Protein and signaling networks in vertebrate photoreceptor cells

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cyclic guanosine monophosphate (cGMP) and Ca(2+). The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase (GRK1) under control of Ca(2+)-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases (GCs) and is regulated by specific neuronal Ca(2+)-sensor proteins called guanylate cyclase-activating proteins (GCAPs). At least one GC (ROS-GC1) was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca(2+)-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated (CNG) channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments

Preparing for life after rugby

The inception of rugby as a workforce in 1995 created a range of new issues surrounding sport as a vocation. With professional rugby often wearing the glamorous coat of fame and fortune, young athletes sacrifice education and learning additional life-skills in pursuit of well-paid contracts and glitzy lifestyles unaware of the realities rugby as a profession holds. One such reality is the relatively short lifespan of a professional rugby career and the fact that transition to a whole new career is firstly inevitable and secondly a very challenging process. Traditional retirement has been associated with the end of a long working career, making comprehensive lifestyle- and financial planning part of the preparation process. This process helps the retiree anticipate and understand the expected demands of life beyond a working career. In rugby however, the retirement experience of a player can be extremely difficult to cope with, especially if the player is not adequately prepared or has not planned for such an event. This leaves players vulnerable for the imminent new phase of life and often leads to physiological - and other challenges players are not able to withstand in a world outside sport (Price, 2007). The aim of this study is to identify the different aspects that influence a professional rugby player’s retirement – and transition experience into a new profession. The researcher believes that an increased understanding of how current and retired professional rugby players perceive/experienced the retirement process would assist current players to better plan and prepare for this phase of life. This ultimately would reduce the anxiety and uncertainty for life after rugby. If players are more relaxed and stress-free about their future, more focus could also be placed on the here-and-now, leading to greater performance on the current field of play. The views of both current and retired professional rugby players were captured through questionnaires distributed all around South Africa. The researcher utilised a mixed mode paradigm of both positivistic and interpretive research methods. This approach enabled him to best compare the views of the two groups and test the developed theories and hypothesis. Ultimately, the research revealed that the presence of the following variables will have a positive influence on a player’s retirement and transition experience: A) Leadership, advice and planning for retirement B) Tertiary education and additional work skills C) Popularity amongst fans and other influential people D) Sufficient wealth and E) A self-selected retirement. With these findings the researcher will develop some specific guidelines for current professional rugby players to help them firstly better prepare for their life beyond sport and secondly successfully switch to a new career. A few valuable recommendations were also made to other stakeholders to better assist and support players in their preparation and transition process

Unitarity constraint for threshold coherent pion photoproduction on the deuteron and chiral perturbation theory

The contribution of the two-step process gamma + d -> p + n -> pi0 + d to the imaginary part of the amplitude for coherent pion production on the deuteron is calculated exploiting unitarity constraints. The result shows that this absorptive process is not negligible and has to be considered in an extraction of the elementary neutron production amplitude from the gamma + d -> pi0 + d cross section at threshold. In addition, it is argued that a consistent calculation of gamma + d -> pi0 + d in baryon chiral perturbation theory beyond next-to-leading order requires the inclusion of this absorptive process.Comment: 11 pages revtex including 2 postscript figure

Differential Palmit(e)oylation of Wnt1 on C93 and S224 Residues Has Overlapping and Distinct Consequences

Though the mechanisms by which cytosolic/intracellular proteins are regulated by the post-translational addition of palmitate adducts is well understood, little is known about how this lipid modification affects secreted ligands, such as Wnts. Here we use mutational analysis to show that differential modification of the two known palmit(e)oylated residues of Wnt1, C93 and S224, has both overlapping and distinct consequences. Though the relative roles of each residue are similar with respect to stability and secretion, two distinct biological assays in L cells show that modification of C93 primarily modulates signaling via a ß-catenin independent pathway while S224 is crucial for ß-catenin dependent signaling. In addition, pharmacological inhibition of Porcupine (Porcn), an upstream regulator of Wnt, by IWP1, specifically inhibited ß-catenin dependent signaling. Consistent with these observations, mapping of amino acids in peptide domains containing C93 and S224 demonstrate that acylation of C93 is likely to be Porcn-independent while that of S224 is Porcn-dependent. Cumulatively, our data strongly suggest that C93 and S224 are modified by distinct enzymes and that the differential modification of these sites has the potential to influence Wnt signaling pathway choice

