for studying the evolution of complex life cycles and, more specifically, how parasites modulate their interactions with their hosts during development.

Complex Ancestries of Lager-Brewing Hybrids Were Shaped by Standing Variation in the Wild Yeast Saccharomyces Eubayanus

PLoS Genetics. 07, 2016  |  Pubmed ID: 27385107

Lager-style beers constitute the vast majority of the beer market, and yet, the genetic origin of the yeast strains that brew them has been shrouded in mystery and controversy. Unlike ale-style beers, which are generally brewed with Saccharomyces cerevisiae, lagers are brewed at colder temperatures with allopolyploid hybrids of Saccharomyces eubayanus x S. cerevisiae. Since the discovery of S. eubayanus in 2011, additional strains have been isolated from South America, North America, Australasia, and Asia, but only interspecies hybrids have been isolated in Europe. Here, using genome sequence data, we examine the relationships of these wild S. eubayanus strains to each other and to domesticated lager strains. Our results support the existence of a relatively low-diversity (π = 0.00197) lineage of S. eubayanus whose distribution stretches across the Holarctic ecozone and includes wild isolates from Tibet, new wild isolates from North America, and the S. eubayanus parents of lager yeasts. This Holarctic lineage is closely related to a population with higher diversity (π = 0.00275) that has been found primarily in South America but includes some widely distributed isolates. A second diverse South American population (π = 0.00354) and two early-diverging Asian subspecies are more distantly related. We further show that no single wild strain from the Holarctic lineage is the sole closest relative of lager yeasts. Instead, different parts of the genome portray different phylogenetic signals and ancestry, likely due to outcrossing and incomplete lineage sorting. Indeed, standing genetic variation within this wild Holarctic lineage of S. eubayanus is responsible for genetic variation still segregating among modern lager-brewing hybrids. We conclude that the relationships among wild strains of S. eubayanus and their domesticated hybrids reflect complex biogeographical and genetic processes.

Speciation Driven by Hybridization and Chromosomal Plasticity in a Wild Yeast

Nature Microbiology. Jan, 2016  |  Pubmed ID: 27571751

Hybridization is recognized as a powerful mechanism of speciation and a driving force in generating biodiversity. However, only few multicellular species, limited to a handful of plants and animals, have been shown to fulfil all the criteria of homoploid hybrid speciation. This lack of evidence could lead to the interpretation that speciation by hybridization has a limited role in eukaryotes, particularly in single-celled organisms. Laboratory experiments have revealed that fungi such as budding yeasts can rapidly develop reproductive isolation and novel phenotypes through hybridization, showing that in principle homoploid speciation could occur in nature. Here, we report a case of homoploid hybrid speciation in natural populations of the budding yeast Saccharomyces paradoxus inhabiting the North American forests. We show that the rapid evolution of chromosome architecture and an ecological context that led to secondary contact between nascent species drove the formation of an incipient hybrid species with a potentially unique ecological niche.

Molecular and Cellular Bases of Adaptation to a Changing Environment in Microorganisms

Proceedings. Biological Sciences. Oct, 2016  |  Pubmed ID: 27798299

Environmental heterogeneity constitutes an evolutionary challenge for organisms. While evolutionary dynamics under variable conditions has been explored for decades, we still know relatively little about the cellular and molecular mechanisms involved. It is of paramount importance to examine these molecular bases because they may play an important role in shaping the course of evolution. In this review, we examine the diversity of adaptive mechanisms in the face of environmental changes. We exploit the recent literature on microbial systems because those have benefited the most from the recent emergence of genetic engineering and experimental evolution followed by genome sequencing. We identify four emerging trends: (i) an adaptive molecular change in a pathway often results in fitness trade-off in alternative environments but the effects are dependent on a mutation's genetic background; (ii) adaptive changes often modify transcriptional and signalling pathways; (iii) several adaptive changes may occur within the same molecular pathway but be associated with pleiotropy of different signs across environments; (iv) because of their large associated costs, macromolecular changes such as gene amplification and aneuploidy may be a rapid mechanism of adaptation in the short-term only. The course of adaptation in a variable environment, therefore, depends on the complexity of the environment but also on the molecular relationships among the genes involved and between the genes and the phenotypes under selection.

The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein-Protein Interactions

Cold Spring Harbor Protocols. Nov, 2016  |  Pubmed ID: 27803252

Protein-fragment complementation assays (PCAs) can be used to study protein-protein interactions (PPIs) in any living cell, in vivo or in vitro, in any subcellular compartment or membranes. Here, we present a detailed protocol for performing and analyzing a high-throughput PCA screening to study PPIs in yeast, using dihydrofolate reductase (DHFR) as the reporter protein. The DHFR PCA is a simple survival-selection assay in which Saccharomyces cerevisiae DHFR (scDHFR) is inhibited by methotrexate, thus preventing nucleotide synthesis and causing arrest of cell division. Complementation of cells with a methotrexate-insensitive murine DHFR restores nucleotide synthesis, allowing cell proliferation. The methotrexate-resistant DHFR has two mutations (L22F and F31S) and is 10,000 times less sensitive to methotrexate than wild-type scDHFR, but retains full catalytic activity. The DHFR PCA is sensitive enough for PPIs to be detected for open reading frame (ORF)-PCA fragments expressed off of their endogenous promoters.

Combining the Dihydrofolate Reductase Protein-Fragment Complementation Assay with Gene Deletions to Establish Genotype-to-Phenotype Maps of Protein Complexes and Interaction Networks

Cold Spring Harbor Protocols. Nov, 2016  |  Pubmed ID: 27803253

Systematically measuring the impact of gene deletion on protein-protein interactions is a promising approach to reveal the structural bases of protein interaction networks and to allow a better understanding of how genotypes translate into phenotypes. Genetic and protein-interaction tools in yeast now allow us to explore this third dimension of protein-protein interaction networks. Because it is scalable and quantitative, the protein-fragment complementation assay (PCA) using dihydrofolate reductase (DHFR) as the reporter protein provides an exceptionally powerful tool for such a purpose. Here, we describe a fully automated protocol that combines DHFR PCA for protein-protein interaction measurement and synthetic genetic array (SGA) technology for introducing mutant and other alleles into PCA strains using genetic crosses. In this, PCA strains are crossed with strains carrying a gene deletion and SGA markers, and the recombinant haploid progeny are selected by SGA. The resulting haploid strains, each expressing a DHFR-fragment fusion protein in a gene-specific haploid deletion background, are crossed to measure the interaction between the two recombinant proteins by PCA in a diploid homozygous deletion background. This approach can be used to measure a single protein interaction in a large array of genetic backgrounds or a large number of protein interactions in a small number of genetic backgrounds.

Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells

Cold Spring Harbor Protocols. Nov, 2016  |  Pubmed ID: 27803260

Protein-fragment complementation assays (PCAs) comprise a family of assays that can be used to study protein-protein interactions (PPIs), conformation changes, and protein complex dimensions. We developed PCAs to provide simple and direct methods for the study of PPIs in any living cell, subcellular compartments or membranes, multicellular organisms, or in vitro. Because they are complete assays, requiring no cell-specific components other than reporter fragments, they can be applied in any context. PCAs provide a general strategy for the detection of proteins expressed at endogenous levels within appropriate subcellular compartments and with normal posttranslational modifications, in virtually any cell type or organism under any conditions. Here we introduce a number of applications of PCAs in budding yeast, Saccharomyces cerevisiae These applications represent the full range of PPI characteristics that might be studied, from simple detection on a large scale to visualization of spatiotemporal dynamics.

The TRP Channels Pkd2, NompC, and Trpm Act in Cold-Sensing Neurons to Mediate Unique Aversive Behaviors to Noxious Cold in Drosophila

Current Biology : CB. Dec, 2016  |  Pubmed ID: 27818173

The basic mechanisms underlying noxious cold perception are not well understood. We developed Drosophila assays for noxious cold responses. Larvae respond to near-freezing temperatures via a mutually exclusive set of singular behaviors-in particular, a full-body contraction (CT). Class III (CIII) multidendritic sensory neurons are specifically activated by cold and optogenetic activation of these neurons elicits CT. Blocking synaptic transmission in CIII neurons inhibits CT. Genetically, the transient receptor potential (TRP) channels Trpm, NompC, and Polycystic kidney disease 2 (Pkd2) are expressed in CIII neurons, where each is required for CT. Misexpression of Pkd2 is sufficient to confer cold responsiveness. The optogenetic activation level of multimodal CIII neurons determines behavioral output, and visualization of neuronal activity supports this conclusion. Coactivation of cold- and heat-responsive sensory neurons suggests that the cold-evoked response circuitry is dominant. Our Drosophila model will enable a sophisticated molecular genetic dissection of cold nociceptive genes and circuits.

Mitotic Phosphotyrosine Network Analysis Reveals That Tyrosine Phosphorylation Regulates Polo-like Kinase 1 (PLK1)

Science Signaling. Dec, 2016  |  Pubmed ID: 27965426

Tyrosine phosphorylation is closely associated with cell proliferation. During the cell cycle, serine and threonine phosphorylation plays the leading role, and such phosphorylation events are most dynamic during the mitotic phase of the cell cycle. However, mitotic phosphotyrosine is not well characterized. Although a few functionally-relevant mitotic phosphotyrosine sites have been characterized, evidence suggests that this modification may be more prevalent than previously appreciated. Here, we examined tyrosine phosphorylation in mitotic human cells including those on spindle-associated proteins.? Database mining confirmed ~2000 mitotic phosphotyrosine sites, and network analysis revealed a number of subnetworks that were enriched in tyrosine-phosphorylated proteins, including components of the kinetochore or spindle and SRC family kinases. We identified Polo-like kinase 1 (PLK1), a major signaling hub in the spindle subnetwork, as phosphorylated at the conserved Tyr(217) in the kinase domain. Substitution of Tyr(217) with a phosphomimetic residue eliminated PLK1 activity in vitro and in cells. Further analysis showed that Tyr(217) phosphorylation reduced the phosphorylation of Thr(210) in the activation loop, a phosphorylation event necessary for PLK1 activity. Our data indicate that mitotic tyrosine phosphorylation regulated a key serine/threonine kinase hub in mitotic cells and suggested that spatially separating tyrosine phosphorylation events can reveal previously unrecognized regulatory events and complexes associated with specific structures of the cell cycle.

Identification of the Fitness Determinants of Budding Yeast on a Natural Substrate

The ISME Journal. Apr, 2017  |  Pubmed ID: 27935595

The budding yeasts are prime models in genomics and cell biology, but the ecological factors that determine their success in non-human-associated habitats is poorly understood. In North America Saccharomyces yeasts are present on the bark of deciduous trees, where they feed on bark and sap exudates. In the North East, Saccharomyces paradoxus is found on maples, which makes maple sap a natural substrate for this species. We measured growth rates of S. paradoxus natural isolates on maple sap and found variation along a geographical gradient not explained by the inherent variation observed under optimal laboratory conditions. We used a functional genomic screen to reveal the ecologically relevant genes and conditions required for optimal growth in this substrate. We found that the allantoin degradation pathway is required for optimal growth in maple sap, in particular genes necessary for allantoate utilization, which we demonstrate is the major nitrogen source available to yeast in this environment. Growth with allantoin or allantoate as the sole nitrogen source recapitulated the variation in growth rates in maple sap among strains. We also show that two lineages of S. paradoxus display different life-history traits on allantoin and allantoate media, highlighting the ecological relevance of this pathway.

