Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors

Jon A. Peet, Alvina Bragin, Peter D. Calvert, Sergei S. Nikonov, Shoba Mani, Xinyu Zhao, Joseph C. Besharse, Eric A. Pierce, Barry E. Knox, Edward N Pugh Jr

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52 Scopus citations

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 mitochondrialrich 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.

Original languageEnglish (US)
Pages (from-to)3049-3059
Number of pages11
JournalJournal of Cell Science
Volume117
Issue number14
DOIs
StatePublished - Jun 15 2004
Externally publishedYes

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Keywords

  • Arrestin
  • Protein movement
  • Transgenesis
  • Xenopus

ASJC Scopus subject areas

  • Cell Biology

Cite this

Peet, J. A., Bragin, A., Calvert, P. D., Nikonov, S. S., Mani, S., Zhao, X., Besharse, J. C., Pierce, E. A., Knox, B. E., & Pugh Jr, E. N. (2004). Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors. Journal of Cell Science, 117(14), 3049-3059. https://doi.org/10.1242/jcs.01167