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Tylor Lewis, Peter J Volberding, Joseph C Besharse; Characterization of the zebrafish cone cytoskeleton using a fluorescent tubulin. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):441.
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© ARVO (1962-2015); The Authors (2016-present)
In explanted zebrafish retinas, cone photoreceptors frequently exhibit microtubule-containing neuritic processes at the distal axoneme that extend beyond the outer segment (OS) and axonemes frequently exhibit branch points within the OS (Bader, et al., 2012. Vision Research). In order to better understand microtubule organization in cone photoreceptors, we have used confocal microscopy and transmission electron microscopy (TEM) on developing and adult zebrafish that express fluorescent tubulin specifically in cone photoreceptors.
A line of zebrafish was generated that express tdEOS, a photoconvertible fluorophore, tagged to α-tubulin (tdEOS-tubulin) under control of the transducin-α cone promoter (TaCP), allowing the cone cytoskeleton to be visualized. Cytoskeletal structure was analyzed with confocal microscopy on histological cryosections of larval and adult eyes, combined with confocal microscopy of ex vivo cultures of isolated photoreceptors and intact retinal slices. In conjunction, TEM was performed on adult zebrafish to analyze ultrastructure of putative microtubule-based processes.
In isolated preparations, a splayed structure adjacent to the OS contained microtubules, and was characteristic of the accessory OS described in other teleosts. Myoid elongation, a well-defined retinomotor movement, was observed in isolated photoreceptors and, as expected, was regulated by addition of dopamine. Interestingly, cultured cones and retinal slices formed neuritic projections containing microtubules that emanated from both the cone inner segment (IS) and OS that also appeared to be regulated by dopamine. TEM of intact retina revealed microtubule-based structures associated cone photoreceptors that are embedded within the retinal pigmented epithelium (RPE).
A variety of unique characteristics of the cone cytoskeleton were observed using tdEOS-tubulin, including the accessory OS and myoid elongation regulated by dopamine, characteristic of retinomotor movements. More unconventionally, putative neuritic extensions that were also regulated by dopamine were observed in culture. Evidence in TEM suggests that there are microtubule-based processes associated with cones in vivo that interact with the RPE. Further characterization of the structure and biological function of these microtubule-based processes is ongoing.
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