Research Article 2003 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid, controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments

Jana Mazelova1, Nancy Ransom1, Lisa Astuto-Gribble1, Michael C. Wilson2 and Dusanka Deretic1,3,* 1Department of Surgery, Division of Ophthalmology, 2Department of Neuroscience and 3Department of Cell Biology and Physiology, University of New Mexico, Albuquerque, New Mexico 87131 *Author for correspondence (e-mail: [email protected])

Accepted 2 March 2009 Journal of Cell Science 122, 2003-2013 Published by The Company of Biologists 2009 doi:10.1242/jcs.039982

Summary The biogenesis of cilia-derived sensory organelles, the 25 and syntaxin 3 at the base of the cilium, which results in the photoreceptor rod outer segments (ROS), is mediated by increased delivery of membrane to the ROS. This is particularly rhodopsin transport carriers (RTCs). The small GTPase Rab8 evident in propranolol-treated retinas, in which the DHA- regulates ciliary targeting of RTCs, but their specific fusion sites mediated increase in SNARE pairing overcomes the tethering have not been characterized. Here, we report that the Sec6/8 block, including dissociation of Sec8 into the cytosol. Together, complex, or exocyst, is a candidate effector for Rab8. We also our data indicate that the Sec6/8 complex, syntaxin 3 and SNAP- show that the Qa-SNARE syntaxin 3 is present in the rod inner 25 regulate rhodopsin delivery, probably by mediating docking segment (RIS) plasma membrane at the base of the cilium and and fusion of RTCs. We show further that DHA, an essential displays a microtubule-dependent concentration gradient, polyunsaturated fatty acid of the ROS, increases pairing of whereas the Qbc-SNARE SNAP-25 is uniformly distributed in syntaxin 3 and SNAP-25 to regulate expansion of the ciliary the RIS plasma membrane and the synapse. Treatment with membrane and ROS biogenesis. omega-3 docosahexaenoic acid [DHA, 22:6(n-3)] causes increased co-immunoprecipitation and colocalization of SNAP- Key words: Rhodopsin, SNARE, Fatty acid, Cilium

Introduction co-transported on RTCs to the ROS (Rodriguez de Turco et al., Retinal rod photoreceptor cells are modified neurons with primary 1997). cilia that elaborate a specialized light-sensing organelle, the rod outer The incorporation of rhodopsin into RTCs at the trans-Golgi segment (ROS). The ROS is filled with membranous disks housing network (TGN) is regulated by the small GTPase Arf4, which binds the phototransduction machinery that converts photon absorption to the conserved rhodopsin C-terminal VxPx ciliary-targeting signal by rhodopsin into changes in neurotransmitter release from (Deretic, 2006; Deretic et al., 1998; Deretic et al., 2005) and specialized ribbon synapses, thus transmitting photosensory mediates the assembly of the ciliary-targeting complex, which is information to the visual cortex (Burns and Arshavsky, 2005; Ridge comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf et al., 2003; tom Dieck and Brandstatter, 2006). The ROS disk effector FIP3, and the Arf GTPase-activating ASAP1 membrane are embedded in a fluid milieu comprised of (Mazelova et al., 2009). Tethering and fusion of RTCs with the RIS polyunsaturated phospholipids that are highly enriched in omega- PM at the base of the cilium is in turn regulated by the small GTPase 3 docosahexaenoic acid [DHA, 22:6(n-3)]. DHA plays an important Rab8 in conjunction with phosphatidylinositol (4,5)-bisphosphate function in human health, including brain and retina development, [PtdIns(4,5)P2], actin and the actin-binding proteins moesin and function of sensory membranes and cell survival (Bazan, 2006; Rac1 (Deretic et al., 1995; Deretic et al., 2004; Moritz et al., 2001). Hoffman et al., 2001; Marszalek and Lodish, 2005). However, the Although the subsequent membrane-fusion event is crucial to exceptionally high content of DHA phospholipids in ROS replenish the light-sensing machinery, it remains poorly understood, membranes renders them highly susceptible to light and oxidative as the soluble N-ethylmaleimide-sensitive factor attachment protein damage (Anderson and Penn, 2004). Thus, ROS membranes are receptor (SNARE) proteins that drive the RTC fusion have yet to continuously removed through daily shedding and phagocytosis by be identified. retinal pigment epithelial (RPE) cells (Besharse, 1986). The renewal GTPases, Rab effectors and SNAREs are major components of light-sensitive ROS membranes is mediated by rhodopsin of the intracellular machinery that is responsible for targeted transport carriers (RTCs), which travel vectorially through the cell membrane delivery (Cai et al., 2007; Sudhof, 2007). In particular, body, the rod inner segment (RIS), to the base of the cilium and Rabs recruit the effectors that promote membrane-tethering fuse with the specialized domain that separates the ciliary membrane interactions (Seabra and Wasmeier, 2004; Zerial and McBride, from the surrounding RIS plasma membrane (PM), delivering 2001). Rabs also function by concentrating and activating SNAREs, rhodopsin to the ROS (Deretic and Papermaster, 1991; Papermaster accessory proteins and lipids at the sites of membrane fusion (Starai et al., 1986). Along with rhodopsin, DHA phospholipids are also et al., 2007). Rab8 regulates polarized trafficking through 2004 Journal of Cell Science 122 (12) cytoskeleton remodeling, which is necessary for membrane also reveal a previously unrecognized role for omega-3 DHA in outgrowth and the formation of cellular protrusions (Ang et al., modulating signal transduction in retinal photoreceptors by 2003; Peranen et al., 1996; Sato et al., 2007; Wandinger-Ness and enhancing syntaxin-3 incorporation into SNARE complexes at RTC Deretic, 2008). Moreover, Rab8 is essential for the formation of fusion sites to promote expansion of the ciliary membrane and ROS primary cilia, the highly conserved organelles that project from the biogenesis. surfaces of many cells (Nachury et al., 2007; Omori et al., 2008; Yoshimura et al., 2007). In retinal photoreceptors, Rab8 regulates Results RTC fusion and biogenesis of the cilia-derived light-sensing Syntaxin 3 is present in the RIS PM and is highly concentrated organelles, the ROS; Rab8 mutants cause defects in membrane in the vicinity of the cilium tethering and accumulation of RTCs below the cilium, leading to To establish the candidate SNAREs for RTC fusion in retinal rod cell death and rapid retinal degeneration (Deretic et al., 1995; photoreceptors, we examined the distributions of syntaxin 3 and Moritz et al., 2001). This suggests that the regulation of rhodopsin syntaxin 4, which segregate in PM domains of polarized epithelial ciliary targeting by