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1.
Biol Bull ; 233(1): 3-20, 2017 08.
Article in English | MEDLINE | ID: mdl-29182506

ABSTRACT

The American horseshoe crab Limulus polyphemus (Linnaeus, 1758) is one of four extant species of xiphosuran chelicerates, the sister group to arachnids. Because of their position in the arthropod family tree and because they exhibit many plesiomorphic characteristics, Xiphosura are considered a proxy for the euchelicerate ancestor and therefore important for understanding the evolution and diversification of chelicerates and arthropods. Limulus polyphemus is the most extensively studied xiphosuran, and its visual system has long been a focus of studies critical for our understanding of basic mechanisms of vision and the evolution of visual systems in arthropods. Building upon a wealth of information about the anatomy and physiology of its visual system, advances in genetic approaches have greatly expanded possibilities for understanding its biochemistry. This review focuses on studies of opsin expression in L. polyphemus, which have been significantly advanced by the availability of transcriptomes and a recent high-quality assembly of its genome. These studies show that the repertoire of expressed opsins in L. polyphemus is far larger than anticipated, that the regulation of their expression in rhabdoms is far more complex than anticipated, and that photosensitivity may be distributed widely throughout the L. polyphemus central nervous system. The visual system of L. polyphemus is now arguably the best understood among chelicerates, and as such, it is a critical resource for furthering our understanding of the evolution and diversification of visual systems in arthropods.


Subject(s)
Gene Expression Regulation , Horseshoe Crabs/genetics , Horseshoe Crabs/metabolism , Opsins/genetics , Animals , Biological Evolution , Genome , Horseshoe Crabs/classification , Ocular Physiological Phenomena/genetics , Transcriptome
2.
Integr Comp Biol ; 56(5): 809-819, 2016 11.
Article in English | MEDLINE | ID: mdl-27444526

ABSTRACT

The eyes and photoreceptors of the American horseshoe crab Limulus polyphemus have been studied since the 1930s, and this work has been critical for understanding basic mechanisms of vision. One of the attractions of Limulus as a preparation for studies of vision is that it has three different types of eyes-a pair of later compound, image-forming eyes and two types of simple eyes, a pair of median ocelli, and three pair of larval eyes. Each eye type is tractable for experimentation. Limulus also has extraocular photoreceptors in its segmental ganglia and tail. The current contribution focuses on photoreceptors in Limulus larval eyes and ocelli and its extraocular photoreceptors with the goal of summarizing what is currently known and not known about their physiology and function and the opsins they express. The Limulus genome encodes a surprisingly large number of opsins (18), and studies of their expression pattern have raised new questions about the role of opsin co-expression, the functions of peropsins expressed outside of eyes, and the physiological relevance of opsins with apparently very low expression levels. Studies of opsin expression in Limulus lead one to wonder whether photoreceptors yet to be discovered might be present throughout its central nervous system.


Subject(s)
Horseshoe Crabs/anatomy & histology , Horseshoe Crabs/physiology , Opsins/metabolism , Animals , Eye/anatomy & histology , Ocular Physiological Phenomena , Photoreceptor Cells, Invertebrate/physiology , United States , Vision, Ocular/physiology
3.
Genome Biol Evol ; 8(5): 1571-89, 2016 06 03.
Article in English | MEDLINE | ID: mdl-27189985

ABSTRACT

Horseshoe crabs are xiphosuran chelicerates, the sister group to arachnids. As such, they are important for understanding the most recent common ancestor of Euchelicerata and the evolution and diversification of Arthropoda. Limulus polyphemus is the most investigated of the four extant species of horseshoe crabs, and the structure and function of its visual system have long been a major focus of studies critical for understanding the evolution of visual systems in arthropods. Likewise, studies of genes encoding Limulus opsins, the protein component of the visual pigments, are critical for understanding opsin evolution and diversification among chelicerates, where knowledge of opsins is limited, and more broadly among arthropods. In the present study, we sequenced and assembled a high quality nuclear genomic sequence of L. polyphemus and used these data to annotate the full repertoire of Limulus opsins. We conducted a detailed phylogenetic analysis of Limulus opsins, including using gene structure and synteny information to identify relationships among different opsin classes. We used our phylogeny to identify significant genomic events that shaped opsin evolution and therefore the visual system of Limulus We also describe the tissue expression patterns of the 18 opsins identified and show that transcripts encoding a number, including a peropsin, are present throughout the central nervous system. In addition to significantly extending our understanding of photosensitivity in Limulus and providing critical insight into the genomic evolution of horseshoe crab opsins, this work provides a valuable genomic resource for addressing myriad questions related to xiphosuran physiology and arthropod evolution.


