Ribbon Complex Research Articles

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses.

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior–Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.

ArfGAP3 Is a Component of the Photoreceptor Synaptic Ribbon Complex and Forms an NAD(H)-Regulated, Redox-Sensitive Complex with RIBEYE That Is Important for Endocytosis

Ribbon synapses are tonically active synapses in the retina and inner ear with intense vesicle traffic. How this traffic is organized and regulated is still unknown. Synaptic ribbons, large presynaptic structures associated with numerous synaptic vesicles, appear to be essential for this process. The base of the synaptic ribbon is anchored at the active zone and is a hotspot of exocytosis. The synaptic ribbon complex is also important for vesicle replenishment. RIBEYE is a unique and major component of synaptic ribbons. It consists of a unique A-domain and an NAD(H)-binding, C-terminal B-domain. In the present study, we show that the Arf-GTPase activating protein-3 (ArfGAP3), a well characterized regulator of vesicle formation at the Golgi apparatus, is also a component of the synaptic ribbon complex in photoreceptor synapses of the mouse retina and interacts with RIBEYE as shown by multiple, independent approaches. ArfGAP3 binds to RIBEYE(B)-domain in an NAD(H)-dependent manner. The interaction is redox sensitive because NADH is more efficient than the oxidized NAD(+) in promoting ArfGAP3-RIBEYE interaction. RIBEYE competes with the GTP-binding protein Arf1 for binding to ArfGAP3. Thus, binding of RIBEYE(B) to ArfGAP3 could prevent inactivation of Arf1 by ArfGAP3 and provides the synaptic ribbon with the possibility to control Arf1 function. The interaction is relevant for endocytic vesicle trafficking because overexpression of ArfGAP3 in photoreceptors strongly inhibited endocytotic uptake of FM1-43.

A Local, Periactive Zone Endocytic Machinery at Photoreceptor Synapses in Close Vicinity to Synaptic Ribbons

Photoreceptor ribbon synapses are continuously active synapses with large active zones that contain synaptic ribbons. Synaptic ribbons are anchored to the active zones and are associated with large numbers of synaptic vesicles. The base of the ribbon that is located close to L-type voltage-gated Ca(2+) channels is a hotspot of exocytosis. The continuous exocytosis at the ribbon synapse needs to be balanced by compensatory endocytosis. Recent analyses indicated that vesicle recycling at the synaptic ribbon is also an important determinant of synaptic signaling at the photoreceptor synapse. To get insights into mechanisms of vesicle recycling at the photoreceptor ribbon synapse, we performed super-resolution structured illumination microscopy and immunogold electron microscopy to localize major components of the endocytotic membrane retrieval machinery in the photoreceptor synapse of the mouse retina. We found dynamin, syndapin, amphiphysin, and calcineurin, a regulator of activity-dependent endocytosis, to be highly enriched around the active zone and the synaptic ribbon. We present evidence for two clathrin heavy chain variants in the photoreceptor terminal; one is enriched around the synaptic ribbon, whereas the other is localized in the entry region of the terminal. The focal enrichment of endocytic proteins around the synaptic ribbon is consistent with a focal uptake of endocytic markers at that site. This endocytic activity functionally depends on dynamin. These data propose that the presynaptic periactive zone surrounding the synaptic ribbon complex is a hotspot of endocytosis in photoreceptor ribbon synapses.

Targeted Expression of Anoctamin Calcium-Activated Chloride Channels in Rod Photoreceptor Terminals of the Rodent Retina

In the vertebrate retina, calcium-activated chloride channels are expressed in photoreceptor synaptic terminals. These channels are involved in the control of transmitter release in the dark. The search for their molecular identity has recently lead to the localization of the protein anoctamin 2 (also TMEM16B) in the outer plexiform layer of the rodent retina. Since both rod and cone photoreceptors have their terminals in this layer, it was not clear which of these express anoctamin 2. Here, we examine rod spherules and cone pedicles for expression of anoctamin 2. Expression of anoctamin genes was studied in the rat eye using RT-PCR. Immunohistochemical experiments were used to localize anoctamins and chloride transporters with their regulatory kinases. Photoreceptor synaptic proteins, as well as the lectins Peanut agglutinin and Griffonia simplicifolia agglutinin, were used to distinguish retinal structures. Anoctamin 1, 2, and 10 were found to be expressed in the eye. Anoctamin 2 was expressed as a splice variant that includes exon 15 of the genomic structure. The protein is exclusively expressed in rod terminals and is not present in cone pedicles. Expression is not clustered at the ribbon complex, but spread across the presynaptic membrane where it colocalizes with the plasma membrane calcium pump. The electroneutral chloride transporter NKCC1 is expressed in photoreceptor terminals, together with its regulatory kinases SPAK and OSR1. Rod photoreceptor terminals possess the molecular machinery for chloride accumulation and for the generation of calcium-dependent chloride currents conducted through anoctamin 2 channels. We discuss this finding in the framework of the established hypothesis that calcium-activated chloride channels are part of a feedback inhibition mechanism that limits transmitter release in the dark.

Protein Composition of Immunoprecipitated Synaptic Ribbons

The synaptic ribbon is an electron-dense structure found in hair cells and photoreceptors. The ribbon is surrounded by neurotransmitter-filled vesicles and considered to play a role in vesicle release. We generated an objective, quantitative analysis of the protein composition of the ribbon complex using a mass spectrometry-based proteomics analysis. Our use of affinity-purified ribbons and control IgG immunoprecipitations ensure that the identified proteins are indeed associated with the ribbon complex. The use of mouse tissue, where the proteome is complete, generated a comprehensive analysis of the candidates. We identified 30 proteins (comprising 56 isoforms and subunits) associated with the ribbon complex. The ribbon complex primarily comprises proteins found in conventional synapses, which we categorized into 6 functional groups: vesicle handling (38.5%), scaffold (7.3%), cytoskeletal molecules (20.6%), phosphorylation enzymes (10.6%), molecular chaperones (8.2%), and transmembrane proteins from the presynaptic membrane firmly attached to the ribbon (11.3%). The 3 CtBP isoforms represent the major protein in the ribbon whether calculated by molar amount (30%) or by mass (20%). The relatively high quantity of phosphorylation enzymes suggests a very active and regulated structure. The ribbon appears to comprise a concentrated cluster of proteins dealing with vesicle creation, retention and distribution, and consequent exocytosis.

Synaptic Ribbons: Machines for Priming Vesicle Release?

Synaptic ribbons are specialized organelles that hold vesicles close to the active zone of sensory synapses, but their function is mysterious. Acute disruption of the ribbon complex using light has now revealed that it has a role in priming synaptic vesicles for fusion.

Characterization of the cystine/glutamate transporter in the outer plexiform layer of the vertebrate retina

The cystine/glutamate transporter (Xc-) is widely expressed in the central nervous system and is thought to play a role in glutamatergic neurotransmission by releasing low levels of glutamate. Although previous studies have localized the transporter throughout the retina, we now present results from localization and functional studies within the first synaptic layer, i.e. the outer plexiform layer. Using light and electron microscopy, we have localized the Xc- transporter to the ribbon complex of both rod and cone photoreceptors in the rat, cow, lamprey, chicken and monkey retina, suggesting that the pre-synaptic expression of the Xc- transporter on the photoreceptor ribbon complex is phylogenetically preserved. The Xc- transporter does not appear to be located within the ribbon synapse of the inner plexiform layer. Developmentally, the evolution of distinct ribbon-shaped Xc- labelling in the outer plexiform layer parallels the known morphological and electrophysiological maturation of photoreceptors. Using the cation channel probe agmatine, we tracked cation fluxes within bipolar cells and therefore indirectly determined the modulation of glutamate release from photoreceptors. Such cystine-driven alteration in agmatine entry into bipolar cells can be modified by a specific metabotropic glutamate receptor 6 antagonist [(RS)-alpha-cyclopropyl-4-phosphonophenylglycine] and an Xc- transport inhibitor [(S)-4-carboxyphenylglycine]. The phylogenetic preservation of the transporter, its ultrastructural localization to the ribbon synapse and functional modulation of post-receptoral neurons collectively support a role for the Xc- transporter in glutamate neurotransmission in the outer retina of vertebrates. We have therefore proposed a model of glutamate release in the photoreceptor synapse that incorporates the Xc- transporter, which complements the established vesicular-mediated glutamate release.

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13–1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13–1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation.

Molecular dissection of the photoreceptor ribbon synapse

The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel α1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

Sewing Ribbons on Graphs in Space

An open ribbon is a square with one side called the seam. A closed ribbon is a cylinder with one boundary component called the seam. We sew an open (resp. closed) ribbon onto a graph by identifying the seam with an open (resp. closed) walk in the graph. A ribbon complex is a graph with a finite number of ribbons sewn on. We investigate when a ribbon complex embeds in 3-dimensional Euclidean space. We give several characterizations of such spatial complexes which lead to algorithms. We examine special cases where (1) each edge of the graph is incident with at most three ribbons, and (2) every ribbon is closed together with a connectivity condition.

An elementary information processing component in the circuitry of the retina generating the on-responses

A pattern of neural connections that is a compulsory feature of photoreceptor terminals and is referred to as the synaptic ribbon complex was analyzed, and by combination of the structural information and information gained by intracellular recordings from photoreceptors, horizontal cells, and bipolar cells, it is possible to explain how the hyperpolarizing effect of light stimulating the photoreceptors is changed to a depolarization of depolarizing bipolar cells. The sign reversal is accomplished by the hyperpolarizing action of the horizontal cells on the photoreceptors, which blocks the transmission between the photoreceptors and the bipolar cells. This blocking action is controlled by the photoreceptor and it functions like a gate that is opened only when the photoreceptor is stimulated by light. The synaptic ribbon complex offers an example of an elementary information processing component with three input channels to the bipolar cells with each channel contributing a different piece of information and with the processing occurring presynaptically. Additional processing of information occurs within the dendritic tree through interactions of the responses of the individual dendritic endings to different types of input. This interaction can involve partial blocking of the conduction within the dendritic tree, making the interaction considerably more complex than simple summation. The responses recorded intracellularly from neurons reveal only the end result of the processing of information at the level of that neuron.

Ver�nderungen in der �u�eren plexiformen Schicht der Kaninchenretina nach langer Gabe von Vincristin

Rabbits which had been intravenously injected with Vincristine for six weeks showed changes in the outer plexiform layer of the retina after one year, which suggests the destruction of receptor cells, bipolar ganglion cells, and horizontal cells. Compensated cell damage accompanied by loss of microtubuli in the cytoplasma, autolytic vacuoles, granular material deposits, and membrane changes of the ribbon complex were frequently observed. A comparison between axons and dendrites showed a predominance of damaged dendritis over axons. Because short-term studies have not produced the same results, different regeneration capacities, which might have their origin in the endoplasmatic reticulum, are discussed.

Circuitry analysis of the outer plexiform layer in the rabbit retina: I. Extension of analysis of circuits interpreted to be associated with directional sensitivity and edge detection

The earlier analysis of the circuitry of the outer plexiform layer in the rabbit retina [ Sjostrand, 1969 , Sjostrand, 1974 , Sjostrand, 1976 , Sjostrand, 1976 ] has been extended with respect to the circuitry that was proposed to be associated with directional sensitivity and edge detection. This circuitry involved one bipolar cell, here referred to as bipolar cell 1, which was connected to horizontal cells according to a characteristic pattern that appeared suitable for a detector for directional sensitivity and for an edge. The same characteristics of the pattern of connections were found repeated at the contacts of this bipolar cell at a second photoreceptor cell of the β type (α and β type cells are usually identified with rod and cone cells, respectively). The similarities involve the presence of collateral branches of bipolar cell 1 that contact horizontal cell processes outside the subsynaptic neuropil, connections in the subsynaptic neuropil which involve both bipolar cell and horizontal cell processes, contacts with efferent bipolar cells in the subsynaptic neuropil, contact relations with horizontal cell processes at synaptic ribbon complexes, and contacts with the photoreceptor cells. Collateral branches are a unique feature of bipolar cell 1 among all bipolar cells contacting these two photoreceptor cells. As in the previous papers, the interpretation of the functional significance of the circuitry was based on distinguishing between two types of horizontal cell-bipolar cell connections. Horizontal cells were thus classified as contributing either processes that showed a structural polarity or processes that were lacking any structural polarity. In five synaptic ribbon complexes analyzed at photoreceptor cell β2, one of the two horizontal cell endings of each complex was contributed by a horizontal cell process with structural polarity, while the other was contributed by a process lacking structural polarity. This is in agreement with the most common arrangement observed in earlier analysis ( Sjostrand, 1974 ). It could also be confirmed that the bipolar cell process to a synaptic ribbon complex was a side branch of the end branch which itself was received in a separate invagination of the plasma membrane of the terminal. There were five horizontal cell processes with structural polarity that contacted or were contacted by bipolar cell 1 in synaptic ribbon complexes, in the subsynaptic neuropil, and outside the subsynaptic neuropil. All these processes showed the same polarity which agreed with the polarity of corresponding horizontal cell processes' contacts at photoreceptor cell β1. The systematic repetition of similar contact relations of bipolar cell 1 at both photoreceptor cells and the fact that the similarities involved a characteristic, nonrandom but rather specific type of contact pattern support the concept that the neurons are connected according to well-defined patterns designed for a particular type of information processing. As a consequence, circuit analysis of this kind together with electrophysiological analysis should form a concrete basis for revealing how information is processed in nervous centers. The subsynaptic neuropil showed an even more complex circuitry at this photoreceptor cell than at photoreceptor cell β1, supporting the earlier conclusion that the subsynaptic neuropil is the site of a major part of retinal circuitry ( Sjostrand, 1974 , Sjostrand, 1976 , Sjostrand, 1976 ). A pairing of contacts of endings of neurons was observed to be a general rule for contacts not only in the synpatic ribbon complexes but also in the subsynaptic neuropil and outside the subsynaptic neuropil. For horizontal cells this pairing involved one horizontal cell process with polarity paired with one lacking polarity like the most common combination at synaptic ribbon complexes. From a functional point of view the horizontal cell processes lacking structural polarity were interpreted to be involved in neural adaptation of the retina while the horizontal cell processes with structural polarity were interpreted to act in an inhibitory way on bipolar cells on the basis of local, one-sided conditions of illumination of the retina. This functional interpretation was the basis for proposing that the observed circuitry could be associated with directional sensitivity and edge detection. These proposals appear to have gained additional support from the fact that the same type of connections were found at both photoreceptor cell β1 and β2.

A search for the circuitry of directional selectivity and neural adaptation through three-dimensional analysis of the outer plexiform layer of the rabbit retina

The circuitry of a part of the outer plexiform layer in the rabbit retina was studied by means of three-dimensional analysis based on electron micrographs of serial sections. 1. A methodology was worked out for such analysis involving large volumes of nervous tissue. Such analysis was evaluated as feasible and technically possible to pursue. 2. The analysis involved the complete circuitry of one β-type photoreceptor cell and was extended to involve all contact relations within the serially sectioned volume of retinal tissue of all processes contacting this photoreceptor cell. 3. The β-type photoreceptor cell was contacted by ten bipolar cell processes and nine horizontal cell processes. Several different contact relations were observed: contacts through endings at synaptic ribbon complexes, through invaginated endings not contacting synaptic ribbon complex components, through endings making superficial contacts without being invaginated. 4. A thorough analysis of the topographic relationships at synaptic ribbon complexes showed a consistent pattern of arrangement of horizontal cell and bipolar cell endings. 5. At the base of the photoreceptor cell terminal, the processes contacting the terminal formed a compact aggregation of branching processes. This structure was referred to as the subsynaptic neuropil and was proposed to play a role in the circuitry of the outer plexiform layer as indicated by the presence of special end branches confined to this region. 6. Bipolar cell endings were observed containing large amounts of synaptic vesicles and other cytoplasmic structures and were proposed to furnish presynaptic endings of efferent bipolar cells. These cells would allow a feedback from the inner plexiform layer to the outer plexiform layer. 7. These bipolar cell endings contacted horizontal cell endings lateral to the synaptic ribbon complexes. 8. An interpretation of the functional significance of the synaptic ribbon complexes was discussed. According to this interpretation, these complexes would form parts of the circuitry for neural adaptation of the retina. 9. Tight junctions and gap junctions were observed between the largest horizontal cell processes. 10. One bipolar cell process of the ten analyzed differed from the others by sending off lateral branches contacting several horizontal cells as well as one bipolar cell of the efferent type. 11. This bipolar cell was associated with a circuit that could possibly furnish the basis for directional selectivity of certain retinal units.