Manipulation of phototransductive membrane turnover by crab photoreceptors in vitro: effects of two protein kinase activators, SC-9 and phorbol ester in the presence of a protein phosphatase inhibitor, okadaic acid.

1. Retinae of crabs, Leptograpsus variegatus, held on a 12:12 h light-dark cycle were prepared for culture in vitro shortly before light-off. After an hour in darkness to permit the assembly of "night" rhabdoms, retinae were exposed to various combinations of drugs: 1 microM okadaic acid (OKA); 60 microM SC-9; 10 microM phorbol, 12,13-diacetate (PDA). 2. The effects of the specific protein phosphatase inhibitor, OKA, are confirmed as light-dependent. Rhabdom sizes were not compromised by OKA, nor by either of the two protein kinase activators, SC-9 or PDA when each was deployed alone in darkness. 3. In combination with OKA, PDA induced demolition of rhabdoms by abnormal macropinocytosis of microvillar membranes. 4. Combined with OKA, SC-9 induced a transient reduction of rhabdoms, followed by overgrowth to abnormal sizes. Overgrowth was blocked by the transcription inhibitor actinomycin D. 5. Disparate consequences of combining OKA with SC-9 or PDA imply that more than one protein kinase C may be involved.

The distribution of actin immunoreactivity in rhabdomeres of tipulid flies in relation to extracellular membrane shedding.

Rhabdomeres of tipulid flies lose membrane during turnover from a 'shedding zone' composed of microvillar tips. These distal domains lack intramicrovillar cytoskeletons and appear to be empty sacs of membrane. Recent concerns about the role of ninaC mechano-enzymes in the architecture of dipteran rhabdomeral microvilli and the dynamic role that they may play in the creation of shedding zones demand an examination of the distribution of actin in tipulid rhabdomeres. We compared rhabdomeres from tipulid retinae incubated before fixation for immunocytochemistry in a buffer without additives and a stabilising buffer that contained a cocktail of cysteine protease inhibitors; both were challenged by an anti-actin antibody for immunogold labelling after embedding in LR White Resin. Shedding zones thus processed collapse to structureless detritus. Stabilised and unstabilized shedding zones were immunonegative to anti-actin. To ensure that the negative results were not consequent upon conformational changes generated by the processing protocol, we examined microvilli of degenerating rhabdomeres of the Drosophila light-dependent retinal degeneration mutant rdgBKS222 (which separate and collapse without creating a shedding zone) and found the detritus they generate to be immunopositive to anti-actin. Stabilised and unstabilized regions of basal regions of tipulid rhabdomeres were equally immunopositive. We infer that (a) actin is absent from shedding zones; (b) actin is not degraded by microvillar cysteine proteases. The implications of these conclusions are discussed in relation to some functional models of arthropod photoreceptor microvilli.

Induction of retinal degeneration in a crab by light and okadaic acid in vitro: comparison with the Drosophila light-dependent retinal degeneration mutant w rdgBKS222.

Retinae of the crab Leptograpsus which had been maintained on a 12-h light/12-h dark cycle were cultured in vitro and exposed to 1 microM okadaic acid (OKA) at 0.75 h before light onset. Control retinae were subjected to the same routine and sampled at the same times without OKA treatment. At the concentration used, OKA totally inhibits types 1 and 2A protein phosphatases, minimally inhibits type 2B, and does not affect type 2C. 1 microM OKA provoked a diminution of rhabdom diameter measured at the level of the photoreceptor nuclei in the dark, some ommatidial cartridges being stripped of rhabdomeral microvilli altogether. After 1-h illumination (225-320 lux), further reduction of rhabdom diameter was modest in control retinae but precipitate in those treated with OKA. After 2 h, control rhabdom diameters showed a further, not significant, decline, but OKA had induced a resynthesis of massive structures with the light-microscopic appearance of rhabdoms. Electron microscopy revealed that they were heterogeneous and of the following kinds: (1) a minority of rhabdoms with normally disposed but distorted microvilli; (2) rhabdoms in the throes of events that parody normal assembly; and (3) rhabdomal volumes occupied by saccular organelles or by pleats or ruffles of irregular architecture. The cytoplasm of all such receptors was packed with free and bound ribosomes and endomembranes. The sequence of events parallels that seen during light-induced degeneration of photoreceptors of the Drosophila mutant w rdgBKS222. Preliminary experiments show that a protein kinase activator SC-9 mimics many of these effects in the dark in the presence of 1 microM OKA. As a working hypothesis, it is proposed that light activates protein kinases via diacylglycerols generated by the phototransduction cascade, and that in both crab retinas challenged with OKA and retinas of rdg BKS222 activation of a nuclear regulatory protein by hyperphosphorylation provokes a runaway transcription whose selectivity and extent remain to be determined.

Isolation of rod outer segments on Percoll gradients: effect of specific protease inhibition.

Rat rod outer segments (ROSs) were isolated by vortexing retinas and separating the detached components on performed Percoll gradients. A lighter band of 20 x 10(6) unsealed ROSs per ten retinas, and a heavier band of 60 x 10(6) sealed ROSs per ten retinas were obtained from each 12 ml gradient. The yield of sealed ROSs (but not unsealed ROSs) was increased up to twofold in the presence of the specific cysteine protease inhibitor, Ep-475. Aprotinin, pepstatin, PMSF, TPCK and EGTA plus EDTA had no effect. These results indicate that during isolation, ROSs are vulnerable to damage by cysteine protease activity either from damaged retinal cells or from within.

Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors.

Infiltration of compound eyes of crayfish, Cherax destructor, with the thiol protease inhibitor Ep-475 or with trifluoperazine prior to fixation for electron microscopy was found to stabilize an axial filament of 6-12 nm diam within each rhabdomeral microvillus of the photoreceptors. Rhabdoms isolated from retinal homogenates by sucrose gradient centrifugation under conditions that stabilize cytoskeletal material contained large amounts of a 42-kd polypeptide that co-migrated with insect flight muscle actin in one- and two-dimensional PAGE, inhibited pancreatic DNase l, and bound to vertebrate myosin. Vertebrate skeletal muscle actin added to retinal homogenates did not co-purify with rhabdoms, implying that actin was not a contaminant from nonmembranous structures. DNase l inhibition assays of detergent-lysed rhabdoms indicated the presence of large amounts of filamentous actin provided ATP was present. Monomeric actin in such preparations was completely polymerizable only after 90 min incubation with equimolar phalloidin. More than half of the actin present could be liberated from the membrane by sonication, indicating a loose association with the membrane. However, a large proportion of the actin was tightly bound to the rhabdomeral membrane, and washing sonicated membrane fractions with solutions of a range of ionic strengths and nonionic detergents failed to remove it. Antibodies to scallop actin only bound to frozen sections of rhabdoms after gentle permeabilization and very long incubation periods, probably because of steric hindrance and the hydrophobicity of the structure. The F-actin probe nitrobenzoxadiazol phallacidin bound to rhabdoms and labeled F-actin aggregates in other retinal components, but rhabdom fluorescence was not abolished by preincubation with phalloidin. The biochemical data indicate the existence of two distinct actin-based cytoskeletal systems, one being closely membrane associated. The other may possibly constitute the axial filament, although the evidence for this is equivocal.

Actin in cellular components of the basement membrane of the compound eye of a blowfly.

The so-called 'basement membrane' of arthropod compound eyes is known to be of heterogeneous origin (Odselius and Eloffson 1981). A major contribution in Diptera with open rhabdoms is provided by a pigmented component which lies at the basal end of the extracellular space of each ommatidium and fills it, the glial plug. Ancillary components consist of the expanded tips of cone cell processes. Each glial plug exhibits two distinct regions: ramifying processes extend into the extracellular space and contain numerous pigment granules, while proximally the cytoplasm is devoid of granules but packed with bundles of cross-linked microfilaments that bind the fluorescent F-actin probe NBD-phallacidin strongly and antibodies to scallop actin weakly. Cone cell expansions also contain microfilaments and exhibit the same binding properties. The proximal faces of the cells of the glial plugs and of the cone cell expansions are covered with a coarsely fibrillar extracellular matrix. Some actin bundles appear to be attached to the plasma membranes at their ends, although the reality of this arrangement is still in question. Cellular components of the basement membrane are bonded together by their extracellular matrices, so that collectively they provide a reinforced network that retains the retina. Bundles of axons from the photoreceptors and tracheae that supply the retina with tracheoles pass through the spaces in this network.