Subject(s)
Evolution, Molecular , Horseshoe Crabs/genetics , Opsins/genetics , Phylogeny , Amino Acid Sequence , Animals , Eye/metabolism , Genome , Multigene Family/genetics , Opsins/classification
4.
J Exp Biol ; 218(Pt 3): 466-79, 2015 Feb 01.
Article in English | MEDLINE | ID: mdl-25524988

ABSTRACT

The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) that are expressed in Limulus lateral compound and larval eyes. Physiological studies showed that visible light-sensitive photoreceptors are also present in median ocelli, but the visible light-sensitive opsins they express were unknown. In the current study we characterize three newly identified, visible light-sensitive rhabdomeric opsins (LpOps6, -7 and -8) that are expressed in median ocelli. We show that they are ocellar specific and that all three are co-expressed in photoreceptors distinct from those expressing LpUVOps1. Our current findings show that the pattern of opsin expression in Limulus eyes is much more complex than previously thought and extend our previous observations of opsin co-expression in visible light-sensitive Limulus photoreceptors. We also characterize a Limulus peropsin/RGR (LpPerOps1). We examine the phylogenetic relationship of LpPerOps1 with other peropsins and RGRs, demonstrate that LpPerOps1 transcripts are expressed in each of the three types of Limulus eyes and show that the encoded protein is expressed in membranes of cells closely associated with photoreceptors in each eye type. These finding suggest that peropsin was in the opsin repertoire of euchelicerates.


Subject(s)
Compound Eye, Arthropod/metabolism , Horseshoe Crabs/metabolism , Light , Opsins/metabolism , Photoreceptor Cells, Invertebrate/metabolism , Animals , Cell Membrane/metabolism , Horseshoe Crabs/radiation effects , Phylogeny
5.
J Exp Biol ; 217(Pt 17): 3133-45, 2014 Sep 01.
Article in English | MEDLINE | ID: mdl-24948643

ABSTRACT

The eyes of the horseshoe crab, Limulus polyphemus, are a model for studies of visual function and the visual systems of euarthropods. Much is known about the structure and function of L. polyphemus photoreceptors, much less about their photopigments. Three visible-light-sensitive L. polyphemus opsins were characterized previously (LpOps1, 2 and 5). Here we characterize a UV opsin (LpUVOps1) that is expressed in all three types of L. polyphemus eyes. It is expressed in most photoreceptors in median ocelli, the only L. polyphemus eyes in which UV sensitivity was previously detected, and in the dendrite of eccentric cells in lateral compound eyes. Therefore, eccentric cells, previously thought to be non-photosensitive second-order neurons, may actually be UV-sensitive photoreceptors. LpUVOps1 is also expressed in small photoreceptors in L. polyphemus ventral larval eyes, and intracellular recordings from these photoreceptors confirm that LpUVOps1 is an active, UV-sensitive photopigment. These photoreceptors also express LpOps5, which we demonstrate is an active, long-wavelength-sensitive photopigment. Thus small photoreceptors in ventral larval eyes, and probably those of the other larval eyes, have dual sensitivity to UV and visible light. Interestingly, the spectral tuning of small ventral photoreceptors may change day to night, because the level of LpOps5 in their rhabdoms is lower during the day than during the night, whereas LpUVOps1 levels show no diurnal change. These and previous findings show that opsin co-expression and the differential regulation of co-expressed opsins in rhabdoms is a common feature of L. polyphemus photoreceptors.


