Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors.
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.
Peet
Jon A
JA
Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA.
Bragin
Alvina
A
Calvert
Peter D
PD
Nikonov
Sergei S
SS
Mani
Shoba
S
Zhao
Xinyu
X
Besharse
Joseph C
JC
Pierce
Eric A
EA
Knox
Barry E
BE
Pugh
Edward N
EN
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
D019393:Arrestin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins