Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

active transport via molecular motors. In principle, the mode of translocation of each protein could be different between the lightand dark-induced directions. Most investigators argue against the involvement of molecular motors. One major argument is that all of these proteins (or at least individual subunits in the case of transducin) are fairly soluble and any of their relocation by molecular motors may be negated by the subsequent diffusion throughout the entire cellular volume. Another argument is that, although protein translocation by motors is rapid, it could be easily saturated by the very large number of translocation protein molecules. On the other hand, intracellular diffusion of soluble proteins in rods is also sufficiently rapid to explain the observed protein translocation rates and, unlike molecular motors, could not be saturated by the amount of protein molecules undergoing lightinduced translocation. Yet, it should be noted that, based on the impediment of translocation by the cytoskeleton disrupting drugs, some believe that motor systems are involved, particularly in protein translocations in the dark-induced directions.

However, diffusion alone cannot account for the phenomenon because it does not explain any disequilibrium of protein distributions with the free cytoplasmic volume of the rod or cone. Thus, while diffusion serves as the mode of protein movement, the observed patterns of light-dependent protein redistribution may be explained by light-dependent appearance or disappearance of specific protein-binding sites in individual subcellular compartments. The next two sections illustrate these ideas in regard to transducin and arrestin.

Specific Mechanisms of Protein

Translocation

Transducin

Recent reports suggest that transducin translocation could be explained by the differences in the membrane affinities of its abg heterotrimer compared to the individual a- and bg-subunits. The heterotrimer is strongly membraneassociated due to the combined action of two lipid modifications: a farnesyl group on the g-subunit and an acyl group on the a-subunit. Thus, in the dark-adapted rod, transducin heterotrimer is predicted to be concentrated on the disk membranes of the outer segment. When transducin is activated in light, a-subunits bind GTP and dissociate from the bg-subunits (Figure 2). Both subunits become more soluble since each has only one lipid modification, allowing them to diffuse throughout the cytoplasm. Inevitably, GTP hydrolysis by the a-subunit would result in the restoration of the poorly soluble trimer. When this happens in the outer segment, transducin becomes re-attached to the disk membranes; but when it

Translocation

GTP

Figure 2 The putative role of transducin subunit dissociation in its translocation. When transducin is activated by photoexcited rhodopsin (R*), its a- and bg-subunits become separated from one another and the membrane affinity of each is less than that of the heterotrimer. In this state, subunits may dissociate from photoreceptor disk membranes and translocate from the outer segment. The efficiency of translocation is dependent on the time during which transducin subunits stay apart before re-formation of the trimer. This time is determined by the rate of the GTP hydrolysis on the a-subunit and is dependent on the absolute amount of activated transducin. Reproduced from Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Jr., and Arshavsky, V. Y. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends in Cell Biology 16: 560–568.

happens in the inner segment, the trimer may adhere to the membranous structures there, causing its transient accumulation. The central assumption of this hypothesis, that transducin subunits move apart from one another, is supported by the difference in their translocation rates and by transducin translocation being facilitated by transgenic or pharmacological manipulations promoting transducin subunits to remain in the dissociated state. The translocation of transducin bg-subunit is further enhanced by the protein called phosducin. Phosducin reduces membrane association of the bg-subunit, which presumably allows it to more easily diffuse throughout the cytoplasm.

Another important mechanistic feature of transducin translocation is its light-intensity threshold. This threshold reflects the fact that the cellular content of transducin significantly exceeds that of RGS9, a protein responsible for rapid inactivation of transducin. Light of the threshold intensity produces activated transducin in the amount exceeding the capacity of RGS9 to inactivate it. Consequently, a fraction of activated transducin stays in the active, GTP-bound form (with a- and bg-subunits dissociated) for a longer time, sufficient for dissociation from the disk membranes and diffusion through the photoreceptor cytoplasm. Accordingly, the knockout of RGS9 allows transducin translocation at a lower light intensity, whereas RGS9 overexpression shifts the threshold to brighter light.

Arrestin

It was first suggested that arrestin is equilibrated throughout the rod cytoplasm in the dark and is trapped in the outer segment in light upon binding to photoexcited rhodopsin. However, recent observations argue that the dark-adapted distribution of arrestin does not match the distribution of the free cytoplasm volume, indicating that most arrestin is bound to sites located in the inner segment. One hypothesis is that these sites are formed by microtubules. It was further proposed that arrestin translocation is explained by a simple competition between constitutive, low-affinity microtubule sites in the inner segment and transient, high-affinity sites in the outer segment formed upon rhodopsin photoexcitation. However, quantitative measurements indicated that, at the minimal light intensity sufficient to trigger arrestin translocation, the number of translocated arrestin molecules exceeds the number of photoactivated rhodopsin molecules by 30-fold. Even less rhodopsin activation is required to trigger arrestin translocation in knockout mice lacking RGS9 where transducin activation persists longer than normally. Therefore, triggering arrestin translocation is not dependent on the absolute amount of rhodopsin excited by light, but is rather dependent on arrestin release from the inner segment sites by a yet-to- be-identified signaling mechanism downstream from phototransduction.

Proteins’ Return in the Dark

The rates of transducin and arrestin return to their darkadapted locations upon switching from light to dark are much slower than their movement in the light-induced direction, with most measurements indicating that it takes at least an hour. This may be slow enough to be within the capacity of molecular motors, and indeed transducin and arrestin return can be blocked by cytoskeleton disrupting drugs. The specificity of these treatments remains unknown and artifacts such as clogging the connecting cilium could not be ruled out. On the other hand, arrestin and transducin return could also be explained by a combination of diffusion, removal of the light-induced binding sites, and restoration of the dark-adapted sites. Current evidence is insufficient to discriminate between the potential roles of molecular motors and diffusion in transducin and arrestin return to their dark-adapted cellular distributions.

See also: Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Inactivation in Rods; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods.

Further Reading

Artemyev, N. O. (2008). Light-dependent compartmentalization

of transducin in rod photoreceptors. Molecular Neurobiology 37: 44–51.

Brann, M. R. and Cohen, L. V. (1987). Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina. Science 235: 585–587.

Broekhuyse, R. M., Tolhuizen, E. F., Janssen, A. P., and Winkens, H. J. (1985). Light induced shift and binding of S-antigen in retinal rods.

Current Eye Research 4: 613–618.

Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N. Jr., and Arshavsky, V. Y. (2006). Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends in Cell Biology 16: 560–568.

Fain, G. L. (2006). Why photoreceptors die (and why they don’t). BioEssays 28: 344–354.

Hanson, S. M., Francis, D. J., Vishnivetskiy, S. A., Klug, C. S., and Gurevich, V. V. (2006). Visual arrestin binding to microtubules involves a distinct conformational change. Journal of Biological Chemistry 281: 9765–9772.

Kerov, V., Chen, D. S., Moussaif, M., et al. (2005). Transducin activation state controls its light-dependent translocation in rod photoreceptors. Journal of Biological Chemistry 280: 41069–41076.

Nair, K. S., Hanson, S. M., Mendez, A., et al. (2005). Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein–protein interactions. Neuron 46:

555–567.

Philp, N. J., Chang, W., and Long, K. (1987). Light-stimulated protein movement in rod photoreceptor cells of the rat retina. FEBS Letters 225: 127–132.

Reidel, B., Goldmann, T., Giessl, A., and Wolfrum, U. (2008). The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton. Cell Motility and the Cytoskeleton 65: 785–800.

Slepak, V. Z. and Hurley, J. B. (2008). Mechanism of light-induced translocation of arrestin and transducin in photoreceptors: Interaction-restricted diffusion. IUBMB Life 60: 2–9.

Sokolov, M., Lyubarsky, A. L., Strissel, K. J., et al. (2002). Massive light-driven translocation of transducin between the two major compartments of rod cells: A novel mechanism of light adaptation. Neuron 34: 95–106.

Strissel, K. J., Lishko, P. V., Trieu, L. H., et al. (2005). Recoverin undergoes light-dependent intracellular translocation in rod photoreceptors. Journal of Biological Chemistry 280: 29250–29255.

Strissel, K. J., Sokolov, M., Trieu, L. H., and Arshavsky, V. Y. (2006). Arrestin translocation is induced at a critical threshold of visual signaling and is superstoichiometric to bleached rhodopsin. Journal of Neuroscience 26: 1146–1153.

Whelan, J. P. and McGinnis, J. F. (1988). Light-dependent subcellular movement of photoreceptor proteins. Journal of Neuroscience Research 20: 263–270.

Glossary

Arhabdomeric cell – The secondary visual cell in Limulus median eyes that is not photoreceptive but is electrically coupled to photoreceptors and generates action potentials in response to photoreceptor depolarization.

Chelicerate – A type of arthropod belonging to the group Chelicerata that includes horseshoe crabs, scorpions, spiders, ticks, and mites.

Circadian clock – A biological clock that oscillates with a period of about 24 h even under constant conditions.

Compound eye – An eye that contains a few to many separate photoreceptive units.

Eccentric cell – A secondary visual cell in Limulus lateral eye that is not photoreceptive but is electrically coupled to photoreceptors and generates action potentials in response to photoreceptor depolarization.

Lateral inhibition – A mechanism of information processing in nervous systems that increases contrast and resolving power.

Ocellus – A type of eye with a single lens. Octopamine – A major amine neurotransmitter in invertebrates that is the phenol analog of norepinephrine.

Ommatidium – The photoreceptive unit of compound eyes.

Quantum bump – The response of photoreceptors to a single photon of light.

Rhabdom – A photoreceptive membrane typically elaborated by arthropod photoreceptors that is composed of tightly packed microvilli.

The retinas of animals that live in cyclic light environments typically undergo rhythmic, daily changes in structure and function. These changes underlie the ability of animals to detect images over the large daily fluctuations in ambient illumination, and therefore they are critical for normal vision. Some of these changes, such as light and dark adaptation, are driven solely by fluctuations in illumination, while others are driven solely by signals from internal circadian clocks. Still other changes require interactions between lightand clock-driven biochemical cascades.

In the American horseshoe crab Limulus polyphemus, a chelicerate arthropod, circadian changes in vision are

dramatic, and as a result, Limulus can see at night nearly as well as it can during the day. Since the circadian organization of the Limulus visual system is also advantageous for experimental manipulation, Limulus has been used to examine in detail the separate and combined effects of diurnal light and the clock on the retina.

The first part of this article describes the organization of the Limulus visual system and the circadian input to the eyes. The second part describes the impact of the circadian clock on Limulus retinal structure, the photoresponse, photosensitive membrane shedding, and gene expression. The synaptic mechanisms through which the clock influences the eyes are described as well as a clockdriven biochemical cascade in photoreceptors that may underlie some of the observed circadian changes in visual function.

Organization of the Limulus Visual

System

Limulus has three major types of eyes: lateral, median, and rudimentary (Figure 1). The eyes that are obvious by examining the dorsal side of an adult animal are the lateral compound eyes (LE) on the dorsolataral carapace, and a pair of median eyes located on either side of the median dorsal spine near the front of the animal. Rudimentary eyes are found at three different locations, and in an adult animal they are largely hidden from view. Lateral rudimentary eyes are located below the cornea at the posterior edge of each LE, and a pair of fused median rudimentary eyes is located below the carapace between the two median ocelli. Ventral rudimentary eyes consist of a pair of optic nerves that extend anteriorly from the brain just below the cuticle on the ventral side of the animal. They terminate in an end organ located below a wart-like structure visible on the ventral cuticle in front of the mouth.

Lateral Compound Eyes

In an adult animal, each LE contains over 1000 conical lenses, and below each lens there is a single ommatidium, or mini-retina, that is aligned with the long axis of the overlying lens (Figure 2(a)) Each ommatidium is composed of 8–12 photoreceptors clustered like the sections of an orange around the dendrite of usually one secondary visual cell called the eccentric cell (Figures 2(b) and 2(c)). The photoreceptors are electrically coupled to the dendrite of the eccentric cell. Two major non-neuronal cells types

Dorsal

Ventral

Ventral eye end organ

Mouth

are present in ommatidia: cone cells and pigment cells. Cone cell processes occupy the aperture at the base of the lens and separate the lens from photoreceptors. Pigment cells surround each ommatidium and form partitions between photoreceptors.

LE photoreceptors respond with graded depolarizations to visible light, and the eccentric cells to which they are electrically coupled, encode these graded depolarizations as action potentials. Both photoreceptors and eccentric cells have axons that project to optic ganglia in the brain (lamina and medulla) through the lateral optic nerve (Figure 3(a)). However, because the lateral optic nerve in an adult animal

is long, often over 10 cm, it is unlikely that the graded photoreceptor potentials reach the brain. Therefore, information from LEs is thought to reach the brain only through the activity of the eccentric cells.

In addition to projecting to the brain, eccentric cells extend axon collaterals laterally within the eye to form a neural plexus just below the ommatidia (Figure 2(a)). Within this plexus, eccentric cell collaterals from neighboring and even distant ommatidia make reciprocal inhibitory synapses which are the basis for lateral inhibition in the eye. Lateral inhibition, a fundamental mechanism of information processing in nervous systems that increases contrast

and resolving power, was first detected experimentally in Limulus LE. In vertebrate retinas, this same mechanism is known as center-surround inhibition.

Median Eyes

Median eyes are called ocelli because they have a single spherical lens (Figure 4). In median eye retinas, groups of 5–11 photoreceptors are typically associated with at least one secondary visual cell or arhabdomeric cell, but unlike LE ommatidia, these groups are not highly organized and difficult to discern anatomically. The photoreceptors and arhabdomeric cells are embedded in guanophores, cells that contain reflective guanine crystals, and the retina is surrounded by pigment cells.

An interesting feature of median eyes is that they contain two different types of photoreceptors: one that responds to visible light and another that is sensitive to ultraviolet (UV) light. These two photoreceptor types have not yet been distinguished anatomically. Median eye arhabdomeric cells are thought equivalent to LE eccentric cells because, similar to eccentric cells, they are electrically coupled to photoreceptors and generate action potentials in response to graded photoreceptor depolarizations. Median eye photoreceptors and arhabdomeric cells both project axons to ocellar ganglia in the brain through the median optic nerve (Figure 3(a)), but only the action potentials of the arhabdomeric cell are thought to reach the brain. Interestingly, arhabdomeric cell action potentials have been detected only in response to UV illumination.

Rudimentary Eyes

The lateral, median, and ventral rudimentary eyes differentiate in the embryo before the more complex LEs and median ocelli, and they presumably provide photic information to the developing embryo. Each rudimentary eye consists of a cluster of large photoreceptors (100 160 mm) that are sensitive to visible light. Ventral eye photoreceptors are typically scattered along the length of the ventral optic nerves as well as clustered near the brain and in the end organ (Figure 3(a)).

Similar to the photoreceptors in lateral and median eyes, rudimentary eye photoreceptors produce graded depolarizations in response to illuminations, but they have no associated secondary visual cells. Rudimentary photoreceptors project axons to the brain through the lateral, median, or ventral optic nerves, and although their axon diameters are considerably larger than those of lateral and median eye photoreceptors, it still seems unlikely that their graded depolarizations reach the brain in an adult animal. An exception may be the graded

End organ

eRh

RB

E

N

Ocellus

Figure 4 Schematic of a median ocellus cut perpendicular to the carapace. Most of the area proximal to the lens is filled with photoreceptor cells. The dark areas below the lens represent the rhabdoms, which are not highly organized in this eye.

Arhabdomeric cells are interspersed among the photoreceptors. Pigment cells surround the structure and reflective guanophores lie nearer the base of the retina. Modified from Jones, J. N. and Brown, J. E. (1971). Z. Zellforsch 118: 297–309, with permission from Springer Verlag.

depolarizations produced by ventral photoreceptors located close to the brain.

The Photoreceptors

The photoreceptors are fundamentally similar in the three types of Limulus eyes. All are composed of two major compartments: a rhabdomeral lobe and an arhabdomeral lobe

(Figures 2(b), 3(b), and 3(c)). The rhabdomeral lobe contains the photosensitive membrane or rhabdom, which in Limulus, as in other arthropods, is composed of tightly packed actin-rich microvilli. The arhabdomeral lobe is not photosensitive and contains the nucleus and metabolic machinery of the cell. As described above, all Limulus photoreceptors produce graded depolarizations in response to illumination, and with the exception of the median eye UV-sensitive photoreceptors, all respond to visible light with an absorption maximum of about 525 nm. Finally, all Limulus photoreceptors, as well as the eccentric cells of the LE and arhabdomeric cells of the median eye, release the inhibitory neurotransmitter histamine. Histamine is also the neurotransmitter used by the photoreceptors of insects and crustaceans.

The rhabdoms in the three types of eyes are organized differently, however, and they are highly organized only in LE ommatidia. In LEs, the rhabdoms of neighboring photoreceptors are fused so that in a cross section of an ommatidium, they appear like an asterisk with the eccentric cell dendrite at the center (Figures 2(c) and 2(c)). The highly complex rhabdoms in median ocelli may project proximally into neighboring photoreceptors or infoldings of photoreceptor membranes may form internal rhabdoms (Figure 4). Rudimentary photoreceptors typically have both external and internal rhabdoms (Figures 3(b) and

3(c)) and often have more than one rhabdomeral lobe. In addition, rhabdoms of adjacent rudimentary photoreceptors frequently fuse forming a double layer of microvilli.

Among Limulus photoreceptors, those of the ventral eyes are the best studied, and their physiology is probably the most thoroughly characterized of any rhabdomeral photoreceptor. Ventral photoreceptors are also significant in the history of vision research. The first intracellular recordings of a photoresponse were made from ventral eye photoreceptors, and physiological studies of these photoreceptors provided the first evidence for the importance of Ca++ in light adaptation and a role for phospholipids in the photoresponse.

Circadian Organization of the Limulus

Visual System

The circadian organization of the Limulus visual system is significantly different from that of vertebrates. In vertebrates, one or more circadian oscillators are present within the retina, and in some species, circadian oscillators are present in photoreceptors. In Limulus, the circadian oscillators that influence the eyes are located in the brain, not in the eyes. The Limulus brain contains bilateral circadian oscillators that remain synchronous through neuronal coupling, and they drive the activity of efferent neurons that project from the brain to all of the eyes.

The cell bodies of clock-driven efferent neurons that project to the eyes are clustered in the cheliceral ganglia located on either side of the base of the brain, and there are about 20 efferent neurons in each ganglion. The axon of each efferent neuron is thought to branch several times in the brain and its branches to project bilaterally out all of the optic nerves and innervate all of the eyes (Figure 5(a)). In the eyes, the efferent axons have neurosecretory-like terminals containing both clear vesicles and large, dense, crystalline granules. In LE ommatidia, efferent terminals innervate all cell types (Figure 5(b)). In median eyes, efferent terminals contact photoreceptors near the rhabdom, and in rudimentary eyes, efferent axons project to the rhabdomeral lobes of photoreceptors where they terminate directly adjacent to rhabdoms (Figures 3(b) and 5(c)). Circadian efferent neurons, similar to those described in the Limulus visual system, also innervate the eyes of scorpions and spiders; therefore, the type of circadian regulation described for Limulus eyes appears to be a feature common to chelicerate arthropods, but not to crustaceans and insects.

In Limulus, efferent neurons innervating the eyes are active at night and silent during the day. They begin firing bursts of action potentials about 45 min before sunset. These bursts, which are synchronous in all optic nerves, reach a maximum frequency of about 2 Hz during the early evening. The burst rate slows after midnight, and then stops after dawn. This pattern of efferent nerve activity persists in constant darkness, and its phase (the time of day it starts and

stops) can be shifted by changing the time of light onset. Thus, efferent nerve activity is clearly circadian.

Effects of the Clock on Limulus Eyes

Clock input increases the sensitivity of Limulus eyes to light at night, and this effect is most dramatic in LEs. In animals maintained in cyclic light, LEs become about 1 million times more sensitive to light at night compared to the day from the combined effects of dark adaptation and clock input. The effect of the clock is to roughly double the sensitivity obtained with dark adaptation alone.

In animals maintained in constant darkness, the onset of efferent nerve activity near dusk correlates with the increase in LE sensitivity. If the lateral optic nerve is cut during the day, severing the efferent axons and thus preventing clock signals from reaching the LE during the night, there is no nighttime increase in sensitivity. If the lateral optic nerve is cut during the night, interrupting clock input to the eyes, LE sensitivity falls rapidly toward the daytime level. However, if the distal end of a cut lateral optic nerve is stimulated electrically to activate the efferent axons, LE sensitivity increases even during the subjective day. Thus, increased LE sensitivity directly correlates with the activity of the efferent neurons.

In LEs, where the effects of the clock have been studied most thoroughly, the clock influences almost every aspect of retinal function, and studies with these eyes have revealed that the full range of some of the diurnal changes observed involves complex interactions between effects of clock and light (Table 1).

Structure

Much of the nighttime increase in LE sensitivity can be attributed to the changes in the structure of ommatidia (Figure 5(b)). As the LE transitions into the nighttime state, the aperture at the base of the lens, which is constricted during the day, widens and shortens permitting more photons to reach the underlying photoreceptors. The rhabdom also becomes shorter and wider, adjusting to the larger aperture. These structural changes, which increase photon catch during the night, persist with reduced amplitude in constant darkness in response to clock input alone. However, if clock input is eliminated, all of these rhythmic structural changes are abolished, even in cyclic light. Without clock input, ommatidia assume a structure that is never seen in an intact animal. Thus, these structural changes require clock input, and the effects of the clock are enhanced by cyclic light.

