Joachim Schwemer

Frequenin—A novel calcium-binding protein that modulates synaptic efficacy in the drosophila nervous system

The T(X;Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca(2+)-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca(2+)-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca(2+)-dependent modulation of synaptic efficacy.

Photoregeneration and Sensitivity Control of Photoreceptors of Invertebrates

It is a well known fact that the metarhodopsins of cephalopods are thermostable and photoreconvertible into rhodopsin (Fig. 1). In the octopus species Eledone moschata the wavelength maxima of the rhodopsin (R) and acid metarhodopsin (M) are separated by about 50 nm. Due to the large distance between the wavelength maxima, this R-M-system is well suited for photokinetic studies (1–3). Monochromatic illumination causes a photoequilibrium between R (11-cis R., isoR) and its acid M. The equilibrium is determined solely by the ratio betweeen the absorption coefficients of the two pigments. Illumination at the isosbestic point yields 50% R and 50% M. Minimal R-concentration (38%) is obtained at 440 nm. At longer wavelength the M is quantitatively reconverted to R.

Renewal of visual pigment in photoreceptors of the blowfly

SummarySpectrophotometric measurements of photoreceptors 1–6 in the blowfly demonstrate that rhodopsin undergoes a continuous renewal. This involves, in the dark, the slow degradation of rhodopsin whereas metarhodopsin is degraded at a much faster rate. The effect of light is to reduce the rate at which metarhodopsin is degraded, i.e. the rate is inversely related to the intensity of the light. Rhodopsin synthesis is dependent on the presence of 11-cis retinal which is formed via a photoreaction from all-trans retinal resulting from the breakdown of rhodopsin and/or metarhodopsin: the biosynthesis of rhodopsin is therefore a light dependent process. Light of the blue/violet spectral range was found to mediate the isomerization of all-trans retinal into the 11-cis form. It is proposed that this stereospecificity is the result of all-trans retinal being bound to a protein. On the basis of the results a visual pigment cycle is proposed.

Photoregeneration and the Adaptation Process in Insect Photoreceptors

Nearly half a century ago it was suggested that absolute sensitivity of photoreceptors depends on the rhodopsin content of rod outer segments in a simple photochemical way: a decrease in rhodopsin concentration causes a proportional decrease in the probability of quantal absorption and hence leads to a proportional loss in sensitivity. From measurements of rhodopsin concentration and sensitivity in the eyes of man (RUSHTON et al., 1955; RUSHTON, 1961) and rat (DOWLING, 1960, 1963) it became evident that this early hypothesis was not true: in both human and rat eyes, an approximately linear relationship between log sensitivity and rhodopsin concentration was found. The slope \( \left( {\frac{{\Delta \log s}}{{\Delta {c_R}}}} \right)\)of this relation varies from species to species. The highest value, \( \left( {\frac{{{R_{\Delta \log s}}}}{{\Delta {c_R}}}} \right) = 6\) was reported for the retina of the albino rat. In this retina, a decrease of 17% in rhodopsin concentration reduces the sensitivity to 1/10; a decrease of 34% reduces the sensitivity to 1/100 and so on (WEINSTEIN et al., 1967). Whereas the sensitivity is greatly decreased, the probability of quantal absorption is only slightly reduced.

UV-Sehfarbstoff bei Insekten

SummaryFrom insect eyes, an u.v.-visual pigment “A” (λmax 345 nm) was extracted by 2% aqueous digitonin (pH 5.2). Upon prolonged u. v. irradiation, A is converted to a stable product “B” (λmax 480 nm), which reconverts completely to A when illuminated with light of longer wavelengths. When the pH of B is raised to 9.3, B is converted to “C” and absorbs at 375 nm. Experiments with NH2OH lead us to the assumption that retinal is the prosthetic group of this pigment.

Three visual pigments inDeilephila elpenor (Lepidoptera, Sphingidae)

SummarySelective irradiation of digitonin extracts (pH 6.1) from dark-adapted eyes of the Sphingid mothDeihphila elpenor revealed three visual pigments with λmax at about 345, 440 and 520 nm. Prom the reaction of the chromophoric group with hydroxylamine it is concluded that these pigments are based on retinaldehyde, and therefore can be classed with rhodopsins. At −15 °C, each of the rhodopsins can completely (P 520) or partially (P 440 and P 345) be converted by light to acid metarhodopsin absorbing maximally at 480 ± 10 nm, which decay at room temperature to retinaldehyde and protein moiety. Spectrophotometric measurements on isolated retinae (pH 7.4) indicate that the metarhodopsins, in contrast to the findings on extracts, are thermostable in the photoreceptor membrane at room temperature. The results are compared with electrophysiological and microspectrophotometrical data. The different properties of the metarhodopsins in detergent solution and in the isolated retina are discussed.

Vitamin a deficiency reduces the concentration of visual pigment protein within blowfly photoreceptor membranes

Visual pigment extracts prepared from rhabdomeric membranes of vitamin A deficient blowflies contain a 5-10 times lower concentration of rhodopsin than extracts from flies which were raised on a vitamin A rich diet. Spectrophotometry showed that digitonin-solubilized rhodopsin from blowfly photoreceptors R1-6 has an absorbance maximum at about 490 nm, but no unusually enhanced beta-band in the ultraviolet. The extracts did not contain detectable concentrations of other visual pigments nor was there any evidence for the presence of photostable vitamin A derivatives. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the concentration of opsin in the rhabdomeric membrane is significantly reduced in vitamin A deficient flies compared to normal flies. The results indicate that the synthesis of opsin or its incorporation into the photoreceptor membrane is regulated by the chromophore concentration in the receptor cell. Furthermore, our findings open up the possibility that differences in the spectral absorption and excitability of photoreceptors from normal and vitamin A deficient flies result from the differing opsin content of the rhabdomeres.

Studies on the insect visual pigment sensitive to ultraviolet light: Retinal as the chromophoric group

Abstract 1. The nature of the chromophoric group of the ultraviolet visual pigment of the neuropteran Ascalaphus macaronius Scop. was investigated. Analysis of the carotenoid content of the retina by the reaction with SbCl 3 indicated the presence of retinal. 2. Thin-layer chromatography of ethanol-ether extracts from retinae on silica gel demonstrated the presence of retinal isomers with R F values corresponding to all- trans -retinal and II- cis - and/or 13- cis -retinal. Other retinal isomers and retinol were not detected. 3. Thermal denaturation released retinal from the ultraviolet visual pigment A and its photoproduct B. Analysis of the compounds resulting from thermal denaturation indicated that all- trans -retinal was predominately released. Formation of cattle rhodopsin from cattle opsin and ultraviolet visual pigment A denatured by Ag + indicated that the stereoconfiguration of retinal in the native pigment is II- cis . 4. As a consequence of the retinal nature of the chromophore, the ultraviolet visual pigment of A. macaronius was designated “rhodopsin 345”, and photoproducts B and C correspond to acid and alkaline metarhodopsin, respectively. The stereoconfiguration of retinal in rhodopsin 345 and metarhodopsin, as well as the linkage of the chromophore in respect of the hypsochromic shift of rhodopsin 345, were discussed.

Light-inducedtrans-cis isomerization of retinal by a protein from honeybee retina

SummaryTwo retinal-binding proteins (RBP-A and RBP-B) isolated from the honeybee retina were further purified by ion-exchange chromatography. Whereas RBP-A seems to be denatured by this procedure, RBP-B remains intact with respect to its photochemical characteristics (Fig. 3a). Analysis of the geometric isomers of retinal bound to RBP-B by high performance liquid chromatography demonstrated that all-trans retinal was the chromophore of the non-irradiated RBP-B. Irradiation converted RBP-B (λmax 440 nm) into a photoproduct (λmax 370 nm) the chromophore of which was 11-cis retinal, i.e., light isomerized all-trans retinal almost exclusively to the 11-cis form (Fig. 3b). Irradiation of a solution of RBP-B in the presence of excess all-trans retinal also led to the formation of 11-cis retinal indicating that RBP ‘catalyzes’ the photoisomerization of all-trans retinal. The physiological significance of RBP-B is discussed with respect to the renewal of rhodopsin.

Morphological correlates of visual pigment turnover in photoreceptors of the fly, Calliphora erythrocephala

SummaryThe content of visual pigment in one eye of Calliphora ‘chalky’ was measured spectrophotometrically and related to the ultrastructure of the other eye and the density of membrane particles as revealed by the freeze-fracture technique. Electron microscopy of thin-sectioned material showed that the manifestations of synthesis and breakdown of photoreceptor membrane were most prominent in flies kept in blue light, in comparison to flies kept in green light or darkness, in which only a moderate breakdown of membrane was evident.By subjecting flies to different light regimes it was found that the density of membrane particles was related to the content of visual pigment, but not on a one-to-one basis. In particular, the particle density in flies with a low (< 10%) rhodopsin content, produced by raising flies on a vitamin A-deficient diet or by exposure to green light, was still about 35% of that of normal flies. Taken together the results indicate that all rhodopsin molecules are particles but that some particles represent another protein that most probably serves to maintain the structural integrity of the photoreceptor membrane. Furthermore, membrane synthesis can take place in the absence of rhodopsin synthesis.