Subject(s)
Horseshoe Crabs/chemistry , Horseshoe Crabs/radiation effects , Opsins/chemistry , Photoreceptor Cells, Invertebrate/chemistry , Photoreceptor Cells, Invertebrate/physiology , Ultraviolet Rays , Amino Acid Sequence , Animals , Compound Eye, Arthropod/chemistry , Compound Eye, Arthropod/physiology , Eye/metabolism , Gene Expression Regulation/radiation effects , Light , Opsins/metabolism , Vision, Ocular/physiology
6.
J Exp Biol ; 216(Pt 10): 1837-49, 2013 May 15.
Article in English | MEDLINE | ID: mdl-23393287

ABSTRACT

Dark and light adaptation in photoreceptors involve multiple processes including those that change protein concentrations at photosensitive membranes. Light- and dark-adaptive changes in protein levels at rhabdoms have been described in detail in white-eyed Drosophila maintained under artificial light. Here we tested whether protein levels at rhabdoms change significantly in the highly pigmented lateral eyes of wild-caught Limulus polyphemus maintained in natural diurnal illumination and whether these changes are under circadian control. We found that rhabdomeral levels of opsins (Ops1-2), the G protein activated by rhodopsin (G(q)α) and arrestin change significantly from day to night and that nighttime levels of each protein at rhabdoms are significantly influenced by signals from the animal's central circadian clock. Clock input at night increases Ops1-2 and G(q)α and decreases arrestin levels at rhabdoms. Clock input is also required for a rapid decrease in rhabdomeral Ops1-2 beginning at sunrise. We found further that dark adaptation during the day and the night are not equivalent. During daytime dark adaptation, when clock input is silent, the increase of Ops1-2 at rhabdoms is small and G(q)α levels do not increase. However, increases in Ops1-2 and G(q)α at rhabdoms are enhanced during daytime dark adaptation by treatments that elevate cAMP in photoreceptors, suggesting that the clock influences dark-adaptive increases in Ops1-2 and G(q)α at Limulus rhabdoms by activating cAMP-dependent processes. The circadian regulation of Ops1-2 and G(q)α levels at rhabdoms probably has a dual role: to increase retinal sensitivity at night and to protect photoreceptors from light damage during the day.


Subject(s)
Arrestin/metabolism , Circadian Rhythm/radiation effects , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Horseshoe Crabs/metabolism , Horseshoe Crabs/radiation effects , Light , Opsins/metabolism , Actins/metabolism , Animals , Circadian Clocks , Colforsin/pharmacology , Compound Eye, Arthropod/cytology , Compound Eye, Arthropod/drug effects , Compound Eye, Arthropod/metabolism , Compound Eye, Arthropod/radiation effects , Cyclic AMP/metabolism , Dark Adaptation/drug effects , Dark Adaptation/radiation effects , Darkness , Octopamine/pharmacology , Retina/cytology , Retina/drug effects , Retina/metabolism , Retina/radiation effects , Rhodopsin/metabolism
7.
J Neurochem ; 119(4): 772-84, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21895655

ABSTRACT

As class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation.


Subject(s)
Myosin Type III/chemistry , Myosin Type III/metabolism , Protein Kinases/metabolism , Amino Acid Sequence , Amino Acids , Animals , Humans , Mass Spectrometry , Mice , Myosin Type III/classification , Myosin Type III/genetics , Peptides/metabolism , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis, Protein , Substrate Specificity
8.
Exp Eye Res ; 89(2): 224-37, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19332056

ABSTRACT

Class III myosins are important for the function and survival of photoreceptors and ciliary hair cells. Although vertebrates possess two class III myosin genes, myo3A and myo3B, recent studies have focused on Myo3A because mutations in the human gene are implicated in progressive hearing loss. Myo3B may compensate for defects in Myo3A, yet little is known about its distribution and function. This study focuses on Myo3B expression in the mouse retina. We cloned two variants of myo3B from mouse retina and determined that they are expressed early in retinal development. In this study we show for the first time in a mammal that both Myo3B and Myo3A proteins are present in inner segments of all photoreceptors. Myo3B is also present in outer segments of S opsin-immunoreactive cones but not M opsin dominant cones. Myo3B is also detected in rare cells of the inner nuclear layer and some ganglion cells. Myo3B may have diverse roles in retinal neurons. In photoreceptor inner segments Myo3B is positioned appropriately to prevent photoreceptor loss of function caused by Myo3A defects.


Subject(s)
Eye Proteins/metabolism , Myosin Heavy Chains/metabolism , Myosin Type III/metabolism , Retina/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Eye Proteins/genetics , Eye Proteins/immunology , Immune Sera , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Myosin Heavy Chains/genetics , Myosin Heavy Chains/immunology , Myosin Type III/genetics , Myosin Type III/immunology , Retina/growth & development , Retinal Cone Photoreceptor Cells/metabolism , Retinal Ganglion Cells/metabolism , Retinal Photoreceptor Cell Outer Segment/metabolism , Tissue Distribution
9.
Dev Dyn ; 237(8): 2209-19, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18651657

ABSTRACT

Pax6 regulates eye development in many animals. In addition, Pax6 activates atonal transcription factors in both invertebrate and vertebrate eyes. Here, we investigate the roles of Pax6 and atonal during embryonic development of Limulus polyphemus rudimentary lateral, medial and ventral eyes, and the initiation of lateral ommatidial eye and medial ocelli formation. Limulus eye development is of particular interest because these animals hold a unique position in arthropod phylogeny and possess multiple eye types. Furthermore, the molecular underpinnings of eye development have yet to be investigated in chelicerates. We characterized a Limulus Pax6 gene, with multiple splice products and predicted protein isoforms, and one atonal homologue. Unexpectedly, neither gene is expressed in the developing eye types examined, although both genes are present in the lateral sense organ, a structure of unknown function.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Eye Proteins/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Horseshoe Crabs/genetics , Nerve Tissue Proteins/genetics , Paired Box Transcription Factors/genetics , Repressor Proteins/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Eye/embryology , Eye Proteins/chemistry , Eye Proteins/metabolism , Homeodomain Proteins/chemistry , Homeodomain Proteins/metabolism , Horseshoe Crabs/embryology , Immunohistochemistry , In Situ Hybridization , Molecular Sequence Data , Myosin Type III/genetics , PAX6 Transcription Factor , Paired Box Transcription Factors/chemistry , Paired Box Transcription Factors/metabolism , Phylogeny , Protein Structure, Tertiary , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Sequence Homology, Amino Acid
10.
Biochemistry ; 46(48): 13907-19, 2007 Dec 04.
Article in English | MEDLINE | ID: mdl-17990896

ABSTRACT

Class III unconventional myosins are critical for the normal function of auditory hair cells and the function and maintenance of photoreceptors; however, the roles of class III myosins in these sensory cells are unknown. Class III myosins are unique in that they have a kinase domain at their N-terminus; thus, they may have both signaling and motor functions. In the horseshoe crab Limulus polyphemus, enhanced phosphorylation of an abundant, photoreceptor specific class III myosin at night correlates with well-characterized circadian changes in photoreceptor structure and function. Thus, the Limulus visual system may be particularly useful for investigating the properties, modulation, and functions of a class III myosin. Previously, we showed that two sites within the actin interface of full-length Limulus myosin III expressed in baculovirus are substrates for both cyclic AMP-dependent protein kinase and autophosphorylation. In the current study, mass spectrometry was used to show that these same sites are phosphorylated in the endogenous protein extracted from Limulus lateral eye, and that enhanced phosphorylation at these sites occurs in vivo in response to natural circadian clock input to these eyes. These findings demonstrate in vivo changes in myosin III phosphorylation in response to a natural stimulus. This phosphorylation may modulate myosin III-actin interactions.