Kinetics of cone specific G-protein signaling in avian photoreceptor cells

Cone photoreceptor cells of night-migratory songbirds seem to process the primary steps of two different senses, vision and magnetoreception. The molecular basis of phototransduction is a prototypical G protein-coupled receptor pathway starting with the photoexcitation of rhodopsin or cone opsin thereby activating a heterotrimeric G protein named transducin. This interaction is well understood in vertebrate rod cells, but parameter describing protein–protein interactions of cone specific proteins are rare and not available for migratory birds. European robin is a model organism for studying the orientation of birds in the earth magnetic field. Recent findings showed a link between the putative magnetoreceptor cryptochrome 4a and the cone specific G-protein of European robin. In the present work, we investigated the interaction of European robin cone specific G protein and cytoplasmic regions of long wavelength opsin. We identified the second loop in opsin connecting transmembrane regions three and four as a critical binding interface. Surface plasmon resonance studies using a synthetic peptide representing the second cytoplasmic loop and purified G protein α-subunit showed a high affinity interaction with a KD value of 21 nM. Truncation of the G protein α-subunit at the C-terminus by six amino acids slightly decreased the affinity. Our results suggest that binding of the G protein to cryptochrome can compete with the interaction of G protein and non-photoexcited long wavelength opsin. Thus, the parallel presence of two different sensory pathways in bird cone photoreceptors is reasonable under dark-adapted conditions or during illumination with short wavelengths

Changes in the fatty acid composition of brown shrimp, Crangon crangon, after boiling

Brown shrimp, Crangon crangon (L.), is the most valuable target of coastal fisheries in the southern North Sea. Annual landings exceeded 30,000 tons in the last decade, yielding up to 100 Mio Euro. The shrimp are boiled immediately after capture onboard the fishing vessel for preservation and easy peeling. After landing, the shrimp are collected by traders and exported for manual peeling. Only the muscle of the pleon is returned and sold as regional delicacy. The remains, comprising the cephalothorax, the shell of the pleon, and, in case, adhering eggs, account for up to 70% of the total body mass. This potential resource, for example as aquaculture feed, has not yet been considered for exploitation. In this respect, the fatty acid (FA) profile and the share of essential FAs are crucial quality factors. Since boiling alters the quality of shrimp, this study evaluates changes in the FA composition of shrimp muscle and remains by comparing frozen and boiled samples. Major FAs in C. crangon were the saturated palmitic acid (PA, 16:0), accounting for 16.6%–19.1% of total fatty acids (TFAs), and the long-chain polyunsaturated FAs (LC-PUFAs) eicosapentaenoic acid (EPA, 20:5(n-3), 16.1–21.6%TFA ) and docosahexaenoic acid (DHA, 22:6(n-3), 11.5–13.6%TFA ). Frozen muscle and frozen remains showed similar FA profiles. Boiling changed the FA profile. PA, EPA, and DHA decreased by up to 25%, whereas palmitoleic acid 16:1(n-7) and oleic acid 18:1(n-9) increased by 2% to 3% each. Boiled muscle and boiled remains showed similar FA profiles. Despite the loss of FAs, the boiled shrimp remains are suggested to be a suitable PUFA supplement for aquaculture feeds, deserving further investigation

The Centrosomal Protein Pericentrin Identified at the Basal Body Complex of the Connecting Cilium in Mouse Photoreceptors

