May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Quantification of light–induced arrestin–EGFP redistribution in living Xenopus rods
Author Affiliations & Notes
  • P.D. Calvert
    Kirby Ctr for Molec Ophthalmol, University of Pennsylvania, Philadelphia, PA
  • J. Peet
    Kirby Ctr for Molec Ophthalmol, University of Pennsylvania, Philadelphia, PA
  • A. Bragin
    Kirby Ctr for Molec Ophthalmol, University of Pennsylvania, Philadelphia, PA
  • S.S. Nikonov
    Kirby Ctr for Molec Ophthalmol, University of Pennsylvania, Philadelphia, PA
  • S. Mani
    Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY
  • B.E. Knox
    Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY
  • E.N. Pugh, Jr.
    Kirby Ctr for Molec Ophthalmol, University of Pennsylvania, Philadelphia, PA
  • Footnotes
    Commercial Relationships  P.D. Calvert, None; J. Peet, None; A. Bragin, None; S.S. Nikonov, None; S. Mani, None; B.E. Knox, None; E.N. Pugh, Jr., None.
  • Footnotes
    Support  EY–02660
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3448. doi:
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      P.D. Calvert, J. Peet, A. Bragin, S.S. Nikonov, S. Mani, B.E. Knox, E.N. Pugh, Jr.; Quantification of light–induced arrestin–EGFP redistribution in living Xenopus rods . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3448.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose:To quantify the redistribution of GFP–tagged arrestin triggered by rhodopsin bleaching in living, physiologically active photorecptors. Methods: A Xenopus rod arrestin–EGFP fusion protein or EGFP was expressed in transgenic Xenopus rods under the rhodopsin promoter. Living fragments of retinas from late stage tadpoles or froglets were placed into a recording chamber and the cellular distribution of EGFP or arrestin–EGFP was quantified by confocal microscopy before and after illumination. Results: On average transgenic Xenopus rods expressed ∼ 11 amols of arrestin–EGFP, corresponding to ∼ 1 per 30 native arrestin molecule. The distribution of arrestin–EGFP was distinct from that of EGFP, which distributed uniformly in the rod cytoplasm and was unaffected by illumination. In darkness, 65% of the arrestin–EGFP appeared in the inner segment. After bleaching, arrestin was redistributed (as reported previously by many investigators), such that it was primarily in the outer segment, in some cases condensed into regularly spaced bands traversing the long axis of the rod. The redistribution was complete within 30 minutes and began to reverse by 60 minutes after the bleach. The total mass of arrestin–EGFP before and after bleaching was conserved.Conclusions: The distinct distributions of EGFP and arrestin–EGFP in dark adapted rods requires the existence of a mechanism that operates in the dark to maintain arrestin in a disequilibrium, so that arrestin is concentrated in the inner segment, rather than being uniformly distributed throughout the rod cytoplasm. Its redistribution to the outer segment on illumination must therefore involve a rapid, light–driven change in the molecular mechanism underlying this confinement.

Keywords: microscopy: confocal/tunneling • photoreceptors • transgenics/knock–outs 
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