The cytoskeleton of microvilli of leech photoreceptors. A stable bundle of actin microfilaments.

The microvilli of leech photoreceptors have diameters in the range of 60-100 nm. Each contains a bundle of microfilaments extending into the photoreceptor soma as a rootlet (Walz 1979 b). Apparent thicknesses of individual filaments are estimated to be 4-5 nm, consonant with those of identified actin filaments in the basement membranes of blowflies (Blest and De Couet 1983). Frozen sections of leech photoreceptors labelled with antibodies against scallop actin exhibited strong binding to the rootlet region but not to the microvilli, which are destroyed by the severe saponin or acetone extraction needed to permeabilise the preparation. NBD-phallacidin binds strongly but non-specifically to the photoreceptors and does not allow positive identification of F-actin. The cytoskeletons of the microvilli and rootlets are adequately preserved by conventional routines of fixation, and similar results were obtained when retinae were pretreated with either 0.5 mM Ca2+, 10 mM EGTA, 2 mM Ep-475 (a specific inhibitor of thiol proteases) or 2 mM Ep-475 combined with 0.5 mM Ca2+. Unlike the axial cytoskeletons of arthropod photoreceptor microvilli, those of the leech are stable to cellular insult.

The local deletion of a microvillar cytoskeleton from photoreceptors of tipulid flies during membrane turnover.

The distal regions of the photoreceptor microvilli of tipulid flies are shed to extracellular space during membrane turnover. Before abscission, the microvillar tips undergo a transformation: they become deformed, and after conventional fixation for electron microscopy are relatively electron-lucent compared to the stable, basal microvillar segments. We now show that the electron-lucent segment is an empty bag of membrane whose P-face after freeze-etch preparation appears as densely particulate as the remainder of the microvillus. Transformation is achieved by the local deletion of a microvillar cytoskeleton which consists of a single, axial filament linked to the plasma membrane by side-arms. The filament may be partially preserved by the chelation of Ca2+; the provision of a divalent cation (Mg2+ or Ba2+) stabilizes the side-arms during subsequent fixation, as has been shown previously for the rhabdomeral cytoskeleton of blowflies. Incubation of the isolated retina in the presence of 0.25 mM Ca2+ at room temperature for 10-20 min causes proteolysis of the cytoskeleton which is blocked by as little as 0.5 mM of the thiol protease inhibitors Ep-475 and Ep-459. Loss of the cytoskeleton is accompanied by deformation of all regions of the microvilli. Local deletion of the cytoskeleton from the transformed zone of the normal rhabdom is sufficient to explain deformation of the microvillar tips, but not their subsequent abscission. The intimate association between a Ca2+-activated thiol protease and the cytoskeleton implied by the great rapidity of proteolysis calls for a reassessment of published studies of membrane turnover by radioautography, and of the nature of light-induced damage to arthropod photoreceptor membranes.

A new mechanism for transitory, local endocytosis in photoreceptors of a spider, Dinopis.

Intermediate segment outgrowth (ISOs) are transitory specialisations of the plasma membrane of intermediate segments of the posterior median photoreceptors of Dinopis. Local regions form outgrowths into the glial partitions separating the receptors and remain connected to their parent intermediate segments by narrow necks. ISOs, only a few micrometers in diameter, are sites of intense endocytosis. Coated pits in their plasma membranes give rise to saccular internalisations. Unusual, slender, endocytotic tubules either pinch off coated vesicles, or become detached to yield a tubular detritus. Products of endocytosis are assembled to yield multivesicular and dense bodies, which are usually surrounded by smooth saccules derived from the endoplasmic reticulum of the intermediate segment. ISOs also contain arrays of tubules, thought to be stacks of haemocyanin molecules. There are usually at least 10 times the number of empty ISOs as full and active outgrowths. The number of active ISOs increases rapidly at dawn to peak at about 3 h after sunrise and then rapidly declines. The present sample suggests that the number of empty ISOs increases steadily throughout the day. Thus, ISOs turn over and are probably rapidly formed and short-lived. The contents of ISOs are observed to be evicted into the intermediate segments, where they presumably join the population of secondary lysosomes and are digested. The cyclical activity of ISOs is greatest after shedding of rhabdomeral membrane at dawn (Blest 1978). ISOs are thought to be concerned with the return to the receptors of a minor fraction of rhabdomeral material lost to extracellular space during shedding of the microvillar membrane.

The posterior median eyes of the dinopid spider Menneus.

The posterior median (pm) eyes of the dinopid spider Menneus unifasciatus L. Koch are described and compared with the pm eyes of Dinopis, which are highly specialised for night vision. The lenses of Menneus have F-numbers of 0.72 compared to 0.58 in Dinopis, the distance between receptors is ca. 4.0 microns compared to 20-22 microns for Dinopis, and image quality is matched to receptor spacing. The lens of Menneus is simple, while that of Dinopis comprises two components of different refractive indices (Blest and Land 1977). Receptive segments of the pm eyes of Dinopis are hexagonal in transverse section and those of adjacent cells are tightly contiguous, allowing the possibility of both optical and electrical coupling (Blest 1978). Receptive segments of Menneus are separated from each other by glial processes containing little pigment, and each segment possesses two rhabdomeres on opposite faces of the cell. Rhabdomere volumes undergo a daily cycle similar to that described for Dinopis, but of relatively minor extent. It is shown that the pm eye of Dinopis could have evolved from that of Menneus by a simple series of transformations, and that a gain of two logarithmic units of sensitivity can be attributed to changes in optical design alone.

The sources of acid hydrolases for photoreceptor membrane degradation in a grapsid crab.

Dawn photoreceptor breakdown in the crab Leptograpsus variegatus is analysed at the ultrastructural level. Coated vesicles derived from microvilli are assembled as mutlivesicular bodies (mvbs), which degrade to multilamellar bodies (mls) and are lysed. Cytochemical markers for hydrolases were a fluoride-inhibited beta-glycerophosphatase and a fluoride-insensitive p-nitrophenyl phosphatse, with indistinguishable distributions when localised at pH 5.0. These enzymes are injected into the secondary lysomes from two sources: (i) immediately after dawn Golgi bodies are highly active, and differentiate a transtubular network, from which tubules and vesicles detach, and can be seen fusing with mvbs and mlbs. (ii) Saccules derived from the rough endoplasmic reticulum (RER) provide a second source and are most often seen in association with late mlbs. Both kinds of primary lysosome rare give AcPh-positive responses when free in the cytosol, but are seen to do so as they make contact with their secondary lysosomal targets. Lipid droplets and lipofuscin bodies are interpreted as the residual products of breakdown. These results are discussed in relation to previous findings on photoreceptor membrane breakdown in a dinopid spider. Attention is drawn to the implied diversity of organisation of lysosomal compartments in receptors which internalise membranes of similar compositions.

Extracellular shedding of photoreceptor membrane in the open rhabdom of a tipulid fly.

The compound eyes of the Australia tipulid fly, Ptilogyna, shed the bulk of their rhabdomeral membrane to extracellular space during turnover. The rhabdomeres of the retinulae lie in a common extracellular space (ECS), which is subdivided in the proximal retina. Before dawn, a distal region of the microvilli in each rhabdomere differentiates and becomes less electron-dense after conventional fixation. The differentiated region then dilates and develops an irregular profile. A few hours after dawn, the transformed tips break off and form a detritus in the ECS. The degraded membrane is internalised back into the retinula cells by mass endocytosis. Retinulae develop pseudopodia at sites bordering the ECS and engulf the membrane detritus, which comes to lie first of all in vacuoles within the receptor cells and then forms very large multivesicular bodies. The latter transform to multilamellar and residual bodies and are, presumably, lysed. Surrounding these secondary lysosomes are rough endoplasmic reticulum and smooth tubular systems, tentatively considered on comparative grounds to provide hydrolases. The literature concerning the ultrastructure of compound eyes offers a small number of instances where extracellular shedding can be suspected for morphological reasons. Attention is drawn to analogies with the shedding of photoreceptor membranes in vertebrate retinae.

Photoreceptor membrane breakdown in the spider Dinopis: localisation of acid phosphatases.

The ultrastructural localisation of acid phosphatases (AcPhs) during the normal daily breakdown of rhabdomere membrane in Dinopis has been examined using beta-glycerophosphate and p-nitrophenyl phosphate as substrates. Results are related to the classification of organelles in the receptors given by Blest, Powell and Kao (1978). Weak and infrequent reactions are obtained in multivesicular bodies (mvbs) and multilamellar bodies (mlbs) derived from them. Residual bodies (rbs) begin to react strongly as they lyse. Source of AcPhs is endoplasmic reticulum which has barely differentiated towards the GERL configuration; it becomes reactive as it is incorporated into secondary lysosomes. GERL tubules, Y-bodies and vesicles respond erratically and weakly, and are also incorporated into rbs. No evidence was found for a significant participation of Golgi bodies in these processes, and acid phosphatase cytochemistry fails to reveal a topographical relationship between GERL in these cells and Golgi saccules. Coated vesicle clusters found in the predawn receptive segments are AcPh-negative; this implies that their previous identification as GERL-derived "Nebenkerne" carrying hydrolytic enzymes to newly-formed mvbs (Blest, Kao and Powell, 1978) is dubious. Isolation bodies and autophagic vacuoles enclosing other organelles in pathological receptors give strong reactions while adjacent secondary lysosomes derived from rhabdomere membrane and associated GERL give weak ones. It is concluded that rhabdomere-derived rb lysis is more tightly regulated than other autophagic processes, and it is suggested that a high degree of control is necessary in a receptor which may repeat the autophagy of a large mass of transductive membrane at least 60--100 times in the course of its working life.

Exocytotic shedding and glial uptake of photoreceptor membrane by a salticid spider.

Receptors in the anterior lateral eyes of salticid spiders possess paired rhabdomeres. The tips of the rhabdomeral microvilli lie adjacent to non-pigmented glial processes. Photoreceptor membrane is lost during turnover by a hitherto undescribed process: individual microvilli lengthen at their tips, taper, and are received by corresponding, coated endocytotic pits in the glial membrane. Pits detach as coated vesicles with coherent fragments of microvilli within them, lose their coats, and accumulate in the glial processes as disorderly membranous detritus. Some microvillus membrane disintegrates before local endocytosis, and appears to get into the glial arms distant from the parent rhabdomere by invaginations which are either endocytotic clefts or a tubulo-cisternal system, but whose precise nature is not yet clear. No photoreceptor membrane is lost by pinocytosis into the receptor cytoplasm. Analogies between the behaviour of this system and the phagocytosis of shed vertebrate photoreceptor membrane are briefly discussed.

A new fixative solution to precede the reduced silver impregnation of arthropod central nervous systems.

Arthropod central nervous tissue is fixed for 1 hr at 20 C in 8% pure formic acid in 1:1 n-butanol/n-propanol prepared immediately before use (FBP), then washed for 15-30 min in 90% ethanol, and embedded in paraffin wax. Impregnation is by modified Ungewitter techniques in which the silver bath is preceded by mercury/cobalt mordanting, or by modified Holmes' methods following similar mordanting procedures. The methods yield high resolution of axons with minimal background staining, while the staining of neuronal somata is suppressed. They succeed with brains of crustacea and Odonata and other difficult materials. Tissues fixed in FBP are hard and require care in sectioning.