Ying G , Gerstner CD , Frederick JM , Boye SL , Hauswirth WW , Baehr W .

P30 EY014800 NEI NIH HHS , R01 EY008123 NEI NIH HHS , R01 EY019298 NEI NIH HHS

	Fig 1. Generation of Rab11a retina-specific KO mouse (retRab11a-/-).(A) Schematic of Rab11a gene-trap (GT) allele, conditional allele and KO allele. P1, P2, P3 represent primers for genotyping wild-type (WT), conditional and KO alleles. Exons are shown as yellow rectangles. The gene trap contains a splice-acceptor (SA), an IRES site, a βGeo cassette, and a poly-A site. The gene trap is flanked by two FRT sites (green triangle), a loxP site (red triangle) at the 5’-end of the cassette and another downstream in intron 3. Location of 5’-long arm and 3’-short arm are indicated. (B) Long range PCR confirmation of 3’ and 5’ recombination arm in ES cells and GT animals. (C) PCR confirms conditional deletion of exons 2 and 3 in retRab11a-/- retina but not in tail. retRab11a+/- mouse was used as control. (D) Rab11b mRNA is not up-regulated in retRab11a-/- retina. +/- and -/- refer to Rab8a heterozygous and homozygous knockouts, respectively. GAPDH was used as internal control. (E) Representative retina sections showing of Rab11a and Rab11b immunostaining in retina. Rab11a is localized mainly in punctuate IS spots in WT but is negative in retRab11a-/- KO (center). Rab11b staining pattern is similar to Rab11a (right). Scale bar, 20 μm.
	Fig 2. Electroretinography (ERG) and OS protein localization in adult retRab11a-/- mice.(A) Representative scotopic ERG traces at -10, 0 and 10 db from 5M-old retRab11a+/- and retRab11a-/- mice. (B) and (C) Quantification of scotopic a- and b-wave amplitude at multiple light intensities. No significant difference was observed between control and KO animals (p> 0.15, n = 5 for each group, one-way ANOVA). (D) Representative photopic ERG trace at 0, 5 and 10 db from 5M-old control and KO mice. (E) Quantification of photopic b-wave amplitude at multiple light intensities. No significant difference was observed between control and KO animals (p> 0.15, n = 5 for each group, one-way ANOVA). (F) Immunostaining of rhodopsin, GC1, CNGA1/A3, PDE6, S-opsin, ML-opsin, ribeye and SV2 using 3M-old animals, showing that all proteins localize correctly in KO as in heterozygous control. Scale bar, 20 μm.
	Fig 3. OS protein localization in adult retRab11a-/- mice injected with Rab11b(S25N) AAV.(A) Alignment of Rab11a and Rab11b protein sequences. One critical GTP binding domain is located in exon 2. Position of S25 in Rab11b is indicated in Rab11b. Both proteins contain geranylgeranylation motifs at the C-terminus. (B) Immunostaining results show that cMyc-Rab11b(S25N) localizes mainly at IS and OS (red channel, top left panel). Rhodopsin, PDE6, GC1, CNGA1/A3, ML- and S-opsin localize correctly to OS, and SV2 localizes correctly to the OPL. Green channel in all panels detect szGreen fluorescence. Scale bar, 20 μm.
	Fig 4. Normal ERG and rhodopsin localization in adult rodRab8a-/- animals.(A) Representative scotopic ERG trace at 10 db from 4M-old rodRab8a+/- and rodRab8a-/- mice. (B) and (C), Quantification of scotopic a-wave and b-wave amplitudes at multiple light intensities. No significant difference was observed between control and KO animals (p> 0.17, n = 5 for each group, one-way ANOVA). (D), Immunostaining of rhodopsin, PDE6, GC1, CNGA1/A3, GCAP1 and prominin. All proteins localize correctly to OS. Scale bar, 20 μm.
