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Results for “Light Signal Transduction”

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PubMed1994

An in vivo assay for members of the cAMP response element-binding protein family of transcription factors.

The CREB/ATF family of dimeric basic/leucine zipper (bZIP) transcription factors have been implicated in signal transduction by several agents including intracellular cAMP. The ubiquity of the cAMP-signaling pathway in mammalian cells has made it difficult to assay individual family members in their native form and dissect their roles in signal transduction. In light of observations that ATF1 exhibits several cell-specific characteristics and is allosterically regulated, the above problem has represented a particular limitation for studying ATF1. To address this we have exploited the modular nature of the bZIP domain to design fusion proteins that closely resemble native ATF1 but have altered DNA binding and dimerization specificity. The activity of these novel proteins can be monitored following their introduction into a cAMP-responsive cell type. Thus we describe an assay for members of the CREB family that should be generally applicable in a range of cell types and to other bZIP protein families.

Amino Acid Sequence↗

PubMed2006

The mitochondrial CMSII mutation of Nicotiana sylvestris impairs adjustment of photosynthetic carbon assimilation to higher growth irradiance.

The CMSII mutant of Nicotiana sylvestris, which lacks a functional mitochondrial complex I, was used to investigate chloroplast-mitochondria interactions in light acclimation of photosynthetic carbon assimilation. CMSII and wild-type (WT) plants were grown at 80 micromol m(-2) s(-1) photosynthetic active radiation (PAR; 80) and 350 micromol m(-2) s(-1) PAR (350). Carbon assimilation at saturating PFD was markedly higher in WT 350 leaves as compared with WT 80 leaves, but was similar in CMS 80 and CMS 350 leaves, suggesting that the mutant is unable to adjust photosynthesis to higher growth irradiance. WT 350 leaves showed several general characteristic light acclimation responses [increases in leaf specific area (LSA), total chlorophyll content, and chlorophyll a/b ratio, and a higher light compensation point]. In contrast, a similar chlorophyll content and chlorophyll a/b ratio were measured for both CMS 80 and CMS 350 leaves, while LSA and the light compensation point acclimated as in the WT. The failure of CMSII to adjust photosynthesis to growth PFD did not result from lower quantum efficiency of PSII, lower whole-chain electron transport rates (ETRs), or lower ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and sucrose phosphate synthase (SPS) capacities. Excess ETR not used for carbon assimilation was even higher in CMS 350 than in WT 350. Since photochemical fluorescence quenching and the initial activity of NADP malate dehydrogenase (NADP-MDH) were identical in WT 350 and CMS 350 leaves but the activation state of NADP-MDH was different, redox signals from primary ETR are not involved in the signal transduction of light acclimation, while a contribution of stromal redox state cannot be excluded. When mature plants were transferred between 350 and 80 conditions, the mutant showed acclimatory tendencies, although adjustments were not as rapid or as marked as in the WT, and the response of the initial activities of Rubisco and NADP-MDH was impaired or altered. Initial activities of Rubisco and SPS at limiting concentration were also affected in CMS 350 as compared with WT plants when compared at growth irradiance or after in situ activation at 1000 micromol m(-2) s(-1) PAR. The data demonstrate that chloroplast-mitochondria interactions are important in light acclimation, and modulation of the activation state of key photosynthetic enzymes could be an important mechanism in this cross-talk.

Acclimatization↗

PubMed1998

Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man.