Rab8 might be part of a broad and more general cells. We performed these experiments using frog retinas, because role in the regulation of ciliogenesis. the large size of photoreceptor cells in the frog retina and the Fusion of Rab8-positive carriers with the basolateral PM of extensive turnover of components of its light-sensing membrane epithelial cells is driven by the SNARE syntaxin 4 and occurs at (Besharse, 1986), especially when compared with mammals, the sites adjacent to the tight junctions, which are marked by the provides an ideal system to define the roles of individual proteins octameric Sec6/8 complex (Grindstaff et al., 1998; Kreitzer et al., in this process. We first determined that anti-syntaxin antibodies 2003). The highly conserved membrane-tethering Sec6/8 complex, that were directed to mammalian proteins did recognize proteins known as exocyst in yeast, consists of subcomplexes that are of the appropriate molecular weights in frog retinal post-nuclear continuously assembled and disassembled during trafficking (Hsu supernatant (PNS) that was enriched in photoreceptor membranes et al., 2004; Munson and Novick, 2006; Novick and Guo, 2002). (Deretic and Papermaster, 1991). Retinal PNS immunoblots that The Sec6/8 complex is also present in nerve terminals (Hsu et al., were probed with antibodies to mammalian syntaxin 3 and syntaxin 1996), where it is not required for regulated and 4 (Fig. 1A) revealed proteins of ~32 kDa, as expected given the neurotransmitter release, but is thought to target fusion for neurite evolutionary conservation of SNARE proteins. Because retinal outgrowth and receptor trafficking to the synapse (Murthy et al., membrane proteins were not boiled for SDS-polyacrylamide gel 2003; Sans et al., 2003; Vega and Hsu, 2001). The Sec6/8 complex electrophoresis (SDS-PAGE) analysis to avoid rhodopsin is also localized to the primary cilia in polarized epithelial cells aggregation (Deretic and Papermaster, 1991), we also observed (Rogers et al., 2004), and is therefore a candidate for a Rab8 effector syntaxin-3-containing oligomers of higher molecular weight, which at the RTC docking site. probably represent SDS-resistant SNARE complexes (Fig. 1A, The membrane-fusion event through which RTCs deliver asterisks) (also discussed later), as previously reported for similar rhodopsin to the cilium is likely to be mediated by a SNARE preparations of rat brain extracts (Pellegrini et al., 1995). complex (Li et al., 2007; Malsam et al., 2008; Paumet et al., 2004; We next examined the retinal distribution of 3 and 4 Rothman, 2002). SNARE complexes are generally composed of a by confocal microscopy. Syntaxin 3 exhibited a polarized four-helical bundle that bridges opposing membranes and brings distribution and was highly abundant in the RIS PM domain tightly them into close proximity to initiate fusion (Jahn et al., 2003; Sollner, juxtaposed to the ROS (Fig. 1B, arrows). The RIS, schematically 2003; Ungar and Hughson, 2003). Fusion with the PM requires the presented in Fig. 1D, terminates at the adherens junctions (AJ) that formation of a complex between syntaxins (Qa-SNAREs) and form the outer limiting membrane (OLM; Fig. 1B, dotted lines). VAMPs (R-SNAREs) (Fasshauer et al., 1998), each contributing Syntaxin 3 displayed a concentration gradient along the RIS PM one helix to the four-helix SNARE bundle, and Qbc-SNAREs, either before reaching negligible levels at the OLM (Fig. 1B). By contrast, neuronal SNAP-25 or non-neuronal SNAP-23, which provide two syntaxin 4 was undetectable in the photoreceptor PM (Fig. 1C). helices to the central layer of the core complex (Jahn and Scheller, Immunoreactivity for syntaxin 3 was absent from the RPE cells 2006). Although SNARE pairing alone is not sufficient to determine (Fig. 1B), whereas a robust signal for syntaxin 4 was detected in the specificity of organelle fusion in reconstituted systems, cognate their basal PM (Fig. 1C, arrows), as previously reported (Low et SNAREs are correctly paired in biological membranes (Bethani et al., 2002), further confirming the antibody specificity. al., 2007; Brandhorst et al., 2006). SNAREs are targeted to Both SNAREs were absent from the ROS, but were enriched in appropriate membrane domains on the basis of specific sequences the retinal outer plexiform layer (OPL) (Fig. 1B,C), as previously (ter Beest et al., 2005). The polarized distribution of Qa-SNAREs, reported (Hirano et al., 2007; Morgans et al., 1996). Syntaxin 3 was such as syntaxin 3 and syntaxin 4, which localize to the apical and also detected in the calycal processes (CPs) (Fig. 1E, see also Fig. basolateral PM of epithelial cells (Low et al., 1996), is likely to 1K), the villous structures enveloped by the RIS PM that evaginate contribute additional specificity of membrane targeting by from the RIS and surround the base of the ROS. Although syntaxin promoting fusion with only certain target membranes. Recent 3 was highly enriched in the OPL, where synapses of photoreceptor evidence suggests that the local lipid environment, particularly cells are located (Fig. 1E), photoreceptor synaptic terminals phospholipids enriched in omega-3 and omega-6 fatty acids, also appeared devoid of this SNARE, in contrast to a previously contributes to regulate SNARE function (Davletov et al., 2007). published report (Morgans et al., 1996). This was particularly In this study we examined the expression and distribution of the evident when syntaxin-3-labeled retinas were compared with those candidate Q-SNAREs and membrane-tethering factors, and labeled with an antibody to a synaptic-vesicle , identified additional components that are responsible for polarized , which delineates synaptic terminals of rod trafficking of rhodopsin in retinal photoreceptors. We report that photoreceptors (Fig. 1F). As schematically depicted in Fig. 1G, the photoreceptor cells display a unique distribution of the Sec6/8 synaptic terminals of rod and cone photoreceptors are located in tethering complex and the SNAREs syntaxin 3 and SNAP-25. We the outer sublamina (o) of the OPL, whereas the inner sublamina STX3 and SNAP-25 in rhodopsin trafficking 2005

(i) consists of the horizontal and rod and cone bipolar cell processes. Syntaxin 3 was detected only in the inner half (i) of the OPL (below the dashed line in Fig. 1E), but was absent from rod photoreceptor synapses in the outer half (o) of the OPL (above the dashed line in Fig. 1E). The photoreceptor PM was syntaxin-3-positive, but showed no immunoreactivity for syntaxin 4 with either a polyclonal (Fig. 1H) or a monoclonal (Fig. 1I) antibody, suggesting that this SNARE is unlikely to regulate RTC fusion. Syntaxin-4 immunoreactivity was observed only in the ellipsoid area of the RIS (Fig. 1H,I; ‘E’, square bracket). The ellipsoid, which is schematically presented in Fig. 1D, is densely packed with mitochondria, as shown in the electron microscopy (EM) image (Fig. 1K). Interestingly, mitochondrial localization of the R-SNARE VAMP-1B has been reported (Isenmann et al., 1998); our data suggest that, in photoreceptors, syntaxin 4, or a syntaxin-4-like protein, also displays this unusual

