B-type Eph receptors and ephrins induce growth cone collapse through distinct intracellular pathways

Forward and reverse signaling mediated by EphB tyrosine kinase receptors and their transmembrane ephrin-B ligands play important roles in axon pathfinding, yet little is known about the intracellular pathways involved. Here we have used growth cones from the ventral (EphB receptor-bearing) and dorsal (ephrin-B-bearing) embryonic Xenopus retina to investigate the signaling mechanisms in both forward and reverse directions. We report that unclustered, but not clustered, EphB2 ectodomains trigger fast (5-10 min) transient collapse responses in growth cones. This collapse response is mediated by low levels of intracellular cyclic GMP and requires proteasome function. In contrast, clustered, but not unclustered, ephrin-B1 ectodomains cause slow (30-60 min) growth cone collapse that depends on high cGMP levels and is insensitive to inhibition of the proteasomal pathway. Upon receptor-ligand binding, endocytosis occurs in the reverse direction (EphB2-Fc into dorsal retinal growth cones), but not the forward direction, and is also sensitive to proteasomal inhibition. Endocytosis is functionally important because blocking of EphB2 internalization inhibits growth cone collapse. Our data reveal that distinct signaling mechanisms exist for B-type Eph/ephrin-mediated growth cone guidance and suggest that endocytosis provides a fast mechanism for switching off signaling in the reverse direction.     

Monocyte tissue factor induction by activation of beta 2-glycoprotein-I-specific T lymphocytes is associated with thrombosis and fetal loss in patients with antiphospholipid antibodies

Antiphospholipid (aPL) syndrome (APS) is characterized by thromboembolic events, thrombocytopenia, or recurrent miscarriage associated with aPL Abs with specificity for beta2-glycoprotein-I (beta2GPI). We recently reported that at least 44% of patients with the APS possess circulating type 1 (Th1) CD4+ T cells that proliferate and secrete IFN-gamma when stimulated with beta2GPI in vitro. In this study, we show that stimulation of PBMCs from 20 APS patients with beta2GPI induced substantial monocyte tissue factor (TF) (80 +/- 11 TF stimulation index (TF-SI)), whereas no induction was observed using PBMCs from 13 patients with aPL Abs without APS (6 +/- 1 TF-SI) or 7 normal and 7 autoimmune controls (5 +/- 1 and 3 +/- 1 TF-SI, respectively) (p < 0.0001). TF induction on monocytes by beta2GPI was dose dependent and required CD4+ T lymphocytes and class II MHC molecules. Because monocyte TF induction by beta2GPI was observed in all patients with APS, but not in any patient with aPL Abs without APS, this response is a potentially useful predictor for APS in patients with aPL Abs, as well as providing mechanistic insight into thrombosis and fetal loss in these patients.     

GIGANTEA acts in blue light signaling and has biochemically separable roles in circadian clock and flowering time regulation

Circadian clocks are widespread in nature. In higher plants, they confer a selective advantage, providing information regarding not only time of day but also time of year. Forward genetic screens in Arabidopsis (Arabidopsis thaliana) have led to the identification of many clock components, but the functions of most of these genes remain obscure. To identify both new constituents of the circadian clock and new alleles of known clock-associated genes, we performed a mutant screen. Using a clock-regulated luciferase reporter, we isolated new alleles of ZEITLUPE, LATE ELONGATED HYPOCOTYL, and GIGANTEA (GI). GI has previously been reported to function in red light signaling, central clock function, and flowering time regulation. Characterization of this and other GI alleles has helped us to further define GI function in the circadian system. We found that GI acts in photomorphogenic and circadian blue light signaling pathways and is differentially required for clock function in constant red versus blue light. Gene expression and epistasis analyses show that TIMING OF CHLOROPHYLL A/B BINDING PROTEIN1 (TOC1) expression is not solely dependent upon GI and that GI expression is only indirectly affected by TOC1, suggesting that GI acts both in series with and in parallel to TOC1 within the central circadian oscillator. Finally, we found that the GI-dependent promotion of CONSTANS expression and flowering is intact in a gi mutant with altered circadian regulation. Thus GI function in the regulation of a clock output can be biochemically separated from its role within the circadian clock.     

Two sub-states of the red2 state of methyl-coenzyme M reductase revealed by high-field EPR spectroscopy

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme has two structurally interlinked active sites embedded in an α₂β₂γ₂ subunit structure. Each active site has the nickel porphyrinoid F₄₃₀ as a prosthetic group. In the active state, F₄₃₀ contains the transition metal in the Ni(I) oxidation state. The active enzyme exhibits an axial Ni(I)-based continuous wave (CW) electron paramagnetic resonance (EPR) signal, called red1a in the absence of substrates or red1c in the presence of coenzyme M. Addition of coenzyme B to the MCR-red1 state can partially and reversibly convert it into the MCR-red2 form, which shows a rhombic Ni(I)-based EPR signal (at X-band microwave frequencies of approximately 9.4 GHz). In this report we present evidence from high-field/high-frequency CW EPR spectroscopy (W-band, microwave frequency of approximately 94 GHz) that the red2 state consists of two substates that could not be resolved by EPR spectroscopy at X-band frequencies. At W-band it becomes apparent that upon addition of coenzyme B to MCR in the red1c state, two red2 EPR signals are induced, not one as was previously believed. The first signal is the well-characterized (ortho)rhombic EPR signal, thus far called red2, while the second previously unidentified signal is axial. We have named the two substates MCR-red2r and MCR-red2a after their rhombic and axial signals, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Towards a resolution of the stoichiometry of the fibroblast growth factor (FGF)-FGF receptor-heparin complex

The 22 members of the fibroblast growth factor (FGF) family have been implicated in cell proliferation, differentiation, survival, and migration. They are required for both development and maintenance of vertebrates, demonstrating an exquisite pattern of affinities for both protein and proteoglycan receptors. Recent crystal structures have suggested two models for the complex between FGFs, FGF receptors (FGFRs) and the proteoglycan heparan sulphate that mediates signalling, and have provided insight into how FGFs show differing affinities for the range of FGFRs. However, the physiological relevance of the two different models has not been made clear. Here, we demonstrate that the two complexes can be prepared from the same protein components, confirming that neither complex is the product of misfolded protein samples. Analyses of the complexes with mass spectrometry and analytical ultracentrifugation show that the species observed are consistent with the crystal structures formed using the two preparation protocols. This analysis supports the contention that both of the crystal structures reflect the state of the molecules in solution. Mass spectrometry of the complexes suggests that the stoichiometry of the complexes is 2 FGF1:2 FGFR2:1 heparin, regardless of the method used to prepare the complexes. These observations suggest that the two proposed complex architectures may both have relevance to the formation of an in vivo signalling complex, with a combination of the two interactions contributing to the formation of a larger focal complex.     

Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of <Emphasis Type="Italic">Solemya velum</Emphasis>: <Emphasis Type="Italic">cbbLSQO</Emphasis>

Chemoautotrophic endosymbionts residing in Solemya velum gills provide this shallow water clam with most of its nutritional requirements. The cbb gene cluster of the S. velum symbiont, including cbbL and cbbS, which encode the large and small subunits of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), was cloned and expressed in Escherichia coli. The recombinant RubisCO had a high specific activity, 3 mol min^–1 mg protein ^–1, and a K _CO2 of 40.3 M. Based on sequence identity and phylogenetic analyses, these genes encode a form IA RubisCO, both subunits of which are closely related to those of the symbiont of the deep-sea hydrothermal vent gastropod Alviniconcha hessleri and the photosynthetic bacterium Allochromatium vinosum. In the cbb gene cluster of the S. velum symbiont, the cbbLS genes were followed by cbbQ and cbbO, which are found in some but not all cbb gene clusters and whose products are implicated in enhancing RubisCO activity post-translationally. cbbQ shares sequence similarity with nirQ and norQ, found in denitrification clusters of Pseudomonas stutzeri and Paracoccus denitrificans. The 3 region of cbbO from the S. velum symbiont, like that of the three other known cbbO genes, shares similarity to the 3 region of norD in the denitrification cluster. This is the first study to explore the cbb gene structure for a chemoautotrophic endosymbiont, which is critical both as an initial step in evaluating cbb operon structure in chemoautotrophic endosymbionts and in understanding the patterns and forces governing RubisCO evolution and physiology.     

Sequence of the Core Antenna Domain from the Anoxygenic Phototroph <Emphasis Type="Italic">Heliophilum fasciatum</Emphasis>: Implications for Diversity of Reaction Center Type I

Broad variation among anoxygenic reaction centers makes it essential to consider a wide variety when considering the origins of photosynthesis. The photosynthetic core antenna domain in the gene pshA from Heliophilum fasciatum was sequenced doubling the number of core sequences available from heliobacteria. The sequence shares a pattern of hydrophobicity and histidine residues with the core antenna domain of pshA from Heliobacillus mobilis. Sequence identity between the two pshA sequences was 68%, indicating heliobacterial reaction centers show similar diversity to photosystem I throughout cyanobacteria and plastids. Thus, the diversity of anoxygenic phototrophic reaction centers may be greater than previously thought.