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 ∼40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

Arrestin Translocation in Rod Photoreceptors

“…These results are consistent with quantitative studies showing that the mass of arrestin-GFP is conserved during translocation from the RIS to the ROS in response to light (Peet et al 2004). This study did not quantify the translocation from ROS to RIS during dark adaptation.…”

Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina

“…9). EGFP is a highly soluble protein, whose distribution in cells, including photoreceptors, reflects the water space [47]. The dominant water spaces of mouse cones are the pedicles in the OPL and the cell body and inner segment, which for most cones span the ELM (Fig.…”

Dephosphorylation during Bleach and Regeneration of Visual Pigment in Carp Rod and Cone Membranes

“…To obtain estimates of the CCP/OSP, we assumed that the cytoplasmic volume in the outer segment of a rod and a cone is half of the outer segment envelope volume (27), which is the volume determined from the outer dimension of an outer segment. In case responsible proteins for dephosphorylation are localized only in the outer segment, the CCP/OSP can be calculated by dividing the above protein content in a single rod or cone (7.2 pg/rod and 188 pg/cone) by half of the outer segment envelope volume.…”