Receptor demise from alteration of glycosylation site in Drosophila opsin: Electrophysiology, microspectrophotometry, and electron microscopy

Abstract

In the δAsn20 Drosophila stock, the N-linked glycosylation site of opsin in Rl-6 receptors (Rhl) is absent. We used electroretinography (ERG), microspectrophotometry (MSP), and electron microscopy (EM) to quantify visual cell defects. Positive controls, w9, had wild type Rhl. MSP revealed minimal photopigment in δAsn20 for 6 days posteclosion; w9 had near normal visual pigment. ERG sensitivity and prolonged depolarizing afterpotential (PDA) were compared for δAsn20 and w9. δAsn20's Rl-6 function is decreased 100–fold at eclosion and diminishes until only R7/8 functions at 11 days. What little rhodopsin is routed to the rhabdomere functions. Morphometry showed smaller Rl-6 rhabdomeres in δAsn20 for 8 days posteclosion. Rhabdomeres in w9 were normal. A negative control, ninaE0117, a deletion of the Rhl gene, also has small rhabdomeres. δAsn20 and ninaE0117 lack the extreme rhabdomere elimination of ora (outer rhabdomeres absent), a nonsense mutant interrupting Rhl's coding sequence. δAsn20 and ora have surplus membrane while ninaE0117 does not. Freeze fracture reveals that δAsn20's rhabdomeric P-face particle count is as low as for vitamin A deprivation, consistent with an opsin defect. High particle density, organized into rows, is present in adjacent plasmalemma where surplus membrane accumulates. In summary, δAsn20 interferes with either synthesis, deployment, or maintenance of opsin.

Carotenoid replacement inDrosophila: freeze-fracture electron microscopy

“…The present freezefracture micrographs depict microvillar coated pits like those observed in the crayfish (Eguchi & Waterman, 1976); we also see origins of endocytosis from the plasmalemma in the form of plasmalemma pock marks (cf. Brown et al, 1994;Blest & Eddey, 1984). Such turnover in Drosophila is likely to be continuous (Sapp et al, 1991b) with little variability in visual parameters over a light/dark cycle (Chen et al, 1992).…”

“…Standard freeze-fracture techniques were used Katz et al, 1991;Brown et al, 1994). Heads were dissected in a fixative of t% glutaraldehyde in 0.17 M sodium cacodylate buffer (pH 7.4) at room temperature for at least 20 min.…”

“…Vitamin A deprivation is one of the conditions that can greatly reduce the density of P-face particles -most of which represent rhodopsin molecules -in rhabdomeric microvilli (Boschek & Hamdorf, 1976;Harris et al, 1977;Schwemer & Henning, 1984;. Also, particle density is decreased in Drosophila with opsin mutations (Schinz et al, 1982;Stark & Carlson, 1983;Brown et al, 1994), digitonin-extracted crayfish receptors (Fernandez & Nickel, 1976), and ultraviolet (UV) and green light-treated wasp ocelli (Pabst & Kral, 1989). In striking contrast with the insect situation, vitamin A depletion does not alter the density of P-face particles in the outer segment disc membranes of vertebrate photoreceptors (Engbretson et al, 1979;Katz et al, 1991).…”

Control ofDrosophilaRetinoid and Fatty Acid Binding Glycoprotein Expression by Retinoids and Retinoic Acid: Northern, Western and Immunocytochemical Analyses

“…∆Asn #! was engineered to lack Rh1's glycosylation site (O'Tousa, 1992) resulting in Rh1 deficiency (Brown et al, 1994 ;O'Tousa, 1992). To test the effect of receptor deletion from the compound eye, the white-eyed glass mutant (w gl none ) was used (Stark and Carlson, 1991 ;Stark, Sapp and Carlson, 1989).…”

“…4 (Leonard et al, 1992) and ninaE ora (Harris, Stark and Walker, 1976) and smaller for ninaE ∆ Asn#! (Brown et al, 1994 ;Stark et al, 1995). The retina (but not the Semper cells) is severely disrupted in gl (Stark and Carlson, 1991 ;Stark et al, 1989).…”