Genetic regulation of the development of mating projections in Candida albicans.

Candida albicans is a major human fungal pathogen, capable of switching among a range of morphological types, such as the yeast form, including white and opaque cell types and the GUT (gastrointestinally induced transition) cell type, the filamentous form, including hyphal and pseudohyphal cell types, and chlamydospores. This ability is associated with its commensal and pathogenic life styles. In response to pheromone, C. albicans cells are able to form long mating projections resembling filaments. This filamentous morphology is required for efficient sexual mating. In the current study, we report the genetic regulatory mechanisms controlling the development of mating projections in C. albicans. Ectopic expression of MTLα1 in "a" cells induces the secretion of α-pheromone and promotes the development of mating projections. Using this inducible system, we reveal that members of the pheromone-sensing pathway (including the pheromone receptor), the Ste11-Hst7-Cek1/2 mediated MAPK signalling cascade, and the RAM pathway are essential for the development of mating projections. However, the cAMP/PKA signalling pathway and a number of key regulators of filamentous growth such as Hgc1, Efg1, Flo8, Tec1, Ume6, and Rfg1 are not required for mating projection formation. Therefore, despite the phenotypic similarities between filaments and mating projections in C. albicans, distinct mechanisms are involved in the regulation of these two morphologies

Bqt2p is essential for initiating telomere clustering upon pheromone sensing in fission yeast

The telomere bouquet, i.e., telomere clustering on the nuclear envelope (NE) during meiotic prophase, is thought to promote homologous chromosome pairing. Using a visual screen, we identified bqt2/im295, a mutant that disrupts telomere clustering in fission yeast. Bqt2p is required for linking telomeres to the meiotic spindle pole body (SPB) but not for attachment of telomeres or the SPB to the NE. Bqt2p is expressed upon pheromone sensing and colocalizes thereafter to Sad1p, an SPB protein. This localization only depends on Bqt1p, not on other identified proteins required for telomere clustering. Upon pheromone sensing, generation of Sad1p foci next to telomeres depends on Bqt2p. However, depletion of Bqt2p from the SPB is dispensable for dissolving the telomere bouquet at the end of meiotic prophase. Therefore, telomere bouquet formation requires Bqt2p as a linking component and is finely regulated during meiotic progression

\u3ci\u3eSolenopsis Invicta\u3c/i\u3e Lipidomics—Fatty Acid Profiling of the Two Social Forms of the Red Imported Fire Ant

Undergraduate Basi

Finding undetected protein associations in cell signaling by belief propagation

External information propagates in the cell mainly through signaling cascades and transcriptional activation, allowing it to react to a wide spectrum of environmental changes. High throughput experiments identify numerous molecular components of such cascades that may, however, interact through unknown partners. Some of them may be detected using data coming from the integration of a protein-protein interaction network and mRNA expression profiles. This inference problem can be mapped onto the problem of finding appropriate optimal connected subgraphs of a network defined by these datasets. The optimization procedure turns out to be computationally intractable in general. Here we present a new distributed algorithm for this task, inspired from statistical physics, and apply this scheme to alpha factor and drug perturbations data in yeast. We identify the role of the COS8 protein, a member of a gene family of previously unknown function, and validate the results by genetic experiments. The algorithm we present is specially suited for very large datasets, can run in parallel, and can be adapted to other problems in systems biology. On renowned benchmarks it outperforms other algorithms in the field.Comment: 6 pages, 3 figures, 1 table, Supporting Informatio

Transcription Factors Mat2 and Znf2 Operate Cellular Circuits Orchestrating Opposite- and Same-Sex Mating in Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1α/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell–cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence

Pheromone-sensing neurons regulate peripheral lipid metabolism in <i>Caenorhabditis elegans</i>

It is now established that the central nervous system plays an important role in regulating whole body metabolism and energy balance. However, the extent to which sensory systems relay environmental information to modulate metabolic events in peripheral tissues has remained poorly understood. In addition, it has been challenging to map the molecular mechanisms underlying discrete sensory modalities with respect to their role in lipid metabolism. In previous work our lab has identified instructive roles for serotonin signaling as a surrogate for food availability, as well as oxygen sensing, in the control of whole body metabolism. In this study, we now identify a role for a pair of pheromone-sensing neurons in regulating fat metabolism in C. elegans, which has emerged as a tractable and highly informative model to study the neurobiology of metabolism. A genetic screen revealed that GPA-3, a member of the Gα family of G proteins, regulates body fat content in the intestine, the major metabolic organ for C. elegans. Genetic and reconstitution studies revealed that the potent body fat phenotype of gpa-3 null mutants is controlled from a pair of neurons called ADL(L/R). We show that cAMP functions as the second messenger in the ADL neurons, and regulates body fat stores via the neurotransmitter acetylcholine, from downstream neurons. We find that the pheromone ascr#3, which is detected by the ADL neurons, regulates body fat stores in a GPA-3-dependent manner. We define here a third sensory modality, pheromone sensing, as a major regulator of body fat metabolism. The pheromone ascr#3 is an indicator of population density, thus we hypothesize that pheromone sensing provides a salient 'denominator' to evaluate the amount of food available within a population and to accordingly adjust metabolic rate and body fat levels

The taste of togetherness.

