title: Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors.
abstract: The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which approximately 40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.
authors.affiliation: Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Jon A
authors.initials: JA
authors.lastname: Peet
authors.forename: Alvina
authors.initials: A
authors.lastname: Bragin
authors.forename: Peter D
authors.initials: PD
authors.lastname: Calvert
authors.forename: Sergei S
authors.initials: SS
authors.lastname: Nikonov
authors.forename: Shoba
authors.initials: S
authors.lastname: Mani
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zhao
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Besharse
authors.forename: Eric A
authors.initials: EA
authors.lastname: Pierce
authors.forename: Barry E
authors.initials: BE
authors.lastname: Knox
authors.forename: Edward N
authors.initials: EN
authors.lastname: Pugh
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019393:Arrestin / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism*; D003623:Dark Adaptation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008027:Light; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000166:cytology* / Q000378:metabolism; D014981:Xenopus
chemical_list: D019393:Arrestin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins