Sunday, November 24, 2024
11/24/2024
Project Summary/Abstract: In this proposal we will address one of the major unanswered questions in vision: the mechanism of rhodopsin delivery and loading into new photoreceptor discs. This perpetual process is particularly robust in rods where, for mammals, the daily delivery of 30 million rhodopsins supports the formation of 80 new discs in each rod. While rhodopsin synthesis and delivery to the rod apical region has been intensively studied, almost nothing is known about what happens afterward, thus representing a critical knowledge gap. Roadblocks to under-standing this process include the complexity of the inner segment-outer segment interface that is below the resolution limit of fluorescence microscopy and the limitations of prior approaches that relied almost exclusively on static analyses in fixed tissues. To overcome these roadblocks, we have implemented live-cell 3D super resolution single particle tracking microscopy (3D-sptPALM) to directly examine the dynamics of individual rhodopsins within discrete photoreceptor compartments in real time. We will exploit a key mouse model, the retinal degeneration slow (rds) mouse, which allows us to intentionally vary the level of outer segment membrane complexity while retaining the photoreceptor cilium. Our preliminary results show the entry of individual rhodopsins into photoreceptor cilia, their transport along the cilium and, remarkably, their exit back into the inner segment, for the very first time. We will address the following fundamental questions: Aim 1: How does rhodopsin enter (and exit) the photoreceptor cilium? Aim 2: How is rhodopsin transported within photoreceptor cilia? Aim 3: How are rhodopsins loaded into new discs?
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