Rod photoreceptors outnumber by 20:1 all other cell types in most vertebrate retinas. Arrestin is a 48 KDa protein expressed specifically in rod photoreceptor cells. Arrestin is the second-most abundant protein in the retina, estimated to be present at 1:10 to 1:2 the quantity of the most abundant retinal protein, rhodopsin, the visual pigment and G-protein coupled receptor that captures light and initiates the rod's light response. Arrestin plays an essential role in the inactivation of light-activated rhodopsin, capping the latter once it is phosphorylated. Arrestin was originally discovered and named retinal S-antigen, because it can evoke uveitis, an autoimmune inflammation of the blood-vessel rich layers of the eye. Arrestin undergoes a remarkable light-dependent migration between the two major compartments of the rod cell: in the dark-adapted eye it is concentrated largely in the inner segment region, while under light-adapted conditions it translocates almost completely to the outer segment, where rhodopsin resides. The proposed work investigates the molecular mechanisms underlying the light-dependent translocation of arrestin in living rod cells of the African clawed frog, Xenopus laevis, using arrestin-EGFP fusion proteins as tracers/biosensors, and 2-photon, confocal microscopy to quantify the protein and its movement. Work by several laboratories has shown such fusion proteins faithfully track the movement of native arrestin, and work from this laboratory has established that the dark-adapted distribution of arrestin is in disequilibrium with the cytoplasm. The proposed work will test several hypothesis about the nature of the arrestin dark disequilibrium and light-driven movement, including that (a) the disequilbrium is effected by a molecular """"""""gate"""""""" in the connecting cilium; (b) the light-driven movement occurs purely by passive (diffusional) redistribution upon massive binding epitope creation by rhodopsin activation; (c) the redistribution is a light-activated process involving intraflagellar transport proteins, which serve to carry proteins from their origin to site of use in polarized, ciliated cells. ? ?
| Calvert, Peter D; Schiesser, William E; Pugh Jr, Edward N (2010) Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium. J Gen Physiol 135:173-96 |
| Nikonov, Sergei S; Brown, Bruce M; Davis, Jason A et al. (2008) Mouse cones require an arrestin for normal inactivation of phototransduction. Neuron 59:462-74 |
| Calvert, P D; Peet, J A; Bragin, A et al. (2007) Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion. J Microsc 225:49-71 |