The larvae of fruit flies produce pheromones to control whether they are attracted to others of the same species or whether they avoid members of a different species

Large-scale Cellular Imaging of Neuronal Activity: a Study of Neural Individuality and a Method for Imaging Mouse Cortex

The brain contains an enormous number of neurons with diverse gene expression, morphology, and connectivity. These neurons exhibit distinct activity in the course of behaviors. The study of neural coding of a specific behavior necessitates recording activity from multifarious neurons in the circuit.One appealing approach is to simultaneously image the activity of a very large neuronal population at cellular resolution. However, recording calcium signals from tens of thousands of neurons at one time is not trivial. The gold standard technique, two-photon laser-scanning microscopy, typically permits recording from hundreds of neurons. Recently, we developed objective-coupled planar illumination (OCPI) microscopy, which uses thin sheets of light to image whole volumes of ∼ 10, 000 neurons within 2 seconds. Mydissertation includes an application and a further methodological development of such a fast large-scale imaging technique: 1) Large-scale functional imaging revealsindividuality, dimorphism, and plasticity of mouse pheromone-sensing neurons.Different individuals exhibit distinctive behaviors, which is presumably attributed to the neuronal differences between brains. However, studying neural individuality, especially at the level of the function of single neurons, requires an effective approach to measure cellular activity of a diverse neuronal population in a circuit. Here using OCPI microscopy, I performed calcium imaging of pheromone-sensing neurons in the intact mouse vomeronasal organ. Exhaustive recording enabled robust detection of 17 functionally-defined neuronal types in each animal. Inter-animal differences were much larger than expected from random sampling, and different cell types showed distinct degrees of variability. One prominent difference was a neuronal type present in males and virtually absent in females, and animals exhibited a corresponding dimorphism in investigatory behavior. Surprisingly, this dimorphism was not innate but generated by plasticity, as exposure to female scents led to both the elimination of this cell type and alterations in behavior. The finding that an all-or-none dimorphism in neuronal types is controlled by experience--even in a sensory system devoted to innate responses--highlights the extraordinary role of nurture in neural individuality. 2) A new generation of OCPI microscopy enables unprecedentedlarge-scalein vivoimaging of mouse brain activity by light-sheet microscopy. I have built a new variant of OCPI microscope, horizontal scanning objective-coupled planar lumination (hsOCPI) microscope, with enhanced imaging volume and speed by ∼ 15 fold compared to OCPI, thereby capable of recording ∼ 100, 000 neurons simultaneously. Using this technique, I imaged the entire nervous system of the larval zebrafish (including the spinal cord) and a square-millimeter patch of mouse cortexex vivo. The miniaturized optics around the specimen allowed in vivo imaging through a cranial window of a head-fixed mouse. This technique is the first application of light-sheet microscopy in calcium imaging of mouse cortexin vivo. The exceptional large-scale sampling of cortical activity with cellular resolution should usher new insights into the functions of brain circuits

A common genetic target for environmental and heritable influences on aggressiveness in Drosophila

Environmental and genetic factors can modulate aggressiveness, but the biological mechanisms underlying their influence are largely unknown. Social experience with conspecifics suppresses aggressiveness in both vertebrate and invertebrate species, including Drosophila. We searched for genes whose expression levels correlate with the influence of social experience on aggressiveness in Drosophila by performing microarray analysis of head tissue from socially isolated (aggressive) vs. socially experienced (nonaggressive) male flies. Among {approx}200 differentially expressed genes, only one was also present in a gene set previously identified by profiling Drosophila strains subjected to genetic selection for differences in aggressiveness [Dierick HA, Greenspan RJ (2006) Nat Genet 38:1023–1031]. This gene, Cyp6a20, encodes a cytochrome P450. Social experience increased Cyp6a20 expression and decreased aggressiveness in a reversible manner. In Cyp6a20 mutants, aggressiveness was increased in group-housed but not socially isolated flies. These data identify a common genetic target for environmental and heritable influences on aggressiveness. Cyp6a20 is expressed in a subset of nonneuronal support cells associated with pheromone-sensing olfactory sensilla, suggesting that social experience may influence aggressiveness by regulating pheromone sensitivity

Imaging pheromone sensing in a mouse vomeronasal acute tissue slice preparation.

Peter Karlson and Martin Lüscher used the term pheromone for the first time in 1959 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules secreted and/or contained in biological fluids, such as urine, a liquid known to be a main source of pheromones. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions, hierarchical organisations and sexual interactions and are consequently directly correlated with the survival of a given species. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO), a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs). Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize. Two main vomeronasal receptor families, V1Rs and V2Rs, are composed respectively by 240 and 120 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs) and formyl peptide receptors (FPRs) are also expressed in VSNs. Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons TRPC2 independent transduction channels have been suggested. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex. Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy