April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Light-induced Raft In The Disc Membrane Of Rod Photoreceptor
Author Affiliations & Notes
  • Fumio Hayashi
    Biology, Kobe University, Nada-Ku, Japan
  • Natsumi Saito
    Biology, Kobe University, Nada-Ku, Japan
  • Keiji Seno
    Biology, Hamamatsu University School of Medicine, Hamamatsu, Japan
  • Footnotes
    Commercial Relationships  Fumio Hayashi, None; Natsumi Saito, None; Keiji Seno, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 30. doi:
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      Fumio Hayashi, Natsumi Saito, Keiji Seno; Light-induced Raft In The Disc Membrane Of Rod Photoreceptor. Invest. Ophthalmol. Vis. Sci. 2011;52(14):30.

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

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Purpose: : To characterize the light-induced raft around photoexcited-rhodopsin/transducin- complex in retinal disc membrane.

Methods: : We used single molecule imaging technique to examine the diffusivity and behavior of rhodopsin (R), transducin (Gt), and Ca2+-S-modulin (S) on the disc membrane of bullfrog rod photoreceptor outer segment (ROS). R was probed by Cy7-labeled Fab’ of IgG(1D4). The α subunit of Gt (G αt) and S was directly labeled with HiLyte Fluor-750 and Cy7, respectively. Labeled proteins were reconstituted to the disc membrane, and visualized on a TIRF microscope. The effective diffusion coefficients of proteins were determined from the 2-dimensional trajectories of proteins. We also evaluate the raftophilicity, i.e. affinity to raft, of proteins and their complexes in terms of their partitioning to the detergent-resistant membrane (DRM). To disrupt the raft in the disc membrane, we used methyl-b-cyclodextrin (MCD).

Results: : Both R and photoexcited R (R*) undergo diffusion in the disc membrane with an effective diffusion coefficient (D100ms) of ~0.3 µm2/s. The D100ms of Gt in dark-adapted disc membrane is ~0.7 µm2/s, much higher than rhodopsin. However, Gt complexed with R* (R*/Gt-complex) in light-exposed disc membrane shows slower diffusion rate than R or R*, i.e. 0.18 µm2/s. Since MCD restores the D100ms, raftophilic interaction seems to hinder the diffusion of the complex. GTP also releases the constraint. Artificially dimerized rhodopsin (R2) crosslinked by IgG exhibits low diffusivity and high raftophilicity. Further, the R2 in DRM is partially resistant to MCD. These characters of R2 are very similar to those of R*/Gt complex. Thus, we examined the stoichiometric ratio between R* and Gt in the complex residing in DRM, and found that R*:Gt=2:1. In addition, D100ms of Ca2+-S-modulin was unexpectedly dependent on light, GTP and MCD-treatment of the disc membrane.

Conclusions: : Light-induced binding of Gt to R* stabilizes the dimer of rhodopsin, which can organize raft around it. This raft must be very small and transient entity. This light-induced raft may participate in recruitment of regulatory proteins to the vicinity of R* to shape up photoresponse in rod photoreceptor.

Keywords: photoreceptors • signal transduction • lipids 

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