All ETDs from UAB

Advisory Committee Chair

Alecia K Gross

Advisory Committee Members

David Bedwell

Gwendalyn King

Timothy Kraft

Lori L McMahon

David Sweatt

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The health and maintenance of rod photoreceptors depends on the high-fidelity vectorial transport of rhodopsin from its site of synthesis in the inner segment to its final resting place in the outer segment disks. Multiple molecular interactions play a role in this process, including the interaction of the small GTPase rab11a with rhodopsin that was first described in vertebrates in amphibians. We found that the interaction of the small GTPase rab11a and rhodopsin is conserved in mammalian retina. This interaction is direct, is abrogated by human disease-causing truncation mutations in rhodopsin, and does not depend on the nucleotide binding status of rab11a. The nucleotide binding status of rab11a does affect subcellular localization, as shown in transgenic Xenopus laevis tadpole retina. We show for the first time that rab11a interacts with the outer segment disk membranes and ciliary axoneme in a nucleotide binding status dependent fashion. Expression of the dominant-negative rab11a mutant N124I in rods leads to shorter outer segments, cell death, and ectopic process formation. Knockdown of rab11a in Xenopus rods leads to ectopic process formation and progressive degeneration, mimicking the human retinitis pigmentosa phenotype seen with rhodopsin truncation mutations. In addition, we also observed the interaction of the protein NUDC with the rhodopsin-rab11a complex during trafficking. NUDC localizes to the inner segment and ciliary axoneme in Xenopus rods. The interaction of rab11a and NUDC, as measured by bimolecular fluorescence complementation, also occurs in the inner segment and ciliary axoneme. While the L280P mutation in NUDC did not affect its subcellular localization, it did change the area of interaction to be exclusively in the distal inner segment. The interaction of rab11a and NUDC appears to be a novel molecular component of golgi-to-cilium trafficking. In addition, we examined the retinal phenotype of the klotho knockout mouse. We found that aged klotho knockout mice have reduced and delayed electroretinogram (ERG) responses in the absence of photoreceptor cell death. Protein levels of phototransduction components were not altered in the klotho mice but labeling for sugar modifications with wheat germ agglutinin increased in the synaptic layers of the retina.

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