Phosducin-like protein 1 is essential for G-protein assembly and signaling in retinal rod photoreceptors

G protein β subunits perform essential neuronal functions as part of G protein βγ and Gβ(5)-RGS (Regulators of G protein Signaling) complexes. Both Gβγ and Gβ(5)-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a co-chaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesis in vivo, we deleted the Phlp1 gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gβγ dimer formation was decreased 50-fold, resulting in a more than 10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gβ(5) and RGS9-1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstrate in vivo that PhLP1 is required for the folding and assembly of both Gβγ and Gβ(5)-RGS9

Data from: A cure for the blues: opsin duplication and subfunctionalization for short-wavelength sensitivity in jewel beetles (Coleoptera: Buprestidae)

Background: Arthropods have received much attention as a model for studying opsin evolution in invertebrates. Yet, relatively few studies have investigated the diversity of opsin proteins that underlie spectral sensitivity of the visual pigments within the diverse beetles (Insecta: Coleoptera). Previous work has demonstrated that beetles appear to lack the short-wavelength-sensitive (SWS) opsin class that typically confers sensitivity to the “blue” region of the light spectrum. However, this is contrary to established physiological data in a number of Coleoptera. To explore potential adaptations at the molecular level that may compensate for the loss of the SWS opsin, we carried out an exploration of the opsin proteins within a group of beetles (Buprestidae) where short-wave sensitivity has been demonstrated. RNA-seq data were generated to identify opsin proteins from nine taxa comprising six buprestid species (including three male/female pairs) across four subfamilies. Structural analyses of recovered opsins were conducted and compared to opsin sequences in other insects across the main opsin classes—ultraviolet, short-wavelength, and long-wavelength. Results: All nine buprestids were found to express two opsin copies in each of the ultraviolet and long-wavelength classes, contrary to the single copies recovered in all other molecular studies of adult beetle opsin expression. No SWS opsin class was recovered. Furthermore, the male Agrilus planipennis (emerald ash borer—EAB) expressed a third LWS opsin at low levels that is presumed to be a larval copy. Subsequent homology and structural analyses identified multiple amino acid substitutions in the UVS and LWS copies that could confer short-wavelength sensitivity. Conclusions: This work is the first to compare expressed opsin genes against known electrophysiological data that demonstrate multiple peak sensitivities in Coleoptera. We report the first instance of opsin duplication in adult beetles, which occurs in both the UVS and LWS opsin classes. Through structural comparisons of known insect opsins, we suggest that opsin duplication and amino acid variation within the chromophore binding pocket explains sensitivity in the short-wavelength portion of the visible light spectrum in these species. These findings are the first to reveal molecular complexity of the color vision system within beetles

Development and Testing of a Bacterial Biosensor for Toluene-Based Environmental Contaminants

A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator xylR from the TOL plasmid of Pseudomonas putida mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, P(u). A reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of XylR and P(u). When Escherichia coli cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited K(1/2) values ranging from 39.0 ± 3.8 μM for 3-xylene to 2,690 ± 160 μM for 3-methylbenzylalcohol (means ± standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays

Data from: A cure for the blues: opsin duplication and subfunctionalization for short-wavelength sensitivity in jewel beetles (Coleoptera: Buprestidae)

Background: Arthropods have received much attention as a model for studying opsin evolution in invertebrates. Yet, relatively few studies have investigated the diversity of opsin proteins that underlie spectral sensitivity of the visual pigments within the diverse beetles (Insecta: Coleoptera). Previous work has demonstrated that beetles appear to lack the short-wavelength-sensitive (SWS) opsin class that typically confers sensitivity to the “blue” region of the light spectrum. However, this is contrary to established physiological data in a number of Coleoptera. To explore potential adaptations at the molecular level that may compensate for the loss of the SWS opsin, we carried out an exploration of the opsin proteins within a group of beetles (Buprestidae) where short-wave sensitivity has been demonstrated. RNA-seq data were generated to identify opsin proteins from nine taxa comprising six buprestid species (including three male/female pairs) across four subfamilies. Structural analyses of recovered opsins were conducted and compared to opsin sequences in other insects across the main opsin classes—ultraviolet, short-wavelength, and long-wavelength. Results: All nine buprestids were found to express two opsin copies in each of the ultraviolet and long-wavelength classes, contrary to the single copies recovered in all other molecular studies of adult beetle opsin expression. No SWS opsin class was recovered. Furthermore, the male Agrilus planipennis (emerald ash borer—EAB) expressed a third LWS opsin at low levels that is presumed to be a larval copy. Subsequent homology and structural analyses identified multiple amino acid substitutions in the UVS and LWS copies that could confer short-wavelength sensitivity. Conclusions: This work is the first to compare expressed opsin genes against known electrophysiological data that demonstrate multiple peak sensitivities in Coleoptera. We report the first instance of opsin duplication in adult beetles, which occurs in both the UVS and LWS opsin classes. Through structural comparisons of known insect opsins, we suggest that opsin duplication and amino acid variation within the chromophore binding pocket explains sensitivity in the short-wavelength portion of the visible light spectrum in these species. These findings are the first to reveal molecular complexity of the color vision system within beetles