Journal: PLoS Genetics

Article Title: Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors

doi: 10.1371/journal.pgen.1007150

Figure Lengend Snippet: Rab8 coats opsin-carrier-vesicles and targets them to the periciliary membrane, where their fusion is mediated by the Exocyst and by SNAREs including SNAP25 and Syntaxin 3 (and by an as yet undefined v-SNARE). CC2D2A at the transition zone is required for correct localization of SNAP25 to the periciliary membrane and provides a docking point for incoming vesicles through its interaction with NINL, which also binds the dynein motor and the Rab8 effector MICAL3. Thus, CC2D2A plays a role in concentrating all components required for correct vesicle fusion at the periciliary membrane.

Article Snippet: Primary antibodies were rabbit anti-Syntaxin3 (1:400, Alomone labs, ANR-005), rabbit anti-SNAP25 (1:1000, StressGen Biotechnologies, VAP-SV002), mouse monoclonal anti-Rab8a (1:100, Novus Biologicals, clone 3G1), rabbit anti-Cacna1fa (1:5000, a gift from Michael Taylor, University of Wisconsin [ ]), rabbit anti-blue opsin (1:250, gift from David Hyde) and rabbit anti green opsin (1:400, gift from David Hyde, University of Notre Dame).

Techniques: Membrane, Docking Assay

Journal: Journal of Cell Science

Article Title: An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

doi: 10.1242/jcs.222034

Figure Lengend Snippet: VAMP7 is localized at the Golgi/TGN and on ciliary-targeted RTCs, and forms a complex with syntaxin 3 and SNAP-25. (A) Diagram of the photoreceptor cell. Ciliary targeting of rhodopsin through the RIS proceeds from the Golgi (G) in the myoid region (M), through the ellipsoid region (E), which is crowded with mitochondria, to the base of the cilium (arrow), for the delivery to ROS. AJ, adherens junction. (B) Confocal optical section of a retina labeled with mouse (m) anti-VAMP7 (green), annotated as in A. G, Golgi; m, mitochondria. The dotted line represents the outer limiting membrane (OLM), comprising adherens junctions (AJ) between photoreceptors and retinal glial cells (Müller cells). Nuclei (N) were stained with TO-PRO-3 (blue). (C) Two confocal sections from a Z-stack of a retina labeled with anti-VAMP7 (m) (green) and rabbit (r) anti-ASAP1 (red). Scale bar: 5 µm (for B), 7 µm (for C). (D) Following pulse-chase labeling and retinal subcellular fractionation, the distribution of rhodopsin was determined by SDS-PAGE and autoradiography (of [35S]rhodopsin). A duplicate gel was tested by immunoblotting on a single blot, as indicated. (E–G) VAMP7+rhodopsin interaction sites (red dots) detected by PLA using anti-rhodopsin mAb11D5(m) and anti-VAMP7(m) conjugated to PLUS and MINUS probes, respectively, as described in the Materials and Methods (E). PLA for VAMP7(m)+ASAP1(r) (F). PLA for VAMP7(m) and phosphorylated tyrosine (p-tyr)(r) (G). (H–J) PLA with a single antibody to rhodopsin (H), ASAP1 (I) or VAMP7 (J). No red fluorescence was detected. (K,L) PLA for VAMP7(m) and syntaxin 3 (STX3)(r) (K), or SNAP-25(r) (L). Cells were visualized by DIC. Arrows in K and L indicate sites of colocalization. Scale bar: 5 µm (for E–G,K,L), 10 µm (H–J). (M) Post-nuclear supernatant (PNS) (0.1 retina) was immunoprecipitated (IP) with anti-syntaxin 3(r) and IPs were analyzed by immunoblotting (IB) as indicated.

Article Snippet: Rabbit polyclonal antibodies were: anti-syntaxin 3 (110 033, Synaptic Systems, Germany), anti-ASAP1 ( Randazzo et al., 2000 ) (a gift from Paul Randazzo, National Cancer Institute, Bethesda, MD), anti-Arf4 ( Mazelova et al., 2009a ), anti-Rab8 (R5530, Sigma), anti-Rabin8 ( Hattula et al., 2002 ) (a gift from Johan Peranen, University of Helsinki, Finland), anti-phosphotyrosine (ab50722, Abcam), anti-Rab6 (sc-310, Santa Cruz Biotechnology), anti-VARP (24034-1-AP, Proteintech), anti-LAMP1 (ab24170, Abcam), anti-βCOP (G-2279, Sigma), anti-LC3 (ab128025, Abcam), anti-GST (G7781, Sigma), anti-6His (10001-0-AP, Proteintech), anti-GFP (ab290, Abcam); rabbit and goat anti-FIP3 antibodies, and FIP3F1 were gifts from Rytis Prekeris, University of Colorado, Denver, CO. Duolink in situ Detection Reagent Red (excitation, 594 nm; emission, 624 nm) was from Sigma-Aldrich (DUO92008).