Another diurnal structural change observed in LE photoreceptors that probably contributes to increased nighttime sensitivity is the migration of photoreceptor screening pigment granules. However, unlike the

End organ

Ventral photoreceptor cell body

Median optic

Ventral optic nerve

nerve

Lateral optic nerve

Lamina

Medulla

Cheliceral

ganglion

R lobe

Photoreceptor

cell body

Efferent

axon

Afferent

axon

(c)

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cells

Pigment granules Eccentric cell dedrite

Retinular

cell

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(b)fibers

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cell

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axon

structural changes in the aperture described above, pigment granule migration appears to be regulated entirely by the clock and is not significantly influenced by light. Photoreceptor pigment granules cluster near the junction of the rhabdomeral and arhabdomeral lobes during the day and disperse toward the periphery of the cell during the night (Figure 5(b)). This rhythm continues undiminished in eyes maintained in constant darkness and is

eliminated in eyes deprived of clock input even when the eyes are exposed to cyclic light.

Physiology

Clock-driven physiological changes in photoreceptors also contribute to increased LE sensitivity. Clock input at night increases the amplitude (gain) and duration of the

Table 1 Lateral eye responses to clock input are influenced by light and mimicked by octopamine and elevated cAMP.

aInfusions of OA into the LE in situ increase LE sensitivity. Since increased sensitivity is largely due to a wider aperture and repositioned rhabdom, the effects of OA on these two parameters are inferred.

response to a single photon (the quantum bump) and decreases the frequency of spontaneous membrane depolarizations recorded in the dark (noise). Thus, the clock increases the signal-to-noise ratio of photoreceptors at night. The onset of these physiological changes correlates directly with the onset of efferent nerve activity and occurs well before the structural changes described above are detected.

Rhabdom Shedding

Similar to the photoreceptors of vertebrates, invertebrate photoreceptors shed some of their photosensitive membrane every day. Limulus photoreceptors exhibit two distinct mechanisms for rhabdom shedding: light-triggered transient membrane shedding and light-driven shedding (Figure 6). Both result in the internalization of photosensitive membrane into photoreceptors. Light-driven shedding is similar to the clathrin-mediated endocytosis of activated G-protein coupled receptors described in many other systems, including insect photoreceptors. It continues throughout the day in the light and is mediated by clathrin and arrestin. The clock does not influence this process. On the other hand, light-triggered transient shedding is a unique, synchronous process that rapidly removes membrane from the rhabdom at dawn. During light-triggered transient shedding, large whorls of rhabdomeral membrane are internalized from the base of rhabdomeral microvilli and trafficked into multivesicular bodies. The process is triggered by the dim light of dawn and is complete within an hour after first light. The mechanism for membrane internalization during transient shedding is not yet known. However, importantly from the perspective of this discussion,

Figure 6 Schematic of transient rhabdom shedding and lightdriven shedding. Transient rhabdom shedding must be primed by clock input during the night. It is triggered by the first light of dawn and characterized by the formation of large whorls of rhabdomeral membrane, which subsequently are processed in densely packed multivesicular bodies (MVBs). Light-driven shedding does not require clock input. This progressive process, which continues throughout the day, requires

brighter light and involves the clathrin-mediated endocytosis of microvillar membrane from the base of the microvilli. The membranes endocytosed by this process aggregate and form loosely packed MVB. From Sacunas et al. (2002). Journal of Comparative Neurology 449: 26–42, with permission from John Wiley & Sons, Inc.

although transient shedding is triggered by light and probably involves the activation of protein kinase (PK) C, it occurs only after photoreceptors have received at least 3 h of clock input and the activation of cyclic adenosine monophosphate (cAMP)-dependent PK.

Gene Expression

In addition to driving structural and physiological changes in photoreceptors and priming transient rhabdom shedding, clock input influences the expression of at least one gene important for the photoresponse. Specifically, clock input to the LE controls an early step in the expression of the gene for visual arrestin (varr). Varr is the protein in photoreceptors responsible for quenching the phototransduction cascade, and as described above, it is also involved in the internalization of rhabdomeral membrane during light-driven shedding. In LEs maintained in constant darkness, clock input during the subjective night causes varr messenger RNA (mRNA) levels in photoreceptors to fall. Circadian fluctuations in varr mRNA levels may lead to a circadian fluctuation in varr

protein levels and thus contribute to some of the circadian changes in the photoresponse described above.