Rhodopsin kinase (RK), a specialized G-protein-coupled receptor kinase expressed in retina, is involved in quenching of light-induced signal transduction in photoreceptors. The role of RK in recovery after photoactivation has been explored in vitro and in vivo experimentally but has not been specifically defined in humans. We investigated the effects on human vision of a mutation in the RK gene causing Oguchi disease, a recessively inherited retinopathy. In vitro experiments demonstrated that the mutation, a deletion of exon 5, abolishes the enzymatic activity of RK and is likely a null. Both a homozygote and heterozygote with this RK mutation had recovery phase abnormalities of rod-isolated photoresponses by electroretinography (ERG); photoactivation was normal. Kinetics of rod bleaching adaptation by psychophysics were dramatically slowed in the homozygote but normal final thresholds were attained. Light adaptation was normal at low backgrounds but became abnormal at higher backgrounds. A slight slowing of cone deactivation kinetics in the homozygote was detected by ERG. Cone bleaching adaptation and background adaptation were normal. In this human in vivo condition without a functional RK and probable lack of phosphorylation and arrestin binding to activated rhodopsin, reduction of photolyzed chromophore and regeneration processes with 11-cis-retinal probably constitute the sole pathway for recovery of rod sensitivity. The role of RK in rods would thus be to accelerate inactivation of activated rhodopsin molecules that in concert with regeneration leads to the normal rate of recovery of sensitivity. Cones may rely mainly on regeneration for the inactivation of photolyzed visual pigment, but RK also contributes to cone recovery.

Animals↗

PubMed1996

Participation of GTP-binding protein in the photo-transduction of Paramecium bursaria.

Analysis of the flow of light information in Paramecium, especially the participation of GTP-binding protein, was carried out. Injection of GDP-beta-S into Paramecium cell abolished the light-induced inward current. The partially purified rhodopsin-like protein (RLP) that we obtained activated frog rod outer segments (ROS) GTPase. A GTP-binding protein, with Mw 57,000 was detected by immunostaining with anti-rat G alpha rabbit IgG. These results suggest that the light information flows from the light-captured RLP to the GTP-binding protein.

Animals↗

PubMed1995

A synthetic peptide corresponding to residues 645-653 in the carboxyl terminal cytoplasmic domain of the rat testicular follicle stimulating hormone receptor modulates G protein coupled-receptor signaling in rat testis membranes and in intact cultured rat Sertoli cells.

We examined the involvement of the carboxyl terminal cytoplasmic domain (residues 645-653) of the rat testicular FSH receptor in FSH signal transduction utilizing light membranes prepared from immature rat testes, intact cultured rat Sertoli cells, and a synthetic peptide approach. This region of the FSH receptor was selected because of its structural similarity to receptor-G protein contact sites identified in other G protein-coupled receptors. FSHR- (645-653) peptide amide promoted guanine nucleotide exchange in rat testis membranes, presumably via its interaction with membrane-associated G protein, but did not inhibit FSH binding to testis membrane receptors. When incubated with intact cultured Sertoli cells from immature rat testes, FSHR- (645-653) peptide amide consistently and significantly stimulated basal cAMP and estradiol biosynthesis. The peptide had no effect on forskolin stimulation of cAMP and estradiol, but inhibited FSH stimulation of each. FSH binding to receptor was unaffected by the peptide, these results suggest peptide interaction with receptor-associated G protein. The effects of FSHR- (645-653) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. These results suggest that peptide effects in intact Sertoli cells were related to peptide entry into the cell, presumably during receptor-mediated endocytosis of FSH, or by diffusion. Synthetic peptide amides not satisfying structural criteria for G protein coupling had no effect on either guanine nucleotide exchange or estradiol biosynthesis, even at concentrations significantly higher than used for FSHR- (645-653) peptide amide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2001

Extraocular phototransduction and circadian timing systems in vertebrates.

It is widely accepted that, for organisms with eyes, the daily regulation of circadian rhythms is made possible by light transduction through those organs. Yet, it has been demonstrated repeatedly in recent years that ocular light receptors that mediate vision, at least in mammals, are not the same photoreceptors involved in circadian regulation. Moreover, it has been recognized for many years that circadian regulation can occur in organisms without eyes. In fact, extraocular circadian phototransduction (EOCP) appears to be a phylogenetic rule for the vast majority of species. EOCP has been reported in every nonmammalian species studied to date. In mammals, however, the story is very different. This paper presents findings from studies that have examined specifically the capacity for EOCP in vertebrate species. In addition, the literature addressing noncircadian aspects of extraocular phototransduction is briefly discussed. Finally, possible mechanisms underlying EOCP are discussed, as are some of the implications of the presence, or absence, of EOCP across phylogeny.

Amphibians↗

PubMed1997

Cloning and characterization of LRG5, a gene involved in blue light signaling in Chlamydomonas gametogenesis.

