Squid Photoreceptors Research Articles

Purification of visual arrestin from squid photoreceptors and characterization of arrestin interaction with rhodopsin and rhodopsin kinase

Invertebrate visual signal transduction involves photoisomerization of rhodopsin, activating a guanine nucleotide binding protein (G protein) of the G(q) class, iG(q), which stimulates a phospholipase C, increasing intracellular Ca2+. Arrestin binding to photoactivated rhodopsin is a key mechanism of desensitization. We have previously reported the cloning of a retina-specific arrestin cDNA from Loligo pealei displaying 56-64% sequence similarity to other reported arrestin sequences. Here, we report the purification of the 55-kDa squid visual arrestin. Purified squid visual arrestin is able to inhibit light-activated GTPase activity dose-dependently in arrestin-depleted rhabdomeric membranes and associate with the membrane in a light-dependent manner. Membrane association can be partially inhibited by inositol 1,2,3,4,5,6-hexakisphosphate (IP6), a soluble analog of the membrane lipid phosphatidylinositol 3,4,5-triphosphate. In reconstitution assays, we demonstrate arrestin phosphorylation by squid rhodopsin kinase, a novel function among the G protein-coupled receptor kinase family. Phosphorylation of purified arrestin requires squid rhodopsin kinase, membranes, light-activation, and the presence of Ca2+. This is the first large-scale purification of an invertebrate arrestin and biochemical demonstration of arrestin function in the invertebrate visual system.

Discriminative responses of squid ( Loligo pealeii) photoreceptors to polarized light

Cephalopods behaviorally respond to polarized light. Electrophysiology experiments with the squid, Loligo pealeii, demonstrated that spike responses from individual photoreceptors are a cosine 2 function of the e-vector orientation of a polarized stimulus. The discrimination limit to this polarization sensitivity depended upon the difference between the orientation of a polarized stimulus with a preferred e-vector. The limit ranged from 2° to 9.2° with a direct stimulus in the dark or 4.8°–22.1° with non-directed background illumination and the cells were least discriminative at the preferred orientations. This limit can be explained partly by the variability in anatomical alignment of microvilli in the photoreceptors around a dominant axis. A few light-sensitive retinal fibers showed no polarization sensitivity. The coding of polarization information suggests that light intensity is transformed into an average spike rate. This average results from silent periods interspersed between bursts of spikes, each burst possessing a consistent interspike interval. The variations in the length and frequency of silent periods depend upon the difference between the polarization e-vector and a preferred e-vector orientation. The minimal discriminated orientation of a squid photoreceptor agrees well with the minimum behavioral discrimination of polarized light by another cephalopod, the octopus.

Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA.

It is now well established that the distal structural/functional domains of the neuron contain 2a diverse population of mRNAs that program the local synthesis of protein. However, there is still a paucity of information on the composition and function of these mRNA populations in the adult nervous system. To generate empirically, hypotheses regarding the function of the local protein synthetic system, we have compared the mRNAs present in the squid giant axon and its parental cell bodies using differential mRNA display as an unbiased screen. The results of this screen facilitated the identification of 31 mRNAs that encoded cytoskeletal proteins, translation factors, ribosomal proteins, molecular motors, metabolic enzymes, nuclear-encoded mitochondrial mRNAs, and a molecular chaperone. Results of cell fractionation and RT-PCR analyses established that several of these mRNAs were present in polysomes present in the presynaptic nerve terminal of photoreceptor neurons, indicating that these mRNAs were being actively translated. Findings derived from in vitro transfection studies established that these isolated nerve terminals had the ability to translate a heterologous reporter mRNA. Based upon these data, it is hypothesized that the local protein synthetic system plays an important role in the maintenance/remodelling of the cytoarchitecture of the axon and nerve terminal, maintenance of the axon transport and mRNA translation systems, as well as contributing to the viability and function of the local mitochondria.