Subject(s)
Actins/metabolism , Biological Clocks , Circadian Rhythm , Myosin Type III/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Binding Sites , Chromatography, Liquid , Horseshoe Crabs , Molecular Sequence Data , Phosphorylation , Tandem Mass Spectrometry
11.
Biochemistry ; 46(14): 4280-93, 2007 Apr 10.
Article in English | MEDLINE | ID: mdl-17367164

ABSTRACT

Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. This enhanced nighttime phosphorylation occurs in response to signals from an endogenous circadian clock and correlates with dramatic changes in photoreceptor structure and function. We seek to understand the role of Limulus myosin III and its phosphorylation in photoreceptors. Here we determined the sites that become phosphorylated in Limulus myosin III and investigated its kinase, actin binding, and myosin ATPase activities. We show that Limulus myosin III exhibits kinase activity and that a major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, an important actin binding region. We also identify the phosphorylation of an additional protein kinase A and autophosphorylation site near loop 2, and a predicted phosphorylation site within loop 2. We show that the kinase domain of Limulus myosin III shares some pharmacological properties with protein kinase A, and that it is a potential opsin kinase. Finally, we demonstrate that Limulus myosin III binds actin but lacks ATPase activity. We conclude that Limulus myosin III is an actin-binding and signaling protein and speculate that interactions between actin and Limulus myosin III are regulated by both second messenger mediated phosphorylation and autophosphorylation of its myosin domain within and near loop 2.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Horseshoe Crabs/metabolism , Myosin Type III/chemistry , Myosin Type III/metabolism , Actins/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Chromatography, Liquid , Escherichia coli/genetics , Genetic Vectors , Kinetics , Molecular Sequence Data , Mutation , Myosin Type III/genetics , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spodoptera/cytology , Spodoptera/metabolism , Tandem Mass Spectrometry
12.
Vis Neurosci ; 21(5): 715-24, 2004.
Article in English | MEDLINE | ID: mdl-15683559

ABSTRACT

The visual arrestins in rhabdomeral photoreceptors are multifunctional phosphoproteins. They are rapidly phosphorylated in response to light, but the functional relevance of this phosphorylation is not yet fully understood. The phosphorylation of Limulus visual arrestin is particularly complex in that it becomes phosphorylated on three sites, and one or more of these site are phosphorylated even in the dark. The purpose of this study was to examine in detail the light-stimulated phosphorylation of each of the three sites in Limulus visual arrestin in intact photoreceptors. We found that light increased the phosphorylation of all three sites (S377, S381, and S396), that S381 is a preferred phosphorylation site, and that S377 and S381 are highly phosphorylated in the dark. The major effect of light was to increase the phosphorylation of S396, the site located closest to the C-terminal and very close to the adaptin binding motif. We speculate that the phosphorylation of this site may be particularly important for regulating the light-driven endocytosis of rhabdomeral membrane.


Subject(s)
Arrestin/metabolism , Horseshoe Crabs , Photic Stimulation , Photoreceptor Cells, Invertebrate/metabolism , Photoreceptor Cells, Invertebrate/radiation effects , Animals , Dark Adaptation , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Endocytosis/physiology , Light , Peptide Fragments/metabolism , Peptide Mapping , Phosphorylation , Receptors, G-Protein-Coupled/metabolism
13.
Vis Neurosci ; 20(3): 241-8, 2003.
Article in English | MEDLINE | ID: mdl-14570246

ABSTRACT

Limulus photoreceptors utilize the phosphoinositide pathway to generate light-induced single photon events (quantum bumps) that sum to form the depolarizing receptor potential. The protein kinase C (PKC) activator, (-)-indolactam V (ILV) rapidly desensitizes the light response in Limulus ventral nerve photoreceptors. Within 10 min of extracellular application, 100 nM (-)-ILV caused a decrease in the mean amplitude of quantum bumps to 38% of control values. PKC activation by (-)-ILV also causes photosensitive membrane disorganization and endocytosis. To investigate whether this precedes desensitization of the electrical response, we fixed cells after 10-min incubation with 25 microM (-)-ILV, a concentration sufficient to cause a 1000-fold desensitization of the receptor potential. The photosensitive microvilli of these photoreceptors remained narrow, densely packed, and well organized. Increasing the incubation time to 60 min did, however, induce disorganization and swelling of the microvilli and endocytosis of the photosensitive membrane, as previously reported. Measurement of membrane capacitance did not indicate a significant reduction in membrane area accompanying desensitization by (-)-ILV. PKC-induced reduction in light sensitivity therefore precedes the detection of ultrastructural changes in the rhabdomeral membrane and is not due to a net loss of membrane.


Subject(s)
Endocytosis/physiology , Horseshoe Crabs/physiology , Light , Photoreceptor Cells/drug effects , Photoreceptor Cells/radiation effects , Protein Kinase C/physiology , Retina/physiology , Animals , Enzyme Activation/physiology , Fluorescent Antibody Technique , Indoles/pharmacology , Lactams/pharmacology , Microscopy, Electron , Microvilli/ultrastructure , Photoreceptor Cells/ultrastructure , Protein Kinase C/metabolism , Retina/cytology , Retina/ultrastructure , Time Factors
14.
Vis Neurosci ; 20(5): 523-34, 2003.
Article in English | MEDLINE | ID: mdl-14977331

ABSTRACT

Much has been learned from studies of Limulus photoreceptors about the role of the circadian clock and light in the removal of photosensitive membrane. However, little is known in this animal about mechanisms regulating photosensitive membrane renewal, including the synthesis of proteins in, and associated with, the photosensitive membrane. To begin to understand renewal, this study examines diurnal changes in the levels of mRNAs encoding opsin, the integral membrane protein component of visual pigment, and the relative roles of light and the circadian clock in producing these changes. We show that at least two distinct opsin genes encoding very similar proteins are expressed in both the lateral and ventral eyes, and that during the day and night in the lateral eye, the average level of mRNA encoding opsinl is consistently higher than that encoding opsin2. Northern blot assays showed further that total opsin mRNA in the lateral eyes of animals maintained under natural illumination increases during the afternoon (9 & 12 h after sunrise) in the light and falls at night in the dark. This diurnal change occurs whether or not the eyes receive input from the circadian clock, but it is eliminated in eyes maintained in the dark. Thus, it is regulated by light and darkness, not by the circadian clock, with light stimulating an increase in opsin mRNA levels. The rise in opsin mRNA levels observed under natural illumination was seasonal; it occurred during the summer but not the spring and fall. However, a significant increase in opsin mRNA levels could be achieved in the fall by exposing lateral eyes to 3 h of natural illumination followed by 9 h of artificial light. The diurnal regulation of opsin mRNA levels contrasts sharply with the circadian regulation of visual arrestin mRNA levels (Battelle et al., 2000). Thus, in Limulus, distinctly different mechanisms regulate the levels of mRNA encoding two proteins critical for the photoresponse.


Subject(s)
Circadian Rhythm/physiology , Gene Expression Regulation/physiology , Rod Opsins/metabolism , Animals , Autoradiography , Blotting, Northern/methods , Blotting, Southern , Denervation/methods , Eye/cytology , Eye/metabolism , Functional Laterality , Horseshoe Crabs , Molecular Sequence Data , Photoreceptor Cells, Invertebrate/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Rod Opsins/genetics , Time Factors
15.
Microsc Res Tech ; 58(4): 345-55, 2002 Aug 15.
Article in English | MEDLINE | ID: mdl-12214301

ABSTRACT

Much is known about the anatomy of Limulus retinal efferent neurons and the structural and functional consequences of their activation. Retinal efferent axons arise from cell bodies located in the cheliceral ganglia of the brain, and they project out all of the optic nerves. Their unique neurosecretory-like terminals contact all cell types in lateral eye ommatidia, the retinular cells of the median eye, and the internal rhabdom of ventral photoreceptors. Lateral and median rudimentary photoreceptors are also innervated. The activity of the efferents is circadian. They are active during the subjective night and inactive during the subjective day. Activation of the efferents drives dramatic and diverse changes in the structure and function of Limulus eyes and causes the sensitivity and responsiveness of the eyes to light to increase at night. Relatively little is known about the molecular mechanisms that produce these structural and functional changes, but one efferent-activated biochemical cascade has been identified. The biogenic amine octopamine is released from efferent terminals, and an octopamine-stimulated rise in cAMP in photoreceptors, with a subsequent activation of cAMP-dependent protein kinase, mediates many of the known effects of efferent input. A photoreceptor-specific protein, myosin III, is phosphorylated in response to efferent input; this protein may play a role in the efferent stimulated changes in photoreceptor structure and function. Anatomical, biophysical, biochemical, and molecular approaches are now being effectively combined in studies of Limulus eyes; thus, this preparation should be particularly useful for further detailed investigations of mechanisms underlying the modulation of primary sensory cells by efferent input.


