Discs of mammalian rod photoreceptors form through the membrane evagination mechanism

Photoreceptor discs are membrane organelles harboring components of the visual signal transduction pathway. The mechanism by which discs form remains enigmatic and is the subject of a major controversy. Classical studies suggest that discs are formed as serial plasma membrane evaginations, whereas a recent alternative postulates that discs, at least in mammalian rods, are formed through intracellular vesicular fusion. We evaluated these models in mouse rods using methods that distinguish between the intracellular vesicular structures and plasma membrane folds independently of their appearance in electron micrographs. The first differentiated membranes exposed to the extracellular space from intracellular membranes; the second interrogated the orientation of protein molecules in new discs. Both approaches revealed that new discs are plasma membrane evaginations. We further demonstrated that vesiculation and plasma membrane enclosure at the site of new disc formation are artifacts of tissue fixation. These data indicate that all vertebrate photoreceptors use the evolutionary conserved membrane evagination mechanism to build their discs.     

Current issues in invertebrate phototransduction

Investigation of phototransduction in invertebrate photoreceptors has revealed many physiological and biochemical features of fundamental biological importance. Nonetheless, no complete picture of phototransduction has yet emerged. In most known cases, invertebrate phototransduction involves polyphosphoinositide and cyclic GMP (cGMP) intracellular biochemical signaling pathways leading to opening of plasma membrane ion channels. Excitation is Ca²⁺-dependent, as are adaptive feedback processes that regulate sensitivity to light. Transduction takes place in specialized subcellular regions, rich in microvilli and closely apposed to submicrovillar membrane systems. Thus, excitation is a highly localized process. This article focuses on the intracellular biochemical signaling pathways and the ion channels involved in invertebrate phototransduction. The coupling of signaling cascades with channel activation is not understood for any invertebrate species. Although photoreceptors have features that are common to most or all known invertebrate species, each species exhibits unique characteristics. Comparative electrophysiological, biochemical, morphological, and molecular biological approaches to studying phototransduction in these species lead to fundamental insights into cellular signaling. Several current controversies and proposed phototransduction models are evaluated.     

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.     

Light- and guanosine 5'-3-O-(thio)triphosphate-sensitive localization of a G protein and its effector on detergent-resistant membrane rafts in rod photoreceptor outer segments

Detergent-resistant membrane microdomains in the plasma membrane, known as lipid rafts, have been implicated in various cellular processes. We report here that a low-density Triton X-100-insoluble membrane (detergent-resistant membrane; DRM) fraction is present in bovine rod photoreceptor outer segments (ROS). In dark-adapted ROS, transducin and most of cGMP-phosphodiesterase (PDE) were detergent-soluble. When ROS membranes were exposed to light, however, a large portion of transducin localized in the DRM fraction. Furthermore, on addition of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to light-bleached ROS, transducin became detergent-soluble again. PDE was not recruited to the DRM fraction after light stimulus alone, but simultaneous stimulation by light and GTPgammaS induced a massive translocation of all PDE subunits to the DRM. A cholesterol-removing reagent, methyl-beta-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in part, to the association of PDE with the DRM. By contrast, transducin was not extracted by the depletion of cholesterol. These data suggest that transducin and PDE are likely to perform their functions in phototransduction by changing their localization between two distinct lipid phases, rafts and surrounding fluid membrane, on disc membranes in an activation-dependent manner.     

Relationship of cholesterol content to spatial distribution and age of disc membranes in retinal rod outer segments

The initial events of visual transduction occur on disc membranes which are sequestered within the photoreceptor outer segment. In rod cells, the discs are stacked in the outer segment. Discs are formed at the base of the rod outer segment (ROS) from evaginations of the plasma membrane. As new discs form, older discs move toward the apical tip of the rod, from which they are eventually shed and subsequently phagocytosed by the adjacent pigment epithelium. Thus, disc membranes within a given rod cell are not of uniform age. We have recently shown that disc membranes are not homogeneous with respect to cholesterol content (Boesze-Battaglia, K., Hennessey, T., and Albert, A. D. (1989) J. Biol. Chem. 264, 8151-8155). In the present study, freshly isolated bovine retinas were incubated with [3H]leucine for 4 h in order to allow sufficient time for the radiolabeled proteins to become incorporated into the basal-most (newest) discs. Osmotically intact discs were then isolated. After the addition of digitonin, the discs were fractionated based on cholesterol content, and radioactivity (indicative of newly synthesized protein) was measured. Discs which exhibited high cholesterol content also exhibited high radio-activity. These results demonstrate that the cholesterol heterogeneity of ROS disc membranes is related to the age, and thus the position, of the discs in the ROS.     

Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of α and β subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel β-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only ∼ 8% of disks and ∼ 12% of ROS plasma membrane.     

Photoreceptor disc morphogenesis: The classical evagination model prevails

Vision begins in photoreceptor outer segments with light captured by opsins in continually synthesized disc membranes. The process by which rod photoreceptor discs are formed has been controversial. In this issue, Ding et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201508093) show conclusively that rod discs are formed by plasma membrane evagination.The vertebrate retina contains two types of photoreceptors, rod cells and cone cells, whose outer segments initiate phototransduction under night and daytime conditions, respectively. The outer segments of these cells lack ER, Golgi, and mitochondria and are filled with hundreds to a few thousand flattened membrane organelles, called photoreceptor discs, which are loaded with the molecular machinery of phototransduction. The structural organization of outer segments differs between rods and cones. Although cone outer segments contain “open” discs that are infoldings of the plasma membrane, rod outer segments possess “closed” discs that are completely separated from the plasma membrane.In 1967, in a paper that has been cited nearly 800 times, Richard Young reported the seminal finding that rod and cone outer segments are continually renewed (Young, 1967). Young’s classic experiment was elegantly simple: he injected [³H]methionine into a rat, mouse, and frog and performed autoradiograms of the excised retina on various days after the injection. He observed that the radiolabeled band moved along the outer segment as time after injection increased and ultimately disappeared at the apex of the cell (Fig. 1, republished from Young, 1967). (As Young was at the University of California, Los Angeles, this result was given the memorable moniker of “the UCLA marching band.”) Young’s seminal insight that outer segments are continually rebuilt posed a problem that has challenged photoreceptor cell biologists ever since: How are rod disc membranes initially formed? In this issue, Ding et al. present a compelling resolution to this question. Specifically, their work differentiates between currently competing models to determine whether rod discs are formed by evagination of plasma membrane at the base of the outer segment or by fusion of intracellular vesicles transported to the outer segment.Open in a separate windowFigure 1.Photoreceptor outer segments are continually renewed. Rats were injected with [³H]methionine, and radioautographs of photoreceptor cells were performed on various days after the injection. As time after injection increases (images 2–7), the radiolabel components are displaced from the inner segment along the outer segment toward the apex of the cell, revealing that the outer segment is continually renewed (figure republished from Young, 1967).The classic hypothesis of disc morphogenesis is that they are formed by evagination of basal outer segment plasma membrane (Steinberg et al., 1980). This hypothesis is based largely on evidence that one surface of the most basal discs of rods is open to the extracellular space, as shown by EM (Carter-Dawson and LaVail, 1979; Steinberg et al., 1980), with lipophilic dye fluorescence (Laties et al., 1976), and by analysis of membrane capacitance (Rüppel and Hagins, 1973). In addition, rods and cones might be expected to share a common machinery of disc formation. Because most cone discs are well established by EM, lipophilic dye imaging, and electrophysiology to be continuous with the plasma membrane, nascent rod discs would seem likely to also be part of the plasma membrane. Thus, according to the classic hypothesis, new discs in both photoreceptor types are formed from outgrowths (evaginations) of the plasma membrane at the outer segment base. In both photoreceptor types, discs would begin life with one face exposed to the extracellular space, but at some point after formation, rod discs would pinch off from the outer segment plasma membrane to become self-contained and fully separated from the plasma membrane, whereas cones discs remain open. On the contrary, the vesicle fusion hypothesis postulates that nascent discs are born completely internalized in rods. Photoreceptor outer segments are now understood to be the plus end of a modified primary cilium (Bloodgood, 2009) and are joined to their inner segments by a narrow ciliary tube called the connecting cilium. This realization, combined with evidence of vesicles in the connecting cilium seen in electron micrographs, has been taken to support the model that vesicles are actively transported through the connecting cilium and generate nascent discs by membrane fusion at the base of the outer segment (Chuang et al., 2007, 2015).Ding et al. (2015) addressed these competing hypotheses with two distinct approaches. First, they treated sections of retinas of mice perfused with a membrane-staining mixture of tannic acid and uranyl acetate and performed EM. Because tannic acid penetrates intact membranes poorly, this treatment distinguishes between membranes exposed to the extracellular space and intracellular membrane structures. The researchers found that, like the plasma membrane, a small number of basal rod discs were intensely stained by tannic acid, whereas the staining of fully internalized discs was weak, confirming that newly formed rod discs are open to the extracellular space. Consistently and strikingly, EM analysis also revealed a single basal disc face (approximately five to seven discs north of the most basal disc) that is contiguous with the plasma membrane. Second, Ding et al. (2015) performed EM with an immunogold-tagged antibody raised against an intracellular epitope of peripherin, a protein that plays an essential role in disc stacking (Arikawa et al., 1992; Goldberg, 2006). Quantification of gold particle counts showed that the peripherin antibody closely associated intracellularly with the edges of fully internalized discs but was negligibly associated with the surface of nascent discs identified as facing the extracellular space, suggesting that peripherin redistributes along the rod disc edge upon its separation from the plasma membrane and enclosure into the outer segment. Finally, Ding et al. (2015) performed experiments using the fixation techniques reported by other investigators and demonstrated that artifacts of tissue fixation were responsible for the erroneous interpretation that basal discs are fully internalized and for the evidence supporting the vesicular fusion hypothesis.Other tools, such as superresolution microscopy of living rods stained with lipophilic dyes or fluorescent antibodies raised against epitopes on the extracellular face of the rod plasma membrane, could further test aspects of the evagination model of disc formation. Nonetheless, the work of Ding et al. (2015) unequivocally shows that basal rod discs are open to the extracellular space and provides a new system and conceptual framework for the investigation of the fundamental biological mechanism of plasma membrane evagination. As outer segment discs exhibit a specialized composition of lipids and phototransduction proteins, further work will also focus on how disc lipids and proteins are transported from the inner segment to the basal outer segment. The current hypotheses about such transport include (a) vesicular transport through the connecting cilium followed by fusion with the outer segment plasma membrane; (b) directed transport through the connecting cilium membrane after vesicle fusion at the base of the connecting cilium in the inner segment; and (c) exocytotic release from the inner segment followed by endocytotic capture in the outer segment. As the molecular details of disc formation and specialization become clearer, Richard Young’s “UCLA marching band” (Young, 1967) will continue to have a broad conceptual impact on the cell biology of photoreceptor development and cilia.     

The cGMP-gated channel and related glutamic acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes

The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of alpha- and beta-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the beta-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel beta-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel.exchanger complex in the rod outer segment plasma membrane.     

Lipid microdomain polarization is required for NADPH oxidase-dependent ROS signaling in Picea meyeri pollen tube tip growth

The polarization of sterol-enriched lipid microdomains has been linked to morphogenesis and cell movement in diverse cell types. Recent biochemical evidence has confirmed the presence of lipid microdomains in plant cells; however, direct evidence for a functional link between these microdomains and plant cell growth is still lacking. Here, we reported the involvement of lipid microdomains in NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) signaling in Picea meyeri pollen tube growth. Staining with di-4-ANEPPDHQ or filipin revealed that sterol-enriched microdomains were polarized to the growing tip of the pollen tube. Sterol sequestration with filipin disrupted membrane microdomain polarization, depressed tip-based ROS formation, dissipated tip-focused cytosolic Ca²⁺ gradient and thereby arrested tip growth. NOX clustered at the growing tip, and corresponded with the ordered membrane domains. Immunoblot analysis and native gel assays demonstrated that NOX was partially associated with detergent-resistant membranes and, furthermore, that NOX in a sterol-dependent fashion depends on membrane microdomains for its enzymatic activity. In addition, in vivo time-lapse imaging revealed the coexistence of a steep tip-high apical ROS gradient and subapical ROS production, highlighting the reported signaling role for ROS in polar cell growth. Our results suggest that the polarization of lipid microdomains to the apical plasma membrane, and the inclusion of NOX into these domains, contribute, at least in part, to the ability to grow in a highly polarized manner to form pollen tubes.     

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.     

Proteomic analysis of the retinal rod outer segment disks

The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segment consists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins that reside in disks has not been achieved yet. We present the first comprehensive proteomic analysis of purified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separation of disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks were isolated from bovine retinal rod outer segments by a method that minimizes contamination from inner segment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additional set of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional (1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism were found, among which are the five complexes of oxidative phosphorylation. Results from biochemical, Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest that F 1F o-ATP synthase is located and catalytically active in ROS disk membranes. This study represents a step toward a global physiological characterization of the disk proteome and provides information necessary for future studies on energy supply for phototransduction.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Light-Induced Tyrosine Phosphorylation of Rod Outer Segment Membrane Proteins Regulate the Translocation,Membrane Binding and Activation of Type II α Phosphatidylinositol-5-Phosphate 4-Kinase

Type II phosphatidylinositol 5-phosphate 4-kinase (PIPKIIα) catalyzes the synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P₂), an essential lipid second messenger that may be involved in the regulation of phototransduction, neuroprotection, and morphogenesis in the vertebrate retina. Here we report that in rodent and transgenic frogs, the light-mediated activity and membrane binding of PIPKIIα in rod outer segments (ROS) is dependent on tyrosine phosphorylation of ROS proteins. The greater type II α PIP kinase activity in the light-adapted ROS membrane results from light-driven translocation of PIPKIIα from the rod inner segment to ROS, and subsequent binding to the ROS membrane, thus improving access of the kinase to its lipid substrates. These results indicate a novel mechanism of light regulation of the PIPKIIα activity in photoreceptors, and suggest that the greater PIPKIIα activity in light-adapted animals and the resultant accumulation of PI-4,5-P₂ within the ROS membrane may be important for the function of photoreceptor cells.     

Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

THE STRIATED MUSCULATURE OF BLOOD VESSELS : II. Cell Interconnections and Cell Surface

The interconnections and the surfaces of the striated muscle cells which occur in thoracic and in lung veins of the mouse were studied with the electron microscope. The osmium-fixed tissues were embedded in methacrylate or in araldite and sectioned with a Porter-Blum microtome. Many preparations were stained before embedding with phosphotungstic acid or after sectioning with uranyl acetate. Typical intercalated discs are observed in this muscle. They are similar to the discs found in heart muscle. These intercalated discs represent boundaries between separate muscle cells. Along the discs, cells are joined in planes normal to their myofilaments. The same cells are also joined in planes parallel to the myofilaments by means of lateral interconnections. These lateral cell boundaries are in continuity with the intercalated discs. Three morphologically distinct parts occur within the lateral cell interconnections: One is characterized by small vesicles along the plasma membrane, the second part has the structure of desmosomes, and a third part represents an external compound membrane (formed by the two plasma membranes of the adjoining cells) and is termed "quintuple-layered cell interconnection." Small vesicles and plasma membrane enfoldings along the free surface of muscle cells are interpreted as products of a pinocytosis (phagocytosis) process. Some of them are seen to contain small membrane-bounded bodies or granules. The free cell surface shows a characteristic outer dense layer ("basement membrane") which accompanies the plasma membrane. The topographic relation of this dense layer with the plasma membrane seems to vary in different preparations. The significance of this variation is not well understood. On two occasions a typical arrangement o vesicles and tubules was observed at Z band levels, just beneath the plasma membrane. These structures are believed to represent endoplasmic reticulum. Their possible significance for the conduction of excitation is discussed.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.