Signal transduction: Clamping down on Src activity

AbstractRecent high-resolution structures of members of the Src family of protein-tyrosine kinases illustrate how a series of cooperative intramolecular interactions represses the catalytic activity of these kinases, but allows for their rapid activation by a variety of regulatory inputs

Molecular machines or pleiomorphic ensembles: signaling complexes revisited

Signaling complexes typically consist of highly dynamic molecular ensembles that are challenging to study and to describe accurately. Conventional mechanical descriptions misrepresent this reality and can be actively counterproductive by misdirecting us away from investigating critical issues

Fast rebinding increases dwell time of Src homology 2 (SH2)-containing proteins near the plasma membrane

Receptor tyrosine kinases (RTKs) control a host of biological functions by phosphorylating tyrosine residues of intracellular proteins upon extracellular ligand binding. The phosphotyrosines (p-Tyr) then recruit a subset of ∼100 Src homology 2 (SH2) domain-containing proteins to the cell membrane. The in vivo kinetics of this process are not well understood. Here we use total internal reflection (TIR) microscopy and single-molecule imaging to monitor interactions between SH2 modules and p-Tyr sites near the cell membrane. We found that the dwell time of SH2 modules within the TIR illumination field is significantly longer than predictions based on chemical dissociation rate constants, suggesting that SH2 modules quickly rebind to nearby p-Tyr sites after dissociation. We also found that, consistent with the rebinding model, the effective diffusion constant is negatively correlated with the respective dwell time for different SH2 domains and the dwell time is positively correlated with the local density of RTK phosphorylation. These results suggest a mechanism whereby signal output can be regulated through the spatial organization of multiple binding sites, which will prompt reevaluation of many aspects of RTK signaling, such as signaling specificity, mechanisms of spatial control, and noise suppression

vbyCaHbeta CCD Photometry of Clusters. VIII. The Super-Metal Rich, Old Open Cluster NGC 6791

CCD photometry on the intermediate-band vbyCaHbeta system is presented for the metal-rich, old open cluster, NGC 6791. Preliminary analysis led to [Fe/H] above +0.4 with an anomalously high reddening and an age below 5 Gyr. A revised calibration between (b-y)_0 and [Fe/H] at a given temperature shows that the traditional color-metallicity relations underestimate the color of the turnoff stars at high metallicity. With the revised relation, the metallicity from hk and the reddening for NGC 6791 become [Fe/H] = +0.45 +/- 0.04 and E(b-y) = 0.113 +/- 0.012 or E(B-V) = 0.155 +/- 0.016. Using the same technique, reanalysis of the photometry for NGC 6253 produces [Fe/H] = +0.58 +/-0.04 and E(b-y) = 0.120 +/- 0.018 or E(B-V) = 0.160 +/- 0.025. The errors quoted include both the internal and external errors. For NGC 6791, the metallicity from m_1 is a factor of two below that from hk, a result that may be coupled to the consistently low metal abundance from DDO photometry of the cluster and the C-deficiency found from high dispersion spectroscopy. E(B-V) is the same value predicted from Galactic reddening maps. With E(B-V) = 0.15 and [Fe/H] = +0.45, the available isochrones predict an age of 7.0 +/- 1.0 Gyr and an apparent modulus of (m-M) = 13.60 +/- 0.15, with the dominant source of the uncertainty arising from inconsistencies among the isochrones. The reanalysis of NGC 6253 with the revised lower reddening confirms that on both the hk and m_1 metallicity scales, NGC 6253, while less than half the age of NGC 6791, remains at least as metal-rich as NGC 6791, if not richer.Comment: Accepted for Astronomical Journal. 42 p. latex file includes 11 figures and 3 tables, one of which is a short version of a data table to appear in online AJ in its entiret

The trough-system algorithm and its application to spatial modeling of Greenland subglacial topography

This is the published version. Copyright 2014 Herzfeld et al.Dynamic ice-sheet models are used to assess the contribution of mass loss from the Greenland ice sheet to sea-level rise. Mass transfer from ice sheet to ocean is in a large part through outlet glaciers. Bed topography plays an important role in ice dynamics, since the acceleration from the slow-moving inland ice to an ice stream is in many cases caused by the existence of a subglacial trough or trough system. Problems are that most subglacial troughs are features of a scale not resolved in most ice-sheet models and that radar measurements of subglacial topography do not always reach the bottoms of narrow troughs. The trough-system algorithm introduced here employs mathematical morphology and algebraic topology to correctly represent subscale features in a topographic generalization, so the effects of troughs on ice flow are retained in ice-dynamic models. The algorithm is applied to derive a spatial elevation model of Greenland subglacial topography, integrating recently collected radar measurements (CReSIS data) of the Jakobshavn Isbræ, Helheim, Kangerdlussuaq and Petermann glacier regions. The resultant JakHelKanPet digital elevation model has been applied in dynamic ice-sheet modeling and sea-level-rise assessment

Full-Scale System for Quantifying Loads and Leak Rates of Seals for Space Applications

NASA is developing advanced space-rated vacuum seals in support of future space exploration missions to low-Earth orbit and other destinations. These seals may be 50 to 60 in. (127 to 152 cm) in diameter and must exhibit extremely low leak rates to ensure that astronauts have sufficient breathable air for extended missions to the International Space Station or the Moon. Seal compression loads must be below prescribed limits so as not to overload the mechanisms that compress them during docking or mating, and seal adhesion forces must be low to allow two mated systems to separate when required. NASA Glenn Research Center has developed a new test apparatus to measure leak rates and compression and adhesion loads of candidate full-scale seals under simulated thermal, vacuum, and engagement conditions. Tests can be performed in seal-on-seal or seal-on-flange configurations at temperatures from -76 to 140 F (-60 to 60 C) under operational pressure gradients. Nominal and off-nominal mating conditions (e.g., incomplete seal compression) can also be simulated. This paper describes the main design features of the test apparatus as well as techniques used to overcome some of the design challenges

Integration of linear and dendritic actin nucleation in Nck-induced actin comets

The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails-dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, form-in-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens.Fil: Surtayeva, Sofya. University of Connecticut School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Velle, Katrina B.. University of Connecticut; Estados UnidosFil: Campellone, Kenneth G.. University of Connecticut; Estados UnidosFil: Talman, Arthur. Yale School of Medicine; Estados UnidosFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Agaisse, Hervé. Yale School of Medicine; Estados UnidosFil: Wu, Yi I.. University of Connecticut School of Medicine; Estados UnidosFil: Loew, Leslie M.. University of Connecticut School of Medicine; Estados UnidosFil: Mayer, Bruce J.. University of Connecticut School of Medicine; Estados Unido