Photoreceptor outer segment membranes, the site of initial photon capture initiating phototransduction, undergo renewal with total replacement occurring at ten day intervals throughout life. A challenge to cellular integrity concerns the post biosynthetic delivery of replacement proteins from the inner, to the outer, segment over the lifetime of the photoreceptor. Complex trafficking pathways require multiple components, such as acyl-binding proteins, Rab GTPases and centrins. This application will identify: i) key Rab GTPases involved in vesicular transport of membrane protein, and ii) centrins participating in ciliary transport.
Aim 1 will focus on Rab GTPases in photoreceptor vesicular transport. Rab (Ras analog in brain) proteins are members of the Ras supergene family involved mainly in membrane protein trafficking. More than 70 distinct Rab polypeptides have been identified.
Aim 1 a focuses on Rab8 and Rab11 isoforms which are known to be important for photoreceptor trafficking in Xenopus and Drosophila models. Surprisingly, a mouse Rab8a/Rab11a double knockout does not produce a defective trafficking phenotype. Therefore, we will focus on closely-related isoforms to identify key Rab proteins responsible for membrane protein organization and rhodopsin trafficking.
Aim 1 b investigates Rab28, shown recently associated with recessive human cone/rod dystrophy.
Aim 2 will examine the function of centrosomal proteins, called "centrins," which are 20kD EF- hand calcium binding proteins (four EF-hand motifs) of the calmodulin superfamily. Centrins were first described in unicellular green algae where they are associated with the flagellar basal apparatus. Photoreceptor centrins (isoforms 1-4) are located in the ciliary lumen and basal body where they associate with transducin via its T??-subunits. One hypothesis is that centrins may be involved in regulation of transducin translocation from the outer, to the inner, segment or vice-versa. As preliminary results, we found that centrin1 is required for the nucleus-basal body connection during mouse spermiogenesis, and centrin2 regulates trafficking of ACIII and channel subunits in olfactory sensory neurons.
This aim seeks to identify the roles of photoreceptor centrin3 and centrin4 which associate strongly with the basal body and/or connecting cilium axoneme. We expect to generate mouse models of syndromic disease with retina degeneration, e.g., Joubert or Senior-Loken syndromes.
We propose to generate several knockout and/or knockdown mouse models to elucidate the function of key proteins involved in membrane protein trafficking. The candidates for our research include Rab GTPases (Rab8, Rab11, Rab28) and centrins (Cetn3, Cetn4). Aim 1: Rab (ras analogs in brain) proteins are membrane organizers that participate in membrane protein transport. More than 70 distinct Rab proteins are expressed in mammalian cells. Rab proteins are prenylated, i.e., modified by the addition of hydrophobic molecules posttranslationally, which is important for insertion into membrane. For example, incorrectly processed Rab27 is responsible for the etiology of choroideremia, a blinding disorder of human patients. We intend to identify Rab proteins essential for rhodopsin trafficking in photoreceptors, starting with Rab8 and Rab11 isoforms, as they have been shown previously to organize rhodopsin trafficking in Xenopus and Drosophila. A third Rab GTPase, Rab28, was shown recently to be associated with autosomal recessive cone-rod dystrophy (arCORD). Accordingly, we propose to generate a Rab28-/- mouse model, study the effect of Rab28-deficiency on phototransduction/membrane trafficking, and test vectors for gene replacement therapy. Aim 2. Centrins are ancient Ca2+-binding proteins present in all animals and some plants. Centrins are associated with connecting cilia and the microtubule organizing centers (basal bodies) of photoreceptors. As centrin isoforms (1-4) interact with transducin, a hypothesis was proposed in which centrins regulate transducin trafficking. In preliminary knockout mouse experiments, however, we found that centrin 1 participates in tail formation during spermiogenesis, and centrin 2 facilitates trafficking of membrane protein in olfactory sensory neurons. Our difficulty in demonstrating a photoreceptor phenotype in centrin1, centrin2 and centrin1/2 knockouts is likely due to isoform redundancy. In proposing to examine centrin3 or centrin4 function on a centrin1/2 double knockout background, we expect that loss of three or all four centrins may replicate complex and severe phenotypes, such as Bardet-Biedl, Meckel-Gruber (MKS) or Joubert (JBTS) syndromes.
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