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First published online 26 July 2005
doi: 10.1242/jcs.02502

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Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules

¹ Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA
² University of Zürich, Institute of Physiology, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

View larger version (31K): [in a new window]   Fig. 1. dVps16A physically interacts with Dor and Car. (A) Cytosol from S2 cells was fractionated by sucrose density gradient centrifugation (10-20%). Proteins in each fraction were detected by western blotting with the indicated antibodies. Sedimentation coefficients (S20w) are indicated for two markers. (B) Proteins were immunoprecipitated from S2-cell cytosol with anti-Dor, anti-Car, anti-dVps16A, or anti-Boss antibodies as controls. Immunoprecipitates were detected on western blots with the indicated antibodies to Dor, dVps16 or Car.

View larger version (56K): [in a new window]   Fig. 2. dVps16A and dVps16B bind different Vps33 homologs. (A) The domain of highest similarity between the two Vps16 homologs in the Drosophila genome is the Vps16-C domain [Pfam04840 in the Conserved Domain Database (Marchler-Bauer et al., 2005)]. (B) Sequences of Vps16 proteins were aligned using ClustalW and a phylogenetic tree was constructed using 1000 bootstraps. Accession numbers: Tetraodon nigroviridis Vps16B (TnVps16: CAF92974, zebrafish Vps16B (DrVps16B: AAQ945721), human Vps16s (Hsvps16A: AAG346781 and HsVps16B: AAD096241) and mouse Vps16s (MmVps16A: AAH256261 and MmVps16B: BAC326801), Anopheles gambia (AgVps16A: XP_310557.1 and AgVps16B: XP_321923.1), Drosophila melanogaster (DmVps16A: NP_649877.1 and DmVps16B: AAF569461) Drosophila pseudoobscura (DpVps16B: EAL281241), Arabidopsis thaliana VACUOLELESS1 (AtVps16: AAM981051) and Saccharomyces cervisiae Vps16p (ScVps16p: NP_015280.1). (C) Myc-tagged Rop, dVps33B or Car proteins were co-expressed in S2 cells with HA-tagged dVsp16A and detected in input samples by immunoblots (IB) with anti-dVps16A or anti-Myc antibodies. Proteins were immunoprecipitated with anti-dVps16A antibodies. Immunoprecipitated proteins (IP) were detected with anti-Myc antibodies. (D) HA-tagged Rop, dVps33B or Car proteins were co-expressed in S2 cells with a Myc-tagged dVsp16A and detected in input samples by immunoblots (IB) with anti-HA or anti-Myc antibodies. Proteins were immunoprecipitated with anti-Myc antibodies. Immunoprecipitated proteins (IP) were detected with anti-HA antibodies.

View larger version (57K): [in a new window]   Fig. 3. A small domain in dVps16A is required for binding to Car. The binding of different dVps16A truncations (outlined in panel E) to Car was evaluated by co-immunoprecipitation experiments. (A) HA-tagged truncations of dVps16A were co-expressed with HA-Car and in whole cell extracts (input) detected with HA antibodies. After IP with anti-Car antibodies only dVps16A-C was co-immunoprecipitated. (B) HA- or Myc-tagged truncations of dVps16A were co-expressed with HA-Car protein and detected in input samples with anti-Vps16 antibodies. After IP with anti-dVps16A antibodies, HA-Car was co-immunoprecipitated only with full-length dVps16A or dVps16A-C. (C) HA-tagged Rop, dVps33B or Car were co-expressed in S2 cells with the Myc-tagged dVsp16A-C domain and detected in input samples with anti-dVps16A or anti-HA antibodies. After IP with anti-dVsp16A antibodies, proteins were detected with anti-HA antibodies. (D) Myc-tagged truncations of dVps16 were co-expressed with HA-Car. After IP with anti-Myc antibodies co-precipitated Car was detected with anti-Car antibodies. (E) The summary of the co-immunoprecipitation results indicates that only a small domain of dVps16A from aa 489-555 is required for binding to Car.

View larger version (41K): [in a new window]   Fig. 4. dVps16 proteins bind to Dor. The binding of different dVps16A truncations (outlined in panel E) to Dor was evaluated by co-immunoprecipitation experiments. (A) Myc-tagged full-length or truncated dVps16A proteins were co-expressed with Myc-Dor and detected with Myc antibodies in whole cell extracts (Input). After IP with anti-Dor antibodies, only the co-immunoprecipitated full-length dVps16A protein (FL) or the C-terminal domain (-C) were detected by anti-Myc antibodies. (B,C) Myc-tagged truncated dVps16A proteins were co-expressed with Myc-Dor and in input samples detected with Myc antibodies. (B) After IP with anti-Dor antibodies, only dVps16A-C2 (-C2) and dVps16A-C3 (-C3) were co-immunoprecipitated as detected by anti-Myc antibodies, but not the shorter dVps16A-C1 (-C1). (C) After IP with anti-Dor antibodies, only the dVps16A-C (C) truncation co-immunoprecipitated, but not the shorter dVps16A-C2 or -C3 peptides. (D) Myc-tagged Dor and dVps16B were expressed alone or together in S2 cells. After IP with anti-Dor antibodies, dVps16B was only pulled down when co-expressed with Dor protein. (E) The summary of the co-immunoprecipitation results indicates that a small domain of dVps16A from amino acid 489 to 633 is required for binding to Dor.

