Light‐sensitive coupling of rhodopsin and melanopsin to G_i/oand G_qsignal transduction inCaenorhabditis elegans

Abstract

Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase Cβ. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.

Inherent Instability of the Retinitis Pigmentosa P23H Mutant Opsin

“…WT and P23H Bovine Rhodopsin Constructs and Generation of TG Worm Lines-For the pan-neuronal bovine opsin ((b)opsin) expression construct (Genscript, Piscataway, NJ), codonoptimized (32) (b)opsin cDNA (sequence reference for (b)opsin, NP_001014890) was driven by the H20 promoter in a pBluscript KS(ϩ) vector (28,30,33). The pan-neuronal bovine opsin P23H expression construct itself was generated from WT (b)opsin vector by site-directed mutagenesis.…”

“…1B) together with a DNA vector encoding DsRed fluorescent protein under the same promoter. Selective genomic integration was indicated by DsRed fluorescence in the nervous system (28,30), and the TG worm line with the greatest P23H opsin expression was used for this study. Previously described TG C. elegans expressing WT bovine opsin (WT worms) were used as controls (28,30).…”

“…Selective genomic integration was indicated by DsRed fluorescence in the nervous system (28,30), and the TG worm line with the greatest P23H opsin expression was used for this study. Previously described TG C. elegans expressing WT bovine opsin (WT worms) were used as controls (28,30). As quantified by quantitative RT-PCR, expression of WT opsin and P23H opsin in the two TG worm lines was comparable at the mRNA level ( Fig.…”

Optogenetic analyses of neuronal network function and synaptic transmission in Caenorhabditis elegans

“…Furthermore, light-inducible enzymes, e.g., adenylate cyclases, are in use [12,50], and light-inducible protein-protein interactions have been established [32]. GPCR signal transduction can be made light-inducible using rhodopsins from vertebrates (bovine iso-rhodopsin, which is coupled to Gα i/o and human melanopsin, which is coupled to Gα q ); so far, however, these systems have not been sufficiently characterized [7]. Recently, optogenetic actuators have been combined with a genetically encoded Ca 2+ sensor that has spectral compatibility with ChR2 and allows the independent and simultaneous use of both tools according to the wavelength utilized (.…”

“…8). Furthermore, these processes can be analyzed to a certain extent in a time-dependent manner, being determined by the time gap between light stimulation and freezing (a few milliseconds up to sever- 7 8 Cell-specific mRNA profiling of the nociceptive neuron PVD, including optogenetic analysis of PVD defects following knockdown of candidate genes. mRNAs from PVD can be specifically isolated by pulldown of a FLAG tag-labeled polyA binding protein (green) that is specifically expressed in PVD.…”