Tubulin Mrna and Protein Expression are Cyclic in Retinas Exposed to Light

Abstract

Abstract: : Purpose:We have shown that after light or dark adaptation (LA, DA, respectively), rhabdomeres in the octopus retina undergo reorganization and the photopigments rhodopsin and retinochrome redistribute. The abundance of other retinal proteins, such as S–crystallin, is also affected by LA and DA (Zuniga et al., Curr. Eye Res. 28:343–50, 2004). Rhabdomere reorganization and protein movements likely involve the cytoskeleton so we are focusing on signaling pathways that may affect the expression and/or distribution of tubulin and actin in LA and DA retinas. In this study we investigated tubulin levels in LA retinas to determine expression levels of mRNA and protein. Methods: Octopuses were DA for 3–4 hours and eyecups collected in the dark to use as controls. The remaining octopuses were moved to the light and sacrificed at 15–minute intervals for up to two hours. Sclera and lens were removed from all the eyecups, the retinas homogenized, mRNA isolated, and cDNA synthesized for quantitative RT–PCR. Retinal homogenates were prepared from some eyecups to use in Western blotting analyses. Relative comparisons of tubulin mRNA levels were made using 100 hundred ng of cDNA from the retinas sampled at the 15 min time points using a Bio–Rad iCycler and SYBR Green fluorescence. For quantitative Western blot analyses, an equal amount of protein was loaded in all lanes, proteins separated on a 12% SDS–PAGE gel and transferred to PVDF membranes. Results were quantified using a Bio–Rad Versadoc 3000. Results:Tubulin mRNA and protein levels in retinas that were DA and then moved to the light and sampled at 15 min intervals show a cyclic expression of tubulin mRNA and protein. For qRT–PCR, we compared the threshold points of all lighting conditions to the DA control and results show fold inductions of –0.64, 0.31, 0.73, 0.82, –1.15, –0.57, –0.95, 0.72, respectively, measured at 15 minute time intervals up to two hours. Western blots also show that the amount of α– tubulin measured as percent volume fluctuates in a cyclic pattern with an identical time course to mRNA levels. Conclusions:Our study shows a quantitative difference in the expression levels of tubulin mRNA and protein after DA animals are moved to the light. Both mRNA and protein show a cyclic expression rising, falling and rising again within the 2 hour sampling period. We believe there is a more complex regulation of tubulin than previously believed.

Keywords: cytoskeleton • gene/expression • photoreceptors