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- Dynamic Behavior of Rod Photoreceptor DisksDynamic Behavior of Rod Photoreceptor Disks
Biophysical Journal, Volume 83, Issue 3, 1 September 2002, Pages 1403-1412
Chunhe Chen, Yunhai Jiang and Yiannis Koutalos
AbstractEukaryotic cells use membrane organelles, like the endoplasmic reticulum or the Golgi, to carry out different functions. Vertebrate rod photoreceptors use hundreds of membrane sacs (the disks) for the detection of light. We have used fluorescent tracers and single cell imaging to study the properties of rod photoreceptor disks. Labeling of intact rod photoreceptors with membrane markers and polar tracers revealed communication between intradiskal and extracellular space. Internalized tracers moved along the length of the rod outer segment, indicating communication between the disks as well. This communication involved the exchange of both membrane and aqueous phase and had a time constant in the order of minutes. The communication pathway uses ∼2% of the available membrane disk area and does not allow the passage of molecules larger than 10 kDa. It was possible to load the intradiskal space with fluorescent Ca and pH dyes, which reported an intradiskal Ca concentration in the order of 1M and an acidic pH 6.5, both of them significantly different than intracellular and extracellular Ca concentrations and pH. The results suggest that the rod photoreceptor disks are not discrete, passive sacs but rather comprise an active cellular organelle. The communication between disks may be important for membrane remodeling as well as for providing access to the intradiskal space of the whole outer segment.
Abstract | Full Text | PDF (444 kb) - Analysis of FRET Signals in the Presence of Free Donors and ...Analysis of FRET Signals in the Presence of Free Donors and Acceptors
Biophysical Journal, Volume 94, Issue 3, 1 February 2008, Pages 986-1000
Jakub Wlodarczyk, Andrew Woehler, Fritz Kobe, Evgeni Ponimaskin, Andre Zeug and Erwin Neher
AbstractA method for spectral analysis of Förster resonance energy transfer (FRET) signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Förster energy transfer efficiency , unless one of the interactors is in large excess and perfectly labeled. Instead, analysis of donor quenching yields a product of the form , where is the fraction of donor-type molecules participating in donor-acceptor complexes and is the labeling probability of the acceptor. Similarly, analysis of sensitized emission yields a product involving . The analysis of intramolecular FRET (e.g., of tandem constructs) yields the product . We use our method to determine these values for a tandem construct of cyan fluorescent protein and yellow fluorescent protein and compare them with those obtained by standard acceptor photobleaching and fluorescence lifetime measurements. We call the method lux-FRET, since it relies on linear unmixing of spectral components.
Abstract | Full Text | PDF (352 kb) - Breaking the Covalent Bond— A Pigment Property that Contribu...Breaking the Covalent Bond— A Pigment Property that Contributes to Desensitization in Cones
Neuron, Volume 46, Issue 6, 16 June 2005, Pages 879-890
Vladimir J. Kefalov, Maureen E. Estevez, Massahiro Kono, Patrice W. Goletz, Rosalie K. Crouch, M. Carter Cornwall and King-Wai Yau
SummaryRetinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11- retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11- retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin—about 10% of total opsin—in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11- retinal, estimated to be only a tiny fraction (∼0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an ∼2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.
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Copyright © 1979 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 28, Issue 2, 281-291, 1 November 1979
doi:10.1016/S0006-3495(79)85176-0
Research Article
Fluorescence redistribution after photobleaching. A new multipoint analysis of membrane translational dynamics
D.E. Koppel
Abstract
A theoretical formulation and experimental methodology are presented for a new multipoint analysis of membrane translational dynamics. The redistribution of fluorescent probe after a localized photobleaching pulse is monitored at several locations by a focused laser beam sequentially scanned through the bleached area. The spatial information so obtained provides a unique sensitivity to possible systematic flow and a direct internal calibration of the characteristic transport distance. These capabilities are demonstrated with experimental data on a reconstituted multibilayer system.