When Nuclear-encoded Proteins and Mitochondrial RNAs Do Not Get Along, Species Split Apart

EMBO Reports. Jan, 2017  |  Pubmed ID: 27986790

Population Genomics Reveals Structure at the Individual, Host-tree Scale and Persistence of Genotypic Variants of the Undomesticated Yeast Saccharomyces Paradoxus in a Natural Woodland

Molecular Ecology. Feb, 2017  |  Pubmed ID: 27988980

Genetic diversity in experimental, domesticated and wild populations of the related yeasts, Saccharomyces cerevisiae and Saccharomyces paradoxus, has been well described at the global scale. We investigated the population genomics of a local population on a small spatial scale to address two main questions. First, is there genomic variation in a S. paradoxus population at a spatial scale spanning centimetres (microsites) to tens of metres? Second, does the distribution of genomic variants persist over time? Our sample consisted of 42 S. paradoxus strains from 2014 and 43 strains from 2015 collected from the same 72 microsites around four host trees (Quercus rubra and Quercus alba) within 1 km(2) in a mixed hardwood forest in southern Ontario. Six additional S. paradoxus strains recovered from adjacent maple and beech trees in 2015 are also included in the sample. Whole-genome sequencing and genomic SNP analysis revealed five differentiated groups (clades) within the sampled area. The signal of persistence of genotypes in their microsites from 2014 to 2015 was highly significant. Isolates from the same tree tended to be more related than strains from different trees, with limited evidence of dispersal between trees. In growth assays, one genotype had a significantly longer lag phase than the other strains. Our results indicate that different clades coexist at fine spatial scale and that population structure persists over at least a one-year interval in these wild yeasts, suggesting the efficacy of yearly sampling to follow longer term genetic dynamics in future studies.

Major Host Transitions Are Modulated Through Transcriptome-wide Reprogramming Events in Schistocephalus Solidus, a Threespine Stickleback Parasite

Molecular Ecology. Feb, 2017  |  Pubmed ID: 27997044

Parasites with complex life cycles have developed numerous phenotypic strategies, closely associated with developmental events, to enable the exploitation of different ecological niches and facilitate transmission between hosts. How these environmental shifts are regulated from a metabolic and physiological standpoint, however, still remain to be fully elucidated. We examined the transcriptomic response of Schistocephalus solidus, a trophically transmitted parasite with a complex life cycle, over the course of its development in an intermediate host, the threespine stickleback, and the final avian host. Results from our differential gene expression analysis show major reprogramming events among developmental stages. The final host stage is characterized by a strong activation of reproductive pathways and redox homoeostasis. The attainment of infectivity in the fish intermediate host-which precedes sexual maturation in the final host and is associated with host behaviour changes-is marked by transcription of genes involved in neural pathways and sensory perception. Our results suggest that un-annotated and S. solidus-specific genes could play a determinant role in host-parasite molecular interactions required to complete the parasite's life cycle. Our results permit future comparative analyses to help disentangle species-specific patterns of infection from conserved mechanisms, ultimately leading to a better understanding of the molecular control and evolution of complex life cycles.

Gene Duplication Can Impart Fragility, Not Robustness, in the Yeast Protein Interaction Network

Science (New York, N.Y.). Feb, 2017  |  Pubmed ID: 28183979

The maintenance of duplicated genes is thought to protect cells from genetic perturbations, but the molecular basis of this robustness is largely unknown. By measuring the interaction of yeast proteins with their partners in wild-type cells and in cells lacking a paralog, we found that 22 out of 56 paralog pairs compensate for the lost interactions. An equivalent number of pairs exhibit the opposite behavior and require each other's presence for maintaining their interactions. These dependent paralogs generally interact physically, regulate each other's abundance, and derive from ancestral self-interacting proteins. This reveals that gene duplication may actually increase mutational fragility instead of robustness in a large number of cases.

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