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Ya-Chu Hsu, Jen-Zen Chuang, Jason Chen, Yu-Ting Yan, Yun Z. Le, Ching-Hwa Sung; Sara Regulates Opsin Trafficking And Outer Segment Morphogenesis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):747.
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© ARVO (1962-2015); The Authors (2016-present)
Mammalian outer segments of retinal photoreceptors turn over every 10 days. Disposed outer segment membranes and proteins are engulfed and degraded by adjacent pigmented epithelia. Directional and regulated transport of membrane and proteins from the cell body to renew the outer segment is necessary. The cellular mechanism that controls renewal processes remains elusive. Previously we demonstrated that SARA, a FYVE domain-containing protein, is required for biogenesis of nascent discs at basal outer segments in rods. We proposed Sara promotes fusion of rhodopsin-bearing vesicles to form new discs. In this study we investigate how SARA regulates outer segment morphogenesis in vivo using Sara knockout mice.
SARA-knockout mice were generated to assess SARA’s functions in retinas. In addition, SaraF/F mice were bred to tissue specific Cre mice to generate rod-specific and cone-specific knockouts.
Sara localizes to the base of rod outer segments and is also present in cone photoreceptors. Sara null mice are viable and fertile. Western blots of retina lysates confirmed the loss of Sara expression in Sara null mice. Lack of Sara did not change the gross histology and lamination of the retinas. Outer segments in Sara null retinas were able to form but are structurally fragile and prone to damages compared to wildtype counterparts. Rhodopsin and cone opsins are severely mislocalized, and peanut agglutinin (PNA) labeling was misplaced to the outer nuclear layer. Morphology of cone pedicles appeared abnormal in the absence of Sara. To tease out how Sara function contributes to rods and cones, we generated tissue- specific knockouts (KO), namely rod-KO and cone-KO. In rod-KO rhodopsin was mislocalized as early as postnatal day 17 and could be detected at 3 months. Cone cell development appeared unaffected in rod-KO. In cone-KO retinas, cone outer segments were shortened and thinner than those in the wildtype. PNA labeling showed reduced intensity. We are interested in examining the expression of known Sara-interacting molecules in KO retinas. To directly examine how Sara regulates rhodopsin trafficking in vivo, we will pulse-express rhodopsin reporters in Sara KO using in vivo retinal transfection.
Opsin unable to target to the outer segment efficiently suggested that Sara regulates outer segment renewal. Our data indicated that Sara is critical for the disc insertion of opsin molecules of both rods and cones. Reduced expression of PNA in cone-KO suggested Sara may regulate the secretion of inter-photoreceptor materials.
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