QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 23 February 2005
doi: 10.1242/dev.01704

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Satoh, A. K. Articles by Ready, D. F. Search for Related Content PubMed PubMed Citation Articles by Satoh, A. K. Articles by Ready, D. F.

Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors

¹ Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
² Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA
³ Graduate School of Frontier Biosciences, Osaka University, Toyonaka, Osaka 560-0043, Japan

View larger version (122K): [in a new window]   Fig. 1. TRP and Rh1 accumulate in large cytoplasmic vesicles during photoreceptor maturation. Confocal cross-sections of staged pupal eyes (% pd=% pupal development) stained for F-actin using rhodamine-phalloidin (red) and either (A) anti-TRP or (B) anti-Rh1 (green). Actin-rich rhabdomeres stain red at all stages; overlapping green immunostain (Rh1 or TRP) renders rhabdomeres yellow. The yellow-red boundary marks the rhabdomere base, the cytoplasmic ends of microvilli and the origin of the actin-rich rhabdomere terminal web (RTW). (A) TRP concentrates in large cytoplasmic vesicles during its synthetic peak, between 50 and 80% pd. (B) Following the onset of Rh1 expression at about 70% pd, the protein concentrates in large cytoplasmic (Rh1-containing large) vesicles (RLVs), before it is detected in rhabdomeres at 70% pd. (C) TRP and Rh1 colocalize in RLVs of 74% pd photoreceptors. (D) A side view of an ommatidium isolated from a newly eclosed wild-type fly shows RLVs associate with one or more small dots of F-actin. (E) Most RLVs appear tethered to the RTW via actin patches. (F) A side view of a live ommatidium isolated from a 6-day-old fly expressing GFP-actin driven by Rh1Gal4. Actin patches decorate the RTW of living cells. Scale bar: 2 µm in A-D; 1 µm in E; 5 µm in F.

View larger version (80K): [in a new window]   Fig. 2. RLVs are MVBs. (A) Immunoelectron microscopy localizes Rh1 to MVBs in 87% pd photoreceptors. (B) Confocal projections of ommatidia isolated from newly eclosed Rh1-Gal4, UAS-Rab7-GFP flies. Rab7-GFP outlines RLVs, whereas Rh1 stains more uniformly. Arrows indicate corresponding RLVs. RLVs, ER and Golgi (not shown) are distributed evenly throughout photoreceptors, unlike cell types in which pericentriolar cytoplasm is a focus for endosomal organization. (C) Hrs localizes to RLVs in 78% pd ommatidia. Arrows indicate corresponding RLVs. Scale bar: 500 nm in A; 4 µm in B; 2 µm in C.

View larger version (88K): [in a new window]   Fig. 3. Rh1 internalization is light dependent. (A) In dark-reared flies, Rh1 accumulates in the rhabdomere shortly after the onset of its expression, about 70% pd. There are few RLVs in developing (74% pd and 90% pd) and newly-eclosed (0 day) flies. (B) Many RLVs appear within 30 minutes of light exposure (DarkLight 30 minutes), approximating the distribution of RLVs in 12L/12D raised flies fixed 4 hours after light (Light). RLVs are absent 13 hours after flies are returned to the dark following light exposure at 78% pd (LightDark 13 hours). Scale bar: 2 µm.

View larger version (116K): [in a new window]   Fig. 4. Rab11 immunolocalization. (A) In a confocal cross-section of a newly-eclosed adult eye, triple stained for Rab11 (green), F-actin (red) and CFP-galactosyl transferase (blue), Rab11 immunolocalizes to vesicles and tubules distributed throughout the cytoplasm; Rab11-positive vesicles frequently associate with the RTW (arrows). Rab11 also associates with Golgi (arrowheads). (B) The cis-Golgi marker, Rab1, localizes to the convex side of CFP-stained Golgi, while Rab11 localizes to the concave, trans side. (C) The projection of five 0.3 µm interval serial z-sections of a 30% pd photoreceptor Golgi triple stained for Rab1, Rab11 and the CFP-Golgi marker localizes Rab11 to trans-Golgi. Arrows show the edges of staining. (D) The time course of Rh1 and Rab11 localizations after blue light irradiation. Arrows and arrowheads indicate colocalization of Rab11 and Rh1 at the rhabdomere base and in cytoplasmic organelles. (E) Rh1 and Rab11 intensely colocalize at the base of rhabdomere 60 minutes after blue light. (F) Most of Rh1 positive organelles are Golgi bodies. Scale bar: 2 µm in A-E.