Fig. 1. Syntaxin 3 is a RIS PM protein. (A) Membrane proteins from one retina were separated by SDS-PAGE and immunoblotted with polyclonal anti- syntaxin-3 (STX3) and -4 (STX4) antibodies, which specifically recognize proteins of ~32 kDa. Asterisks indicate species of higher molecular mass, probably SNARE complexes. (B) A confocal optical section (0.7 μm) of frog retina labeled with syntaxin 3 (red), which is highly concentrated in the RIS PM (arrows), but is absent from the ROS, and from the RPE, as reported (Low et al., 2002). AJ, adherens junctions that form the outer limiting membrane (OLM, dotted line). The retinal layers are: ONL, outer nuclear; OPL, outer plexiform; INL, inner nuclear. Syntaxin 3 is also abundant in the OPL. Asterisks indicate the protruding RIS of green rods, a minor subpopulation that accounts for ~5% of total rods. Nuclei are stained with TO-PRO-3 (blue). (C) Syntaxin 4 (green) is present in the basal membranes of RPE cells (arrows), as reported (Low et al., 2002), and in the RIS ellipsoid region (‘E’, square bracket). (D) A schematic diagram of the photoreceptor cell. Cilium (‘C’) protrudes from the cell body (RIS) and elaborates the ROS, which is filled with membranous disks. Newly synthesized rhodopsin traverses the Golgi (‘G’) and the TGN in the myoid (‘M’) of the RIS, where it is incorporated into transport carriers (RTCs), which travel though the ellipsoid (‘E’) and fuse with the RIS PM in the vicinity of the basal body (BB). m, mitochondria; N, nucleus; S, synapse. (E) Syntaxin 3 is highly concentrated in the vicinity of cilia (arrows), in the RIS PM including CPs (arrow, see also panel K). It is also abundant in the inner half (i) of the OPL (square bracket, see also panel G), below the dashed line that indicates the border of the inner and outer (o) sublamina of the OPL. (F) Anti-synaptophysin antibody (SYP, green) labels synaptic terminals of rod photoreceptors located in the outer half (o) of the OPL, above the dashed line. Newly synthesized synaptophysin in the Golgi is also detected. (G) A schematic diagram showing the OPL, which encompasses the synapses of rods (‘R’) and cones (‘C’) in the outer sublamina (o) and the rod bipolar (RB), cone bipolar (CB) and horizontal cells (H) in the inner sublamina (i). (Modified, with permission, from http://webvision.med.utah.edu.) (H) A polyclonal anti-syntaxin-4 (green) antibody labels the RIS ellipsoid region. (I) A monoclonal anti-syntaxin-4 (green) antibody shows identical localization as the polyclonal anti-syntaxin-4 antibody. Polyclonal anti-syntaxin-3 (red) labels the RIS periciliary PM (arrows). (J) A similar membrane domain to that labeled by polyclonal anti- syntaxin-3 is labeled with an antibody to a ciliary protein, whirlin (red, arrows). (K) An EM image detailing RIS structure. The ellipsoid region is filled with densely packed mitochondria (m). RTCs traverse this region and fuse at the base of the cilium (‘C’), at the periciliary ridge complex (PRC, arrow). CP, calycal processes. (L) Anti-whirlin specifically detects the PRC (arrow). (M) Syntaxin 3 cofractionates on linear sucrose density gradients with the RIS PM identified by β2 Na,K-ATPase. Quantification of the blots confirms their parallel distribution. By contrast, 40% of syntaxin 4 is found in a fraction with buoyant density higher than the RIS PM. (N) Antibody to α- Na,K-ATPase recognizes specifically a protein of ~110 kDa. (O) Na,K-ATPase α-subunit (green) is evenly distributed in the RIS PM. Asterisk indicates RIS of green rods. (P) Syntaxin 3 (red) colocalizes with α-Na,K-ATPase (green) in the RIS PM (yellow, arrow), including CPs. Syntaxin-3 immunoreactivity decreases along the lateral RIS PM to undetectable levels (arrowhead) and only α-Na,K-ATPase is detected in the proximity of the OLM. Scale bar (shown in P): 10 μm (B,C), 5 μm (E,F,P), 3 μm (H-J), 1.5 μm (K), 2 μm (L) and 7 μm (O). 2006 Journal of Cell Science 122 (12) mitochondrial localization. In double-labeled confocal sections, syntaxin 3 was also found to be abundant in the periciliary RIS PM (Fig. 1I, arrows). In amphibian photoreceptors, this area includes the periciliary ridge complex (PRC) (Fig. 1K), a highly specialized membrane domain consisting of symmetrical ridges and grooves that comprise the RTC fusion sites (Peters et al., 1983). Accordingly, we identified the PRC using an antibody to a scaffold protein, whirlin (USH2D) (Fig. 1J,L, arrows), a component of the protein network that is disrupted in human Usher syndrome (Liu et al., 2007b; Maerker et al., 2008). Whirlin immunoreactivity was localized within the syntaxin-3-enriched domain (compare Fig. 1J,L with Fig. 1I), demonstrating the predominant association of this SNARE with RTC fusion sites. We next examined the distribution of syntaxins 3 and 4 among retinal subcellular fractions separated on sucrose density gradients. The distribution of syntaxin 3 closely paralleled that of the β2 subunit of Na,K-ATPase, a marker for the RIS PM (Schneider et al., 1991), suggesting that they were present in the same membrane (Fig. 1M). By contrast, ~ 40% of syntaxin 4 was found in a gradient fraction with a higher buoyant density, consistent with its association with other membrane organelles. Because the fraction containing syntaxin 4 was previously shown to be enriched in mitochondria (Deretic and Papermaster, 1991), this further supports the mitochondrial association of syntaxin 4. To confirm the colocalization of syntaxin 3 and Na,K-ATPase in the RIS PM, we employed an antibody specific for the α-Na,K-ATPase subunit, which recognized a protein of ~110 kDa in retinal PNS (Fig. 1N), consistent with the α3 isoform expressed in photoreceptors. By confocal microscopy, α-Na,K-ATPase was detected in the RIS PM, including in CPs (Fig. 1O), as well as in the inner retinal layers. Na,K-ATPase extensively colocalized with syntaxin 3 in the RIS PM in the proximity of the cilium (Fig. 1P, arrow). However, Na,K- ATPase was uniformly distributed between the base of the cilium and the OLM, whereas syntaxin-3 staining gradually diminished and reached negligible levels well above the OLM (Fig. 1P, arrowhead). Overall, the distribution of syntaxin 3 suggests that it might be selectively partitioned to serve as a SNARE for RTC fusion with the RIS PM.