Techniques: Labeling, Staining, Pulse Chase, Fractionation, SDS Page, Autoradiography, Western Blot, Fluorescence, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

doi: 10.1242/jcs.222034

Figure Lengend Snippet: Aberrant vesicular structures containing GFP–VAMP7-R150E are deficient in typical regulators of intracellular trafficking. (A–C) Transgenic X. laevis expressing the GFP–VAMP7-R150E mutant and examined by CLEM. Retinas were first examined by confocal microscopy (A) and then processed and analyzed by EM (B). Six cells in the confocal optical section (a–f) were matched to the cells (a–f) in the EM micrograph. The asterisk marks a control photoreceptor (f), not expressing GFP–VAMP7-R150E. The boxed area in B is magnified in C. m, mitochondria, Ly, lysosome. (D–F) Transgenic retinas expressing GFP–VAMP7-R150E fusion protein (green) labeled with anti-LAMP1 (r) (D), anti-GFP (r) (E), or anti-βCOP (r) (F). Arrows point to localization of the proteins examined. (G) PLA between: Rab6 (r) and anti-GFP (m) detecting GFP–VAMP7-R150E. (H,I) EM of the photoreceptor cell expressing the R150E mutant (H). The boxed area is magnified in I. Arrows point to vesicular structures with electron-dense content. (J–M) Transgenic GFP–VAMP7-R150E retinas labeled with anti-IRBP (m) (J), anti-ASAP1 (r) (K), anti-FIP3 (r) (L) and anti-VARP (r) (M). (N) PLA between syntaxin 3 (r) and anti-GFP (m). (O) Retinas labeled with anti-peripherin (m) (red). (P,Q) The f1 generation of GFP–VAMP7-R150E-expressing retinas stained with WGA (red). (R) Labeling with anti-LC3 (r) (red, arrows). (S) Epithelial cells outside of Xenopus eye labeled with anti-LC3 (r) (red, arrows point to autophagosomes). (T,U) Labeling with anti-ATG16L1 (m) (red, arrow) (T), or anti-ubiquitin (m) (U). Cells were visualized by DIC. (V) The f1 generation of GFP-VAMP7-Y45E-expressing retinas stained with WGA (red). Scale bar: 25 µm (for A); 5 µm (for B,D–H); 10 µm (for J–N); 1 µm (for C,I); 12 µm (for O,R,S,T,U); 20 µm (for Q); 50 µm (for P,V). M, myoid region; E, ellipsoid region; G, Golgi, N, nucleus; m, mouse antibody; r, rabbit antibody.

Article Snippet: Rabbit polyclonal antibodies were: anti-syntaxin 3 (110 033, Synaptic Systems, Germany), anti-ASAP1 ( Randazzo et al., 2000 ) (a gift from Paul Randazzo, National Cancer Institute, Bethesda, MD), anti-Arf4 ( Mazelova et al., 2009a ), anti-Rab8 (R5530, Sigma), anti-Rabin8 ( Hattula et al., 2002 ) (a gift from Johan Peranen, University of Helsinki, Finland), anti-phosphotyrosine (ab50722, Abcam), anti-Rab6 (sc-310, Santa Cruz Biotechnology), anti-VARP (24034-1-AP, Proteintech), anti-LAMP1 (ab24170, Abcam), anti-βCOP (G-2279, Sigma), anti-LC3 (ab128025, Abcam), anti-GST (G7781, Sigma), anti-6His (10001-0-AP, Proteintech), anti-GFP (ab290, Abcam); rabbit and goat anti-FIP3 antibodies, and FIP3F1 were gifts from Rytis Prekeris, University of Colorado, Denver, CO. Duolink in situ Detection Reagent Red (excitation, 594 nm; emission, 624 nm) was from Sigma-Aldrich (DUO92008).

Techniques: Transgenic Assay, Expressing, Mutagenesis, Confocal Microscopy, Labeling, Staining