	Fig 5. Normal ERG and opsin localization in adult coneRab8a-/- animals.(A) Representative photopic ERG trace at 15 db from 3M-old coneRab8a+/- and coneRab8a-/- mice. (B) Quantification of photopic b-wave amplitude at multiple light intensities. No significant difference was observed between control and KO animals (p> 0.17, n = 5 for each group, one-way ANOVA). C, Immunostaining results showed that cone arrestin, cone transducin gamma, ML-opsin, S-opsin and prominin1 all localize correctly in KO as in control. Scale bar, 20 μm.
	Fig 6. Correct localizations of OPL and inner retina proteins in Rab8a global KO mice.Immunostaining results showed that distribution of OPL proteins ribeye, Vglut1, synaptophysin, ribeye and pan-PMCA, as well as inner retina marker PKCα, calbindin, CaM kinase II (CamKII), tyrosine hydroxylase (TH) and choline acetyltransferase (CHAT), is indistinguishable between P21 Rab8a+/- and Rab8a -/- controls. Scale bar, 30 μm.
	Fig 7. Rhodopsin localizes normally in adult Rab8a KO mice injected with Rab8b(T22N) AAV.(A) Alignment of Rab8a and Rab8b amino acid sequences. Exon 1 contains the GTP binding domain. Exon 2 is deleted in the germline and conditional knockouts. T22, preventing GTP binding when mutated, is shown. (B) Genotyping confirms deletion of exon2 in retRab8a-/- retinas but not tails. retRab8a+/- tissues were used as controls. (C) Immunofluorescence showed that Rab8a is mainly localized as punctate signal in IS and OPL in WT but absent in retRab8a-/- photoreceptor cells. (D) Rab8b(T22N) is mainly localized at IS and OPL. (E) Rhodopsin, GC1, CNGA1/A3 and ribeye are localized correctly in 4M-old retRab8a-/- and retRab8a+/- animals injected with dnRab8b (Rab8b(T22N)) AAV. Scale bars, 20 μm (C and D) and 30 μm (E).
	Fig 8. Rhodopsin localizes normally in adult Rab11a and Rab8a double KO mice.Immunostaining of rhodopsin, GC1, CNGA1/A3, PDE6, ML-opsin, S-opsin, ribeye and SV2 using 3M-old animals showed that all proteins localize correctly in dKOs (retRab8a-/-; retRab11a-/-) as in controls (retRab8a+/-; retRab11a-/-). Scale bar, 20 μm.
	Fig 9. Rhodopsin correctly targets to the OS in adult Rab8a retina cKO mice injected with GFP-Rab8b(T22N), GFP-Rab10(T23N), cMyc-Rab11a(S25N) and cMYc-Rab11b(S25N) AAV.One month old retRab8a-/- mice were injected subretinally with mixed Rab11a(S25N), 11b(S25N), 8b(T22N) and 10(T23N) AAVs or Rab10(T23N) only-AAV. Three months post-injection, rhodopsin, PDE6, GC1, CNGA1/A3 and ML-opsin all localize correctly in injected animals. Scale bar, 30 μm.

