Orientation of acidic polysaccharides and rhodopsin-oligosaccharides in frog retinal rod outer segments.

Using topo-optical staining reactions, the presence and molecular order of three structural components of outer segments of frog retina were studied. These components included (1) an acidic polysaccharide texture, (2) free aldehyde groups which arise during formalin fixation and (3) the oligosaccharide chains of rhodopsin. Quantitative measurements of the dye binding and birefringence effects arising from the individual structural components in rod outer segments were made. Results indicated that all three structural components had a rather well-defined orientation within the ROS. The spherulites phagocytized from the apical ends of ROSs by the pigment epithelium also demonstrate preferred orientation of the three structural components investigated.     

The multifunctional sensory complex in the antennae ofAllacma fusca (Insecta)

Summary The antennal-tip sensory complex inAllacma fusca (Collembola) was reconstructed from serial ultrathin sections. The complex contains 16 sensory cells which belong to three spatially separated subunits: (1) a sensory hair; (2) a cuticular protrusion, containing two highly-specialized dendritic outer segments from a single sensory cell encapsulated by an enveloping cell and a gland cell (Figs. 4 and 5); and (3) two sensory cells with partly lamellated, double dendritic outer segments located well away from the cuticular surface (Fig. 3). A phylogenetic evaluation reveals a mosaic of apparently primitive characters, and trends toward higher structural complexity as well as toward reduction (Table 1). There is support for a hypothesis that the receptors of the sensory hair are chemosensitive. The functional interpretation of the other components of the complex is problematic due to their unusual structural properties. The sensory cell beneath the cuticular protrusion is considered to be proprioceptive.Supported by the Deutsche Forschungsgemeinschaft (Al 56/6)     

Histological techniques for study of photoreceptor orientation

An histological method for the study of photoreceptor orientation in primate eyes is described. To preserve photoreceptor orientation it is necessary to protect the fragile rod and cone outer segments to the maximum extent possible from mechanical deformation and from injury by solvent extraction. To prevent mechanical deformation the eyes are freeze-dried and embedded in plastic with or without prior vapor fixation. Solvent extraction from the lipidrich outer segment is limited by avoidance or restriction of organic solvents. When large segments of primate eyes are so treated, it is possible to section the plastic blocks along the visual axis, polish the block surface, and view photoreceptor orientation by epi-illumination microscopy. In such specimens a differential orientation of photoreceptors exists with the long axis of photoreceptor inner and outer segments in line with incoming light rays.     

Light-induced dephosphorylation of two proteins in frog rod outer segments: influence of cyclic nucleotides and calcium

Two minor proteins of frog rod outer segments become phosphorylated when retinas are incubated in the dark with 32Pi. The proteins, designated component I (13,000 daltons) and component II (12,000 daltons), are dephosphorylated when retinas are illuminated. The dephosphorylation is reversible; the two proteins are rephosphorylated when illumination ceases. Each outer segment contains approximately 10(6( molecules of components I and II. These remain associated with both fragmented and intact outer segments but dissociate from the outer segment membranes under hypoosmotic conditions. The extent of the light-induced dephosphorylation increases with higher intensities of illumination and is maximal with continuous illumination which bleaches 5.0 x 10(5) rhodopsin molecules/outer segment per second. Light which bleaches 5.0 x 10(3) rhodopsin molecules/outer segment per second causes approximately half-maximal dephosphorylation. This same intermediate level of illumination causes half-suppression of the light-sensitive permeability mechanism in isolated outer segments (Brodie and Bownds. 1976. J. Gen Physiol. 68:1-11) and also induces a half-maximal decrease in their cyclic GMP content (Woodruff et al. 1977. J. Gen. Physiol. 69:667-679). The phosphorylation of components I and II is enhanced by the addition of cyclic GMP or cyclic AMP to either retinas or isolated rod outer segments maintained in the dark. Several pharmacological agents which influence cyclic GMP levels in outer segments, including calcium, cause similar effects on the phosphorylation of components I and II and outer segment permeability. Although the cyclic nucleotide-stimulated phosphorylation can be observed either in retinas or isolated rod outer segments, the light-induced dephosphorylation is observed only in intact retinas.     

Correlation between dendrite diameter and action potential amplitude in sex pheromone specific receptor neurons in male Ostrinia nubilalis (Lepidoptera: Pyralidae)

Outer dendritic segments of olfactory receptor neurons tuned to sex pheromone components were measured morphometrically on the antenna of male European corn borers. Ostrinia nubilalis, to determine if a correlation exists between the diameter of the outer dendritic segment and the spike amplitude. The olfactory sensilla investigated each contained three receptor cells. Two cells were each specific for one of the two pheromone components, (Z)-11-tetradecenyl acetate (Z11-14:OAc) and (E)-11-tetradecenyl acetate (E11-14:OAc). Two strains of cornborers (Z and E) differ as to which of the two pheromone components is the main one. In both strains a large difference could be observed between the spike amplitudes elicited in the receptor cells by the two pheromone components, the main component always eliciting the large spike. In F1-hybrids (EZ) of these two strains, producing both pheromone components in similar quantities, the spike amplitudes were equal in the two pheromone-specific receptor cells. The third cell responded specifically to a behavioural antagonist. (Z)-9-tetradecenyl acetate (Z9-14:OAc) in both the parental and hybrid strains, and always showed the smallest spike amplitude. In a morphometric study, the outer dendritic segments were shown to differ more in diameter between the largest and second largest cell in the two parental strains than in the hybrid strain, while the smallest diameter cell did not differ between the different strains. These results imply that receptor cells with larger diameter produce spikes with greater amplitude. The data also show that all three types of receptor neurons display outer dendritic segments with strong variation in the diameter along the length of the segment, and with a pronounced taper towards the tip.     

cGMP-binding centers in photoreceptor membranes

The binding of cGMP by structural components of bovine rod outer segments was studied. The discs and plasma membranes were shown to contain two types of the specific binding sites for cGMP which are distinct from cyclic GMP phosphodiesterase. The sites have a "high" and "low" (Kd = 0.1 divided by 0.35 and 1.5 divided by 2.0 X 10(-6) M respectively) affinity for cGMP. They belong to membraneous integral proteins presumably associated with phospholipids. Their affinity for cGMP is controlled by GTP and calmodulin.     

The fine structure of thermo-/hygrosensitive sensilla in the silkmoth Bombyx mori: Receptor membrane substructure and sensory cell contacts

Summary The thermo-/hygroreceptive sensilla styloconica of the silkmoth Bombyx mori are located on the tips of the antennal branches. A small poreless cuticular peg is innervated by three sensory cells. The outer dendritic segments of two type-1 receptor cells, the presumed hygroreceptors, almost completely fill the peg lumen and are in close contact with each other. The outer dendritic segment of the third (type-2) receptor cell, the presumed thermoreceptor, forms lamellae below the peg base. The membranes of these lamellae are studded with knobs in orthogonal array, protruding into the extracellular space with the same orientation on facing lamellae. This Bossy Orthogonal Surface Substructure (BOSS) is assumed to play a role in thermoreceptor function. Contacts are observed between the outer dendritic segments, between the inner dendritic segments immediately below the ciliary segments, and between the sensory cell somata. These contacts, which are not found in the olfactory sensilla (s. trichodea and basiconica) of this species, indicate electrical interactions between the three sensory cells of the styloconic sensillum and possibly are involved in the antagonistic and/or bimodal response characteristics of thermo-/hygroreceptor cells.     

Membrane topology of transmembrane proteins: determinants and experimental tools

Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.     

Regulation of signal transfer from beta 1-adrenoceptor to adenylate cyclase by beta gamma subunits in a reconstituted system

The beta gamma subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, beta gamma subunits from these sources have been found to be fully interchangeable in terms of their interaction with alpha subunits of pertussis-toxin-sensitive G proteins. In contrast, however, there are striking differences between these beta gamma subunits with regard to their ability to deactivate fluoride-stimulated Gs. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of beta gamma purified from bovine brain to vesicles containing beta-receptor and Gs results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin beta gamma was virtually without any effect. Likewise, beta gamma from bovine brain, but not transducin beta gamma, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, Gs, C). Thus, the alpha subunit of Gs, but not the alpha subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different beta gamma subunits.     

Outer acrosomal membrane of guinea pig spermatozoa: Isolation and structural characterization

We describe a protocol to isolate a highly enriched fraction of outer acrosomal membrane from guinea pig spermatozoa and present new data on the ultrastructure of this membrane domain. Cauda epididymal spermatozoa were suspended into a low ionic strength buffer and subjected to brief homogenization; this stripped the plasma membrane from the spermatozoa and severed the acrosomal apical segment from the spermatozoon. The crescent-shaped apical segments retained the outer acrosomal membrane and specific components of the acrosomal matrix. Enriched fractions of apical segments were isolated on discontinuous sucrose gradients and the outer acrosomal membrane purified by subsequent centrifugation onto Percoll density gradients. The isolated outer acrosomal membrane did not form vesicles, but instead rolled up into spiral sheets. Both thin section and negatively stained specimens revealed a paracrystalline arrangement of filaments associated with the luminal surface of the membrane. The isolated outer acrosomal membrane revealed a limited number of polypeptides by SDS-PAGE, and the polypeptide pattern was distinct from the plasma membrane fraction. The isolated acrosomal membranes possessed no oubain sensitive Na+, K+-ATPase activity, whereas about 20% of the ATPase activity of the plasma membrane enriched fraction was inhibited by oubain. The potential function of the structural differentiations of the outer acrosomal membrane in the membrane fusion events of the acrosome reaction is discussed.     

The pineal organ of Raja clavata: Opsin immunoreactivity and ultrastructure

Summary The pineal organ of Raja clavata was studied by light and electron microscopy, including the immunocytochemical antiopsin reaction. The pineal organ of the ray consists of three portions: (i) a large proximal pineal, (ii) a long tube-like connecting stalk, and (iii) a short distal terminal enlargement. This latter end-vesicle lies in the deep connective tissue layers of the braincase. All portions of the pineal are composed of pinealocytes, intrinsic neurons, ependymal/glial cells, and bundles of nerve fibers embedded in thin neuropil formations. The inner segments of the pinealocytes protrude into the lumen in all parts of the organ and usually contain basal bodies and numerous mitochondria. Often, two outer segments were found to arise from the basal bodies of a single inner segment. By means of light-microscopic immunocytochemistry the outer segments showed a strong antiopsin reaction.The axons of the pinealocytes form ribbon-containing synapses on dendritelike profiles, which appear to belong to the intrinsic pineal neurons. There are other axo-dendritic synapses established by presynaptic terminals lacking ribbons and containing granular and synaptic vesicles. Pineal neurons may contain granular vesicles approximately 60–100 nm in diameter; their processes contribute to the bundles of unmyelinated axons.The fine structural organization of the pineal organ and the opsin immunoreactivity of the outer segments of the pinealocytes indicate a photoreceptive capacity of the organ. The double outer segments represent a peculiar multiplication of the photoreceptor structures.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft to A. Oksche (Ok 1/24; 1/25: Mechanismen biologischer Uhren)On leave from the 2nd Department of Anatomy, Semmelweis OTE, Budapest, Hungary     

Rhodopsin in the rod outer segment plasma membrane

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.     

COMPARATIVE STUDIES ON THE CELL WALLS OF SEXUAL AND ASEXUAL BANGIA ATROPURPUREA (RHODOPHYTA). II ELECTROPHORETIC PATTERNS OF POLYSACCHARIDES1

Cellulose acetate electrophoresis of the hot water soluble polysaccharide extracts from whole filaments, as well as base, mid and tip segments, of marine asexual and sexual Bangia atropurpurea (Roth) C. Ag. Yielded distinctive patterns which demonstrated that changes occur in the outer cell walls during sexual reproduction. Heterogeneity of the sulfated polysaccharide components isolated from outer cell walls was shown to be specifically related to sexual reproduction. Two components (Band I and II) were detected in extracts from tips of sexual filaments, whole only one (Band I) was present in the vegetative segments of all filaments and in asexual reproductive regions. The faster running component (Band II) was detected during the later stages of sexual development, prior to maturation.     

Mode of action of natural growth inhibitors in radish hypocotyl elongation – influence of raphanusanin on auxin-mediated microtubule orientation

Raphanusanin is a plant growth-inhibiting substance which plays an important role in light growth inhibition and phototropism of radish hypocotyls. We investigated the effect of raphanusanin on indole-3-acetic acid (IAA)-mediated orientation of microtubules (MT) in the outer epidermal cells of radish hypocotyl segments using immunofluorescence microscopy. IAA-mediated MT reorientation preceded cell elongation induced by IAA. A change of IAA-mediated MT orientation from longitudinal to transverse started within less than 15 min after IAA treatment, while significant growth promotion induced by IAA was found within about 30 min. The IAA-mediated transverse MT orientations were significantly inhibited by simultaneously added raphanusanin. We also investigated the effect of raphanusanin on the MT orientation of the segments pretreated with IAA. The change of MT orientation induced by raphanusanin preceded growth inhibition of the segments. Within about 60 min after its application, raphanusanin initiated inhibition of the steady-state elongation pre-induced by IAA, while IAA-mediated transverse MT orientations started to change into longitudinal orientations within less than 30 min after application of raphanusanin. Based on these results, it is suggested that raphanusanin induces growth inhibition through interference with the auxin-mediated MT orientations.     

Isolation and characterization of a macromolecular complex associated with the outer acrosomal membrane of bovine spermatozoa

The acrosomal membrane of mammalian spermatozoa is segregated into domains of different structure and function. The outer acrosomal membrane of the apical and principal segments is the only domain to participate in the membrane fusion events of the acrosome reaction, but the molecular basis for this function is not resolved. In previous studies of bovine spermatozoa, we noted that a unique structural feature of the outer acrosomal membrane was an adherent layer of electron-dense material on its luminal surface (ES Surface, Branton et al., 1975). In this study, we report the isolation of this material and we describe both its structural and biochemical characteristics. Cauda epididymal spermatozoa were extracted with 1% Triton X-100 to solubilize cytoplasmic and membrane components; detergent treatment solubilized the outer acrosomal membrane but not its adherent electron-dense complex. Homogenization released this complex from the spermatozoa and it was then resolved into a homogeneous fraction by centrifugation on Percoll density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction revealed a spectrum of polypeptides including components of 290 kDa, 280 kDa, 260 kDa, 115 kDa, 81 kDa, 58 kDa, and 46 kDa and a family of interrelated components in the 34-12 kDa range. This complex possesses protein kinase activity that phosphorylates specific endogeneous polypeptides in a cAMP-independent manner. In addition, several polypeptides of the 34-12 kDa family specifically bind 125I-calmodulin. One consistent structural response of the isolated complex was that its edges wound into a spiral configuration. We speculate that this membrane-associated assembly plays a functional role in the membrane fusion events of the acrosome reaction.     

Light-dependent translocation of arrestin in rod photoreceptors is signaled through a phospholipase C cascade and requires ATP

Partitioning of cellular components is a critical mechanism by which cells can regulate their activity. In rod photoreceptors, light induces a large-scale translocation of arrestin from the inner segments to the outer segments. The purpose of this project is to elucidate the signaling pathway necessary to initiate arrestin translocation to the outer segments and the mechanism for arrestin translocation.Mouse retinal organotypic cultures and eyes from transgenic Xenopus tadpoles expressing a fusion of GFP and rod arrestin were treated with both activators and inhibitors of proteins in the phosphoinositide pathway. Confocal microscopy was used to image the effects of the pharmacological agents on arrestin translocation in rod photoreceptors. Retinas were also depleted of ATP using potassium cyanide to assess the requirement for ATP in arrestin translocation.In this study, we demonstrate that components of the G-protein-linked phospholipase C (PLC) pathway play a role in initiating arrestin translocation. Our results show that arrestin translocation can be stimulated by activators of PLC and protein kinase C (PKC), and by cholera toxin in the absence of light. Arrestin translocation to the outer segments is significantly reduced by inhibitors of PLC and PKC. Importantly, we find that treatment with potassium cyanide inhibits arrestin translocation in response to light.Collectively, our results suggest that arrestin translocation is initiated by a G-protein-coupled cascade through PLC and PKC signaling. Furthermore, our results demonstrate that at least the initiation of arrestin translocation requires energy input.     

The cGMP-gated channel of the rod photoreceptor cell characterization and orientation of the amino terminus.

The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit Mr of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent Mr of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH2-terminal sequence analysis indicates that the lower Mr of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluorescent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co-translational or post-translational cleavage of the NH2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane.     

The retinal rod outer segment of the frog: detachment, isolation, phosphorus fractions and enzyme activity

Frog retinal rod outer segments were detached from dark adapted retinas by (1) agitation in frog Ringer's solution or (2) by crushing between two glass surfaces. The resulting suspensions were further purified by low and high speed centrifugation procedures in Ficoll density gradients. The density of the outer segments in Ficoll solutions was found to be 1.09. The large frog outer segments, unlike bovine outer segments, are not readily separated from nuclei, which were estimated to comprise 2.6–8% of the material, based on DNA analyses. The RNA/DNA ratio was 0.4–0.5, like that of neuronal nuclei. Representative enzymes of glycolysis (lactic dehydrogenase and glyceraldehyde phosphate dehydrogenase), phosphogluconate oxidation (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the citric acid cycle (malic dehydrogenase) and ATP degradation (ATPase) were assayed. A major part of the malic dehydrogenase activity was probably due to inner segments attached to some of the outer segments. Glyceraldehyde phosphate dehydrogenase and glucose-6-phosphate dehydrogenase (but not lactic dehydrogenase) activities were lower in detached outer segments purified on Ficoll gradients than in samples of outer segment layer microdissected from freeze-dried sections of frog retina, as well as in whole retina. The data suggest that the activities of all the enzymes studied are intrinsically low in rod outer segments.     

Purification and properties of guanylate kinase from bovine retinas and rod outer segments

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside-diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3). The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP in equilibrium GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20-23 kDa for both native enzyme and dodecylsulfate-denatured polypeptide), Km values (13 microM for GMP and 430 microM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s-1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.