The Chlamydomonas reinhardtii mutant lrg2 exhibits a partial defect in the blue-light activated signal transduction chain that controls gametogenesis. By genomic complementation, a cosmid clone that corrects the lrg2 phenotype was isolated. The smallest fragment of this cosmid that gave complementation was used in RFLP analysis, which revealed that the cloned gene was not linked to the LRG2 locus. The sequence of the genomic clone and a cDNA of this gene, which we named LRG5, was determined. Although no significant homology with sequences in the databases was found, the nuclear localization signal and the presence of negatively and positively charged domains suggests a function for the LRG5 protein in the regulation of transcription. The correction of the lrg2 phenotype is probably caused by overexpression of the LRG5 gene in the transformants, as these expressed a level of LRG5 approximately twofold higher than wild-type or mutant strains. Southern blot analysis provided evidence for the existence of homologous sequences in other green algae as well as in higher plants, suggesting a conserved function for the LRG5 protein.

Amino Acid Sequence↗

PubMed2006

Evolving visual pigments: hints from the opsin-based proteins in a phylogenetically old "eyeless" invertebrate.

Visual pigments are photosensitive receptor proteins that trigger the transduction process producing the visual excitation once they have absorbed photons. In spite of the molecular and morpho-functional complexity that has characterized the development of animal eyes and eyeless photoreceptive systems, opsin-based protein family appears ubiquous along metazoan visual systems. Moreover, in addition to classic rhodopsin photoreceptors, all Metazoa have supplementary non-visual photosensitive structures, mainly located in the central nervous system, that sense light without forming an image and that rather regulate the organism's temporal physiology. The investigation of novel non-visual photopigments exerting extraretinal photoreception is a challenging field in vision research. Here we propose the cnidarian Hydra as a useful tool of investigation for molecular and functional differences between these pigment families. Hydra is the first metazoan owning a nervous system and it is an eyeless invertebrate showing only an extraocular photoreception, as it has no recognized visual or photosensitive structures. In this paper we provide an overview of the molecular and functional features of the opsin-based protein subfamilies and preliminary evidences in a phylogenetically old species of both image-forming and non-visual opsins. Then we give new insights on the molecular biology of Hydra photoreception and on the evolutionary pathways of visual pigments.

Animals↗

PubMed1998

Flow cytometric quantitation of human opsonin-dependent phagocytosis and oxidative burst responses to meningococcal antigens.

A one-step flow cytometric (FCM) assay has been developed to quantify both opsonin- and antigen-dependent phagocytosis and intraphagocyte oxidative burst responses. Meningococcal outer membrane structures (OMV) were adsorbed to fluorescent polystyrene beads, opsonized with serum, and exposed to leukocytes. FCM parameters of phagocytosis were evaluated in combinations with oxidative burst indicators. Rhodamine-123 was the most sensitive indicator and was compatible with quantitation of phagocytosis. The phagocytosis and oxidative burst responses induced by OMV beads were dependent on both antigens and opsonins. Increased human opsonic responses against OMV were induced during clinical meningococcal disease. A dissociation was noted between phagocytosis and oxidative burst in individual cells, indicating that functional opsonins against OMV components may differ in their ability to stimulate phagocytosis and oxidative burst responses. The method facilitates evaluation of purified bacterial structures as mediators of opsonin-dependent phagocytosis and intracellular oxidative microbicidal mechanisms, which is of interest in the complex process of selecting bacterial antigens as constituents of certain vaccines.

Adult↗

PubMed2001

Using light scatter signal to estimate bacterial biovolume by flow cytometry.

BACKGROUND: In the past decade, flow cytometry has become a useful and precise alternative to microscopic bacterial cell counts in aquatic samples. However, little evidence of its usefulness for the evaluation of bacterial biovolumes has emerged in from the literature. METHODS: The light scattering and cell volume of starved bacterial strains and natural bacterial communities from the Black Sea were measured by flow cytometry and epifluorescence microscopy, respectively, in order to establish a relationship between light scattering and cell volume. RESULTS: With the arc-lamp flow cytometer, forward angle light scatter (FALS) was related to cell size in both the starved strains and natural communities, although regression parameters differed. We tested the predictive capacity of the FALS verous cell size relationship in a bacterial community from the North Sea. That analysis showed that a reliable bacterial biovolume prediction of a natural bacterial community can be obtained from FALS using a model generated from natural bacterial community data. CONCLUSIONS: Bacterial biovolume is likely to be related to FALS measurements. It is possible to establish a generally applicable model derived from natural bacterial assemblages for flow cytometric estimation of bacterial biovolumes by light scatter.