Subject(s)
Circadian Rhythm/physiology , Eye , Horseshoe Crabs/physiology , Neurons, Efferent/physiology , Ocular Physiological Phenomena , Animals , Eye/anatomy & histology , Eye/innervation , Eye Proteins/genetics , Eye Proteins/metabolism , Horseshoe Crabs/anatomy & histology , Light Signal Transduction/physiology , Photoreceptor Cells, Invertebrate/metabolism
16.
Article in English | MEDLINE | ID: mdl-12160879

ABSTRACT

There is now strong evidence that arthropod photoreceptors use histamine as a neurotransmitter. The synthesis, storage and release of histamine from arthropod photoreceptors have been demonstrated, and the postsynaptic effects of histamine and the endogenous neurotransmitter are similar. However, a full understanding of these photoreceptor synapses also requires knowledge of histamine inactivation and metabolism. Relatively little is known about histamine metabolism in the nervous system of arthropods, and mechanisms appear to differ with the species. This study focuses on histamine metabolism in visual tissues of the horseshoe crab Limulus polyphemus, a chelicerate. We present two major findings: (1) histamine is metabolized to imidazole acetic acid and to gamma-glutamyl histamine. (2) relatively low levels of histamine metabolites accumulate in Limulus visual tissues.


Subject(s)
Histamine/metabolism , Horseshoe Crabs/metabolism , Photoreceptor Cells, Invertebrate/metabolism , Animals , Chromatography, Thin Layer
17.
J Comp Neurol ; 449(1): 26-42, 2002 Jul 15.
Article in English | MEDLINE | ID: mdl-12115691

ABSTRACT

Rhabdom shedding in horseshoe crab lateral eye photoreceptors was studied with anti-opsin and anti-arrestin immunocytochemistry. Two, possibly three, distinct shedding mechanisms were revealed in animals maintained in natural lighting. Transient rhabdom shedding, triggered by dawn, is a brief, synchronous event that removes up to 10% of the rhabdom membrane. Whorls of rhabdomeral membrane break into vesicles and form compact multivesicular bodies. These debris particles are immunoreactive for opsin and are of a relatively uniform size, averaging approximately 2 microm(2) in area. Transient shedding requires that input from circadian efferent fibers to the retina precedes the light trigger, and cutting the optic nerve blocks efferent input and transient shedding. Light-driven rhabdom shedding is a progressive process. Rhabdomeral membrane is removed by coated vesicles that accumulate into loosely packed multivesicular bodies. These debris particles label with antibodies directed against opsin, arrestin, and adaptin, and they have a large distribution of sizes, averaging almost 6 microm(2) in area and ranging up to 25 microm(2) or more. The amount of rhabdomeral membrane removed by light-driven shedding has seasonal variation and depends on latitude. Light-driven shedding does not require circadian efferent input. A possible third shedding mechanism, light-independent shedding, is observed when transient shedding is blocked either by 48 hours of darkness or by cutting the optic nerve. Small particles, averaging 1.8 microm(2) in area, exhibiting opsin but not arrestin immunoreactivity can then be found in the cytoplasm surrounding the rhabdom. The nature of light-independent shedding is not yet clear.


Subject(s)
Horseshoe Crabs/physiology , Photoreceptor Cells, Invertebrate/physiology , Amino Acid Sequence , Animals , Arrestin/analysis , Arrestin/biosynthesis , Darkness , Eye/chemistry , Eye/ultrastructure , Female , Horseshoe Crabs/chemistry , Horseshoe Crabs/ultrastructure , Light , Male , Molecular Sequence Data , Photoreceptor Cells, Invertebrate/chemistry , Photoreceptor Cells, Invertebrate/ultrastructure , Rod Opsins/analysis , Rod Opsins/biosynthesis
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