Disruption of microtubules causes depolarization of syntaxin 3 We next investigated whether the observed concentration gradient Fig. 2. The concentration gradient of syntaxin 3 in the RIS PM is maintained of syntaxin 3 depends on the integrity of microtubules, because the by microtubules. (A-D) Eyecups were incubated without (A,C) or with (B,D) 20 μM nocodazole for 5 hours, to depolymerize ellipsoid microtubules (see disruption of microtubules in polarized epithelial cells causes Fig. 1D). Upon microtubule depolymerization, syntaxin 3 (STX3, red) shifts depolarization of the apical SNARE syntaxin 3, but not of the from the periciliary area underlying the ROS (arrow in A and C) to the lateral basolateral syntaxin 4 (Kreitzer et al., 2003). In photoreceptors, an RIS PM (arrow in D), even extending bellow the OLM (arrow in B). It also array of microtubules that radiates from the basal body below the appears in intracellular vesicles (asterisk in D). Nuclei (N) are stained with cilium is tethered with their plus-end at the RIS PM (see Fig. 1D). TO-PRO-3 (blue). (E,F) The same sections as in C and D rendered with 2.5D software for Zeiss LSM 510. The lowest antigen (STX3) concentration on the These microtubules, which might be responsible for the restricted fluorescence intensity scale is indicated in blue, and the highest in red. distribution of syntaxin 3, are known to be sensitive to anti- (G,H) EM images of the cross-sections of the RIS show abundant microtubules microtubule drugs (Vaughan et al., 1989). Upon nocodazole (MT) in control retinas (G), which are absent following nocodazole treatment treatment to promote microtubule depolymerization, the PM in the (H). (I) In control retinas, Golgi (‘G’) detected by Rab6 (green) extends away from the OLM towards the cilium. (J) Microtubule disruption causes the Golgi proximity of the RTC fusion sites was depleted of syntaxin 3 (Fig. to collapse around the nucleus towards the OLM, nearly reaching the lateral 2A-F). In nocodazole-treated retinas, syntaxin 3 delocalized and RIS PM (outlined). Scale bar (shown in J): 5 μm (A-F,I,J) and 0.5 μm (G,H). was detected even below the OLM (Fig. 2B, arrow), rather than in the periciliary area (Fig. 2A,C,E, arrows). Syntaxin 3 was also seen in intracellular vesicles (Fig. 2D,F, asterisk). The highest density of staining was detected in the lateral portion of the RIS PM (Fig. cisternae, which are probably supported by microtubules, extended 2D,F, arrows), suggesting the microtubule-dependent redistribution away from the OLM in control retinas labeled with Rab6 (Fig. 2I). of syntaxin 3. EM images of the cross-sections of nocodazole-treated In the absence of microtubules, the photoreceptor Golgi, which was retinas at the level of the Golgi confirmed that the treatment was distinguished either by Rab6 staining (Fig. 2J) or GM130 staining effective in depolymerizing RIS microtubules (Fig. 2G,H). Golgi (not shown), collapsed around the nucleus towards the OLM, nearly STX3 and SNAP-25 in rhodopsin trafficking 2007 reaching the lateral RIS PM. Thus, microtubule-dependent change et al., 1989) displayed an identical distribution, albeit a more punctate in Golgi organization accompanied the shift in syntaxin-3 polarity. pattern (not shown). These differences are probably due to the By contrast, microtubule disruption had no effect on syntaxin-4 conformation-dependent differential accessibility of the two antigenic immunoreactivity in photoreceptors, although this SNARE did lose sites. SNAP-25 colocalized with whirlin at the base of the cilium its polarized distribution in the RPE cells (not shown). (Fig. 3B, arrows), with synaptophysin in photoreceptor synapses (Fig. 3A,D-F) and with syntaxin 3 in the periciliary RIS PM (Fig. 3C, SNAP-25 is present in the RIS PM and is concentrated at the arrows). Subcellular fractionation confirmed the presence of SNAP- base of the cilium, along with syntaxin 3 25 and syntaxin 3 in the RIS, and their absence from the isolated Consistent with previous reports in mammalian retinas (Greenlee et purified ROS (Fig. 3G). Notably, they displayed a nearly identical al., 2001; Greenlee et al., 2002), confocal microscopy with a ladder of immunoreactivity in unboiled samples (Fig. 3G), suggesting monoclonal anti-N-terminal antibody showed abundant SNAP-25 that SNAP-25 is associated with syntaxin 3 in these SDS-resistant in the RIS PM and the synapse, CPs, and around the cilium at the complexes in the RIS. Detailed analysis revealed that SNAP- base of the ROS, but not in the ROS PM of the frog (Fig. 3A). 25–syntaxin-3-positive domains encircled the base of the cilium (Fig. Similarly, a polyclonal anti-C-terminal-SNAP-25 antibody (Oyler 3H-J, arrows), consistent with the formation of SNARE complexes specifically at the sites of RTC fusion. Although both SNAREs were highly concentrated in the periciliary area, SNAP-25 displayed uniform RIS PM localization that did not parallel the concentration gradient of syntaxin 3 (Fig. 3K). Nocodazole treatment resulted in extensive colocalization of SNAP-25 with syntaxin 3 in the lateral RIS PM (Fig. 3L, arrows). In control retinas, the pixel intensity plot (Fig. 3K, lower panel) along a line (yellow line in the upper panel) drawn approximately halfway between the ROS and the OLM (dotted line) showed that the lateral RIS PM is largely devoid of syntaxin 3. By contrast, upon nocodazole treatment, comparable pixel intensity for SNAP-25 and syntaxin 3 was observed in the lateral RIS PM (Fig. 3L, arrows, lower panel), consistent with ectopic SNARE pairing when microtubule-directed organization was disrupted.

Fig. 3. SNAP-25 is a photoreceptor RIS PM and synaptic SNARE. (A) Anti-SNAP-25-N-terminal mAb (green) outlines the photoreceptor PM. The CPs, which are in continuum with the RIS PM, also contain SNAP-25. The ROS, which are visible by DIC, are completely devoid of this SNARE. SNAP-25 colocalizes with synaptophysin (SYP, red) in the OPL (detailed in panels D-F). Asterisk, RIS of green rods. (B) SNAP-25 and whirlin colocalize at the base of the cilium (arrows). (C) SNAP-25 and syntaxin 3 colocalize in the RIS at the RTC fusion sites (arrows). Asterisk, RIS of green rods. (D-F) SNAP-25 (green in D) colocalizes with synaptophysin (red in E) in the photoreceptor synapses (yellow in F). However, SNAP-25 is abundant in the cell processes in the OPL (green in F), where synaptophysin is not detected. (G) Immunoblots of subcellular fractions isolated from one retina. Rhodopsin is present both in the RIS and the ROS, whereas syntaxin 3 and SNAP-25 are present only in the RIS. Syntaxin 3 and SNAP-25 monomers are abundant in the RIS and both SNAREs display a nearly identical ladder of immunoreactivity in high-molecular-weight forms (asterisks) in these unboiled samples. (H-J) Higher magnifications of the upper portion of the RIS, enlarged from panel C, show foci along the RIS PM (arrows), containing SNAP-25 and syntaxin 3 (yellow in J). Syntaxin 3 might also be present on vesicular structures in the RIS, as reported in PC12 cells (Darios and Davletov, 2006). (K,L) In control retinas (K), syntaxin 3 (red) is nearly absent from the lateral RIS PM, whereas, in nocodazole-treated retinas (L), it relocates to the lateral RIS PM (arrows) where it extensively colocalizes with SNAP-25 (green). Analysis of pixel colocalization along a line (yellow line) drawn approximately halfway between the ROS and the OLM, shows low levels of syntaxin 3 at the lateral RIS PM in control retinas (K, lower panel) and a near-complete overlap of SNAP-25 with syntaxin 3 (arrows in L, lower panel) in nocodazole-treated retinas. Scale bar (shown in L): 8 μm (A), 5 μm (B,K,L), 7 μm (C-F) and 3 μm (H- J). 2008 Journal of Cell Science 122 (12)

The Sec6/8 tethering complex is concentrated along microfilaments and partially overlaps with syntaxin 3 and the small GTPase Rab8 at RTC fusion sites To examine whether the Sec6/8 complex serves to tether RTCs at fusion sites, we performed confocal microscopy using an antibody to Sec8, as Sec8 is a representative component of this multimeric complex. Anti-Sec8 showed immunoreactivity along the RIS PM (Fig. 4A). Foci of high Sec8 immunoreactivity were also observed below the OLM (Fig. 4A). Importantly, Sec8 localized to the structures in the proximity of RTC fusion sites, tightly juxtaposed to the RIS PM labeled by syntaxin 3 (Fig. 4A, arrow in inset). By contrast, in nocodazole-treated retinas (Fig. 4B), extensive colocalization of Sec8 and syntaxin 3 was evident along the lateral RIS PM (Fig. 4B, arrowheads in inset). Immunolocalization of Sec6 was identical to that of Sec8 (not shown), in agreement with the conservation of the Sec6/8 tethering complex in the frog retina. Sec8 localization was consistent with potential microfilament association. Actin filaments encircle photoreceptor inner segments, extending from the AJs outward to the CPs (Fig. 4C). In confocal sections labeled with phalloidin to reveal microfilaments (Fig. 4D), Sec8 did not colocalize with the filaments, but was concentrated at the sites of actin-cytoskeleton attachment to the RIS PM, including the AJs (Fig. 4D, asterisk). In the same section, Rab8, which is known to associate with photoreceptor actin cytoskeleton (Deretic et al., 1995; Moritz et al., 2001), colocalized with Sec8 at the sites of RTC fusion (Fig. 4D, arrow), as well as in the CPs (Fig. 4D, arrowhead). Colocalization of Rab8 and Sec8 was particularly evident at higher magnification, as Rab8-labeled RTCs were in the close proximity of, and appeared to be tethered to, the Sec8-labeled structures (Fig. 4E, arrows). Thus, the Sec6/8 complex might cooperate with syntaxin 3 and SNAP-25 to regulate the fusion of Rab8-positive RTCs to the membrane.