Aeromonas hydrophila↗

PubMed1997

The genetics of phototransduction and circadian rhythms in Arabidopsis.

A wide range of biological processes, in all eukaryotes and in some prokaryotes, are controlled by rhythms with a period close to 24 hours. The circadian oscillator, which is responsible for generating these rhythms, is controlled by light signals that maintain its synchrony with the environmental day/night cycle. Higher plants exhibit many circadian rhythms, including rhythms in the transcription of specific genes. Molecular tools derived from such clock-controlled genes have led to the identification of several circadian rhythm mutants in the genetic model, Arabidopsis thaliana. The extensive understanding of photoperception in this species will make it a powerful system with which to investigate the light regulation of circadian rhythms. We compare Arabidopsis rhythms to the results from other systems, and discuss these data with respect to the current phototransduction models.

Arabidopsis↗

PubMed2005

Ciliary behavior of a negatively phototactic Chlamydomonas reinhardtii.

With an instrument that can record the motion of both cilia of the unicellular alga Chlamydomonas reinhardtii for many hours, the behavioral differences of its two cilia have been studied to determine their specific role in phototaxis. The organism was held on a fixed micropipette with the plane of ciliary beating rotated into the imaging plane of a quadrant photodetector. The responses to square-wave light patterns of a wide range of temporal frequencies were used to characterize the responses of each cilium. Eighty-one cells were examined showing an unexpectedly diverse range of responses. Plausible common signals for the linear and nonlinear signals from the cell body are suggested. Three independent ciliary measures--the beat frequency, stroke velocity, and phasing of the two cilia--have been identified. The cell body communicates to the cilia the direction of phototaxis the cell desires to go, the absolute light intensity, and the appropriate graded transient response for tracking the light source. The complexity revealed by each measure of the ciliary response indicates many independent variables are involved in the net phototactic response. In spite of their morphological similarity, the two cilia of Chlamydomonas respond uniquely. Probably the signals from the cell body fan out to independent pathways in the cilia. Each cilium modifies the input in its own way. The change in the pattern of the effective and recovery strokes of each cilium associated with negative phototaxis has been demonstrated and its involvement in phototactic turning is described.

Animals↗

PubMed2006

Linear systems analysis of the ciliary steering behavior associated with negative-phototaxis in Chlamydomonas reinhardtii.

In response to light stimulation Chlamydomonas reinhardtii changes the beating frequency, beating pattern, and beating synchrony of the trans and cis cilia to steer the freely-swimming cell relative to light sources. To understand the cell steering behavior the impulse responses of the beating frequency and stroke velocity of each cilium have been obtained with high temporal resolution on cells held with a micropipette. Interestingly the response of each cilium is quite different. The trans cilium responds with less delay than the cis cilium for both beating frequency and stroke velocity. For light stimulation at 2 Hz, the critical cell-rotation frequency, both responses of the trans and cis cilia are about 180 degrees out of phase. The trans-cilium beating frequency response peaks at a stimulus frequency of 5-6 Hz, higher than the cis at 1-2 Hz. The stroke velocities of the trans and cis cilia have the same stimulus-frequency response (2 Hz), but the trans cilium has a shorter delay than the cis. The times to maximum response are much shorter than the time for a rotation of the cell. The use of two different approaches that enable the trans cilium to respond ahead of the cis for both the beating frequency and stroke velocity responses suggests the importance of both responses to phototaxis. Internal cell processing responsible for the time course of the responses is proposed.

Animals↗

PubMed2002

Early development of the retina and pineal complex in the sea lamprey: comparative immunocytochemical study.

