April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Access of Soluble Proteins to Rod Outer Segment Cytoplasmic Domains Depends on Molecular Size, but Transport Through the Ciliary Transition Zone Does Not
Author Affiliations & Notes
  • Mehdi Najafi
    Ophthal & Biochem & Mol Biol, SUNY Upstate Medical University & SUNY Eye Institute, Syracuse, New York
  • Nycole Maza
    Ophthal & Biochem & Mol Biol, SUNY Upstate Medical University & SUNY Eye Institute, Syracuse, New York
  • Peter D. Calvert
    Ophthal & Biochem & Mol Biol, SUNY Upstate Medical University & SUNY Eye Institute, Syracuse, New York
  • Footnotes
    Commercial Relationships  Mehdi Najafi, None; Nycole Maza, None; Peter D. Calvert, None
  • Footnotes
    Support  NIH Grant EY018421, RPB, Lions 20Y1, SUNY Reach
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 35. doi:
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      Mehdi Najafi, Nycole Maza, Peter D. Calvert; Access of Soluble Proteins to Rod Outer Segment Cytoplasmic Domains Depends on Molecular Size, but Transport Through the Ciliary Transition Zone Does Not. Invest. Ophthalmol. Vis. Sci. 2011;52(14):35.

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

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Abstract

Purpose: : Rod photoreceptors adapt to light in part by the transport of light signaling proteins between the outer (OS) and inner (IS) segment compartments. Recent studies have suggested that the ciliary transition zone restricts the movement of proteins into and out of primary cilia via barriers to free diffusion. Moreover, it has been suggested that access of soluble molecules is dependent on size. To examine this possibility we have employed live tissue confocal and multiphoton imaging to measure the distribution and mobility of fluorescent proteins of various sizes expressed in Xenopus photoreceptors.

Methods: : Constructs encoding concatemers of photoactivatable (PA) and enhanced (E) GFP variants that express as double and triple GFPs (2x and 3xGFP) were made. Transgenic Xenopus laevis were generated by REMI. Micro-slices of retina from late stage tadpoles or juvenile frogs were placed into a live tissue imaging chamber on our microscope for quantification of subcellular distribution. Protein mobility was probed by multiphoton photoactivation of 1x, 2x and 3xPAGFP in various rod compartments, followed by confocal monitoring. Diffusion models were employed to quantify and compare mobilities.

Results: : The ratios of the mean fluorescence in the OS to that in the myoid were 0.52±0.02, 0.30±0.02 and 0.13±0.02 for 1x, 2x and 3xGFP, respectively. Flux through the connecting cilium did not vary among these proteins more than expected based on their predicted hydrated radii. 1xGFP fluorescence was homogenous across the OS diameter whereas that of 2x and 3xGFP were lower in the radial OS center.

Conclusions: : The fraction of total fluorescence found in the OS depends on the size of the GFP concatemer. However all GFPs passed through the rod connecting cilium irrespective of size, thus challenging the notion of a size-dependent barrier to soluble protein diffusion at the rod ciliary transition zone. Rather, these results suggest that the interdiskal cytoplasmic milieu imposes a size constraint on molecular access that is the primary determinant of soluble protein distribution in rods.

Keywords: photoreceptors • microscopy: light/fluorescence/immunohistochemistry • signal transduction 
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