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Z. Han, D.S. Papermaster; Proteolysis and Circadian Variation of Localization of Prominin–1 in Wild–Type and Transgenic X. Laevis Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2006;47(13):819.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the proteolysis and circadian variation of subcellular localization of prominin–1 in X. laevis photoreceptors.
Antibody generation and verification: Polyclonal antibodies against N– and C–terminal sequences of X. laevis prominin–1 were generated in rabbits. Antibodies were affinity purified and their specificities were verified by antigen competition. Immunolabeling of tadpole eyes: X. laevis tadpoles were sacrificed at different time points in the 24–hour cycle. Their eyes were fixed in 4% paraformaldehyde for 1 hour and embedded in OCT for cryosectioning. The sections were stained with the affinity–purified antibodies overnight, Alexa Fluor 488 conjugated secondary antibody for 2 hours and examined with a Zeiss 510 confocal microscope. Fractionation of retinal membrane proteins: Isolated X. laevis retinas were sheared by repeated gentle passage through an 18 gauge trochar. Fractions were collected using differential sucrose gradient centrifugation according to Papermaster et. al, (Biochemistry, 1975). Restriction enzyme mediated transgenesis: Plasmids containing the Prominin–1–hrGFP expression cassette were linearized, mixed with the permeablized sperm nuclei and injected into X. laevis eggs as described by Kroll and Amaya (Development, 1996). Transgenic tadpoles expressing the transgene were screened under a fluorescence illuminated dissecting microscope for hrGFP fluorescence (Moritz et. al, IOVS, 1999).
Immunoblotting of fractionated membrane proteins from X. laevis retina using antibodies specific for both N– and C–terminal sequences of prominin–1 reveals multiple bands that represent distinct cleaved forms of prominin–1. Immunoblotting with anti–hrGFP antibody on retinal proteins from transgenic tadpoles expressing prominin–1–hrGFP reveals the same pattern of cleavage, except that the fragments carrying the hrGFP tag shift to higher MW, as expected. The anti prominin–1 N–terminal antibody specifically labels the base of the rod outer segments as a thin band in a circadian manner: The labeling appears strongest in the afternoon and diminishes or disappears at other time points. The same antibody also labels the entire outer segment of cones without obvious circadian variation.
1) Prominin–1 appears to be proteolyzed in X. laevis photoreceptors in vivo. 2) There is a circadian variation in the immunolabeling of prominin–1 at the base of the rod outer segments, but not the outer segment of cones. Prominin–1 appears to be confined to rod basal disks but is distributed throughout cone lamella.
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