Lampreys have a complex life cycle, with largely differentiated larval and adult periods. Despite the considerable interest of lampreys for understanding vertebrate evolution, knowledge of the early development of their eye and pineal complex is very scarce. Here, the early immunocytochemical organization of the pineal complex and retina of the sea lamprey was studied by use of antibodies against proliferating cell nuclear antigen (PCNA), opsin, serotonin, and gamma-aminobutyric acid (GABA). Cell differentiation in the retina, pineal organ, and habenula begins in prolarvae, as shown by the appearance of PCNA-negative cells, whereas differentiation of the parapineal vesicle was delayed until the larval period. In medium-sized to large larvae, PCNA-immunoreactive (-ir) cells were numerous in regions of the lateral retina near the differentiated part of the larval retina (central retina). A late-proliferating region was observed in the right habenula. Opsin immunoreactivity appears in the pineal vesicle of early prolarvae and 3 or 4 days later in the retina. In the parapineal organ, opsin immunoreactivity was observed only in large larvae. In the pineal organ, serotonin immunoreactivity was first observed in late prolarvae in photoreceptive (photoneuroendocrine) cells, whereas only a few of these cells appeared in the parapineal organ of large larvae. No serotonin immunoreactivity was observed in the larval retina. GABA immunoreactivity appeared earlier in the retina than in the pineal complex. No GABA-ir perikaryon was observed in the retina of larval lampreys, although a few GABA-ir centrifugal fibers innervate the inner retina in late prolarvae. First GABA-ir ganglion cells occur in the pineal organ of 15-17 mm larvae, and their number increases during the larval period. The only GABA-ir structures observed in the parapineal ganglion of larvae were afferent fibers, which appeared rather late in development. The time sequence of development in these photoreceptive structures is rather different from that observed in teleosts and other vertebrates. This suggests that the unusual development of the three photoreceptive organs in lampreys reflects specialization for their different functions during the larval and adult periods.

Aging↗

PubMed2002

Protein kinase C-induced disorganization and endocytosis of photosensitive membrane in Limulus ventral photoreceptors.

Protein kinase C (PKC) desensitizes the light response in photoreceptors from the ventral optic nerve of the horseshoe crab Limulus. Photoisomerization of Limulus rhodopsin leads to phosphoinositide hydrolysis, resulting in the production of inositol trisphosphate and diacylglycerol (DAG). Inositol trisphosphate mobilizes intracellular stores of Ca(2+), resulting in photoreceptor excitation in Limulus, while DAG may activate PKC. We investigated whether PKC-mediated desensitization of the photoresponse is accompanied by ultrastructural changes in the rhodopsin-bearing photosensitive membrane (rhabdom) in Limulus ventral photoreceptors. PKC activation by (-)-indolactam V in darkness induces disorganization and swelling of the rhodopsin-containing microvilli and endocytosis of rhabdomeral membrane. The effects of (-)-indolactam V on dark-adapted photoreceptor ultrastructure are reversible, are stereospecific, are blocked by coapplication of PKC inhibitors, and closely match those induced by continuous, bright light. Rhabdom disorganization and endocytosis via PKC activation may, therefore, contribute to desensitization of the light-adapted photoreceptor.

Animals↗

PubMed2001

Short and mid-wavelength cone distribution in a nocturnal Strepsirrhine primate (Microcebus murinus).