Babbey, Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells. 2006, Pubmed

Babbey, Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells. 2006, Pubmed
Bachmann-Gagescu, The ciliopathy gene cc2d2a controls zebrafish photoreceptor outer segment development through a role in Rab8-dependent vesicle trafficking. 2011, Pubmed
Best, Small GTPase Rab11b regulates degradation of surface membrane L-type Cav1.2 channels. 2011, Pubmed
Bok, The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. 1971, Pubmed
Butterworth, Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC). 2012, Pubmed
Chen, Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells. 2001, Pubmed
Deretic, Cytoplasmic domain of rhodopsin is essential for post-Golgi vesicle formation in a retinal cell-free system. 1996, Pubmed
Deretic, rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis. 1995, Pubmed
Farnsworth, Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A. 1994, Pubmed
Feng, A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis. 2012, Pubmed
Furuta, Retina- and ventral forebrain-specific Cre recombinase activity in transgenic mice. 2000, Pubmed
Guo, The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. 1999, Pubmed
Hattula, A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport. 2002, Pubmed
Jiang, Long-term RNA interference gene therapy in a dominant retinitis pigmentosa mouse model. 2011, Pubmed
Kelly, Rab11 proteins in health and disease. 2012, Pubmed
Knödler, Coordination of Rab8 and Rab11 in primary ciliogenesis. 2010, Pubmed
Lakso, Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. 1996, Pubmed
Lapierre, Myosin vb is associated with plasma membrane recycling systems. 2001, Pubmed
Le, Targeted expression of Cre recombinase to cone photoreceptors in transgenic mice. 2004, Pubmed
Li, Rhodopsin-iCre transgenic mouse line for Cre-mediated rod-specific gene targeting. 2005, Pubmed
Li, Myosin V, Rab11, and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors. 2007, Pubmed
Mattapallil, The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. 2012, Pubmed
Mazelova, Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4. 2009, Pubmed , Xenbase
Mazelova, Syntaxin 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. 2009, Pubmed
Moritz, Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods. 2001, Pubmed , Xenbase
Mukhopadhyay, Sensory signaling-dependent remodeling of olfactory cilia architecture in C. elegans. 2008, Pubmed
Murga-Zamalloa, Interaction of ciliary disease protein retinitis pigmentosa GTPase regulator with nephronophthisis-associated proteins in mammalian retinas. 2010, Pubmed
Nachury, A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. 2007, Pubmed
Nickell, Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography. 2007, Pubmed
Omori, Elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8. 2008, Pubmed
Pearring, Protein sorting, targeting and trafficking in photoreceptor cells. 2013, Pubmed , Xenbase
Peränen, Rab8 GTPase as a regulator of cell shape. 2011, Pubmed
Reish, Nucleotide bound to rab11a controls localization in rod cells but not interaction with rhodopsin. 2014, Pubmed , Xenbase
Riveiro-Álvarez, New mutations in the RAB28 gene in 2 Spanish families with cone-rod dystrophy. 2015, Pubmed
Roland, Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3. 2007, Pubmed
Roosing, Mutations in RAB28, encoding a farnesylated small GTPase, are associated with autosomal-recessive cone-rod dystrophy. 2013, Pubmed
Sato, Rab8a and Rab8b are essential for several apical transport pathways but insufficient for ciliogenesis. 2014, Pubmed
Sato, The Rab8 GTPase regulates apical protein localization in intestinal cells. 2007, Pubmed
Satoh, Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors. 2005, Pubmed
Seabra, Deficient geranylgeranylation of Ram/Rab27 in choroideremia. 1995, Pubmed
Silvis, Rab11b regulates the apical recycling of the cystic fibrosis transmembrane conductance regulator in polarized intestinal epithelial cells. 2009, Pubmed
Stenmark, Rab GTPases as coordinators of vesicle traffic. 2009, Pubmed
Tai, Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1. 1999, Pubmed
Troutt, Microtubule polarity and distribution in teleost photoreceptors. 1988, Pubmed
Wang, The Arf and Rab11 effector FIP3 acts synergistically with ASAP1 to direct Rabin8 in ciliary receptor targeting. 2015, Pubmed
Wang, The Arf GAP ASAP1 provides a platform to regulate Arf4- and Rab11-Rab8-mediated ciliary receptor targeting. 2012, Pubmed
Wang, Molecular complexes that direct rhodopsin transport to primary cilia. 2014, Pubmed
Welz, Orchestration of cell surface proteins by Rab11. 2014, Pubmed
Westfall, Retinal degeneration and failure of photoreceptor outer segment formation in mice with targeted deletion of the Joubert syndrome gene, Ahi1. 2010, Pubmed
Westlake, Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. 2011, Pubmed
Wu, Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo. 2005, Pubmed
Yoshimura, Functional dissection of Rab GTPases involved in primary cilium formation. 2007, Pubmed
Yoshimura, Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors. 2010, Pubmed
Young, The renewal of photoreceptor cell outer segments. 1967, Pubmed
Yu, Global ablation of the mouse Rab11a gene impairs early embryogenesis and matrix metalloproteinase secretion. 2014, Pubmed
Zolotukhin, Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. 2002, Pubmed