The expression of other photoreceptor proteins probably is also under circadian control, but the clock does not regulate the expression of all proteins important for the photoresponse. For example, Limulus opsin mRNA levels are regulated by light, not by the clock. In LEs that receive normal clock input and are exposed to cyclic light, opsin mRNA levels rise during the late afternoon and fall during the night. This rhythm continues in LEs deprived of clock input if they are exposed to cyclic light, and it is eliminated in LEs maintained in the dark even when they receive normal clock input.

Biochemical Processes Mediating Clock Effects on Limulus Eyes

Octopamine and the Activation of a cAMP

Cascade

When circadian efferent neurons are active during the night, they release the biogenic amine octopamine (OA) from their terminals, and OA, the phenol analog of norepinephrine, is probably most responsible for initiating the circadian changes observed in the eyes. The application of OA to Limulus eyes mimics many effects of the clock (Table 1). Other molecules are also released from efferent terminals. Specifically, g-glutamyl conjugates of OA and tyramine, the precursor of OA, are released, but their physiological relevance is not yet clear. The presence of crystalline granules in efferent terminals suggests that one or more neuropeptides may be released. Crystalline granules are often associated with peptidergic synaptic terminals in invertebrates, but presumptive neuropeptides in the circadian efferent terminals in Limulus eyes have not been identified.

In Limulus eyes, OA activates membrane receptors that are coupled to adenylyl cyclase stimulating a rise in cAMP in photoreceptors, and any effects of the clock are mediated through the activation of this cAMP cascade (Table 1). There is no evidence for a direct effect of cAMP on Limulus photoreceptor physiology; however, there is good evidence that some of the clock-driven changes in the eye require the activation of cAMP-dependent PKA. Therefore, investigations of mechanisms underlying the circadian regulation of photoreceptor function have focused on identifying and characterizing clock-regulated photoreceptor phosphoproteins.

Clock-Driven Protein Phosphorylation

To date, one clock-regulated phosphoprotein in Limulus photoreceptors has been identified and partially characterized. It is an unconventional myosin III (LpMyo3), a homolog of the Drosophila ninaC gene product that is

NINAC(174)

Human3B

Human3A

Figure 7 Schematic comparing the domain structure of LpMyo3 with that of the class III myosins expressed in Drosophila and humans. One isoform of myo3 has been found in Limulus. The two isoforms of myo3 expressed in Drosophila are splice variants of the same ninaC gene. Human class III myosins are products of two separate genes, myo3A and myo3B. Each protein contains an N-terminal kinase domain, a myosin domain, and one or more IQ calmodulin-binding motifs (black bars). The tail domains are most variable in length and sequence. LpMyo3 is a target of clock-stimulated phosphorylation within an actinbinding region near the C-terminus of its myosin-like domain (red asterisks).

required in Drosophila for normal photoreceptor function and survival. Since their discovery in Drosophila and Limulus photoreceptors, class III myosins also have been detected in photoreceptors of a variety of invertebrates and vertebrates, including octopus, fish, mice, and humans; thus, these proteins may be important for photoreceptor function in both vertebrates and invertebrates.

Class III unconventional myosins are characterized by having an N-terminal kinase domain, a myosin-motor-like domain, one or more IQ calmodulin-binding motifs, and a C-terminal tail of varying lengths (Figure 7). Thus, they are potential signaling molecules as well as potential molecular motors. LpMyo3 is photoreceptor specific, quantitatively a major protein in photoreceptors and distributed throughout the photoreceptor from its cell body to its terminals. During the day in the light, LpMyo3 concentrates over the rays of the photosensitive rhabdom (Figure 2(c)).

LpMyo3 becomes more highly phosphorylated in LEs in vivo in response to activation of the circadian efferent input. Its phosphorylation also becomes elevated when intact photoreceptors are incubated in vitro with OA, drugs that elevate intracellular cAMP levels and that activate PKA, respectively. Since LpMyo3 is also phosphorylated in response to light, this protein may be particularly important as a substrate upon which lightand clock-driven cascades converge.

Some biochemical properties of LpMyo3 are known. It is a kinase that phosphorylates its own myosin domain as well as other substrates, and its substrate specificity is similar, but not identical, to PKA. LpMyo3 also binds actin, but since its actin binding is insensitive to adenosine triphosphate (ATP) and the protein lacks ATPase activity,