Strepsirrhines are of considerable interest for understanding the evolution of cone photoreceptors because they represent the most ancestral living primates. The retina of nocturnal Strepsirrhines is reported to contain a single population of medium/long wavelength (MW/LW) cones whereas short wavelength (SW) cones are totally absent. The area centralis of nocturnal Strepsirrhines also lacks the degree of central specialization seen in the fovea of diurnal primates. In this study of a nocturnal Strepsirrhine, the gray mouse lemur (Microcebus murinus), we used specific antibodies that recognize SW and MW/LW opsins to determine the presence of different cone subtypes and their distribution in relation to that of rods and ganglion cells. The results are compared to two diurnal Haplorhine species, a New World (Callithrix jacchus) and an Old World (Macaca fascicularis) monkey. In the mouse lemur, both antibodies to MW/LW cone opsin (COS-1 and CERN956) label the same population of cones. A small proportion of SW cones is only stained by the JH455 antiserum whereas the monoclonal OS-2 antibody shows negative staining. These two antibodies label the same SW cone population in other primates. The extracellular matrix of all cones is also labeled by the peanut agglutinin (PNA) lectin. In mouse lemur retinal wholemounts, peak cone density is localized at the area centralis and ranged from 7,500 to 8,000 cones/mm(2). SW cones represent less than 0.2 % of the total cone population and are mainly located in the nasal part of the retina. SW cones show an irregular distribution and densities never exceed 49 cones/mm(2). The distribution of neurons in the ganglion cell layer shows a distinct centroperipheral gradient with a peak of 28,000 cells/mm(2) at the area centralis. Rod distribution shows a centroperipheral gradient with the peak (850,000 rods/mm(2)) including and extending slightly dorsal to the area centralis. The theoretical spatial resolution of the mouse lemur (4.9 cycles/degree) is slightly lower to that of other nocturnal primates. The densities of rods, cones, and ganglion cell layer neurons represent a compromise between spatial resolution and sensitivity for both photopic and scotopic vision.

Animals↗

PubMed2001

Diurnal and circadian variation of protein kinase C immunoreactivity in the rat retina.

We studied the dependence of the expression of protein kinase C immunoreactivity (PKC-IR) in the rat retina on the light:dark (LD) cycle and on circadian rhythmicity in complete darkness (DD). Two anti-PKC alpha antibodies were employed: One, which we call PKCalphabeta recognized the hinge region; the other, here termed PKCalpha, recognized the regulatory region of the molecule. Western blots showed that both anti-PKC antibodies stained an identical single band at approximately 80 kD. The retinal neurons showing PKC-IR were rod bipolar cells and a variety of amacrine neurons. After 3 weeks on an LD cycle, PKCalphabeta-IR in both rod bipolar and certain amacrine cells manifested a clear rhythm with a peak at zeitgeber time (ZT) of 06-10 hours and a minimum at ZT 18. No rhythm in total PKC-IR was observed when using the PKCalpha antibody, but, at ZT 06-10 hours, rod bipolar axon terminals showed increased immunostaining. After 48 hours in DD, with either antibody, rod bipolar cells showed increased PKC-IR. The PKCalpha antibody alone revealed that, after 48 hours, AII amacrine neurons, which lacked PKC-IR in an LD cycle, manifested marked PKC-IR, which became stronger after 72 hours. Light administered early in the dark period greatly increased PKCalphabeta-IR in rod bipolar and some amacrine neurons. Our data indicate that light and darkness exert a strong regulatory influence on PKC synthesis, activation, and transport in retinal neurons.

Amacrine Cells↗

PubMed2001

Anatomy and physiology of neurons with processes in the accessory medulla of the cockroach Leucophaea maderae.

The accessory medulla (AMe), a small neuropil in the insect optic lobe, has been proposed to serve a circadian pacemaker function analogous to the role of the suprachiasmatic nucleus in mammals. Building upon considerable knowledge of the circadian system of the cockroach Leucophaea maderae, we investigated the properties of AMe neurons in this insect with intracellular recordings combined with dye injections. Responses of neurons with processes in the AMe to visual stimuli, including stationary white light, moving objects, and polarized light were compared with the responses of adjacent medulla tangential neurons. Neurons with processes in the AMe and additional ramifications in the medulla strongly responded to stationary light stimuli and might, therefore, be part of photic entrainment pathways to the clock. Accessory medulla neurons lacking significant processes in the medulla but with projections to the midbrain or to the contralateral optic lobe, in contrast, responded weakly or not at all to light and, thus, seem to be part of the clock's output pathway. Two types of commissural neurons with tangential arborizations in both medullae were sensitive to polarized light, suggesting a role of these neurons in celestial navigation. Sidebranches in the AMae of one of the two cell types are discussed with respect to a possible involvement of the AMe in polarization vision. Finally, neurons responding to movement stimuli did not arborize in the AMe. The results show that the AMe receives photic input and support a role of this neuropil in circadian timekeeping functions.

Action Potentials↗