Dark Energy Survey Year 1 Results: Cosmological Constraints from Cluster Abundances, Weak Lensing, and Galaxy Correlations.

We present the first joint analysis of cluster abundances and auto or cross-correlations of three cosmic tracer fields: galaxy density, weak gravitational lensing shear, and cluster density split by optical richness. From a joint analysis (4×2pt+N) of cluster abundances, three cluster cross-correlations, and the auto correlations of the galaxy density measured from the first year data of the Dark Energy Survey, we obtain Ω_{m}=0.305_{-0.038}^{+0.055} and σ_{8}=0.783_{-0.054}^{+0.064}. This result is consistent with constraints from the DES-Y1 galaxy clustering and weak lensing two-point correlation functions for the flat νΛCDM model. Consequently, we combine cluster abundances and all two-point correlations from across all three cosmic tracer fields (6×2pt+N) and find improved constraints on cosmological parameters as well as on the cluster observable-mass scaling relation. This analysis is an important advance in both optical cluster cosmology and multiprobe analyses of upcoming wide imaging surveys.

Detection of CMB-Cluster Lensing using Polarization Data from SPTpol.

We report the first detection of gravitational lensing due to galaxy clusters using only the polarization of the cosmic microwave background (CMB). The lensing signal is obtained using a new estimator that extracts the lensing dipole signature from stacked images formed by rotating the cluster-centered Stokes QU map cutouts along the direction of the locally measured background CMB polarization gradient. Using data from the SPTpol 500 deg^{2} survey at the locations of roughly 18 000 clusters with richness λ≥10 from the Dark Energy Survey (DES) Year-3 full galaxy cluster catalog, we detect lensing at 4.8σ. The mean stacked mass of the selected sample is found to be (1.43±0.40)×10^{14}M_{⊙} which is in good agreement with optical weak lensing based estimates using DES data and CMB-lensing based estimates using SPTpol temperature data. This measurement is a key first step for cluster cosmology with future low-noise CMB surveys, like CMB-S4, for which CMB polarization will be the primary channel for cluster lensing measurements.

A lobster phospholipase C-beta that associates with G-proteins in response to odorants.

A cDNA clone encoding a protein of 1116 amino acids with significant homology to beta-isoforms of phospholipase C was isolated from lobster olfactory organ cDNA libraries and named lobPLCbeta. This cDNA hybridized predominantly to a 9 kb transcript in RNA from olfactory organ, pereiopod, brain, and eye-eyestalk and to several smaller minor transcripts only in eye-eyestalk. An antiserum raised to the C terminus of lobPLCbeta detected immunoreactivity in a single 130 kDa band in olfactory aesthetasc hairs, olfactory organ, pereiopod, dactyl, and brain. In eye-eyestalk this 130 kDa band was abundant, and minor bands of 100, 79, and 57 kDa also were detected. In cross sections of the aesthetasc hairs, immunoreactivity was detected in the outer dendritic segments of the olfactory receptor neurons, the site of olfactory transduction. A complex odorant caused lobPLCbeta immunoreactivity to increase in membrane fractions and decrease in soluble fractions of homogenates of aesthetasc hairs. The odorant also increased the amount of lobPLCbeta in immunoprecipitates of Galphaq and Gbeta from homogenates of aesthetasc hairs. These results support the conclusion that lobPLCbeta mediates olfactory transduction.

Ionic currents and ion channels of lobster olfactory receptor neurons.

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.

Distribution of G-protein alpha subunits and neurotransmitter activation of g(alphai) and g(alphaq) in the brain of the lobster Homarus americanus.

Immunocytochemistry using antisera specific for the G-protein alpha subunits G(alphai), G(alphaq), and G(alphas) revealed similar patterns of immunoreactivity in the lobster brain. Immunoreactivity was strongest in neuropil, especially the olfactory and accessory lobes, and was characterized by bundles of fine threads leading to dense concentrations of punctate staining in the glomeruli. This may reflect the concentration of G-protein alpha subunits at synapses. The major differences between the antisera were distinct patterns of staining intensity in subregions of glomeruli of the olfactory and accessory lobes. This result is potentially correlated with previous evidence that these subregions are neurochemically distinct. Neuronal cell bodies contained moderate levels of immunoreactivity at the plasma membrane and faint staining in the cytoplasm. The olfactory globular tract was moderately immunoreactive, but other fiber tracts were weakly immunoreactive. Immunoreactivity in the deutocerebral commissure consisted of small oval cell bodies and strands that formed a reticulated pattern, suggestive of glia. Photoaffinity labelling by using an analog of GTP demonstrated that histamine activated G(alphai) in brain homogenates. Further evidence of G-protein activation was obtained by showing that stimulation with a mixture of neuroactive substances increased the amount of phospholipase C-beta associated with membranes, G(alphaq), and G(beta). The lobster brain, especially in its neuropil regions, is richly endowed with neuromodulatory biochemical pathways involving G(alphai), G(alphaq), and G(alphas).