Squid photoreceptor terminals synthesize calexcitin, a learning related protein

Nerve endings of squid photoreceptor neurons generate large synaptosomes upon homogenization of the optic lobe. Using several independent methods, these presynaptic structures have been shown to synthesize a wealth of soluble, cytoskeletal and nuclear encoded mitochondrial proteins, and to account for essentially all the translation activity of the synaptosomal fraction. We are now presenting evidence that calexcitin, a learning related, Ca 2+-binding protein of the B photoreceptors of Hermissenda crassicornis (a mollusk), is synthesized and subjected to post-translational modifications in the squid photoreceptor terminals. In view of the essential role of presynaptic protein synthesis in long-term memory formation in Aplysia, our data suggest that a comparable role may be played by calexcitin synthesized in the squid photoreceptor terminals.

Protein synthesis in synaptosomes: a proteomics analysis.

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.

Retinal phospholipase C from squid is a regulator of Gq alpha GTPase activity.

The phospholipase C (PLC) pathway is the major signaling mechanism of photoactivation in invertebrate photoreceptors. Here we report the cloning of a cDNA encoding a 140-kDa retinal PLC that is uniquely expressed in squid photoreceptors. This cDNA encodes a protein with multiple distinct modular domains: PH, X and Y catalytic, and C2 domains, as well as G- and P-box motifs and two GTP/ATP binding motifs. The PLC was stimulated by activated squid Gq alpha but not by squid Gq beta gamma or mammalian beta gamma subunits. The PLC was inhibited by monophosphate, diphosphate and triphosphate nucleotides but not cyclic nucleosides. We also tested the ability of PLC-140 to regulate the GTPase activity of Gq alpha in the rhabdomeric membranes. Depletion of PLC-140 from the rhabdomeric membranes decreased the GTP hydrolysis but not GTP gamma S binding to the membranes. Reconstitution of purified PLC-140 with membranes accelerated Gq alpha GTPase activity by fivefold at a concentration of 2.5 microM. Our data suggest that PLC-140 plays an important role in both the activation and inactivation pathways of invertebrate visual transduction.

Highly conserved glutamic acid in the extracellular IV-V loop in rhodopsins acts as the counterion in retinochrome, a member of the rhodopsin family.

Retinochrome is a member of the rhodopsin family having a chromophore retinal and functioning as a retinal photoisomerase in squid photoreceptor cells. Unlike vertebrate rhodopsins, but like many invertebrate rhodopsins, retinochrome does not have a glutamic acid at position 113 to serve as a counterion for the protonated retinylidene Schiff base. Here we investigated possible counterions in retinochrome by site-specific mutagenesis. Our results showed that the counterion is the glutamic acid at position 181, at which almost all the pigments in the rhodopsin family, including vertebrate and invertebrate rhodopsins, have a glutamic or aspartic acid. The remarkable exceptions are the long-wavelength visual pigments that have a histidine that, together with a nearby lysine, serves as a chloride-binding site. Replacement of Glu-181 of bovine rhodopsin with Gln caused a 10-nm red-shift of absorption maximum. Because the position at 181 is in the extracellular loop connecting the transmembrane helices VI and V, these results demonstrate the importance of this loop to function for spectral tuning in the rhodopsin family.

The ordered visual transduction complex of the squid photoreceptor membrane.

The study of visual transduction has given invaluable insight into the mechanisms of signal transduction by heptahelical receptors that act via guanine nucleotide binding proteins (G-proteins). However, the cyclic-GMP second messenger system seen in vertebrate photoreceptor cells is not widely used in other cell types. In contrast, the retina of higher invertebrates, such as squid, offers an equally accessible transduction system, which uses the widespread second messenger chemistry of an increase in cytosolic calcium caused by the production of inositol-(1,4,5)-trisphosphate (InsP3) by the enzyme phospholipase C, and which may be a model for store-operated calcium influx. In this article, we highlight some key aspects of invertebrate visual transduction as elucidated from the combination of biochemical techniques applied to cephalopods, genetic techniques applied to flies, and electrophysiology applied to the horseshoe crab. We discuss the importance and applicability of ideas drawn from these model systems to the understanding of some general processes in signal transduction, such as the integration of the cytoskeleton into the signal transduction process and the possible modes of regulation of store-operated calcium influx.

Direct evidence of Na+/Ca2+ exchange in squid rhabdomeric membranes.

Na+/Ca2+ exchange has been investigated in squid (Loligo pealei) rhabdomeric membranes. Ca2+-containing vesicles have been prepared from purified rhabdomeric membranes by extrusion through polycarbonate filters of 1-micrometer pore size. After removal of external Ca2+, up to 90% of the entrapped Ca2+ could be specifically released by the addition of Na+; this finding indicates that most of the vesicles contained Na+/Ca2+ exchanger. The Na+-induced Ca2+ efflux had a half-maximum value (K1/2) of approximately 44 mM and a Hill coefficient of approximately 1.7. The maximal Na+-induced Ca2+ efflux was approximately 0.6 nmol Ca2+. s-1. mg protein-1. Similar Na+-induced Ca2+ effluxes were measured if K+ was replaced with Li+ or Cs+. Vesicles loaded with Ca2+ by Na+/Ca2+ exchange also released this Ca2+ by Na+/Ca2+ exchange, suggesting that Na+/Ca2+ exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate of Ca2+ efflux enhanced by approximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin, Na+/Ca2+ exchange was characterized by a K1/2 of approximately 25 mM and a Hill coefficient of 1.6. For these vesicles, the maximal Na+-induced Ca2+ efflux was about twice as great as in control vesicles. We conclude that Na+/Ca2+ exchange proteins localized in rhabdomeric membranes mediate Ca2+ extrusion in squid photoreceptors.

Protein-synthesizing machinery in the axon compartment

Contrary to the prevailing view that the axon lacks the capacity to synthesize proteins, a substantial body of evidence points to the existence of a metabolically active endogenous translationa machinery. The machinery appears to be largely localized in the cortical zone of the axon, where, in vertebrate axons, it is distributed longitudinally as intermittent, discrete domains, called periaxoplasmic plaques. Studies, based on translation assays and probes of RNA transcripts in axon models such as the squid giant axon and selected vertebrate axons, provide evidence of locally synthesized proteins, most of which appear to be constituents of the slow axoplasmic transport rate groups. Metabolic and molecular biological findings are consistent with the view that the synthesis of proteins undergoing local turnover in the axonal compartment of macroneurons depends on the activity of an endogenous translational machinery. The documented presence of a metabolically active machinery in presynaptic terminals of squid photoreceptor neurons is also described. Finally, potential sources of axoplasmic RNAs comprising the machinery, which may include the ensheathing cell of the axon, as well as the cognate cell body, are also discussed.

Protein synthesizing units in presynaptic and postsynaptic domains of squid neurons.

Putative protein synthesizing domains, called plaques, are characterized in the squid giant synapse and axon and in terminals of squid photoreceptor neurons. Plaques are oval-shaped formations of about 1 microm in size, which (1) generate signals that have spectroscopic electron energy loss characteristics of ribosomes, (2) exhibit ribonuclease-sensitive binding of YOYO-1, a fluorescent RNA/DNA dye, and (3) in part hybridize with a poly(dT) oligonucleotide. In the giant synapse plaques are abundant in the postsynaptic area, but are absent in the presynaptic terminal. In the cortical layer of the optic lobes, plaques are localized in the large carrot-shaped presynaptic terminals of photoreceptor neurons, where they are surrounded by synaptic vesicles and mitochondria. Biochemical and autoradiographic data have documented that the protein synthetic activity of squid optic lobe synaptosomes is largely due to the presynaptic terminals of the photoreceptor neurons. The identification of ribosomes and poly(A+)-mRNA in the plaques indicates that these structures are sites of local protein synthesis in synaptic domains.

Purification, G protein activation, and partial amino acid sequence of a novel phospholipase C from squid photoreceptors.

