Fig 1.
Vesicle accumulation is progressive from the onset of OS formation in cc2d2a-/- PRs (A-D) Transmission electron microscopy images (TEM) of retinal sections at 60 hpf are indistinguishable between wild-type (wt) (A, C) and cc2d2a-/- (B, D), including with respect to basal body docking (arrowheads in C-D) and extension of the connecting cilium (arrows in C-D). (E-H) Retinal sections at 72 hpf: note the nascent outer segments (OS) in wt (E, G), but the quasi-absence of OSs in cc2d2a-/- with onset of apical accumulation of vesicular structures (F, H, arrowheads). (I-L) Retinal sections at 96 hpf: well-formed OSs are present apical to the mitochondrial cluster in wt (I, K), while in mutant photoreceptors an increased number of vesicles (brackets) are found in the apical portion of the cell together with misshapen membrane stacks (arrowheads) instead of OSs (J, L). Scale bars are 1 μm in all panels. m mitochondria, N nuclei, OS outer segments, wt wild-type, hpf hours post fertilization.
Fig 2.
Accumulated vesicles in cc2d2a-/- PRs are opsin-carrier-vesicles (A-C) 5 dpf correlative light and electron microscopy (CLEM) image of retinal sections stained with BODIPY (magenta) to mark membranes of the outer segment and the mitochondrial cluster and with 4D2 (green) to label rhodopsin and red-green cone opsin. (A’-C’) are the corresponding scanning electron microscopy (SEM) images. Note that while 4D2 staining only localizes at the outer segments of wild-type (wt) PRs (A), it is visible in accumulated vesicles in cc2d2a-/- PRs (B, arrows) and in dysmorphic outer segments (C). Also note normal cilium docking in cc2d2a-/- PRs (C-C’, arrowhead). Scale bars are 2 μm in all panels. m mitochondria, N nuclei, OS outer segments, P pigment in melanosomes, wt wild-type.
Fig 3.
Rab8a and Rab8ba co-localize with endogenous opsins (A-C) 5 dpf cryosections of wild-type (wt) zebrafish expressing mCherry-tagged Rab8a in rods (magenta), stained with the anti-opsin antibody 4D2 (green). (D-F) 5 dpf cryosections of wt zebrafish expressing mCherry-tagged Rab8a in cones (magenta), stained with anti-blue opsin (green). (G-I) 5 dpf cryosections of wt zebrafish expressing mCherry-tagged Rab8ba in rods (magenta), stained with the anti-opsin antibody 4D2 (green). Arrowheads indicate examples of co-localization in all cases. Scale bars are 5 μm in all panels.
Fig 4.
Rab8 and opsin are associated with membrane-bound vesicular structures (A) CLEM image of wild-type (wt) transgenic zebrafish at 3 dpf expressing mCherry-tagged Rab8a in rods stained with anti-mCherry (magenta) and anti-opsin antibody 4D2 (green).(A’) High magnification image of the boxed area in (A) showing a clearly individualizable small round membrane-bound structure (white circle) coated with Rab8a signal on either side (empty arrowheads over the magenta signal) and with opsin signal over the edge of the structure (asterisk over the 4D2 signal), compatible with transmembrane opsins in a Rab8-coated vesicle. (A”) Immunohistochemistry image only from (A’). The white circle is placed where the vesicular structure is observed in the SEM image. (A”‘) SEM image only from (A’), showing the vesicular structure. The empty arrowheads are placed where the Rab8a signal is observed and the asterisk is located over the opsin signal. The additional m-Cherry and opsin signal likely represent a conglomerate of several vesicular structures with multilobulated membranes (arrows in A’ and A”‘). Scale bars: 4 μm in A and 1 μm in A’-A”‘. m mitochondria, N nuclei, OS outer segments, P pigment, wt wild-type.
Fig 5.
Rab8a partially mislocalizes in cc2d2a-/- cones 5 dpf CLEM images of wild-type (wt) (A-A’) and cc2d2a-/- (B-C’) zebrafish expressing mCherry-tagged Rab8a in cones (tg(tacp:mCherry-rab8a), magenta). The mCherry signal is enhanced with anti-mCherry antibody. (A-A’) Note the localization of Rab8a (magenta) to vesicular structures (arrowheads) in wt. (B-B’) In cc2d2a-/- cones, Rab8a (magenta) localizes to accumulated vesicles (v, bracket) as well as to non-membrane-delimited cytoplasmic areas (arrows in C-C’). Scale bars are 3 μm in all panels. OS outer segment, m mitochondria, N nuclei, v vesicular structures, wt wild-type.
Fig 6.
Rab8-particles display dynamic movement patterns and transiently approach the BB (A-B) Time-lapse imaging of a 5 dpf wt zebrafish retina expressing Rab8ba in rods (tg(rhod:mCherry-rab8ba)). The Rab8 particle marked with an arrow in all time frames can be recognized and followed by the Ilastik tracking software (cyan overlay) (A’-B’). This particle transiently approaches the GFP-tagged basal body (C and merge in D). The movement is specific for Rab8 as transiently expressed Rab3aa in rods (rhod:mCherry-rab3aa) (E-F’) exhibits the expected synaptic localization (F’). Scale bars are 10 μm in all images. OS outer segment, IS inner segment, Syn Synapse.
