Boitet ER , Reish NJ , Hubbard MG , Gross AK .

P30 EY003039 NEI NIH HHS , R01 EY019311 NEI NIH HHS , R01 EY030096 NEI NIH HHS

             rod photoreceptor outer segment

	Figure 1- NudC binds to rab11a and rhodopsin in bovine retinas and is present in mouse and frog retinas. A ) IP using an anti‐rab11a pAb antibody as bait followed by SDS‐PAGE revealed 2 proteins binding to rab11a, which were identified by mass spectroscopy as nudC and rhodopsin. B ) Western blot probed for rab11a following IP of bovine retinal lysates using rab11a antibody as bait. C ) Western blots from reverse pulldown experiments using bovine retinal extract and a nudC antibody as bait support an interaction between nudC, rab11a, and rhodopsin. D ) Western blotting confirms nudC is found in retinal extracts prepared from bovines, mice, and frogs. Ab, rab11a polyclonal antibody eluate; C, control IgG; IB, immunoblotting; MW, molecular weight.
	Figure 2- NudC and nudCL280P localizes to the IS and ONL of photoreceptors and can be knocked down upon expression of shRNA against nudC. A–D) IHC of retinal cryosections probing with antibody indicates endogenous nudC (red) is expressed throughout the retinas of mice (A) and is primarily localized to the IS of tree shrew (B ), monkey (C ) and frog (D ) retinas. Photoreceptors are counterstained with WGA (green). NudC stains the axoneme of cone cells (B). A ′–D ′) View of endogenous nudC IHC shown without counterstains. E, F) Expression and localization of mC‐nudC (E , yellow) or mC‐nudCL280P (F , yellow) in transgenic X. laevis , counterstained with WGA (green). E,F ) View of mC‐nudC (E ) or mC‐nudCL280P (F ) localization shown without counterstains. G ) IHC from transgenic tadpole retinas expressing nudC shRNA (green) probing with anti‐nudC (red). ∧, successful depletion of nudC protein in cells expressing nudC shRNA (*). H ) NudC (red) protein levels are unaffected by expression of shRNA targeting firefly luciferase (green). G ′, H ′) View of nudC IHC with nuclear counterstain alone. DAPI (blue), nuclear marker. OPL, outer plexiform layer. Scale bar, 20 µm.
	Figure 3- Dot blots of whole tadpole eyes demonstrate a decrease of nudC in shRNA‐transgenic X. laevis photoreceptors. Expression of mC‐nudCL280P or nudC‐shRNA leads to rhodopsin mislocalization, which is resolved when coexpressing nudC‐shRNA and mC‐nudC. A) Dot blot showing nudC‐expression changes in NTG, mC‐nudC, mC‐nudCL280P, nudC‐shRNA, nudC‐shRNA plus mC‐nudC rescue, and luciferase shRNA control transgenic animals. B ) Quantification of relative nudC amounts in lysate from whole tadpole eyes with expression normalized to GAPDH (n = 4–7 per group; 1‐way AN OVA, F (4,27) = 4.64, P = 0.007; Tukey's HSD test for mC‐nudCL280P vs . hairpin, P < 0.01 (*); all other comparisons, P> 0.05). C–F) IHC for rhodopsin (green) in retina cryosections of transgenic X. laevis expressing mC‐nudC (C , red), mC‐nudCL280P (D , red), nudC‐shRNA (E , red), and coexpression nudC‐shRNA (cyan) and mC‐nudC (red) as a rescue experiment (F ). (D, E ) Intense band of rhodopsin staining at the base of the OS (∧). C ′–F ′) View of rhodopsin IHC without counterstains highlighting protein mislocalization. E) Aberrant rhodopsin in the IS (*). Nuclei marked by DAPI (blue). Scale bars, 20µm.
	Figure 4- Transgenic expression of mC‐nudCL280P but not mC‐nudC causes dysmorphic OS disk membranes. A, B) Ultrathin sagittal sections taken from 2‐wk‐postfertilization wild‐type retinas (A ) and mC‐nudC transgenic retinas (B) show (∧) properly formed rod and cone photoreceptors using TEM. C ) X. laevis mC‐nudCL280P photoreceptors contain overgrown disc membranes and ectopic disc formation (arrow) at 2 wk postfertilization. D–F ), Ultrathin sagittal retinal sections from 4‐wk‐postfertilization wild‐type retinas (D ) and mC‐nudC (E ) contain photoreceptors with normal disks formed (∧) in the OS, whereas mC‐nudCL280P 4‐wk animals (F) show ectopic disk overgrowth (arrow). Scale bars, 500 nm. Asterisk (*) indicates oil droplets. WT, wild type.
	Figure 5- Knockdown of nudC in rod photoreceptors results in overgrowth of disk membranes and ectopic disk formation. A–D) 2‐wk‐postfertilization tadpole ultrathin retina sections imaged by TEM. A) Transgenic tadpoles expressing nudC shRNA show dysregulation of new disk formation at the base of ROSs. An accumulation of tubulovesicular membranes near the base of ROSs was observed (#). A ′) Tubulovesicular membrane accumulation at the base of the OS in nudC shRNA retinas has been resolved; however, overgrowth of discs at the base of the OS is observed (arrows). B) Phenotype associated with expression of nudC shRNA is rescued by coexpression of mC‐nudC with no dysregulation of disk membranes observed. B ′) Rescue of nudC shRNA via coexpression of mC‐nudC results in normal formation of ROSs based on ultrastructure. C) NudC shRNA is not rescued by coexpression of mC‐nudCL280P, and large overgrown disks are present at base of OS (arrow). C ′) Animals coexpressing mC‐nudCL280P and nudC shRNA do not show normal OS disk membranes and have ectopic disks formed at the base of the OS (arrows). D ) Control shRNA directed against firefly luciferase showed no apparent phenotype in X. laevis photoreceptors. A ′–D ′) Ultrathin sections from 4‐wk‐postfertilization tadpole retinas imaged by TEM. D ′) Knockdown of firefly luciferase via shRNA shows no adverse effects of OS disk formation. Carets (∧) indicate normal OS disk ultrastructure. Asterisk (*) shows oil droplets contained in X. laevis photoreceptors. Scale bars, 500 nm.
	Figure 6- Schematic representation of the regulation of newly formed disk membranes by nudC in X. laevis rod photoreceptors. NudC localizes primarily to the IS and synapse in normal rods with uniform disk membranes in the OS. NudCL280P is present in the IS but does not localize to the synapse. Newly formed disks grow ectopically, spiraling out of the OS, either contained within or external to the plasma membrane. Knockdown of nudC results in drastically overgrown disk membranes and the mislocalization of rhodopsin. Arf, ADP ribosylation factor; ASAP, arf‐GAP with SH3 domain, ankryn repeat and pH domain containing protein; NudC, nuclear distribution protein C; rab, ras‐related protein.

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