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Kasey Rose, Rahul Kumar, Jeannie Chen; Rho-Timer, a reporter of rhodopsin biosynthesis and incorporation in to the rod outer segment in transgenic mouse rods. Invest. Ophthalmol. Vis. Sci. 2017;58(8):341.
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Our understanding of how light exposure affect rhodopsin synthesis, trafficking and rod outer segment (ROS) formation in the mammalian rod cell is still incomplete. We expressed a fluorescently tagged rhodopsin, Rho-Timer, to visualize these processes in intact mouse rods.
Timer was attached to rhodopsin’s C-terminus, followed by an additional 11 a.a. of rhodopsin C-terminus repeated at Timer’s C-terminus. Mice expressing Rho-Timer were raised in standard conditions (12 h light:12 h dark) and at 1 month of age they were treated as follows for 10 days: complete darkness (n=12), diurnal (12h light, 12h dark, n=10), constant light (n=18), and 5d dark/5d light (n=11). Dissociated ROS were imaged using a Leica SP8 microscope. ROS lengths were measured with LAS Lite software, and fluorescent intensity profiles and number of fluorescent bands were measured using ImageJ. Northern blots for endogenous rhodopsin and Rho-Timer were performed. One-way ANOVA and post-hoc T-Tests were used for statistical analysis.
One-way ANOVA detected a statistically significant difference in ROS length between treatment groups (p<0.001). ROS length decreased in both 10d light (14.1μm±3.9) and 5d dark/5d light (10.7±1.9 μm) when compared to dark (23.4±3.3 μm) or cyclic light (21.1±1.4 μm). A post-hoc t-test showed that length differed significantly (p<0.001) in dark, light, and 5 d dark/light conditions from diurnal light conditions. An ANOVA on fluorescent banding pattern yielded significant variation among the treatments (p<0.001). Clear patterns of alternating intense and dim fluorescent bands were observed in the ROS of both constant dark and diurnal treatment groups, whereas 10d light and 5d dark/5d light caused a complete or partial loss of fluorescent banding pattern. The number of fluorescent bands are: 10d dark (10.2±1.1), 10d light (5.9±1.2), 5d dark/5d light (5.6±1.6), and cyclic light (9.3±1.2). Finally, no significant difference was found in the RNA expression of rhodopsin and Rho-Timer under light or dark conditions.
Rho-Timer mRNA tracks endogenous rho mRNA. Rho-Timer incorporation into the ROS is regulated at the post-transcription level. Pattern of Rho-Timer distribution in discs appeared to mirror an internal circadian rhythm. Excess light disrupted rho incorporation and ultimately changed disc rho composition.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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