The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton

In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subsequently analyzed by light and electron microscopy. The application of cytoskeletal drugs differentially affected the cytoskeleton in photoreceptor compartments. During dark adaptation the depolymerization of microtubules as well as actin filaments disrupted the translocation of arrestin and transducin in rod photoreceptor cells. During light adaptation only the delivery of arrestin within the outer segment was impaired after destabilization of microtubules. Movements of transducin and arrestin required intact cytoskeletal elements in dark adapting cells. However, diffusion might be sufficient for the fast molecular movements observed as cells adapt to light. These findings indicate that different molecular translocation mechanisms are responsible for the dark and light associated translocations of arrestin and transducin in rod photoreceptor cells.     

Photoreceptor adaptation in intrinsically photosensitive retinal ganglion cells

A rare type of mammalian retinal ganglion cell (RGC) expresses the photopigment melanopsin and is a photoreceptor. These intrinsically photosensitive RGCs (ipRGCs) drive circadian-clock resetting, pupillary constriction, and other non-image-forming photic responses. Both the light responses of ipRGCs and the behaviors they drive are remarkably sustained, raising the possibility that, unlike rods and cones, ipRGCs do not adjust their sensitivity according to lighting conditions ("adaptation"). We found, to the contrary, that ipRGC sensitivity is plastic, strongly influenced by lighting history. When exposed to a constant, bright background, the background-evoked response decayed, and responses to superimposed flashes grew in amplitude, indicating light adaptation. After extinction of a light-adapting background, sensitivity recovered progressively in darkness, indicating dark adaptation. Because these adjustments in sensitivity persisted when synapses were blocked, they constitute "photoreceptor adaptation" rather than "network adaptation." Implications for the mechanisms generating various non-image-forming visual responses are discussed.     

The role of cytoplasmic calcium in photoreceptor light adaptation.

The process of light adaptation in vertebrate rod and cone photoreceptors is believed to involve a diffusible cytoplasmic messenger. Two lines of evidence indicate that photoreceptor light adaptation is mediated by a light-induced fall in cytoplasmic calcium concentration (Ca2+i). First, if changes in calcium concentration are slowed by the incorporation of calcium chelators into the photoreceptor cytoplasm then light adaptation is slowed also. Second, if the normal control of Ca2+i is prevented by simultaneously minimising calcium influx and efflux across the outer segment membrane by means of external solution changes, then all of the manifestations of light adaptation are abolished. Furthermore, recent results show that changes in Ca2+i imposed in the absence of light are sufficient to cause at least some of the manifestations of light adaptation. Together these results indicate that calcium acts as the messenger of light adaptation in the photoreceptors of both lower and higher vertebrates.     

Biochemical mechanism of light adaptation in vertebrate photoreceptors.

Vertebrate photoreceptors can adjust their sensitivity to a wide range of light intensities spanning several orders of magnitude, the phenomenon of which is called light adaptation. Electrophysiological and biochemical studies have revealed that calcium can serve as an intracellular transmitter of light adaptation under the control of cGMP metabolism. After illumination, the cytoplasmic calcium concentration of a photoreceptor decreases, which in turn strongly activates photoreceptor guanylate cyclase. This calcium-dependent effect is mediated by a novel calcium-binding protein (recoverin) and leads to the restoration of the depleted cGMP pool after illumination.     

Photosensory Adaptation in Plants

Photosensory adaptation (range adjustment) is of great importance in plants (including fungi and various microorganisms) that operate in large intensity ranges. The adaptation mechanisms of plants have some features in common with those of vertebrates and invertebrates. As with those systems, plants have biphasic exponential dark-adaptation kinetics, that are much slower than the corresponding light adaptation kinetics. One needs to distinguish between sensor adaptation, which regulates range adjustment, and effector adaptation (habituation), which regulates the motor apparatus of the organism (flagellar movement or cell wall growth). In Phycomyces, and perhaps Stentor, sensor adaptation is mediated by the photoreceptor system. In contrast to vertebrates and invertebrates, dark adaptation can be controlled in some plants by special photoreceptors. In Phycomyces, these can be either photo-products of the actinic photoreceptor(s) or not yet identified receptor pigments. In higher plants phytochrome can alter the state of adaptation.     

