<p>The complex process of visually interpreting our environment begins with the task of detecting the light that enters our eyes. This task is performed by the rod and cone photoreceptors, which both contain a highly evolved sensory cilium called the outer segment. The outer segment is a specialized cellular compartment that contains all of the protein machinery involved in converting the initial light signal into an electrical signal that can be ultimately transmitted to the brain. Outer segments are cylindrical structures that envelop an array of individual, densely packed membrane discs. Discs are renewed throughout the lifetime of a photoreceptor, with older material being shed at the tip and new material added at the base of the outer segment. Many studies conducted over the past 35 years conclude that disc formation starts with evagination of the plasma membrane at the outer segment base, followed by membrane expansion and, in the case of rods, subsequent disc enclosure. Despite the intense interest in the topic, the molecular mechanisms governing how outer segment discs are formed and renewed are not well understood. </p><p>The focus of this dissertation centers on elucidating the molecular role of peripherin/retinal degeneration slow (rds) in outer segment disc morphogenesis, including study of peripherin/rds trafficking from its site of synthesis in the endoplasmic reticulum to its site of function in the outer segment. Peripherin/rds is expressed specifically in photoreceptor outer segments, where it fulfills a critical role in assembling and/or maintaining the structure of this organelle. Mutation or loss of peripherin/rds in humans is often associated with visual impairments, and its knockout in mice results in rudimentary ciliary stumps completely lacking disc structures. </p><p>We found that early outer segment morphogenesis steps in mice lacking peripherin/RDS proceed normally for the first week. However, in the second week of postnatal development at the onset of disc formation, mice lacking peripherin/RDS produce extracellular vesicles next to their connecting cilia rather than discs. We characterized these vesicles and determined that they are enriched in outer segment proteins, are ~230 nm in size, and are formed as outward buds of the plasma membrane. These characteristics allowed us to classify these extracellular vesicles as ciliary ectosomes. Furthermore, we determined that ectosome shedding is arrested upon expression of the peripherin/rds C-terminal cytoplasmic sequence, which allows for the accumulation of excessive membranous material. Thus, we conclude that peripherin/rds transforms the functional dynamics of photoreceptor primary cilium from shedding massive amounts of ectosomes to retaining these membranes in the outer segment to eventually become photoreceptor discs. This novel function of peripherin is performed by its C-terminal cytoplasmic sequence and represents the first step in disc morphogenesis. </p><p>Finally, the morphogenesis study of peripherin/rds is complemented by a study of its trafficking. Understanding how peripherin is delivered from the site of its synthesis is critical for its function at the outer segment. We show that the peripherin/rds targeting sequence is confined within ten amino acid residues, which do not overlap with the putative fusogenic domain, and that only a single amino acid within this region is irreplaceable, a highly conserved valine at position 332.</p><p>Collectively, these studies shed considerable light on the molecular role played by peripherin. Peripherin is a photoreceptor specific protein that transforms the primary sensory cilium into a specialized sensory cilium capable of building the discs required for efficient photon capture. While work in this direction provides a significant advance in our understanding of peripherin’s role in disc morphogenesis, questions such as whether peripherin participates in disc enclosure, remain to be solved.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/11379 |
| Date | January 2015 |
| Creators | Salinas, Raquel Ybanez |
| Contributors | Arshavsky, Vadim |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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