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Rosario Sanchez-Gonzalez, John Willoughby, Abbie M. Jensen; Microtubules Dynamics During Photoreceptor Morphogenesis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):911.
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The sensory compartment (outer segment) of vertebrate photoreceptors is a highly modified cilium supported by a microtubular axoneme. The synthetic machinery supporting phototransduction and outer segment growth is located in the inner segment, and proteins are transported through the connecting cilium into the outer segment. The ciliary microtubules provide structural support for the outer segment and are the tracks for the intraflagellar transport for outer segment cargo. However, little is known about the microtubule dynamics in photoreceptors and whether ciliary microtubule dynamics contribute to outer segment morphogenesis and maintenance. Our study seeks to determine the dynamics of microtubules in the photoreceptors, in particular outer segments.
For transgenesis, we cloned into a zebrafish transposase vector the construct where mCherry-αTubulin (mCherry-Tub) is under the control of the zebrafish hsp70 promoter. To generate mosaic transgenics, we injected this plasmid along with transposase mRNA into one or two-cell zebrafish. Injected larvae were heat-shocked (37oC for 45 minutes) to induce transgene expression. Larvae were examined by immunofluorescence at various times post-heat shock (pHS).
Our preliminary results show that following heat shock induction, mCherry-Tub is expressed in several retinal cell types, including photoreceptors. When we heat-shock 3 days post fertilization (dpf) larvae, we observed mCherry-Tub expression soon after heat shock (within two hours pHS). When we examined photoreceptors in 5 dpf larvae 2 days pHS, mCherry-Tub seems as strong as that observed at 2 hours pHS, suggesting that mCherry-Tub is stable and is not rapidly turned over.
By using this new probe of microtubule dynamics, we should be able to measure microtubule dynamics in photoreceptor outer segments and determine the stability of axonemal microtubules.
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