Abstract
Abstract: :
Purpose: The RP1 protein has been shown to be localized in the connecting cilia of photoreceptors. This location implies a role for RP1 in protein transport and/or outer segment disk morphogenesis. To help elucidate the function of RP1 and the mechanism by which mutations in RP1 lead to photoreceptor cell death, we have determinated more precisely the location of the Rp1 protein using several photoreceptor markers and mice expressing two modified alleles of Rp1. Methods: The location of Rp1 in mouse photoreceptors was compared with several markers, including acetylated α-tubulin, Rom1, peripherin/rds. The distribution of abnormal Rp1 proteins was investigated using two mouse models. Rp1-myc mice were generated by truncating the Rp1 gene after codon 662, which is analogous to the most common mutation in RP1 patients, Arg677Ter. Rp1-exon 2/3 deletion mice were generated by removing exons 2 and 3, which disrupted the N-terminal 265 aa, from the Rp1 gene. These two Rp1 mutant mice were crossed to generate Rp1 double mutant mice (Rp1myc/exon2/3 deletion) expressing both abnormal Rp1 proteins. The relative locations of the two mutant Rp1 proteins was determined by confocal immunomicroscopy. Results: Double immunostaining of Rp1 with acetylated α-tubulin showed that Rp1 was localized in the axonemal microtubules of photoreceptors. The most proximal portion of the Rp1 signal (0.4-1µm in length) did not overlap with the outer segments labels of Rom1, peripherin/rds. The more distal portion of the Rp1 signal extended 5-6 µm along the edge of the outer segments. In the double mutant Rp1myc/exon2/3 deletion mice, both mutant proteins were expressed in the retina, but their locations were distinct. The Rp1-myc protein, which contains the N-terminal 661aa including the doublecortin domain, was detected only in the axoneme of outer segments. The protein generated from the Rp1-exon 2/3 deletion allele, which lacks the doublecortin domain, was located proximally in the connecting cilia. Conclusions: The RP1 protein is a component of the axoneme of rod and cone photoreceptors. This axoneme is part of the connecting cilia, but also extends into the outer segments. The distinct locations of the two mutant Rp1 proteins in the double mutant mice indicates that the doublecortin domain mediates its association with the outer segment portion of the axoneme. We are hopeful that further investigations of the interactions between RP1 and other components of the photoreceptor axoneme will provide insight into the function of RP1.
Keywords: photoreceptors • retina: distal(photoreceptors, horizontal cell • retinal degenerations: cell biology