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Research Article
The roles of Syx5 in Golgi morphology and Rhodopsin transport in Drosophila photoreceptors
Takunori Satoh, Yuri Nakamura, Akiko K. Satoh
Biology Open 2016 5: 1420-1430; doi: 10.1242/bio.020958

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  • Fig. 1.
    Fig. 1.

    Failure of Rh1 accumulation on the rhabdomeres in two insertional mutants of Syx5. (A) Schematic view of Syx5 gene on the genome. Insertion positions for two mutants are indicated with red triangles. Gray bars represent the mRNA sequences, and the blue bars represent the coding sequences. (B-E) Visualization of endogenous Rh1 by Arrestin2::GFP (green) and wild-type cell marker, RFP (red) in Syx5 EP2313 (B), Syx5 EY07901 (C), Syx5 EP2313 ex2 (D), and Syx5 EY07901 ex1 (E) mosaic retinas using a water immersion technique. Scale bar: 5 μm. Numbers of the samples observed were shown in the top left corner of the images.

  • Fig. 2.
    Fig. 2.

    Levels of membrane proteins, Rh1, Na+K+ATPase, TRP, and Chp and a secretary protein, Eys, are reduced in Syx5-deficient photoreceptors. Immunostaining of Syx5 EP2313 (A,C,E), and Syx5 EP2313 ex2 (B,D,F) mosaic retinas. RFP (red) marks wild-type cells. Asterisks indicate Syx5 EP2313, or Syx5 EP2313 ex2 homozygous photoreceptors. (A,B) Immunostaining with anti-Rh1 (blue) and anti-Na+K+ATPase (green) antibodies. (C,D) Immunostaining with anti-TRP (blue) and anti-Eys (green) antibodies. (E,F) Immunostaining with anti-DE-Cad (blue) and anti-Chp (green) antibodies. Scale bar: 5 μm. Numbers of the samples observed were shown in the top-left corner of the composite images.

  • Fig. 3.
    Fig. 3.

    Syx5 localizes on the cis side of Golgi units. Immunostaining (left panels) of the retinas expressing both CFP::GalT and Syx5::Myc (A,D,E) and Syx5::Myc only (B,C) by GMR-Gal4 driver and plots representing their intensities (right) are shown. (A) CFP::GalT (green), Syx5::Myc (blue) and phalloidin (red). Arrows mark co-localization of CFP::GalT and Syx5::Myc. Asterisks mark auto-fluorescence from pigment granules. (B) Rab6 (blue), GM130 (green), and Syx5::Myc (red). (C) Chc (blue), anti-p120 (green), and Syx5::Myc (red). (D) CFP::GalT (blue), Rab1 (green), and Syx5::Myc (red). (E) CFP::GalT (blue), GM130 (green), and Syx5::Myc (red). Arrows in left panels in B-E indicate the medial-Golgi cisternae. Arrows in right panels in B-E indicate the X-axes of the florescent intensity plots. Scale bar: 5 μm in A, 1 μm in B-E. 2 to 4 samples were observed for each of stainings.

  • Fig. 4.
    Fig. 4.

    Marked increase in cis-Golgi markers, GM-130, and Rab1, in Syx5- deficient photoreceptors. Syx5EP2313 (A,C,E,G) or Syx5EP2313 ex2 (B,D,F,H) mosaic retinas were immunostained with one of the following antibodies: anti-GM130 (A,B), anti-Rab1(C,D), anti-MPPE (E,F), or anti-GFP antibody (G,H), along with phalloidin (A-H). RFP (red) marks wild-type cells. Asterisks indicate Syx5EP2313 or Syx5EP2313 ex2 homozygous photoreceptors. In G and H, peripheral photoreceptors in mosaic retinas express GalT::CFP. (A,B) GM130 (blue) and phalloidin (green). (C,D) Rab1 (blue) and phalloidin (green). (E,F) MPPE (blue) and phalloidin (green). (G,H) GFP (blue) and phalloidin (green). Scale bar: 5 μm. Numbers of the samples observed were shown in the top left corner of the composite images.

  • Fig. 5.
    Fig. 5.