Invertebrate visual signal transduction is initiated by rhodopsin activation of a guanine nucleotide binding protein, Gq, which stimulates phospholipase C (PLC) activity. We have previously purified a 140-kDa PLC enzyme from squid photoreceptors that is regulated by squid Gq. In these studies, an additional PLC enzyme was purified from the cytosol of squid photoreceptors and identified as a 70-kDa protein by SDS-polyacrylamide gel electrophoresis. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PLC-70 was optimal at pH 5 in the presence of 100 microM Ca2+ with a specific activity of 10.3 micromol min-1 mg-1. A polyclonal antibody raised against purified PLC-70 did not recognize purified PLC-140, and proteolytic digestion of the two purified enzymes with trypsin or Staphylococcus aureaus V8 protease showed distinct patterns of peptide fragments, indicating that PLC-70 is not a fragment of PLC-140. The partial amino acid sequence of the protein showed homology with PLC21 and norpA isozymes cloned from Drosophila, and mammalian PLC beta isozymes. Reconstitution of purified GTPgammaS-bound soluble squid Gq with PLC-70 resulted in significant enhancement of PIP2 hydrolysis over a range of Ca2+ concentrations and shifted the maximum activation by calcium to 1 microM. These results suggest that cephalopod phototransduction is mediated by Gq activation of more than one cytosolic PLC enzyme.

Isolation, cloning, and characterisation of a trp homologue from squid (Loligo forbesi) photoreceptor membranes.

The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92-kDa component isolated from a detergent-insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full-length cDNA, encoding the 92-kDa component, using both cDNA library screening and 5'-rapid amplification of cDNA ends (5'-RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl-terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl-terminal domain has been shown to be in the regulation of several ligand-gated channels. The carboxyl-terminal domain has been expressed and shown to interact with calmodulin in a calcium-dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.

Protein Synthesis in the Presynaptic Endings of the Squid Photoreceptor Neuron: In vitro and in viva Modulation

Protein Synthesis in the Presynaptic Endings of the Squid Photoreceptor Neuron: <i>In vitro</i> and <i>in viva</i> Modulation

Ca2+Induces an Increase in cGMP-Phosphodiesterase Activity in Squid Retinal Photoreceptors

In invertebrate photoreceptors, light elicits the opening of cationic channels to produce a depolarizing receptor potential. One hypothesis is that cGMP is the agent that gates the channels. It has been previously proposed that the light-induced rise in intracellular Ca2+down-regulates phosphodiesterase activity, thereby eliciting an increase in intracellular cGMP concentration. cGMP-phosphodiesterase activity from squid photoreceptors was monitored both by a continuous fluorescence assay using 2′-o-(N-methylanthraniloyl)-cGMP and by hydrolysis of [3H]cGMP. The activity of cGMP-phosphodiesterase was found to be increased by Ca2+in the physiological range of concentrations. These findings suggest that the previously known light-induced increase in Ca2+in invertebrate photoreceptors cannot directly account for light-elicited down-regulation of cGMP-phosphodiesterase.

Projection Structure of an Invertebrate Rhodopsin

Rhodopsin is the G-protein-coupled membrane receptor that initiates the visual transduction cascade in retinal photoreceptors. In the present study rhodopsin from the dark-adapted retinas of squid (Loligo forbesi) was detergent-extracted, purified, and reconstituted into native squid photoreceptor lipids following proteolytic cleavage of its proline-rich C-terminus. Two-dimensional crystals of C-terminally truncated rhodopsin reconstituted from octyl glucoside solution formed in a p2221lattice (a=44 Å,b=131 Å). Electron micrographs of frozen-hydrated crystals were processed and a projection structure to 8 Å resolution was calculated. The projection map obtained is very similar to maps previously determined for bovine and frog rhodopsins although the crystal packing of the molecules is quite different. Comparison of the maps shows that the arrangement of α-helices in the proteins is very similar despite their great phylogenetic distance; this structure is likely to be present in the whole superfamily of G-protein-coupled receptors. Invertebrate rhodopsins have a large insertion in the helix 5–helix 6 loop. Assignment of an additional density in the squid rhodopsin map to this region supports a previously proposed helix assignment and identifies the end-to-end contacts as helices 1 and 5.