Lobster G-protein coupled receptor kinase that associates with membranes and G(beta) in response to odorants and neurotransmitters.

A cDNA clone (lobGRK2) encoding a protein of 690 amino acids with significant similarity to the GRK2 subfamily of G-protein coupled receptor kinases was isolated. lobGRK2 was widely expressed as a 9-kb major transcript and a protein of 80 kDa. It was most abundant in the brain and the olfactory organ but was absent in the eye/eyestalk. Immunocytochemistry revealed lobGRK2 immunoreactivity in the outer dendritic segments of the olfactory receptor neurons, the site of olfactory transduction. LobGRK2 immunoreactivity was observed in most neuronal structures in the brain, although with varying intensity. It was strongest in neuropil, especially the olfactory and accessory lobes but was also detectable in neuronal cell bodies. Stimulation of brain homogenates with a mixture of neurotransmitters increased the association of lobGRK2 with membranes and with G(beta). Similarly, stimulation of olfactory dendrite homogenates with an odorant mixture caused lobGRK2 to associate with G(beta). These results support the conclusion that lobGRK2 responds to odorants and to neurotransmitters and may act to initiate desensitization by phosphorylating G-protein-coupled receptors in the olfactory organ and the brain, respectively.

Signal transmission in lobster olfactory receptor cells: functional significance of electrotonic structure analysed by a compartmental model.

The electrotonic structure of lobster olfactory receptor cells was evaluated using general purpose simulation software in a compartmental model derived from electron-microscopic reconstruction. The model with non-uniform membrane resistance (Rm) was used to (i) simulate current spread and (ii) determine if the electronic structure of the cell improves signal recognition in the soma. The odor-evoked conductance change in dendrites was calculated according to the Michaelis-Menten equation with the assumption that the outer dendritic segments function as independent stimulus detectors. The inflection point of the concentration-response function measured in the soma was shifted to lower concentrations relative to that measured in the ciliary (outer dendritic) arbor. The shift, which was greater for inputs with lower efficacy (represented in the model by smaller Hill coefficients) and for the dynamic phase of the response than for the steady-state phase, effectively increased the selectivity of the somatic response. Randomized input distributed uniformly to progressively more restricted areas of the ciliary arbor showed that stimulation of larger areas (presumably the entire ciliary arbor) decreased the statistical variability of the somatic response.

Molecular cloning of a G-protein alpha i subunit from the lobster olfactory organ.

A G-protein alpha subunit was cloned from a lobster olfactory organ cDNA library and sequenced. The clone encodes an alpha i subunit based on the 80% identity its predicted amino acid sequence shares with mammalian alpha i subunits. On Northern blots of polyadenylated RNA, the clone hybridized to a 5 kb species from several tissues.

Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors.

Replacing the G-protein-coupling domains of the beta 2-adrenergic receptor with homologous domains of putative olfactory receptors produced chimeric receptors which were able to stimulate pigment dispersion in Xenopus melanophores, a G-protein-mediated pathway. A multiple replacement chimera containing the second, third and C-terminal cytoplasmic domains of receptor OR5 elevated cyclic adenosine 3':5'-monophosphate (cAMP) and suppressed production of inositol phosphates. Co-expression of G alpha olf did not alter the strength of response of this chimera. A novel rat olfactory receptor cDNA (U131) was isolated and sequenced. Expression of U131 and OR5 constructs containing an N-terminal epitope-tag or C-terminal fusion to green fluorescent protein occurred in an intracellular network but not in the plasma membrane of heterologous cells. Similarly treated beta 2-adrenergic receptors were functional and were observed in the plasma membrane and the intracellular network. These results demonstrate that the putative cytoplasmic domains of olfactory receptors are capable of functional interaction with heterologous G-proteins of the G alpha s subtype. Instead, the absence of these receptors from the plasma membrane of heterologous cells appears to explain our inability to determine if odorants can activate the olfactory receptor clones. We hypothesize that the olfactory receptors have requirements for maturation and targeting to the plasma membrane that are different from most other G-protein-coupled receptors.

Functional analysis by imaging of melanophore pigment dispersion of chimeric receptors constructed by recombinant polymerase chain reaction.

