Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells

Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T–EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2′-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T–EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP

The Carnegie Astrometric Planet Search Program

We are undertaking an astrometric search for gas giant planets and brown dwarfs orbiting nearby low mass dwarf stars with the 2.5-m du Pont telescope at the Las Campanas Observatory in Chile. We have built two specialized astrometric cameras, the Carnegie Astrometric Planet Search Cameras (CAPSCam-S and CAPSCam-N), using two Teledyne Hawaii-2RG HyViSI arrays, with the cameras' design having been optimized for high accuracy astrometry of M dwarf stars. We describe two independent CAPSCam data reduction approaches and present a detailed analysis of the observations to date of one of our target stars, NLTT 48256. Observations of NLTT 48256 taken since July 2007 with CAPSCam-S imply that astrometric accuracies of around 0.3 milliarcsec per hour are achievable, sufficient to detect a Jupiter-mass companion orbiting 1 AU from a late M dwarf 10 pc away with a signal-to-noise ratio of about 4. We plan to follow about 100 nearby (primarily within about 10 pc) low mass stars, principally late M, L, and T dwarfs, for 10 years or more, in order to detect very low mass companions with orbital periods long enough to permit the existence of habitable, Earth-like planets on shorter-period orbits. These stars are generally too faint and red to be included in ground-based Doppler planet surveys, which are often optimized for FGK dwarfs. The smaller masses of late M dwarfs also yield correspondingly larger astrometric signals for a given mass planet. Our search will help to determine whether gas giant planets form primarily by core accretion or by disk instability around late M dwarf stars.Comment: 48 pages, 9 figures. in press, Publ. Astron. Soc. Pacifi

Serum 25-Hydroxyvitamin D and Intact Parathyroid Hormone Influence Muscle Outcomes in Children and Adolescents

Increases in 25-hydroxyvitamin D concentrations are shown to improve strength in adults; however, data in pediatric populations are scant and equivocal. In this ancillary study of a larger-scale, multi-sited, double-blind, randomized, placebo-controlled vitamin D intervention in US children and adolescents, we examined the associations between changes in vitamin D metabolites and changes in muscle mass, strength, and composition after 12 weeks of vitamin D3 supplementation. Healthy male and female, black and white children and adolescents between the ages of 9 and 13 years from two US states (Georgia 34°N and Indiana 40°N) were enrolled in the study and randomly assigned to receive an oral vitamin D3 dose of 0, 400, 1000, 2000, or 4000 IU/d for 12 weeks between the winter months of 2009 to 2011 (N = 324). Analyses of covariance, partial correlations, and regression analyses of baseline and 12-week changes (post-baseline) in vitamin D metabolites (serum 25(OH)D, 1,25(OH)2 D, intact parathyroid hormone [iPTH]), and outcomes of muscle mass, strength, and composition (total body fat-free soft tissue [FFST], handgrip strength, forearm and calf muscle cross-sectional area [MCSA], muscle density, and intermuscular adipose tissue [IMAT]) were assessed. Serum 25(OH)D and 1,25(OH)2 D, but not iPTH, increased over time, as did fat mass, FFST, forearm and calf MCSA, forearm IMAT, and handgrip strength (p < 0.05). Vitamin D metabolites were not associated with muscle strength at baseline nor after the 12-week intervention. Changes in serum 25(OH)D correlated with decreases in forearm IMAT, whereas changes in serum iPTH predicted increases in forearm and calf MCSA and IMAT (p < 0.05). Overall, increases in 25(OH)D did not influence muscle mass or strength in vitamin D-sufficient children and adolescents; however, the role of iPTH on muscle composition in this population is unknown and warrants further investigation