View larger version (32K): [in a new window]   Fig. 5. RNAi-induced knockdown of dVps16A. (A) dVps16A is expressed throughout development. Extracts for western blotting were prepared from embryos (E), first and third instar larvae (L1 and L3), pupae (P) and adult females (F) and males (M). (B) Western blots of extracts from wild-type or dVps16A RNAi knockdown larvae (0) or 1-, 2- and 3-day-old pupae (1, 2 and 3). In addition to dVps16A, Dor was drastically reduced after dVps16A knockdown, but Car, dSyntaxin7, and tubulin levels were unchanged. (C) Western blots of extracts from adult wild-type, dor1, dor4 or car1 flies did not show a reduction in dVps16A, although Dor protein levels were reduced in the dor alleles. (D) Western blots from extracts of day-four wild-type or dor8 third instar larvae show the loss of Dor and the reduced expression of dVps16A whereas Car, Hook, dSyn7 or tubulin were unchanged.

View larger version (132K): [in a new window]   Fig. 6. dVps16C is required for eye development. (A-C) Micrographs of compound eyes from wild type (A), ey-Gal4&GMR-Gal4>dVps16A-RNAi (B), or white1118 flies (C). (D-I) Micrograph of sections of plastic embedded wild type (D,G), or ey-Gal4&GMR-Gal4>dVps16A-RNAi (E,H) or white1118 eyes (F,I). Lenses (L) and pseudocones (P) are indicated in (D). Arrows in E point to the dense material next to a malformed pseudocone. Arrowheads in panels G-I point to wild-type (G,I) or degenerate (H) rhabdomeres. Scale bar in I represents 15 µm in panels D-I.

View larger version (150K): [in a new window]   Fig. 7. Ultrastructural defects in compound eyes lacking dVps16A. Electron micrographs show details of compound eyes from wild type (A,E), white1118 (C,F) or ey-Gal4 and GMR-Gal4>dVps16A-RNAi flies (B,D,G). Lenses (L) and pseudocones (P) are indicated in A and B. Primary pigment cells (PPC) face the pseudocones and contain type I granules, which appear electron dense. The larger type II granules are predominant in secondary pigment cells (SPC); they appear translucent because their content of pigments were washed out during the embedding process (A). Both types of pigment granules are absent after dVps16A knockdown (B). Rhabdomeres (Rh) contain the phototransduction cascade and are composed of tightly packed microvilli (C). After dVps16A knockdown (D), most rhabdomeres are only detected as remnants (arrows in D) in degenerating photoreceptors cells full of vacuoles and autophagosomes. The inset in D shows an example of an autophagosome with an internal piece of rough endoplasmic reticulum. (E-G) Photoreceptor cells (R) of neighboring ommatidia are separated by a layer of pigment cells. In wild-type eyes (E), the width of pigment cells (arrows) between photoreceptor cells (R) is dominated by type II pigment granules. These granules are essentially absent in white1118 mutant eyes (F) resulting in a thin layer of pigment cells (between the arrows in F). After dVps16A knockdown (G), pigment cells are full of vacuoles and autophagosomes resulting in expanded width of the pigment cell layer (indicated between the arrows in G). Photoreceptors (R) in panel G were recognized by the remnants of rhabdomeres outside the field shown. Scale bar: 2 µm in A and G, 1 µm in B-F, and 100 nm for the inset in D.

View larger version (97K): [in a new window]   Fig. 8. Cells lacking dVps16A are deficient in lysosomal delivery. Eye discs from wild-type (A,C) or dVps16A knockdown (B,D) third instar larvae were stained for Boss (A,B) or Scabrous (C,D). As indicated in the inset in panel A, the large dots of Boss staining represent its expression on the apical surface of R8 cells in each ommatidium whereas the small dots next to it (arrows in A and B) show the ligand after its internalization into the neighboring R7 cells (Cagan et al., 1992). Note that, after dVps16A knockdown, R8 surface levels are unchanged whereas the level of Boss staining in R7 cells is increased. Similarly, posterior to the furrow (arrowhead in panel C and D), Scabrous protein is rapidly degraded in wild-type cells (C) but after dVps16A knockdown Scabrous levels remain high (D). (E) In wild-type eye discs, a GFP-LAMP fusion protein is targeted to lysosomes and efficiently degraded. (F) After dVps16A knockdown, this degradation is inhibited and GFP-LAMP accumulates in vesicles. All images are projections of confocal z-series. Posterior is to the left. Scale bar: in F is 7 µm in A and B, 20 µm in C and D, and 10 µm in E and F.

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