Light Adaptation in the Ventral Photoreceptor of Limulus

Light adaptation in both the ventral photoreceptor and the lateral eye photoreceptor is a complex process consisting of at least two phases. One phase, which we call the rapid phase of adaptation, occurs whenever there is temporal overlap of the discrete waves that compose a light response. The recovery from the rapid phase of adaptation follows an exponential time-course with a time constant of approximately 75 ms at 21°C. The rapid phase of adaptation occurs at light intensities barely above discrete wave threshold as well as at substantially higher light intensities with the same recovery time-course at all intensities. It occurs in voltage-clamped and unclamped photoreceptors. The kinetics of the rapid phase of adaptation is closely correlated to the photocurrent which appears to initiate it after a short delay. The rapid phase of adaptation is probably identical to what is called the "adapting bump" process. At light intensities greater than about 10 times discrete wave threshold another phase of light adaptation occurs. It develops slowly over a period of ½ s or so, and decays even more slowly over a period of several seconds. It is graded with light intensity and occurs in both voltage-clamped and unclamped photoreceptors. We call this the slow phase of light adaptation.     

Transducin activation state controls its light-dependent translocation in rod photoreceptors

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-alpha subunit (Gtalpha)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtalphaQ200L. An illumination threshold for the Gtalpha movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtalphaQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtalpha, the GtalphaQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtalphaQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtalpha, and apparently, do not require Gtalpha interaction with RGS9 and PDE6.     

Sensitivity changes and facilitation in the photoreceptor of phalangium opilio due to light adaptation. Preliminary notes

These preliminary notes were made on sensitivity changes and facilitation in the photoreceptor of phalangium opilio, due to light adaptation. They show that facilitation is a case opposite to light adaptation. Other measurements are planned in the progress of this work.     

In situ cGMP phosphodiesterase and photoreceptor potential in gecko retina

The possible involvement of phosphodiesterase (PDE) activation in phototransduction was investigated in gecko photoreceptors by comparing the in situ PDE activity with the photoreceptor potential. In the dark, intracellular injection of cGMP into a gecko photoreceptor caused a long-lasting depolarization. An intense light flash given during the depolarization phase repolarized the cell with a short latency comparable to that of the light-evoked hyperpolarizing response, which indicates that the activation of PDE in situ is rapid enough to generate the photoreceptor potential. PDE activity in situ was estimated quantitatively from the duration of the cGMP-induced depolarization, since it was expected that the higher the PDE activity, the shorter the duration. Under steady illumination, the enzyme exhibited a constant activity. On exposure to a light flash, PDE became activated, but recovered in the dark with a time course that was dependent on the intensity of the preceding stimulus. When PDE activity and photoreceptor sensitivity to light were measured in the same cell after a light flash, both recovery processes showed similar kinetics. Theoretical analysis showed that the parallelism in the recovery time courses could be explained if cGMP is the transduction messenger. These results suggest that PDE activation is involved not only in the generation but also in the adaptation mechanisms of the photoreceptor potential.     

A quadruple photoreceptor mutant still keeps track of time

Time measurement and light detection are inextricably linked. Cryptochromes, the blue-light photoreceptors shared between plants and animals, are critical for circadian rhythms in flies and mice [1-3]. WC-1, a putative blue-light photoreceptor, is also essential for the maintenance of circadian rhythms in Neurospora [4]. In contrast, we report here that in Arabidopsis thaliana the double mutant lacking the cryptochromes cry1 and cry2, and even a quadruple mutant lacking the red/ far-red photoreceptor phytochromes phyA and phyB as well as cry1 and cry2, retain robust circadian rhythmicity. Interestingly, the quadruple mutant was nearly blind for developmental responses but perceived a light cue for entraining the circadian clock. These results indicate that cryptochromes and phytochromes are not essential components of the central oscillator in Arabidopsis and suggest that plants could possess specific photosensory mechanisms for temporal orientation, in addition to cryptochromes and phytochromes, which are used for both spatial and temporal adaptation.     

