Pleckstrin Homology Domains: Two Halves Make a Hole?

In a recent issue of Nature, van Rossum et al. report binding of a “split” pleckstrin homology (PH) domain from phospholipase C-γ1 to the TRPC3 ion channel. Through sequence analyses and in vitro studies, they suggest a novel mode of protein-protein interaction in which two PH domain fragments in distinct proteins associate to form an “intermolecular” PH domain that binds inositol phospholipids and is required for ion channel location and function

Cell Signaling by Receptor Tyrosine Kinases

Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses

Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

<p>Abstract</p> <p>Background</p> <p>Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)<it>P</it>₂] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)<it>P</it>₂also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)<it>P</it>₂levels in living cells is critical to understanding the biology of PtdIns(4,5)<it>P</it>₂. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)<it>P</it>₂imaging tools.</p> <p>Results</p> <p>All of the yeast PH domains that have been previously shown to bind PtdIns(4,5)<it>P</it>₂showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)<it>P</it>₂. None of these domains showed any advantage over the PLCδ1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)<it>P</it>₂binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P₃. Detailed comparison of the Tubby and PLCδ1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)<it>P</it>₂and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.</p> <p>Conclusion</p> <p>These results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)<it>P</it>₂changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCδ1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P₃binding may compromise its accuracy to measure PtdIns(4,5)<it>P</it>₂changes. The Tubby domain is more accurate to report on PtdIns(4,5)<it>P</it>₂but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)<it>P</it>₂levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)<it>P</it>₂.</p

Titan Science with the James Webb Space Telescope (JWST)

The James Webb Space Telescope (JWST), scheduled for launch in 2018, is the successor to the Hubble Space Telescope (HST) but with a significantly larger aperture (6.5 m) and advanced instrumentation focusing on infrared science (0.6-28.0 $\mu$m ). In this paper we examine the potential for scientific investigation of Titan using JWST, primarily with three of the four instruments: NIRSpec, NIRCam and MIRI, noting that science with NIRISS will be complementary. Five core scientific themes are identified: (i) surface (ii) tropospheric clouds (iii) tropospheric gases (iv) stratospheric composition and (v) stratospheric hazes. We discuss each theme in depth, including the scientific purpose, capabilities and limitations of the instrument suite, and suggested observing schemes. We pay particular attention to saturation, which is a problem for all three instruments, but may be alleviated for NIRCam through use of selecting small sub-arrays of the detectors - sufficient to encompass Titan, but with significantly faster read-out times. We find that JWST has very significant potential for advancing Titan science, with a spectral resolution exceeding the Cassini instrument suite at near-infrared wavelengths, and a spatial resolution exceeding HST at the same wavelengths. In particular, JWST will be valuable for time-domain monitoring of Titan, given a five to ten year expected lifetime for the observatory, for example monitoring the seasonal appearance of clouds. JWST observations in the post-Cassini period will complement those of other large facilities such as HST, ALMA, SOFIA and next-generation ground-based telescopes (TMT, GMT, EELT).Comment: 50 pages, including 22 figures and 2 table

Dust aerosol, clouds, and the atmospheric optical depth record over 5 Mars years of the Mars Exploration Rover mission

Dust aerosol plays a fundamental role in the behavior and evolution of the Martian atmosphere. The first five Mars years of Mars Exploration Rover data provide an unprecedented record of the dust load at two sites. This record is useful for characterization of the atmosphere at the sites and as ground truth for orbital observations. Atmospheric extinction optical depths have been derived from solar images after calibration and correction for time-varying dust that has accumulated on the camera windows. The record includes local, regional, and globally extensive dust storms. Comparison with contemporaneous thermal infrared data suggests significant variation in the size of the dust aerosols, with a 1 {\mu}m effective radius during northern summer and a 2 {\mu}m effective radius at the onset of a dust lifting event. The solar longitude (LS) 20-136{\deg} period is also characterized by the presence of cirriform clouds at the Opportunity site, especially near LS=50 and 115{\deg}. In addition to water ice clouds, a water ice haze may also be present, and carbon dioxide clouds may be present early in the season. Variations in dust opacity are important to the energy balance of each site, and work with seasonal variations in insolation to control dust devil frequency at the Spirit site.Comment: 60 pages, 12 figures, to be published in Icaru

Mechanism of Activation and Inhibition of the HER4/ErbB4 Kinase

SummaryHER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members

Quantitative Analysis of the Effect of Phosphoinositide Interactions on the Function of Dbl Family Proteins

Normally, Rho GTPases are activated by the removal of bound GDP and the concomitant loading of GTP catalyzed by members of the Dbl family of guanine nucleotide exchange factors (GEFs). This family of GEFs invariantly contain a Dbl homology (DH) domain adjacent to a pleckstrin homology (PH) domain, and while the DH domain usually is sufficient to catalyze nucleotide exchange, possible roles for the conserved PH domain remain ambiguous. Here we demonstrate that the conserved PH domains of three distinct Dbl family proteins, intersectin, Dbs, and Tiam1, selectively bind lipid vesicles only when phosphoinositides are present. While the PH domains of intersectin and Dbs promiscuously bind several multiphosphorylated phosphoinositides, Tiam1 selectively interacts with phosphatidylinositol 3-phosphate (K(D) approximately 5-10 microm). In addition, and in contrast to recent reports, catalysis of nucleotide exchange on nonprenylated Rac1 provided by various extended portions of Tiam1 is not influenced by (a) soluble phosphoinositide head groups, (b) dibutyl versions of phosphoinositides, or (c) lipid vesicles containing phosphoinositides. Likewise, GEF activity afforded by DH/PH fragments of intersectin and Dbs are also not altered by phosphoinositide interactions. These results strongly suggest that unless all relevant components are localized to a lipid membrane surface, Dbl family GEFs generally are not intrinsically modulated by binding phosphoinositides

An advanced expiratory circuit for the recovery of perfluorocarbon liquid from non-saturated perfluorocarbon vapour during partial liquid ventilation: an experimental model

BACKGROUND: The loss of perfluorocarbon (PFC) vapour in the expired gases during partial liquid ventilation should be minimized both to prevent perfluorocarbon vapour entering the atmosphere and to re-use the recovered PFC liquid. Using a substantially modified design of our previously described condenser, we aimed to determine how much perfluorocarbon liquid could be recovered from gases containing PFC and water vapour, at concentrations found during partial liquid ventilation, and to determine if the amount recovered differed with background flow rate (at flow rates suitable for use in neonates). METHODS: The expiratory line of a standard ventilator circuit set-up was mimicked, with the addition of two condensers. Perfluorocarbon (30 mL of FC-77) and water vapour, at concentrations found during partial liquid ventilation, were passed through the circuit at a number of flow rates and the percentage recovery of the liquids measured. RESULTS: From 14.2 mL (47%) to 27.3 mL (91%) of the infused 30 mL of FC-77 was recovered at the flow rates studied. Significantly higher FC-77 recovery was obtained at lower flow rates (ANOVA with Bonferroni's multiple comparison test, p < 0.0001). As a percentage of the theoretical maximum recovery, 64 to 95% of the FC-77 was recovered. Statistically significantly less FC-77 was recovered at 5 Lmin(-1 )(ANOVA with Bonferroni's multiple comparison test, p < 0.0001). Amounts of perfluorocarbon vapour recovered were 47%, 50%, 81% and 91% at flow rates of 10, 5, 2 and 1 Lmin(-1), respectively. CONCLUSION: Using two condensers in series 47% to 91% of perfluorocarbon liquid can be recovered, from gases containing perfluorocarbon and water vapour, at concentrations found during partial liquid ventilation