Fig 7.
Rab8-trafficking kinetics are conserved between different paralogs in wt rods and cones and in cc2d2a-/- photoreceptors (A-D) Quantification for various parameters generated by tracking of tagged Rab8 particles on 10-minute long videos recorded at 1 frame/second. 13 photoreceptors (PRs) per group (5 different conditions) were analyzed: PRs expressing mCherry-Rab8a in wt cones (orange circles), wt rods (orange squares) and cc2d2a-/- rods (blue inverted triangles) as well as PRs expressing mCherry- Rab8ba in wt rods (orange triangles) and cc2d2a-/- rods (blue diamonds). Each dot in the scatter plots represents the average value of all the particles present in 1 PR. Only particles present in ≥10 frames were analyzed. Bars indicate average and standard deviations. (A) Particle displacement measured as the distance between the first set of coordinates and the last set of coordinates. Average displacement was close to 1 μm for all conditions. (B) Maximum speed of particles (largest distance traveled between two consecutive time points) was about 1 μm/s in all conditions. (C) Particle directionality measured as the ratio of distance spanned in the lateral axis over the distance spanned in the apico-basal axis. A predominantly apico-basal movement was observed in all conditions; Rab8ba particles displayed a more lateral movement in cc2d2a-/- compared to wt rods. (D) Total trajectory traveled by particles during the entire duration of the recording. Average trajectory was close to 20 μm for all conditions. Given the much more limited displacement, this indicates that the particles shuffle substantially within the inner segment, as visible on the videos. (E) Average cross-sectional surface area of tracked particles, calculated from the number of pixels constituting each particle. (F) Proportion of puncta coming in proximity with the BB = “contacting the BB”. * p < 0.01, ** p < 0.01, n.s. not significant, Mann-Whitney U-test. Note consistent results between Rab8a-particles in cones and rods, between Rab8a and Rab8ba particles and between wt and cc2d2a-/- for both paralogs for the majority of parameters.
Fig 8.
SNAP25 mislocalizes in cc2d2a-/- PRs (A-C’) 4 dpf cryosections of wild-type (wt) (A-A’), cc2d2a-/- (B-B’) and ift88-/- (C-C’) retinae stained for SNAP25 (green in A’-C’) and BODIPY (magenta). In wt PRs (A-A’) SNAP25 localizes along the plasma membrane, between the mitochondrial cluster and the OS (arrowhead) and at the synapse. In cc2d2a-/- (B-B’), SNAP25 synaptic localization is preserved but apical mislocalization to a membrane-rich compartment (BODIPY, arrows) is obvious. (C and C’) Despite absence of OSs in the ift88-/- mutant, SNAP25 localizes correctly at the apical membrane of PRs. Scale bars are 10 μm in all panels.
Fig 9.
SNAP25 localizes to wt periciliary membrane and mislocalizes to accumulated vesicles of cc2d2a-/- PRs (A-E) 5 dpf CLEM retinal sections stained for SNAP25 (green), acetylated tubulin (magenta) and DAPI (blue, nuclei). (C-E) are higher magnification images of the boxed areas in (A, B) and (C’-E’) are the corresponding SEM images. In wt PRs (A) SNAP25 is found at the inner segment apical membrane, along the calycal processes (empty arrowhead in A) as well as at the periciliary membrane (C-C’, arrow) around the base of the anti-acetylated tubulin-marked cilium (C-C’, arrowhead). In contrast, in cc2d2a-/- PRs (B), SNAP25 prominently mislocalizes in accumulated vesicles (D-D’, arrows) and dysmorphic OSs (E-E’, bracket). Arrowheads point to connecting cilia in (C-D’). Scale bars: 4 μm in A-B, 1 μm in C-E’. OS outer segment, m mitochondria, N nuclei, P pigment, v vesicles, wt wild-type.
Fig 10.
Proteins involved in OCV fusion are affected by loss of Cc2d2a function 4 dpf wild-type (wt) (A-A’) and cc2d2a-/- retinal cryosections (B-B’) and 6 dpf wt (C-C’) and cc2d2a-/- retinal cryosections (D-D’), all stained for Syntaxin3 (Stx3) (grayscale in A-D and green in A’-D’) and counterstained with DiO (A’,B’) or BODIPY (C’,D’) (both magenta) to label membranes. In wt PRs at both developmental times Stx3 localizes along the plasma membrane (arrowhead), between the mitochondrial cluster and the OS and at the synapse (A-A’, C-C’), similar to SNAP25. While minimal Stx3 mislocalization is visible in cc2d2a-/- at 4dpf, a striking decrease in fluorescence intensity is obvious in the mutant (B, D). (E) Western blot on whole eye lysates at 6 dpf confirms decreased protein levels of Stx3 in cc2d2a-/-. Protein levels of SNAP25 and the Exocyst component Exoc4 are also decreased in cc2d2a-/- whole eyes. (F) Relative protein content was determined as the ratio of band intensity relative to the housekeeping protein control (beta-actin), averaged for all replicates and repeated in 3 independent blots. Error bars represent standard deviation. *p<0.05, ** p< 0.01, Student’s t-test. Full western blots are shown in S12 Fig.
Fig 11.
The role of CC2D2A in opsin-carrier-vesicle fusion at the periciliary membrane.
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.