DHA increases colocalization of SNAP-25 and syntaxin 3, promotes their interaction and enhances the delivery of rhodopsin to the ROS Fig. 4. Syntaxin 3 and Sec8 are present in distinct subcellular domains in the vicinity of RTC fusion sites where Rab8 is localized. (A) Anti-Sec8 antibody Syntaxin 3 was recently identified as the molecular target for omega- (green) shows high immunoreactivity along the lateral RIS PM, extending into 3 and omega-6 polyunsaturated fatty acids, which alter its CPs surrounding the ROS, which is consistent with microfilament association conformation thereby promoting formation of the SNARE complex (see panel C). Sec8-positive structures are detected in the proximity of syntaxin- and membrane expansion at the growth cones of PC12 cells (Darios 3-labeled PM (red) nearly exclusively around the sites of RTC fusion (inset, and Davletov, 2006). Given the similarity of the PM addition to arrow). Foci of high Sec8 immunoreactivity are also observed bellow the OLM (dotted line). Nuclei are stained with TO-PRO-3 (blue). (B) In nocodazole- ROS-membrane renewal and the unique importance of omega-3 treated retinas, syntaxin 3 and Sec8 immunoreactivity is apparent along the DHA to photoreceptor physiology, we sought to define the role of lateral RIS PM (inset, arrowheads), which is normally devoid of syntaxin 3. DHA in the context of membrane trafficking required for effective (C) A diagram that schematically represents the relationship between the photoreceptor function. Treatment with 100 μM DHA markedly microfilaments and CPs that evaginate from the RIS and surround the base of the ROS [modified from Deretic et al. (Deretic et al., 1995)]. (D) Sec8 (green) is increased the colocalization of syntaxin 3 and SNAP-25 in the localized in distinct domains that are defined by microfilaments and are revealed periciliary domain (Fig. 5A). This appeared to be primarily due to by phalloidin (blue). Asterisks indicate the sites of microfilament attachment the redistribution of syntaxin 3. Quantitative analysis of syntaxin enriched in Sec8, including the OLM. Rab8 (red), known to associate with the 3 and SNAP-25 distribution performed in three separate experiments actin cytoskeleton, colocalizes with Sec8 at the sites of RTC fusion (arrow), as showed an additional ~20% pixel colocalization in DHA-treated well as in the CPs (arrowhead). Notably, Sec8 shows a much broader distribution. (E) Rab8-postive RTCs (red, arrows) are in close proximity and retinas (Fig. 5B). Moreover, co-immunoprecipitation of syntaxin 3 partially overlap (yellow) with Sec8-labeled structures (green). Scale bar (shown by anti-SNAP-25 antibody was also increased by ~20% after DHA in E): 7 μm (A,B), 4 μm (D) and 1 μm (E). treatment (Fig. 5C), indicating a stimulatory effect of DHA on SNARE pairing in these preparations. Importantly, this increase in SNARE pairing was accompanied by a ~15% increase in rhodopsin We next tested the ability of DHA to overcome the inhibition of delivery to ROS, measured by the incorporation of newly fusion by propranolol, which causes membrane-tethering defects synthesized rhodopsin into isolated purified ROS disk membranes and inhibits RTC fusion (Deretic et al., 2004). As shown in Fig. (Fig. 5D). This significant effect was specific for DHA (P=0.01), 5E, consistent with our published data, propranolol nearly as saturated palmitic acid [PA, 16:0] did not significantly impact completely inhibited rhodopsin delivery to the ROS. DHA treatment rhodopsin delivery (P=0.17). specifically restored it to more than 20% of the control, whereas STX3 and SNAP-25 in rhodopsin trafficking 2009

Fig. 5. DHA specifically increases syntaxin-3–SNAP-25 interaction and stimulates the delivery of rhodopsin to the ROS. (A) DHA- treated retinas display an increase in syntaxin 3 (red) content of the PM surrounding the cilium. Boxed areas in the upper panels are magnified below to demonstrate that, upon DHA treatment, syntaxin 3 and SNAP-25 (green) increasingly colocalize (yellow, merge). (B) Syntaxin-3–SNAP-25 pixel-colocalization analysis was performed within the RIS area indicated by square brackets in panel A, and expressed by Pearson’s coefficient, calculated in three separate experiments. The data from a representative experiment are presented as the means ± s.e.m. of five cells each in control and DHA-treated retinas. DHA significantly increases syntaxin- 3–SNAP-25 pixel colocalization (**P=0.003). (C) Anti-SNAP-25 co-immunoprecipitates 20% more syntaxin 3 from DHA-treated than control retinas (*P=0.02). (D) Rhodopsin delivery to the ROS is stimulated by DHA, but not by palmitic acid (PA). [35S]-Rh (autoradiogram); CB-Rh (Coomassie blue) of the same gel. Autoradiogram quantification data are presented as the means ± s.e. of three separate experiments. Coomassie-blue-stained rhodopsin was quantified and used to correct for gel loading. DHA increases rhodopsin delivery by ~15% (*P=0.01). (E) In retinas treated with propranolol (Ppl), DHA, but not PA, restores rhodopsin delivery to the ROS to ~20% of control. (F) In Ppl-treated retinas, Sec8 (green) is diffusely distributed in the cytoplasm and is no longer associated with microfilaments (see Fig. 4). Rab6 (red) delineates the Golgi (‘G’) and RTCs accumulating bellow the cilium (arrow). (G) In Ppl- treated retinas, syntaxin 3 and SNAP-25 are nearly absent from the RIS PM surrounding the RTC fusion sites (arrows) and are instead concentrated in the lateral RIS PM (arrowheads), similar to the nocodazole-treated retinas (see Fig. 3). (H) DHA partially suppresses the effect of Ppl and restores syntaxin 3 and SNAP-25 to the fusion site (arrow). (I) In a representative experiment, syntaxin-3–SNAP-25 co-immunoprecipitation is increased in DHA-treated, but not in PA- treated, retinas, with or without Ppl. Scale bar (shown in H): 3 μm (upper panels in A), 0.5 μm (lower panels in A) and 5 μm (F-H). palmitic acid had no effect (Fig. 5E). Quantification of overexposed are highly concentrated in the proximity of the RTC fusion sites. autoradiograms (to detect rhodopsin in the ROS of propranolol- The uniform distribution of SNAP-25 throughout the RIS PM and treated retinas) showed that DHA significantly increased the amount photoreceptor synapse indicates that, in addition to syntaxin 3, this of radiolabeled rhodopsin by restoring delivery to the ROS SNARE is likely to also pair with other Qa-SNAREs. Interaction membrane in propranolol-treated retinas (15.6±0.8% vs 31.0±5.3% of SNAP-25 with all PM syntaxins has been demonstrated in PC12 of control in DHA-treated, n=3, P=0.04). Propranolol also led to cells (Bajohrs et al., 2005). One candidate for association in the the redistribution of Sec8 and, by extension, of the tethering complex RIS is the neuronal-specific syntaxin 1A, a prominent partner of into the cytosol (Fig. 5F), most probably by causing the loss of SNAP-25 and a known regulator of fusion (Sudhof, PtdIns(4,5)P2 from the PM fusion sites (Deretic et al., 2004). 