Analysis of functional aspects of the molecular structure of proteins often requires a means to selectively alter structure and subsequently analyze function. We have adapted a method of overlap extension polymerase chain reaction (PCR) to generate multiple domain replacements in G-protein coupled receptors. The examples described herein are beta 2-adrenergic receptors whose G-protein coupling domains have been replaced by homologous domains of olfactory receptors, but the procedure has also been used to produce constructs with mutations, deletions, and fusions of two complete open reading frames. The chimeric olfactory-adrenergic receptors were assayed by functional expression in clonal lines of Xenopus melanophores. The ability of G-protein coupled second messenger pathways to cause translocation of pigment organelles within melanophores allows the use of video microscopy to assay the function of the chimeric receptors. Digital automation of microscope stage, camera, and image processing allows multiple parallel experiments to be performed. Melanophores allow responses mediated by the Gs, Gq and Gi pathways to be assayed with equal efficiency and the specificity of the coupling between chimera (or receptor) and G-protein subtypes can be rapidly determined.

Consensus translational initiation sites of marine invertebrate phyla.

The efficiency of translational initiation depends upon the sequence context surrounding the AUG codon (1, 2, 3). A purine at position -3 contributes critically to context, but other neighboring nucleotides are also important. Nucleotide frequencies at these neighboring positions vary among distant taxa (4, 5). We have analyzed the translational initiation sites of cnidarian, echinoderm, molluscan, annelid, and crustacean sequences in nucleotide sequence databases. These taxa conform to the pattern of a strong preference for a purine at -3, but the frequencies of nucleotides at neighboring positions are characteristic for each taxon. The consensus translational initiation sequences of the marine invertebrate taxa are also different from those of vertebrates and single-celled eukaryotes. These consensus sequences are useful guides for predicting translational initiation sites in cDNA clones.

Histamine directly gates a chloride channel in lobster olfactory receptor neurons.

Biogenic amines mediate many types of intercellular communication in multicellular organisms. Heretofore, little direct evidence has indicated that biogenic amines produce intracellular responses other than by triggering the enzymatic production of second messengers. Our electrophysiological studies of lobster olfactory receptor neurons now reveal that one biogenic amine, histamine, can directly gate an ion channel. The channel responds to histamine concentrations of 1 microM or more, is permeable primarily to Cl-, is more active at depolarized potentials, and has a conductance of 44 pS in the American lobster and 66 pS in the Caribbean spiny lobster. The expression of this ligand-gated channel in olfactory receptor neurons implies that these neurons are targets of a regulatory or feedback process.

Lobster GABA receptor subunit expressed in neural tissues.

A cDNA encoding an ionotropic gamma-aminobutyric acid (GABA) receptor subunit was isolated from a lobster (Homarus americanus) cDNA library. A longer version of this cDNA, containing a 108-bp insert, was also detected. The two cDNAs are predicted to encode alternatively spliced proteins of 485 and 521 amino acids, respectively. The sequences were most similar to the Drosophila RDL (resistance to dieldrin) GABA subunit with 54% identity, and 30-35% identity with vertebrate ionotropic GABA receptor subunits. Only the shorter clone formed functional ion channels when transfected into human embryonic kidney (HEK) 293 cells. GABA caused a Cl(-)-selective current in the presence of GABA that was blocked by picrotoxin. The GABA-induced current was weakly sensitive to the GABA(A) antagonist, bicuculline, but was enhanced by pentobarbital. Expression of the GABA receptor mRNA was highest in brain and the olfactory organ, but was not detected in leg muscle. These data suggest that the isolated cDNAs are likely to encode proteins that comprise subunits of native GABA receptors expressed in olfactory receptor neurons and projection neurons of the olfactory deutocerebrum.

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons.

A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na+, K+ and Cs+, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage- and concentration-dependent block of Na+ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg2+ was greater than 1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg2+ block of Na+ permeation, and demonstrating the channel's presence in arthropods.

Olfactory receptor trafficking involves conserved regulatory steps.

Olfactory receptors are difficult to functionally express in heterologous cells. They are typically retained in the endoplasmic reticulum of cells commonly used for functional expression studies and are only released to the plasma membrane in mature cells of the olfactory receptor neuron lineage. A recently developed olfactory cell line, odora, traffics olfactory receptors to the plasma membrane when differentiated. We found that undifferentiated odora cells do not traffic olfactory receptors to their surface, even though they release the receptors to the Golgi apparatus and endosomes. This behavior differs from other cell lines tested thus far. Differentiated odora cells also properly traffic vomeronasal receptors of the VN1 type, which lack sequence similarity to olfactory receptors. ODR-4, a protein that is necessary for plasma membrane trafficking of a chemosensory receptor in nematodes, facilitates trafficking of rat olfactory receptor U131 in odora and Chinese hamster ovary cells. Olfactory receptor trafficking from the endoplasmic reticulum to the plasma membrane involves at least two steps whose regulation depends on the maturation state of cells in the olfactory receptor neuron lineage. These results also indicate that some components of the regulatory mechanism are conserved.