A method for combined pulse chase labeling and super-resolution microscopy in mouse rod photoreceptor neurons

About This Grant

PROJECT SUMMARY Efficient protein trafficking is vital for photoreceptor and visual health since photoreceptor cell death is directly preceded by protein mistrafficking in numerous blinding diseases. Despite the accrued knowledge of photore- ceptor function, there remain large knowledge gaps about the dynamics of protein trafficking in mammalian pho- toreceptor cells at the mechanistic level. The overall goal of this proposal is to develop a novel fluorescence- based, in vivo pulse-chase technique for tracking specific proteins within mouse photoreceptors. This new tech- nique, which uses SNAP-tag technology to label nascent photoreceptor proteins, fills a long-standing technical gap for studying the temporal dynamics of mammalian photoreceptor cell biology. Furthermore, we will combine nascent protein SNAP-tag labeling with quantitative super-resolution microscopy to analyze photoreceptor pro- tein dynamics on a subcellular scale. Thus, the proposed technique is predicted to be a tool that enables the discovery of fundamental and disease-relevant photoreceptor protein trafficking mechanisms. To test this new technique, rhodopsin trafficking in mouse rod photoreceptors will be used as a model system, since rhodopsin trafficking is essential for healthy rod function, yet much remains to be discovered about mammalian rhodopsin transport. In pilot studies, we observed correct targeting of a rhodopsin-SNAP fusion to rod outer segments following AAV-mediated delivery and live fluorescence labeling via intravitreal injection. Based on this finding and other preliminary data, the proposed studies are highly feasible. Additionally, the research is highly innova- tive since it is a novel application of SNAP-tags in the mouse retina, which will be technically validated and tested throughout the course of this project. The research aims are designed to rigorously validate and optimize in vivo SNAP-tag pulse-chase labeling in the mouse retina and to apply this approach to analyze the subcellular dy- namics of nascent rhodopsin trafficking. In Aim 1, the dosage and clearance rate for SNAP-tag substrate fluo- rescent dyes will be determined following intravitreal injection to mouse retinas that have been transduced with a rhodopsin-SNAP AAV. In Aim 2, the efficiency of rhodopsin-SNAP pulse chase labeling will be determined by measuring the turnover of pulse-labeled rhodopsin in rod outer segments. Then, nascent rhodopsin trafficking in the inner segment will be quantitatively analyzed with super-resolution imaging. Completion of the research aims will 1) yield critically needed methodology for analyzing protein dynamics in photoreceptors and 2) uncover new details about the rhodopsin trafficking kinetics in mouse rods. Discovery of novel molecular mechanisms that promote long-term photoreceptor survival will provide the framework for understanding the subcellular pathology of various blinding diseases. As such, successful completion of this research will greatly inform the optimization of novel vision therapies for clinical applications, which aligns well with the NEI’s mission to “drive innovative research to understand the eye and visual system” and to “prevent and treat vision diseases.”