Path-integral dynamics of water using curvilinear centroids

We develop a path-integral dynamics method for water that resembles centroid molecular dynamics (CMD), except that the centroids are averages of curvilinear, rather than cartesian, bead coordinates. The curvilinear coordinates are used explicitly only when computing the potential of mean force, the components of which are re-expressed in terms of cartesian 'quasi-centroids' (so-called because they are close to the cartesian centroids). Cartesian equations of motion are obtained by making small approximations to the quantum Boltzmann distribution. Simulations of the infrared spectra of various water models over 150-600 K show these approximations to be justified: for a two-dimensional OH-bond model, the quasi-centroid molecular dynamics (QCMD) spectra lie close to the exact quantum spectra, and almost on top of the Matsubara dynamics spectra; for gas-phase water, the QCMD spectra are close to the exact quantum spectra; for liquid water and ice (using the q-TIP4P/F surface), the QCMD spectra are close to the CMD spectra at 600 K, and line up with the results of thermostatted ring-polymer molecular dynamics and approximate quantum calculations at 300 and 150 K. The QCMD spectra show no sign of the CMD 'curvature problem' (of erroneous red shifts and broadening). In the liquid and ice simulations, the potential of mean force was evaluated on the fly by generalising an adiabatic CMD algorithm to curvilinear coordinates; the full limit of adiabatic separation needed to be taken, which made the QCMD calculations 8 times more expensive than partially adiabatic CMD at 300 K, and 32 times at 150 K (and the intensities may still not be converged at this temperature). The QCMD method is probably generalisable to many other systems, provided collective bead-coordinates can be identified that yield compact mean-field ring-polymer distributions.Cambridge University Vice Chancellor's award

Near-Infrared and Star-forming properties of Local Luminous Infrared Galaxies

We use HST NICMOS continuum and Pa-alpha observations to study the near-infrared and star-formation properties of a representative sample of 30 local (d ~ 35-75Mpc) luminous infrared galaxies (LIRGs, infrared 8-1000um luminosities of L_IR=11-11.9[Lsun]). The data provide spatial resolutions of 25-50pc and cover the central ~3.3-7.1kpc regions of these galaxies. About half of the LIRGs show compact (~1-2kpc) Pa-alpha emission with a high surface brightness in the form of nuclear emission, rings, and mini-spirals. The rest of the sample show Pa-alpha emission along the disk and the spiral arms extending over scales of 3-7kpc and larger. About half of the sample contains HII regions with H-alpha luminosities significantly higher than those observed in normal galaxies. There is a linear empirical relationship between the mid-IR 24um and hydrogen recombination (extinction-corrected Pa-alpha) luminosity for these LIRGs, and the HII regions in the central part of M51. This relation holds over more than four decades in luminosity suggesting that the mid-IR emission is a good tracer of the star formation rate (SFR). Analogous to the widely used relation between the SFR and total IR luminosity of Kennicutt (1998), we derive an empirical calibration of the SFR in terms of the monochromatic 24um luminosity that can be used for luminous, dusty galaxies.Comment: Accepted for publication in ApJ. Contact first author for high qualitity version of figure

Ariel - Volume 10 Number 3

Executive Editors Madalyn Schaefgen David Reich Business Manager David Reich News Editors Medical College Edward Zurad CAHS John Guardiani World Mark Zwanger Features Editors Meg Trexler Jim O\u27Brien Editorials Editor Jeffrey Banyas Photography and Sports Editor Stuart Singer Commons Editor Brenda Peterso

Measuring Cation Dependent DNA Polymerase Fidelity Landscapes by Deep Sequencing

High-throughput recording of signals embedded within inaccessible micro-environments is a technological challenge. The ideal recording device would be a nanoscale machine capable of quantitatively transducing a wide range of variables into a molecular recording medium suitable for long-term storage and facile readout in the form of digital data. We have recently proposed such a device, in which cation concentrations modulate the misincorporation rate of a DNA polymerase (DNAP) on a known template, allowing DNA sequences to encode information about the local cation concentration. In this work we quantify the cation sensitivity of DNAP misincorporation rates, making possible the indirect readout of cation concentration by DNA sequencing. Using multiplexed deep sequencing, we quantify the misincorporation properties of two DNA polymerases – Dpo4 and Klenow exo[subscript −] – obtaining the probability and base selectivity of misincorporation at all positions within the template. We find that Dpo4 acts as a DNA recording device for Mn[superscript 2+] with a misincorporation rate gain of ~2%/mM. This modulation of misincorporation rate is selective to the template base: the probability of misincorporation on template T by Dpo4 increases >50-fold over the range tested, while the other template bases are affected less strongly. Furthermore, cation concentrations act as scaling factors for misincorporation: on a given template base, Mn[superscript 2+] and Mg[superscript 2+] change the overall misincorporation rate but do not alter the relative frequencies of incoming misincorporated nucleotides. Characterization of the ion dependence of DNAP misincorporation serves as the first step towards repurposing it as a molecular recording device.Damon Runyon Cancer Research FoundationNational Institutes of Health (U.S.)National Science Foundation (U.S.)McGovern Institute for Brain Research at MITMassachusetts Institute of Technology. Media LaboratoryNew York Stem Cell Foundation (Robertson Neuroscience Investigator Award)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award