    Kinetics of Rh1 transport in Syx5EP2313 mosaic retinas. Syx5EP2313 mosaic retinas were immunostained with antibodies described below. RFP (red) marks wild-type cells. Asterisks indicate Syx5EP2313 homozygous photoreceptors. (A) Immunostaining before BLICS with anti-Rh1 (blue) and anti-Cnx (green) antibodies. Cnx is an ER marker. (B-E) Immunostaining of Syx5EP2313 mosaic retinas 30 min (B), 60 min (C), 120 min (D), and 180 min (E) after BLICS, using anti-Rh1 (blue) and anti-GM130 (green) antibodies. GM130 is a Golgi marker. (F) Immunostaining of Syx5EP2313 mosaic retinas with anti-Rh1 (blue) and anti-Rab7 (green) antibodies 180 min after BLICS. Rab7 is a late endosome marker. Scale bar: 5 μm. Numbers of the samples observed were shown in the top left corner of the composite images.

  • Fig. 6.
    Fig. 6.

    TRP and Eys are degraded by ERAD system in Syx5-deficient photoreceptors. EDEM2 DG03809 and Syx5EP2313 mosaic retinas with a viable EDEM1EP1588 homozygous mutation were immunostained by the indicated antibodies. RFP (red) marks EDEM1EP1588 single mutant cells. Asterisks indicate EDEM1EP1588, EDEM2 DG03809 and Syx5 EP2313 triple deficient photoreceptors. (A) Anti-NinaA (green) and anti-Eys (blue) antibodies. (B) Anti-dPob (green) and anti-TRP (blue) antibodies. (C) Anti- Na+K+-ATPase (green) and anti-Rh1 (blue) antibodies. Scale bar: 5μm. Three samples were observed for each of stainings.

  • Fig. 7.
    Fig. 7.

    Massive accumulation of vesicles between ER and Golgi units in Syx5-deficient photoreceptors. Flies were reared in the dark and the retinal samples were fixed at late pupal stage. To avoid light-dependent Rh1 endocytosis, fixation was performed within 3 min of transferring the pupae to light. (A-C) Electron microscopy of the ommatidia with the following genotypes: wild-type (A), Syx5EP2313 homozygous ommatidia (B), Syx5EP2313 ex2 homozygous ommatidia (C). (D) Golgi units in wild-type photoreceptors. (E) Vesicle clusters in Syx5EP2313 homozygous photoreceptors. (F) Golgi units in Syx5EP2313 ex2 homozygous photoreceptors. (G) Quantifications of the number of the vesicle clusters and the Golgi units in the cross section of a photoreceptor. The wild-type: 0.07 (s.d.±0.05) vesicle clusters and 0.17 (s.d.±0.05) Golgi units, Syx5EP2313/Syx5EP2313: 0.25 (s.d.±0.02) vesicle clusters and no Golgi units, Syx5EP2313 ex2/Syx5EP2313 ex2: 0.07 (s.d.±0.05) vesicle clusters and 0.13 (s.d.±0.05) Golgi units. *P<0.05, **P<0.1 (Student's t-test between samples). Scale bar: 2 μm (A-C), 300 nm (D-F). Three samples were observed for each of genotypes.

  • Fig. 8.
    Fig. 8.

    Proposed model of Syx5 function and three polarized transport pathways. Schematic diagram for polarized transport pathways in wild-type (left) and Syx5EP2313 mutant (right) photoreceptors. Membrane proteins synthesized on the ER membrane are transported to cis-Golgi cisternae by Rab1 (Satoh et al., 1997), Syx5, and possibly by Sec22 (Zhao et al., 2015). Probably after Gos28 dependent processes (Rosenbaum et al., 2014), basolateral membrane proteins are sorted by AP1 at trans-Golgi/TGN (Satoh et al., 2013) and the two apical membrane proteins are transported together to the recycling endosome (RE) by Rab6 (Iwanami et al., 2016). There is another round of sorting for the two apical pathways at the RE. Rab11, dRip11, and MyoV then mediate the transport of post-Golgi vesicles carrying rhabdomere proteins (Li et al., 2007; Satoh et al., 2005). Finally, the exocyst complex tethers these post-Golgi vesicles to the base of the rhabdomeres (Beronja et al., 2005).

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Research Article
The roles of Syx5 in Golgi morphology and Rhodopsin transport in Drosophila photoreceptors
Takunori Satoh, Yuri Nakamura, Akiko K. Satoh
Biology Open 2016 5: 1420-1430; doi: 10.1242/bio.020958
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