Molecular physiology of visual pigment rhodopsin

The key physiological functions of the rhodopsin molecule are reviewed. Molecular mechanisms of visual pigments spectral tuning, photoisomerization of the 11-cis-retinal chromophore that triggers the phototransduction process, formation of physiologically active state of rhodopsin as a G-protein-coupled receptor, rhodopsin visual cycle, and consequences of its impairment are evaluated. Visual pigment rhodopsin performs several functions, providing spectral sensitivity of photoreceptor cells, phototransduction processes and light and dark adaptation. Genetically determined defects of visual pigment molecule and proteins involved into mechanisms of phototransduction and adaptation or into mechanism of visual cycle are directly linked to pathogenesis of different forms of degenerative retina diseases. Understanding the molecular mechanisms of these physiological processes uncovers the way to direct investigation of pathogenesis of these severe eye diseases.     

Light-dependent CK2-mediated phosphorylation of centrins regulates complex formation with visual G-protein

Centrins are Ca2+-binding EF-hand proteins. All four known centrin isoforms are expressed in the ciliary apparatus of photoreceptor cells. Cen1p and Cen2p bind to the visual G-protein transducin in a strictly Ca2+-dependent way, which is thought to regulate light driven movements of transducin between photoreceptor cell compartments. These relatively slow motile processes represent a novel paradigm in light adaptation of photoreceptor cells. Here we validated specific phosphorylation as a novel regulator of centrins in photoreceptors. Centrins were differentially phosphorylated during photoreceptor dark adaptation. Inhibitor treatments revealed protein kinase CK2 as the major protein kinase mediating phosphorylation of Cen1p, Cen2p and Cen4p, but not Cen3p, at a specific target sequence. CK2 and ciliary centrins co-localize in the photoreceptor cilium. Direct binding of CK2 and centrins to ciliary microtubules may spatially integrate the enzyme-substrate specificity in the cilium. Kinetic light-scattering assays revealed decreased binding affinities of phosphorylated centrins to transducin. Furthermore, we show that this decrease is based on the reduction of Ca2+-binding affinities of centrins. Present data describe a novel regulatory mechanism of reciprocal regulation of stimulus dependent distribution of signaling molecules.     

Adaptation of Mammalian Photoreceptors to Background Light: Putative Role for Direct Modulation of Phosphodiesterase

All sensory receptors adapt. As the mean level of light or sound or odor is altered, the sensitivity of the receptor is adjusted to permit the cell to function over as wide a range of ambient stimulation as possible. In a rod photoreceptor, adaptation to maintained background light produces a decrease (or “sag”) in the response to the prolonged illumination, as well as an acceleration in response decay time and a Weber–Fechner-like decrease in sensitivity. Earlier work on salamander indicated that adaptation is controlled by the intracellular concentration of Ca²⁺. Three Ca²⁺-dependent mechanisms were subsequently identified, namely, regulation of guanylyl cyclase, modulation of activated rhodopsin lifetime, and alteration of channel opening probability, with the contribution of the cyclase thought to be the most important. Later experiments on mouse that exploit the powerful techniques of molecular genetics have shown that cyclase does indeed play a significant role in mammalian rods, but that much of adaptation remains even when regulation of cyclase and both of the other proposed pathways have been genetically deleted. The identity of the missing mechanism or mechanisms is unclear, but recent speculation has focused on direct modulation of spontaneous and light-activated phosphodiesterase.     