BACKGROUND: Pericentrin (Pcnt), a conserved protein of the pericentriolar material, serves as a multifunctional scaffold for numerous proteins and plays an important role in microtubule organization. Recent studies indicate that Pcnt mutations are associated with a range of diseases including primordial dwarfism and ciliopathies. To date, three Pcnt splice variants from orthologous genes in mice and humans are known. PRINCIPAL FINDINGS: We generated a specific Pcnt antiserum detecting all known Pcnt splice variants and examined the cellular and subcellular distribution of Pcnt in ciliated tissues of the mouse, the olfactory epithelium and the retina. For the first time, we identified Pcnt and its centrosomal interaction partners at the basal body complex of mouse retinal photoreceptors. Photoreceptors are morphologically and functionally subdivided into the light sensitive outer segment and the inner segment comprising the metabolic function of the cell. The two compartments are linked via a modified, specialized, non-motile cilium, the connecting cilium. Here, Pcnt colocalized with the whole protein machinery responsible for transport processes between the two compartments. Surprisingly, photoreceptors expressed a small Pcnt splice transcript - most likely a modified variant of Pcnt S - which was not present in receptor neurons of the olfactory epithelium. CONCLUSIONS: Our findings suggest distinct functional roles of several Pcnt variants in different ciliated tissues and sensory neurons, like the olfactory epithelium and the retina of the mouse. The individual patchwork of different Pcnt splice transcripts seems to reflect the complexity of Pcnt function, an assumption corroborated by the heterogeneous clinical manifestations associated with mutations in the Pcnt gene

Non-additivity of decoherence rates in superconducting qubits

We show that the relaxation and decoherence rates 1/T_1 and 1/T_2 of a qubit coupled to several noise sources are in general not additive, i.e., that the total rates are not the sums of the rates due to each individual noise source. To demonstrate this, we calculate the relaxation and pure dephasing rates 1/T_1 and 1/T_\phi of a superconducting (SC) flux qubit in the Born-Markov approximation in the presence of several circuit impedances Z_i using network graph theory and determine their deviation from additivity (the mixing term). We find that there is no mixing term in 1/T_\phi and that the mixing terms in 1/T_1 and 1/T_2 can be positive or negative, leading to reduced or enhanced relaxation and decoherence times T_1 and T_2. The mixing term due to the circuit inductance L at the qubit transition frequency \omega_{01} is generally of second order in \omega_{01}L/Z_i, but of third order if all impedances Z_i are pure resistances. We calculate T_{1,2} for an example of a SC flux qubit coupled to two impedances.Comment: 5 pages, 2 figure

Constitutive activation of guanylate cyclase by the G86R GCAP1 variant is due to "locking" cation-\u3c0 interactions that impair the activator-to-inhibitor structural transition

Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca2+] and as an activator at low [Ca2+]. Recently, a novel missense mutation (G86R) was found in GUCA1A, the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the N- and C-domains of GCAP1, resulting in decreased Ca2+-sensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-\u3c0 interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well as lower affinity for both Mg2+ and Ca2+, moreover, substitutions of W94 abolished "phase II" in Ca2+-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca2+-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca2+ supported the presence of two cation-\u3c0 interactions involving R86, which was found to act as a bridge between W94 and W21, thus, locking the hinge region in an activator-like conformation and resulting in the constitutive activation of the target under physiological conditions

Molecular properties of human guanylate cyclase-activating protein 2 (GCAP2) and its retinal dystrophy-associated variant G157R

In murine and bovine photoreceptors, guanylate cyclase activating-protein 2 (GCAP2) activates retinal guanylate cyclases (GC) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog GCAP1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited-retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to three Mg2+ ions with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, non-myristoylated GCAP2 does not bind Mg2+ over the physiological range, and remains as a monomer in the absence of Ca2+. Both myristoylated and non-myristoylated GCAP2 bind Ca2+ with high affinity. At odds with GCAP1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations, and is prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 in human photoreceptors might be mostly implicated in processes other than phototransduction, and suggest a possible molecular mechanism for G157R-associated IRD