RGS Proteins and Septins Cooperate to Promote Chemotropism by Regulating Polar Cap Mobility

Background—Septins are well known to form a boundary between mother and daughter cells in mitosis, but their role in other morphogenic states is poorly understood. Results—Using microfluidics and live cell microscopy, coupled with new computational methods for image analysis, we investigated septin function during pheromone-dependent chemotropic growth in yeast. We show that septins colocalize with the regulator of G-protein signaling (RGS) Sst2, a GTPase-activating protein that dampens pheromone receptor signaling. We show further that the septin structure surrounds the polar cap, ensuring that cell growth is directed toward the source of pheromone. When RGS activity is abrogated, septins are partially disorganized. Under these circumstances the polar cap travels toward septin structures and away from sites of exocytosis, resulting in a loss of gradient tracking. Conclusion—Septin organization is dependent on RGS protein activity. When assembled correctly, septins promote turning of the polar cap and proper tracking of a pheromone gradient

Molecular Characterization of Putative Chordoma Cell Lines

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies

Pheromone- and RSP5-dependent Ubiquitination of the G Protein β Subunit Ste4 in Yeast

Ste4 is the β subunit of a heterotrimeric G protein that mediates mating responses in Saccharomyces cerevisiae. Here we show that Ste4 undergoes ubiquitination in response to pheromone stimulation. Ubiquitination of Ste4 is dependent on the E3 ligase Rsp5. Disrupting the activity of Rsp5 abolishes ubiquitination of Ste4 in vivo, and recombinant Rsp5 is capable of ubiquitinating Ste4 in vitro. We find also that Lys-340 is a major ubiquitination site on Ste4, as pheromone-induced ubiquitination of the protein is prevented when this residue is mutated to an arginine. Functionally, ubiquitination does not appear to regulate the stability of Ste4, as blocking ubiquitination has no apparent effect on either the abundance or the half-life of the protein. However, when presented with a concentration gradient of pheromone, Ste4K340R mutant cells polarize significantly faster than wild-type cells, indicating that ubiquitination limits pheromone-directed polarized growth. Together, these findings reveal a novel stimulus-dependent posttranslational modification of a Gβ subunit, establish Ste4 as a new substrate of the E3 ligase Rsp5, and demonstrate a role for G protein ubiquitination in cell polarization

Cellular Noise Suppression by the Regulator of G Protein Signaling Sst2

G proteins and their associated receptors process information from a variety of environmental stimuli to induce appropriate cellular responses. Generally speaking, each cell in a population responds within defined limits despite large variation in the expression of protein signaling components. Therefore we postulated that noise suppression is encoded within the signaling system. Using the yeast mating pathway as a model we evaluated the ability of a regulator of G protein signaling (RGS) protein to suppress noise. We found that the RGS protein Sst2 limits variability in transcription and morphogenesis in response to pheromone stimulation. While signal suppression is a result of both the GAP (GTPase accelerating) and receptor binding functions of Sst2, noise suppression requires only the GAP activity. Taken together our findings reveal a hitherto overlooked role of RGS proteins as noise suppressors, and demonstrate an ability to uncouple signal and noise in a prototypical stimulus-response pathway

Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK

MAPKs are activated by dual phosphorylation. Much of the MAPK Fus3 is monophosphorylated and acts to inhibit signaling in vivo. Computational models reveal how a kinase scaffold and phosphatase act together to dynamically regulate dual-phosphorylated and monophosphorylated MAPKs and the downstream signal.Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5ND) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or “synthetic,” supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling

Modulation of receptor dynamics by the regulator of G protein signaling Sst2

G protein–coupled receptor (GPCR) signaling is fundamental to physiological processes such as vision, the immune response, and wound healing. In the budding yeast Saccharomyces cerevisiae, GPCRs detect and respond to gradients of pheromone during mating. After pheromone stimulation, the GPCR Ste2 is removed from the cell membrane, and new receptors are delivered to the growing edge. The regulator of G protein signaling (RGS) protein Sst2 acts by accelerating GTP hydrolysis and facilitating pathway desensitization. Sst2 is also known to interact with the receptor Ste2. Here we show that Sst2 is required for proper receptor recovery at the growing edge of pheromone-stimulated cells. Mathematical modeling suggested pheromone-induced synthesis of Sst2 together with its interaction with the receptor function to reestablish a receptor pool at the site of polarized growth. To validate the model, we used targeted genetic perturbations to selectively disrupt key properties of Sst2 and its induction by pheromone. Together our results reveal that a regulator of G protein signaling can also regulate the G protein–coupled receptor. Whereas Sst2 negatively regulates G protein signaling, it acts in a positive manner to promote receptor retention at the growing edge

Search for the standard model Higgs boson decaying to bottom quarks in pp collisions at √s = 7 TeV

A search for the standard model Higgs boson (H) decaying to b[bar over b] when produced in association with weak vector bosons (V) is reported for the following modes: W(μν)H, W(eν)H, Z(μμ)H, Z(ee)H and Z(νν)H. The search is performed in a data sample corresponding to an integrated luminosity of 4.7 fb[superscript −1], recorded by the CMS detector in proton–proton collisions at the LHC with a center-of-mass energy of 7 TeV. No significant excess of events above the expectation from background is observed. Upper limits on the VH production cross section times the H→b[bar over b] branching ratio, with respect to the expectations for a standard model Higgs boson, are derived for a Higgs boson in the mass range 110–135 GeV. In this range, the observed 95% confidence level upper limits vary from 3.4 to 7.5 times the standard model prediction; the corresponding expected limits vary from 2.7 to 6.7 times the standard model prediction.European Organization for Nuclear ResearchUnited States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio

Measurement of the production cross section for pairs of isolated photons in pp collisions at √s = 7 TeV

The integrated and differential cross sections for the production of pairs of isolated photons is measured in proton-proton collisions at a centre-of-mass energy of 7 TeV with the CMS detector at the LHC. A data sample corresponding to an integrated luminosity of 36 pb[superscript −1] is analysed. A next-to-leading-order perturbative QCD calculation is compared to the measurements. A discrepancy is observed for regions of the phase space where the two photons have an azimuthal angle difference Δφ ≲ 2.8 rad.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio

Search for microscopic black hole signatures at the Large Hadron Collider

A search for microscopic black hole production and decay in pp collisions at a center-of-mass energy of 7 TeV has been conducted by the CMS Collaboration at the LHC, using a data sample corresponding to an integrated luminosity of 35 pb[superscript −1]. Events with large total transverse energy are analyzed for the presence of multiple high-energy jets, leptons, and photons, typical of a signal expected from a microscopic black hole. Good agreement with the standard model backgrounds, dominated by QCD multijet production, is observed for various final-state multiplicities and model-independent limits on new physics in these final states are set. Using simple semi-classical approximation, limits on the minimum black hole mass are derived as well, in the range 3.5–4.5 TeV. These are the first direct limits on black hole production at a particle accelerator.European Organization for Nuclear ResearchNational Science Foundation (U.S.)United States. Dept. of Energ

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin