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Figures
- Fig. 1.
Screening and identification of PIG genes as essential components for Rh1 transport. (A) Schematic view of a Drosophila ommatidium cross section showing three different plasma membrane domains for each photoreceptor. (B) Immunostaining of an ommatidial cross section. Three membrane proteins, Rh1 (blue), Crb (purple) and Na+K+-ATPase (green), are localized on the different membrane domains. (C) Electron microscopic cross section of an ommatidium. (D) KY111587 mosaic retina. RFP (red) marks wild-type cells; RFP expression levels in each cell are variable. Arrestin2::GFP (green) shows endogenous Rh1 localization. Scale bar: 10 μm. (E) KY114591 mosaic retina was stained by Rh1 (green) and F-actin (magenta). Asterisks show mutant photoreceptors. Scale bar: 5 μm. (F-I) Rh1 immunostaining of a cross section of an ommatidium from PIG-null mosaic eyes. Asterisks show PIG-null photoreceptors. Scale bar: 5 μm.
- Fig. 2.
Essential roles of PIG genes in Chp transport and rhabdomere formation. (A) PIG-C f05249 mosaic Drosophila eye immunostained by anti-Chp (green) and anti-NinaA antibodies (magenta). Asterisks show PIG-C-null photoreceptors. (B) PIG-V f05618 mosaic ommatidium by electron microscopy. R1, R3 and R7 photoreceptors are wild type; R2, R4, R5 and R6 photoreceptors are PIG-V-null mutants. Adherens junctions (arrowheads) and basolateral membrane (white arrows) are indicated. Inset: High-magnification image of an R6 rhabdomere; black arrows show cross sections of the microvilli. (C) chp2 mutant ommatidium by electron microscopy. (D) Chp immunostaining of chp2 mutant ommatidium. (E) Rh1 immunostaining of chp2 mutant ommatidium. Scale bars: 2 μm.
- Fig. 3.
Kinetics of Rh1 transport. (A,B) Immunostaining of Rh1 before and after BLICS in Drosophila wild-type (A) and PIG-C-mutant ommatidia (B). (C) Immunostaining of Rh1 (green) and Golgi marker, GM130 (magenta) 40 min after BLICS in PIG-C-mutant ommatidia. Asterisks show PIG-C–null photoreceptors. Arrows indicate Golgi units. (D,E) Immunostaining of Rh1 (green) and the endolysosome marker Rab7 (magenta) at 60 minutes after BLICS in wild-type (D) and PIG-C-mutant (E) ommatidia. Arrows show colocalization. (F) Projection image from five slices at 0.5-μm intervals of PIG-C-null mosaic eye with lt1 homozygous background. Asterisks show PIG-C-null lt1 double-mutant photoreceptors. lt1 single-mutant photoreceptors and R4-R6 photoreceptors of the right-hand ommatidium accumulated Rh1 in the rhabdomeres; no cytoplasmic Rh1 staining is visible. Arrows indicate the rhabdomeres of wild-type photoreceptors. (G) PIG-V-null mosaic eye expressing Rip11 dominant-negative proteins by Rh1Gal4 driver. Asterisks show PIG-V-null photoreceptors. Scale bars: 2 μm.
- Fig. 4.
Mislocalization of Na+K+-ATPase and Crb to the rhabdomeres in GPI-deficient photoreceptors. Immunostaining of Drosophila mosaic retina containing both wild-type and mutant photoreceptors. Asterisks show mutant photoreceptors. Mutant alleles and antibodies used are specified below. (A) PIG-C f05249 mosaic retina. Na+K+-ATPase (green) and Rh1 (magenta). (B) PIG-V f05618 mosaic retina. Crb staining. (C) PIG-C f05249 mosaic retina. DE-cadherin (DE-cad) staining. (D) crb11A22 mosaic retina. Na+K+-ATPase (green) and Rh1 (magenta). The stalk membrane in crb11A22 mutant photoreceptor is short, as shown by Pellikka et al. (Pellikka et al. 2002). (E) PIG-C f05249 mosaic retina. TRP (green) and Rh1 (magenta). (F) PIG-C f05249 mosaic retina. Syx1A (green) and Rh1 (magenta). Scale bars: 2 μm.
- Fig. 5.
Deficiencies of Rh1 and Na+K+-ATPase transport in earlier developmental stages. (A-C) Immunostaining of Drosophila mosaic retina containing both wild-type and PIG-C f05249 photoreceptors. Asterisks show mutant photoreceptors. Developmental stages and antibodies used are specified below: (A) 55% pd retina stained by Na+K+-ATPase (green) and TRP (magenta). (B) 73% pd retina stained by Na+K+-ATPase (green) and Rh1 (magenta). (C) 73% pd retina stained by Chp (green) and TRP (magenta). (D,E) Observation of PIG-C f05249 mosaic ommatidium at 58% (D) and 73% (E) by electron microscopy. R5 and R6 photoreceptors in D and R1, R4, R6 and R7 photoreceptors in E are PIG-C-null mutants. Insets are high-magnification images of R6 rhabdomeres. Scale bars: 2 μm.
- Fig. 6.
Lipid raft-independent sorting of Rh1 at the TGN. (A) Filipin staining in Drosophila mosaic retina containing both wild-type and PIG-C-null mutant photoreceptors. Asterisks show PIG-C mutant cells. Scale bar: 5 μm. (B) Immunoblot analysis of the associations of TRP and Rh1 with DRM in wild-type and dominant-negative Rip11-expressing retina. Membranes of homogenates from illuminated wild-type heads (lanes 1 and 3) and illuminated Rip11DN/Rh1Gal4 heads (lanes 2 and 4) were separated into DRMs and detergent-soluble membranes (DSMs). (C) Immunostaining of Na+K+-ATPase (green) and Rh1 (magenta) in brn9PP4, egh1.6P6 double-mutant ommatidium. Scale bar: 2 μm. (D) Formation of GlcT-1 deletion mutants. Two transposon insertion lines, GlcT-1e2644 and GlcT-1e2597, containing the FRT sequence were used to make GlcT-1 deletion mutants using the FLP/FRT method. GlcT-1Δ8 includes a part of synaptojanin in addition to GlcT-1. (E) Immunostaining of a cross section of a GlucT-1Δl8 ommatidium. Rh1 (magenta) and Na+K+-ATPase (green). Scale bar: 2 μm.
- Fig. 7.
Model of protein sorting at the TGN. In the wild-type TGN, one or more GPI-anchored protein localizes to the neck and/or inside budding post-Golgi vesicles destined for the rhabdomeres and recruits rhabdomere proteins but excludes others. By contrast, GPI-deficient TGNs do not contain GPI-anchored proteins and do not perform protein sorting. Consequently, most Rh1 is sent to the endocytotic pathway and degraded by lysosomes whereas small amounts of Rh1, Na+K+-ATPase and Crb are loaded into post-Golgi vesicles for delivery to the rhabdomeres.
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