View larger version (114K): [in a new window]   Fig. 5. Rh1 transport requires Rab11. (A) Rab11-reduced photoreceptors (arrows) in a mosaic eye triple stained for Rh1 (blue), Rab11 (green) and F-actin (red) show disrupted RLVs and Rh1 dispersed throughout the photoreceptor cytoplasm. Rh1 accumulation in rhabdomeres and rhabdomere size is decreased in the Rab11-reduced photoreceptors. (B) Electron micrographs of mosaic ommatidia show photoreceptors with normal MVBs (arrows) and vesicle-filled cells lacking MVBs (arrowheads). Other internal membranes appear normal. (C,D) Higher magnification of abnormal cytoplasmic vesicles shows unusual irregular MVB vesiculation. These appear incomplete, leaving openings, or iterated, resulting in vesicles within endovesicles not seen in normal MVBs. Scale bar: 5 µm in A; 2 µm in B; 500 nm in C,D.

View larger version (102K): [in a new window]   Fig. 6. Rab11 RNAi inhibits Rh1 transport. (A) In control Hs-Gal4 flies, heat shocked at 27% pd, then fixed and stained at 85% pd, Rh1 (blue) transport is normal. Rh1 is present in cytoplasmic RLVs and in rhabdomeres stained red using rhodamine phalloidin. Rab11-positive (green) vesicles are distributed throughout the cytoplasm. (B) In experimental Hs-Gal4, dsRNA flies, Rh1 is dispersed throughout photoreceptor cytoplasm and delivery to the rhabdomere is inhibited. (C) Shown separately, Rab11 immunostaining of control flies is brighter than D, Hs-Gal4, Rab11 dsRNA flies. The R2 photoreceptor (arrow) shows a higher level of Rab11 signal and detectable Rh1 delivery to the rhabdomere. R7, which also shows Rab11 signal here, does not express Rh1; transport of its rhodopsin, either Rh3 or Rh5, cannot be determined. (E) Electron micrographs of Hs-Gal4, UAS-Rab11 dsRNA pupae. Rhabdomere size is small. Abnormal cytoplasmic vesicles (arrowheads) proliferate and MVBs are absent. Scale bar: 2 µm in A-E.

View larger version (131K): [in a new window]   Fig. 7. Dominant-negative Rab11 inhibits TRP and Rh1 transport, and disperses RLVs and MVBs. (A) Wild-type rhabdomeres are TRP immunopositive at 64% pd. (B) Rab11N124I expression beginning at 50% pd prevents TRP transport to the rhabdomere; TRP is dispersed throughout photoreceptor cytoplasm. (C) There is no TRP containing vesicles in Rab5N142I-expressing cells, but TRP is normally transported to the rhabdomere. (D) Rab11N124I and Rab5N142I co-expression beginning at 50% pd prevents TRP transport to the rhabdomere. (E) Wild-type rhabdomeres are Rh1 immunopositive at 83% pd; cytoplasmic Rh1 is present in RLVs. (F) Rab11N124I expression beginning at 70% pd prevents Rh1 transport to the rhabdomere. RLVs are lost and Rh1 accumulates diffusely throughout photoreceptors. (G) RLVs are absent in Rab5N142I-expressing cells, but Rh1 is normally transported to the rhabdomere. (H) Rab11N124I and Rab5N142I co-expression beginning at 70% pd prevents Rh1 transport to the rhabdomere. RLVs are lost and Rh1 accumulates diffusely throughout photoreceptors, similar to Rab11N124I single expression. (I,J) Golgi morphologies are not affected by Rab11N124I expression. (K-N) Electron micrographs of 65% pd pupae heat shocked from 50% pd (K,L) or 83% pd pupae heat shocked from 70% pd (M,N). Rab11N124I expression reduces rhabdomere size and causes proliferation of abnormal cytoplasmic vesicles (arrowheads). MVBs are evident in control photoreceptors (arrows), but are absent in photoreceptors expressing Rab11N124I. IRS is reduced by Rab11N124I expression. (O) Rab11N124I expression decreases RLVs and MVBs in parallel (see Materials and methods). Scale bar: 2 µm in A-N.

View larger version (147K): [in a new window]   Fig. 8. Rab11N124I inhibits endocytosis in Garland cells. (A,B) Confocal cross sections of wild-type or Rab11N124I-expressing Garland cells after 10 minutes TR-avidin uptake. Many vesicles stain in wild-type Garland cells. However, there is no avidin uptake in Rab11N124I expressing Garland cells. (C,D) Electron micrographs of Garland cells show labrynthine channels, deep invaginations of the plasma membrane. These are lost in Rab11N124I-expressing Garland cells. Scale bar: 5 µm in A,B; 500 nm in C,D.