Squid photoreceptor phospholipase C is stimulated by membrane Gq α but not by soluble Gq α

Phospholipase C (PLC) was purified from squid retina. Soluble Gq α, membrane Gq α and G βγ were isolated from GTP γS-treated and light-illuminated photoreceptor membranes. The membrane Gq α stimulated phosphatidyl inositoi-phospholipase C (PI-PLC) activity in a dose-dependent manner. Soluble Gq α and membrane G βγ showed no stimulating effects on PLC. GTP γS-binding was found exclusively in membrane fraction, with very little present in the KCl-soluble fraction which contained soluble Gq α. These results indicate that light-activated rhodopsin activates PLC through membrane-bound Gq α and suggest that the rhodopsin/Gq/PLC cascade might be the pathway of phototransduction in squid photoreceptors.

Current components stimulated by different G-proteins in Limulus ventral photoreceptor.

The components of the light-activated current in ventral photoreceptor of Limulus have different physical and pharmacological properties, indicating the operation of different transduction pathways. We investigated whether these pathways are activated by different G-proteins. An antiserum, directed against the C-terminal sequence QLNLKEYNLV of the alpha subunit of the Gq-protein, which is present in squid photoreceptor, was injected into the ventral photoreceptor of Limulus. This Gq-alpha mediates the light-dependent activation of PLC. Injections of the antibody into the photoreceptor blocked the second component of the light-evoked current in Limulus ventral photoreceptor, but the other components could still be activated. The results support our earlier hypothesis that the transduction pathways of the current components are selectively activated by different G-proteins.

A β-subclass phosphatidylinositol-specific phospholipase C from squid ( Loligo forbesi) photoreceptors exhibiting a truncated C-terminus

A PCR-based strategy has been used to isolate a full length cDNA encoding a phosphatidylinositol-specific phospholipase C from a sized cDNA squid ( Loligo forbesi) retinal library. The predicted protein sequence contains 875 amino acids, with calculated M r 98,181, and has marked similarity with PLC β-isoforms, including conservation of the ‘X’ and ‘Y’ regions. It is unique in having a major C-terminal truncation. A major protein of apparent M r 120,000 estimated by SDS-PAGE has been isolated from squid photoreceptors and identified by partial protein sequence analysis to correspond to the protein sequence predicted from the cDNA clone. This protein has been shown to hydrolyse phosphatidylinositol 4,5-bisphosphate. It is not yet clear whether this represents the major light-activated PLC in squid vision.

Immunoelectron-microscopic study of G-protein distribution in photoreceptor cells of the cephalopod Sepia officinalis

Previous studies suggest that more than one single light-stimulated transduction pathway seems to be present in photoreceptor cells of invertebrates. Accordingly, more than one light-dependent G-protein has been detected in squid photoreceptor cells. Two different antibodies were used to locate the distribution of G-proteins in Sepia photoreceptors. One antiserum (anti-Gα-common) has been raised against the peptide CGAGESGKSTIVKQMK. This sequence is found in most Gα-protein subunits, it is also present in transducin of vertebrates. This sequence however, is conserved only partially in Gαq from photosensory membranes of the squid. The second antiserum (anti-Gαq) has been raised against the peptide QLNLKEYNLV. This sequence is present at the C-terminus of e.g. Gαq from mouse brain and is identical also in squid. Anti-Gαq very strongly interacted with the rhabdomeres of the photoreceptor cells from Sepia. In the non-rhabdomeric cytoplasm of the photoreceptor cells gold granules (bound to the second antibody) were almost absent. With anti-Gα-common, only a small number of gold particles could be found at the rhabdomeres. The non-rhabdomeric cytoplasm was not labelled. Thus, further G-proteins that might be involved in a light dependent phototransduction mechanism besides the phosphoinositide pathway can hardly be expected to belong to the group of ‘common’ G-proteins.