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Han Yen Tan, Mehdi Najafi, Peter D. Calvert; Protein Dynamics In The Light Signaling Microcompartments Of Ciliary Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):741.
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We recently showed that the geometry of the cytoplasmic spaces in the ciliary photoreceptor outer segment (OS), and the size and shape of soluble molecules, governs the steady-state levels of proteins within the OS via "steric volume exclusion" (SVE)1. Here we explore the impact of the geometry of OS microcompartments, directly visualized in live cells, on the dynamics of soluble and membrane-associated proteins.
Monomer EGFP or PAGFP (GFP) and GFP dimers or tetramers (1x-, 2x- and 3xGFP), without and with double geranylgeranyl lipidation motifs (GFP-GerGer)2), or a rhodopsin-GFP fusion protein (Rho-GFP), were expressed in Xenopus rods. Diffusion of the expressed GFPs within visualized signaling microcompartments, defined as disc lobule regions that include the disc membranes bounded by incisures and the associated cytoplasm, was examined by fluorescence recovery after photoconversion (FRAP). A numerical disc diffusion model that includes incisure boundaries was developed and used to assess FRAP data.
The diffusion of all proteins examined was significantly impacted by disc incisures. The local diffusion coefficient, D, of Rho-GFP within lobules was ~0.30 µm2s-1, however, with this D the disc diffusion model could not reproduce the time course of equilibration across the entire disc surface, the model prediction being significantly faster than observation. GFP-(GerGer)2 was tightly membrane bound, had a D similar to Rho-GFP and did not cross incisures. D of soluble GFPs within lobule regions were more steeply molecular size dependent than predicted from Stokes radii, however Stokes behavior returnedupon hypo-osmotic OS swelling. Soluble GFPs were able to pass through theincisures, however their movement was significantly hindered.
Discs and incisures in the ciliary outer segment appear to delineate microcompartments that confine intrinsic and peripheral membrane proteins, as well as soluble proteins. The failure of a disc diffusion model with incisures to reproduce Rho-GFP equilibration across the disc suggests rhodopsin diffusion may be heterogeneous in disc membranes. The size dependence of the diffusivities of soluble GFPs within lobules suggests SVE effects become significant for proteins in the size range of transducin and arrestin. 1Najafi, Maza and Calvert, Proc. Natl. Acad. Sci. in press
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