Abstract
Abstract: :
Purpose:Mutations in RP1 are a common cause of retinitis pigmentosa, yet little is known about the function of the RP1 protein in vision. We recently found that the RP1 protein is located in the connecting cilia of photoreceptor cells (IOVS, Jan 2002), which contain an axoneme comprised of microtubules. Sequence analysis demonstrates that the N-terminal portion of RP1 shows homology to doublecortin (DCX), a microtubule-associated protein with two repeated doublecortin domains (DC). The goal of this study is to explore the potential interaction of the RP1 protein with microtubules. Methods:Co-immunostaining and in vitro microtubule binding assays were used to assess the interaction between RP1 and microtubules in cultured cells and mouse retinas. Five expression constructs were made by subcloning different fragments of human RP1 cDNA into pcDNA3.1/V5-His vector. The constructs contain N-terminal RP1 with two repeated DC domains (N-RP1/2DC), N-terminus with one DC domain (N-RP1/1DC), N-terminus without DC domain (N-RP1/0DC), middle portion of RP1 (M-RP1), and C-terminal RP1 (C-RP1). COS-7 cells were transiently transfected to overexpress the recombinant truncated RP1 proteins. In some experiments, cells were treated with nocodazole to disrupt the cytoskeleton. Results:Double staining with anti-Rp1 and anti-acetylated tubulin antibodies showed that Rp1 was co-localized with the axonemal microtubules in photoreceptor cells. In transfected COS cells, the N-RP1/2DC protein colocalized with the cytoplasmic microtubule network; this colocalization was eliminated in nocodazole treated cells. Some cells transfected with the N-RP1/1DC construct were found to have a mixed labeling pattern of microtubule fibers and diffuse particles especially in the perinuclear region. Cells transfected with the other three constructs showed only diffuse particles in the cytoplasm. Binding assays revealed that Rp1 protein from mouse retina and recombinant N-RP1/2DC or N-RP1/1DC co-sedimented with polymerized microtubules. Conclusion:The RP1 protein appears to associate with microtubules in vivo and in vitro, and this interaction is dependent upon the two DC domains in the N-terminal portion of the protein. Based on these data we hypothesize that the RP1 protein may be part of a multi-protein complex which interacts with the axonemal microtubules of connecting cilia, and contribute to the structure of the cilia or movement of proteins between the inner and outer segment of photoreceptor cells.
Keywords: 561 retinal degenerations: cell biology • 517 photoreceptors