2004); syntaxin 1A is absent from ribbon synapses of photoreceptor Similarly, syntaxin 3 and SNAP-25 also appeared greatly diminished cells (Brandstatter et al., 1996; Ullrich and Sudhof, 1994) but is at the base of the cilium (Fig. 5G, arrows) and colocalized only in present in the RIS of rat photoreceptors (Low et al., 2002). the lateral RIS PM of propranolol-treated retinas (Fig. 5G, Quantitative proteome analysis of the mouse photoreceptor sensory arrowheads). Treatment with DHA did, however, appear to prevent cilium complex estimated 4ϫ105 molecules of SNAP-25, 1.9ϫ105 the loss of these SNAREs from the fusion sites (Fig. 5H, arrow). of syntaxin 3 and 1.2ϫ105 of syntaxin 1B per isolated photoreceptor Accordingly, DHA, but not palmitic acid, increased co- RIS/ROS preparation (Liu et al., 2007a). This supports the notion immunoprecipitation of SNAP-25 by anti-syntaxin-3 antibody even that syntaxin 3 is the major partner for SNAP-25 in photoreceptor in the presence of propranolol (Fig. 5I). These data suggest that cells, and that SNAP-25 pairing with syntaxin 1A, or syntaxin 1B, DHA exerts its physiological role in membrane trafficking to ROS might regulate a distinct trafficking pathway in the RIS. not only through lipid modulation, but also through activation of Our findings suggest that microtubules promote the restricted syntaxin 3, facilitation of SNARE pairing and increased availability distribution of syntaxin 3, which is consistent with the membrane of the fusion machinery at the base of the cilium, which together cytoskeleton playing an essential role in concentrating regulators promote RTC fusion, ciliary membrane addition and delivery of of RTC fusion around the cilium. In epithelial cells, syntaxins 3 rhodopsin to the sites of light capture. and 4 are present in separate small clusters even before the establishment of cell polarity, and the integrity of syntaxin-3 Discussion clusters depends on intact microtubules and on the presence of In this study we provide evidence for the role of syntaxin 3 and cholesterol (Low et al., 2006). The enrichment of cholesterol in the SNAP-25 as the Q-SNAREs for the fusion of incoming carriers, periciliary region (Andrews and Cohen, 1983) and the involvement RTCs, with the RIS PM, and therefore as regulators of ciliogenesis of microtubules in clustering syntaxin 3 in photoreceptors suggest and ROS biogenesis in photoreceptors. Syntaxin 3 and SNAP-25 that similar cellular mechanisms are likely to be crucial in 2010 Journal of Cell Science 122 (12) developing and maintaining syntaxin-3 polarity. Re-distribution of with DHA is able to increase membrane delivery to the cilium and syntaxin 3 to the lateral RIS PM of nocodazole-treated retinas, where the ROS in a system that already operates at an extremely high SNAP-25 and the Sec6/8 tethering complex are already present in level, synthesizing and delivering an equivalent of 3 μm2 of the proximity of the delocalized Golgi, suggests that this more membrane a minute (Besharse, 1986). Although the effect of DHA peripheral membrane domain could become competent for fusion on the coupling of syntaxin 3 and SNAP-25 was relatively small, with RTCs under such circumstances. Involvement of microtubules it was measurable and reproducible, which is consistent with in rhodopsin trafficking through the RIS has been unclear, because physiological relevance in photoreceptor function. Unlike syntaxin they are not absolutely required for delivery of rhodopsin to the 1, which readily pairs with SNAP-25, syntaxin 3 is unable to form ROS (Vaughan et al., 1989). The absence of microtubules does not a complex with SNAP-25, except in the presence of polyunsaturated prevent the delivery of post-Golgi carriers to the PM, indicating fatty acids (Connell et al., 2007). Strikingly, even when bound to that they only provide fast and directional movement (Hirschberg the SNARE regulator Munc18, syntaxin 3 is activated by DHA et al., 1998). Thus, upon disruption of microtubule machinery in (Connell et al., 2007). Thus, the reported stoichiometry of Munc18- photoreceptor cells, if the Golgi and the cognate SNAREs 1 and syntaxin 3 (1.93ϫ105 vs 1.94ϫ105 per RIS/ROS) (Liu et al., responsible for RTC fusion are mislocalized, RTCs could be 2007a) suggests that they might form a ‘dormant’ syntaxin- mistargeted and could fuse with inappropriate photoreceptor 3–Munc18 complex, the activation of which depends crucially on membranes such as the lateral RIS PM, or even the synapse. the lipid environment. In this context, the physiological role of DHA Although this would not abrogate delivery of rhodopsin to the ROS might be to selectively pair syntaxin 3 with SNAP-25, which has entirely (Vaughan et al., 1989), it could result in delivery of an access to primary cilia on its own, to open the gate for rhodopsin rhodopsin to RIS PM domains that are normally devoid of delivery and the addition of the ciliary membrane material. rhodopsin, which would be detrimental to cell polarity and retinal Similar to SNARE overexpression (Starai et al., 2007), the function. Consistent with this idea, the loss of photoreceptor increase in availability of activated SNAREs in retinas treated with polarity is highly correlated with the disease progression and propranolol and DHA appears to reduce the need for tethering blindness in patients with retinitis pigmentosa (Berson et al., 2002; factors because Sec6/8 complex dissociates from the membranes Li et al., 1995). and yet fusion is enhanced. Free omega-3 and omega-6 fatty acids The peripheral actin network at the base of the cilium plays a have been demonstrated to directly activate syntaxin 3 by increasing crucial role in the targeted delivery of ROS membranes and in its α-helical content (Darios and Davletov, 2006). However, in photoreceptor morphogenesis (Chaitin, 1992; Williams et al., 1988). photoreceptors, the total amount of unesterified DHA is small The capture of incoming Rab8-positive RTCs and their tethering because of its rapid acylation into phospholipids. Only when at the fusion sites organized by PtdIns(4,5)P2 is regulated by moesin retinas are incubated with high concentrations of DHA, similar to and Rac1, through their cooperative regulation of the actin the concentrations used in this study, does the majority of DHA cytoskeleton (Deretic et al., 2004). The actin network and the actin- remain unesterified (Rodriguez de Turco et al., 1991). Even so, the based motors are essential for Sec6/8 and the exocyst localization, physiological uptake of free DHA from the RPE by the and for polarized exocytosis (Hsu et al., 1999). Our finding that photoreceptors (Rodriguez de Turco et al., 1994) leads to preferential Rab8 and Sec8 are associated with microfilaments at RTC fusion incorporation into the RIS PM domains proximal to ROS that we sites at the base of photoreceptor cilium is consistent with emerging now show are enriched in syntaxin 3. In addition, regulated evidence that Rab8 is a master regulator of ciliogenesis (Nachury localized release of DHA is likely to be required for SNARE