Response transfer functions of Limulus ventral photoreceptors: interpretation in terms of transduction mechanisms

In recent years, our knowledge of the biochemical mechanisms underlying the transduction process in photoreceptors has expanded rapidly. However, a full picture of the temporal dynamics of these mechanisms remains elusive. To study the dynamics in the Limulus ventral photoreceptor, we measure its light-evoked transfer function under voltage clamp. Comparison of this transfer function to biochemically realistic theoretical models of transduction provides insights into the photoreceptor dynamics. This comparison supports the suggestion that the low-frequency behaviour of the Limulus photoreceptor, corresponding to light and dark adaptation, is that of a nonlinear negative feedback loop. The main reactions of this loop have time constants between about 1 and 40 s. Such a feedback loop does not account, however, for the high-frequency behaviour of the responses, which implies the existence of a further, fast-acting, mechanism.This work was supported by grants from the Binational Science Foundation (BSF) Jerusalem, Israel and the Israel Academy of Sciences and Humanities, by NIH grant EY 1428, and by NSF grant DMS 8505442     

Ca(2+)-binding proteins in the retina: structure, function, and the etiology of human visual diseases

The complex sensation of vision begins with the relatively simple photoisomerization of the visual pigment chromophore 11-cis-retinal to its all-trans configuration. This event initiates a series of biochemical reactions that are collectively referred to as phototransduction, which ultimately lead to a change in the electrochemical signaling of the photoreceptor cell. To operate in a wide range of light intensities, however, the phototransduction pathway must allow for adjustments to background light. These take place through physiological adaptation processes that rely primarily on Ca(2+) ions. While Ca(2+) may modulate some activities directly, it is more often the case that Ca(2+)-binding proteins mediate between transient changes in the concentration of Ca(2+) and the adaptation processes that are associated with phototransduction. Recently, combined genetic, physiological, and biochemical analyses have yielded new insights about the properties and functions of many phototransduction-specific components, including some novel Ca(2+)-binding proteins. Understanding these Ca(2+)-binding proteins will provide a more complete picture of visual transduction, including the mechanisms associated with adaptation, and of related degenerative diseases.     

Pikachurin Protein Required for Increase of Cone Electroretinogram B-Wave during Light Adaptation

In normal eyes, the amplitude of the b-wave of the photopic ERGs increases during light adaptation, but the mechanism causing this increase has not been fully determined. The purpose of this study was to evaluate the contribution of receptoral and post-receptoral components of the retina to this phenomenon. To accomplish this, we examined the ERGs during light adaptation in Pikachurin null-mutant (Pika -/-) mice, which have a misalignment of the bipolar cell dendritic tips to the photoreceptor ribbon synapses. After dark-adaptation, photopic ERGs were recorded from Pika -/- and wild type (WT) mice during the first 9 minutes of light adaptation. In some of the mice, post-receptoral components were blocked pharmacologically. The photopic b-waves of WT mice increased by 50% during the 9 min of light adaptation as previously reported. On the other hand, the b-waves of the Pika -/- mice decreased by 20% during the same time period. After blocking post-receptoral components, the b-waves were abolished from the WT mice, and the ERGs resembled those of the Pika -/- mice. The extracted post-receptoral component increased during light adaptation in the WT mice, but decreased for the first 3 minutes to a plateau in Pika -/- mice. We conclude that the normal synaptic connection between photoreceptor and retinal ON bipolar cells, which is controlled by pikachurin, is required for the ERGs to increase during light-adaptation. The contributions of post-receptoral components are essential for the photopic b-wave increase during the light adaptation.     

Polyphosphoinositide hydrolysis in response to light stimulation of rat and chick retina and retinal rod outer segments

Phosphoinositides of chick and rat retina were labelled with [3H]inositol. Exposure of retinal preparations to light for 30 s caused loss of labelled phosphatidylinositol 4,5-bisphosphate and to a smaller extent of the other phosphoinositides. Similar light-induced changes were seen when rod outer segment preparations were used and, when these were illuminated in calcium-free media, phosphatidylinositol 4,5-bisphosphate was the only lipid affected. No inositol 1,4,5-trisphosphate was seen after either 30 s or 5 s of illumination of retina or 30 s illumination of rod outer segments. It is concluded that this compound plays no direct part in vertebrate photoreceptor light transduction, though phosphoinositide metabolism might relate to adaptation mechanisms.