RNAI AND MORPHOLINO AS TOOLS TO STUDY SIGNALING IN LIMULUS VENTRAL PHOTORECEPTORS.

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2006-07-12
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Deshpande, Monika Manohar
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Payne, Richard
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ABSTRACT Title of Document: RNAI AND MORPHOLINO AS TOOLS TO STUDY SIGNALING IN LIMULUS VENTRAL PHOTORECEPTORS. Monika M Deshpande, Doctor of Philosophy, 2006 Directed By: Dr. Richard Payne, Neuroscience and Cognitive Science program Limulus ventral photoreceptors have been studied using electrophysiological, immunocytochemical, and biochemical techniques. However, genetic manipulation has eluded Limulus research because the animal takes about 10 years to mature. As an alternative, we decided to explore the possibility of using RNA interference (RNAi), and morpholino to reduce the levels of a target protein. The purpose of this study was to test whether use of these techniques would result in any physiological changes in the ventral photoreceptors. As a target we chose arrestin, a protein that binds to and quenches metarhodopsin, the activated form of rhodopsin. Injecting arrestin RNAi, or arrestin morpholino into the cells had a profound effect. The rate of spontaneous quantal events ('bumps') in the injected cells had a 5-fold increase as compared to bump rates of control cells at 24 hrs after injection. Because high levels of metarhodopsin are thought to be present in ventral photoreceptors even in darkness, this result is consistent with an increase in the amount of unquenched metarhodopsin in the cells. To show that the increase in spontaneous dark bumps was a result of unquenched metarhodospin, we treated RNAi -injected cells with hydroxylamine, a bleaching agent that destroys metarhodopsin. The bump rate after this treatment was down to pre-injection levels confirming our hypothesis that the excess bumps were generated by unquenched metarhodopsin. Another target protein selected was opsin. Opsin bound to the chromophore forms rhodopsin, and rhodopsin photoconverts to metarhodopsin. Therefore, opsin reduction was expected to lead to a reduction in sensitivity to light. sensitivity to light decreased by about 30 fold at 96 hrs after opsin morpholino injections. In another set of experiments, we fixed the injected cells with the arrestin/opsin antibodies. We measured the fluorescence from thee secondary antibodies targeted against the arrestin/opsin primary antibodies to get an insight into the protein levels. We observed a 2.5 fold decrease in fluorescent counts in arrestin morpholino injected cells and a 17 fold decrease in opsin injected cells.
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