pairing et al., 2007; Yoshimura et al., 2007). Because the Sec6/8 complex and ultimately for membrane delivery to the ROS. The mechanism also localizes to the primary cilia in epithelial cells (Rogers et al., that targets the localized production of free DHA in the RIS by 2004), it is likely that it functions as one of the Rab8 effectors in phospholipase A2 thus becomes an important issue (Darios et al., ciliogenesis. In this process, Rab8 might cooperate with Rab11, 2007), especially because it also produces another fusion stimulator, which regulates budding of RTCs (Mazelova et al., 2009). In lysophospholipid, that remains in the membrane. This mechanism Drosophila, the Sec6/8 complex interacts with Rab11 and regulates also has to prevent rapid reacylation and excessive incorporation rhodopsin transport to the rhabdomeres (Beronja et al., 2005). Our of DHA phospholipids into membranes that could destabilize data suggest that this interaction might be conserved in vertebrates cholesterol-rich domains, which provide diffusion barriers and that the formation of this tightly regulated signaling network, separating the ciliary membrane from the surrounding PM (Reiter together with specific membrane-fusion proteins at the base of the and Mostov, 2006; Vieira et al., 2006). DHA metabolism and cilium, ensures the rapid and directional membrane expansion for homeostasis could therefore profoundly affect ROS biogenesis and the sustained biogenesis of light-sensing organelles. photoreceptor polarity, which are frequently severely compromised It is interesting that, when transfected into polarized epithelial in retinal degenerative diseases (Bazan, 2006; Hoffman et al., 2001). cells (which express SNAP-23 in a non-polar fashion), SNAP-25 The crucial role that DHA plays in retinal health is further localizes both to the PM and, unlike SNAP-23, also to primary cilia underscored by the Age-Related Eye Disease Study 2 (AREDS2), (Low et al., 1998), similar to its localization in photoreceptor cells. which examines the effects of high supplemental doses of DHA Our data indicate that, together with SNAP-25, syntaxin 3 regulates on the development of Age-Related Macular Degeneration ciliogenesis and ROS biogenesis, a conclusion that is in keeping (http://www.nei.nih.gov/neitrials/index.aspx). with studies suggesting a role for syntaxin 3 in membrane expansion Although the precise molecular interactions remain to be (Darios and Davletov, 2006). Photoreceptor connecting cilium delineated, identification of the candidate Sec6/8 tethering complex corresponds to the transition zone of primary cilia, which is and the candidate PM SNAREs syntaxin 3 and SNAP-25, together considered a gateway for the admission of specific proteins to this with the ability to modulate their interaction with DHA, has privileged intracellular compartment (Rosenbaum and Witman, provided a framework for future investigation to establish the 2002). Because SNARE proteins act as gatekeepers for the addition function of these proteins in rhodopsin trafficking, ciliogenesis, of the ciliary membrane material, it is remarkable that treatment photoreceptor polarity and health. STX3 and SNAP-25 in rhodopsin trafficking 2011

Materials and Methods References Confocal microscopy Anderson, R. E. and Penn, J. S. (2004). Environmental light and heredity are associated Confocal microscopy was performed on dark-adapted Rana berlandieri frog retinas with adaptive changes in retinal DHA levels that affect retinal function. Lipids 39, 1121- as described (Deretic et al., 2004). Isolated eyecups were either fixed immediately 1124. with 4% paraformaldehyde or incubated for 5 hours at 22°C in oxygenated medium Andrews, L. D. and Cohen, A. I. (1983). Freeze-fracture studies of photoreceptor with, or without, 20 μM nocodazole (Vaughan et al., 1989), 0.5 mM propranolol membranes: new observations bearing upon the distribution of cholesterol. J. Cell Biol. 97, 749-755. (Deretic et al., 2004) or 100 μM DHA (Darios and Davletov, 2006). Following 4% Ang, A. L., Folsch, H., Koivisto, U. M., Pypaert, M. and Mellman, I. (2003). The rab8 paraformaldehyde fixation overnight, 100-μm sections were cut and labeled with rabbit GTPase selectively regulates AP-1B-dependent basolateral transprot in polarized Madin- polyclonal antibodies to: syntaxin 3 (1:100; Synaptic Systems), syntaxin 4 (1:100; Darby canine kidney cells. J. Cell Biol. 163, 339-350. Synaptic Systems), C-terminal SNAP-25 [371, 1:1000 (Oyler et al., 1989)], Bajohrs, M., Darios, F., Peak-Chew, S. Y. and Davletov, B. (2005). Promiscuous synaptophysin [1:100, a gift of F. Valtorta (Deretic and Papermaster, 1991; Valtorta interaction of SNAP-25 with all plasma membrane syntaxins in a neuroendocrine cell. et al., 1988)] and whirlin [1:100, antibody to frog whirlin, a kind gift of Tiansen Li Biochem. J. 392, 283-289. (Tiansen Li and Jun Yang, Mass Eye and Ear Infirmary, Boston, MA, personal Bazan, N. G. (2006). Cell survival matters: docosahexaenoic acid signaling, neuroprotection communication)]; and/or with mouse monoclonal antibodies to: syntaxin 4 (1:100; and photoreceptors. Trends Neurosci. 29, 263-271. BD Biosciences), N-terminal SNAP-25 [SMI 81, 1:1000 (Philip Washbourne and Beronja, S., Laprise, P., Papoulas, O., Pellikka, M., Sisson, J. and Tepass, U. (2005). M.C.W., unpublished)], Rab8 (1:100; BD Biosciences), Sec8 [8S2E12, 1:100, a gift Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor of S. C. Hsu (Wang et al., 2004)] and α-Na,K-ATPase (M7-PN-E9, 1:100, GeneTex), cells. J. Cell Biol. 169, 635-646. and with Alexa-Fluor-633-conjugated phalloidin (1:30; Molecular Probes). Primary Berson, E. L., Rosner, B., Weigel-DiFranco, C., Dryja, T. P. and Sandberg, M. A. antibodies were detected with Cy2 and Cy3 goat anti-rabbit IgG or goat anti-mouse (2002). Disease progression in patients with dominant retinitis pigmentosa and rhodopsin IgG (1:200; Jackson ImmunoResearch). All sections were counterstained with the mutations. Invest. Ophthalmol. Vis. Sci. 43, 3027-3036. nuclear stain TO-PRO-3 (1:1000; Molecular Probes). Confocal optical sections Besharse, J. C. (1986). Photosensitive membrane turnover: differentiated membrane (0.7 μm) were obtained on a Zeiss 510 laser scanning confocal microscope (Carl domains and cell-cell interaction. In The Retina: A Model for Cell Biological Studies Zeiss) using a 488 nm argon ion laser for Cy2, 543 nm HeNe laser for Cy3 and 633 (ed. R. Adler and D. Farber), pp. 297-352. New York: Academic Press. nm HeNe laser for Alexa-Fluor-633 or TO-PRO-3 excitation. Digital images were Bethani, I., Lang, T., Geumann, U., Sieber, J. J., Jahn, R. and Rizzoli, S. O. (2007). prepared using Adobe Photoshop CS (Adobe Systems). Colocalization analysis The specificity of SNARE pairing in biological membranes is mediated by both proof- (Pearson’s coefficient) was calculated using SlideBook Image Analysis software reading and spatial segregation. EMBO J. 26, 3981-3992. Brandhorst, D., Zwilling, D., Rizzoli, S. O., Lippert, U., Lang, T. and Jahn, R. (2006). (Intelligent Imaging Innovations). Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity. Proc. Natl. Acad. Sci. USA 103, 2701-2706. Electron microscopy Brandstatter, J. H., Wassle, H., Betz, H. and Morgans, C. W. (1996). The plasma Following 5 hours of incubation with 20 μM nocodazole or in the control media, membrane protein SNAP-25, but not syntaxin, is present at photoreceptor and bipolar retinas were fixed in 4% formaldehyde and 1% glutaraldehyde in 0.12 M cacodylate cell synapses in the rat retina. Eur. J. Neurosci. 8, 823-828. buffer pH 7.5 for 1 hour at 22°C, post-fixed in OsO4 and embedded in Epon. Thin Burns, M. E. and Arshavsky, V. Y. (2005). Beyond counting photons: trials and trends sections were examined in a Philips CM-100 transmission electron microscope. in vertebrate visual transduction. Neuron 48, 387-401. Cai, H., Reinisch, K. and Ferro-Novick, S. (2007). Coats, tethers, Rabs, and SNAREs Pulse-chase labeling, preparation of PNS and retinal subcellular work together to mediate the intracellular destination of a transport vesicle. Dev. Cell fractionation 12, 671-682. Frogs were dark-adapted for 2 hours before the experiment. Isolated retinas were Chaitin, M. H. (1992). Double immunogold localization of opsin and actin in the cilium of developing mouse photoreceptors. Exp. Eye Res. 54, 261-267. incubated for 1 hour at 22°C in oxygenated medium with [35S]-Express protein labeling μ Connell, E., Darios, F., Broersen, K., Gatsby, N., Peak-Chew, S. Y., Rickman, C. and mixture (25 Ci/retina), followed by a 2-hour chase. In some experiments, incubation Davletov, B. (2007). Mechanism of arachidonic acid action on syntaxin-Munc18. EMBO medium contained 0.5 mM propranolol (Deretic et al., 2004), 100 μM DHA or 100 μ Rep. 8, 414-419. M palmitic acid (PA). Darios, F. and Davletov, B. (2006). Omega-3 and omega-6 fatty acids stimulate cell Retinal fractionation and preparation of PNS enriched in photoreceptor biosynthetic membrane expansion by acting on syntaxin 3. Nature 440, 813-817. membranes were performed as described (Deretic and Papermaster, 1991). ROS were Darios, F., Connell, E. and Davletov, B. (2007). Phospholipases and fatty acid signalling sheared using a 14-gauge needle, separated from the remainder of the retina by in exocytosis. J. Physiol. 585, 699-704. flotation on high density (34%) sucrose and purified on a step sucrose gradient Davletov, B., Connell, E. and Darios, F. (2007). Regulation of SNARE fusion machinery (Papermaster and Dreyer, 1974). After gradient centrifugation at 100,000 gav in a by fatty acids. Cell Mol. Life Sci. 64, 1597-1608. SW40 rotor (Beckman-Coulter) for 30 minutes, purified ROS membranes were Deretic, D. (2006). A role for rhodopsin in a signal transduction cascade that regulates collected from the 1.11-1.13 g/ml sucrose interface, diluted with 10 mM Tris acetate, membrane trafficking and photoreceptor polarity. Vision Res. 46, 4427-4433. pH 7.4, and sedimented for 20 minutes at 20,000 rpm in a JA25.5 rotor (Beckman- Deretic, D. and Papermaster, D. S. (1991). Polarized sorting of rhodopsin on post-Golgi Coulter). Following ROS removal, retinal pellets were homogenized in 0.25 M membranes in frog retinal photoreceptor cells. J. Cell Biol. 113, 1281-1293. sucrose, and retinal fragments and nuclei were sedimented at 4,000 rpm in a JA25.5 Deretic, D., Huber, L. A., Ransom, N., Mancini, M., Simons, K. and Papermaster, D. rotor for 4 minutes, generating PNS that was enriched in photoreceptor biosynthetic S. (1995). rab8 in retinal photoreceptors may participate in rhodopsin transport and in membranes. In some experiments, PNS was centrifuged at 80,000 rpm for 1 hour in rod outer segment disk morphogenesis. J. Cell Sci. 108, 215-224. a TLA 100.3 rotor (Beckman-Coulter) to sediment RIS membrane proteins, or at Deretic, D., Schmerl, S., Hargrave, P. A., Arendt, A. and McDowell, J. H. (1998). 17,500 g for 10 minutes at 4°C in a JA25.5 rotor to pellet large organelles, including Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence 90% of photoreceptor RIS PM (Deretic et al., 2004). Pellets were resuspended in QVS(A)PA. Proc. Natl. Acad. Sci. USA 95, 10620-10625. Deretic, D., Traverso, V., Parkins, N., Jackson, F., Rodriguez De Turco, E. B. and 0.25 M sucrose and, following fractionation on linear 20-39% (w/w) sucrose Ransom, N. (2004). Phosphoinositides, ezrin/moesin and rac1 regulate fusion of gradients, subcellular fraction pools were created as described (Deretic et al., 2004). rhodopsin transport carriers in retinal photoreceptors. Mol. Biol. Cell 15, 359-370. Deretic, D., Williams, A. H., Ransom, N., Morel, V., Hargrave, P. A. and Arendt, A. SDS-PAGE and immunoblotting (2005). Rhodopsin C terminus, the site of mutations causing retinal disease, regulates Membrane proteins were analyzed by SDS-PAGE as described (Deretic and trafficking by binding to ADP-ribosylation factor 4 (ARF4). Proc. Natl. Acad. Sci. USA Papermaster, 1991). Gels were stained by Phast Gel Blue R (Amersham Pharmacia 102, 3301-3306. Biotech) and imaged with a GS-700 Imaging Densitometer (BioRad). The images Fasshauer, D., Sutton, R. B., Brunger, A. T. and Jahn, R. (1998). Conserved structural of the radiolabeled proteins were generated by autoradiography at –85°C using Kodak features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R- BioMax MR film. Gels were blotted onto Immobilon-P membranes and blots were SNAREs. Proc. Natl. Acad. Sci. USA 95, 15781-15786. probed with anti-Na,K-ATPase-β2 (1:100; BD Biosciences), or the antibodies listed Greenlee, M. H., Roosevelt, C. B. and Sakaguchi, D. S. (2001). Differential localization above, followed by the secondary antibodies conjugated to HRP. Bound antibodies of SNARE complex proteins SNAP-25, syntaxin, and VAMP during development of were detected using a chemiluminescent Western Lightning immunodetection system the mammalian retina. J. Comp. Neurol. 430, 306-320. (Perkin Elmer Life Sciences). The distribution of detected antigens was quantified Greenlee, M. H., Wilson, M. C. and Sakaguchi, D. S. (2002). Expression of SNAP-25 using Multianalyst software (BioRad). during mammalian retinal development: thinking outside the synapse. Semin. Cell Dev. Biol. 13, 99-106. Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Boulan, We thank Flavia Valtorta, Shu-Chan Hsu and Tiansen Li for their E., Scheller, R. H. and Nelson, W. J. (1998). Sec6/8 complex is recruited to cell-cell gifts of antibodies, and Andy Williams for his help with the experiments. contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial Supported by the NIH/NEI grant EY-12421 to D.D. M.C.W. was cells. Cell 93, 731-740. supported by MH-48989. Fluorescence Microscopy Facility at UNM Hirano, A. A., Brandstatter, J. H., Vila, A. and Brecha, N. C. (2007). Robust syntaxin- 4 immunoreactivity in mammalian horizontal cell processes. Vis. Neurosci. 24, 489-502. is supported by NCRR, NSF, NCI and the UNM Cancer Center. Hirschberg, K., Miller, C. 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