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Sungsu Lee, Ivayla I Geneva, Han Yen Tan, Peter D Calvert; Super Resolution Tracking of Single G-Protein Coupled Receptors within Ciliary Membranes Reveals Transient Confinement. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4807.
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© ARVO (1962-2015); The Authors (2016-present)
Current understanding of the mechanisms underlying G-Protein Coupled Receptor (GPCR) transport within ciliary compartments is limited and several competing models exist, including motor-driven transport, local binding and free diffusion. An analysis of the two dimensional mean square displacement (MSD) of single GPCRs within ciliary membranes as a function of time step magnitude was performed to assess the predominant modes of movement.
Using previously established protocols, murine inner medullary collecting duct (IMCD3) cells were transfected with a somatostatin receptor 3 (SSTR3-EGFP) fusion construct or a Rhodopsin chimera (Rhoi3S-EGFP) where the third intracellular loop of rod opsin was replaced with that of SSTR3, both with an N-terminal myc tag. After 24 hours, the cover slip on which cells were grown was incubated with a primary antibody to the myc tag, and a quantum-dot conjugated secondary antibody to the primary antibody. The cells were imaged using an inverted Olympus fluorescence microscope, an optosplit to image the green and red channels simultaneously, an EMCCD camera (Andor) and image acquisition software (Nikon). MSD as a function of tau was determined using a custom MATLAB code. A model of GPCR transport that included random walk, motor driven transport, local binding and transient confinement, with spatial boundary conditions as defined by the particular cilium observed, was employed to analyze the results.
The MSD(tau) analysis revealed that the displacement of the GPCRs at small taus followed the prediction for free diffusion. However at larger taus, the relation fell significantly below the MSD(tau) relationship predicted for pure diffusion or motor transport. A mobility index (MI), defined as the ratio of the MSD from the experiment to the model prediction, was calculated to quantify the magnitude of deviation. The results showed a MI of 0.62 ± 0.05 for SSTR3 and 0.67 ± 0.09 for Rhoi3S.
The MSD analysis clearly demonstrates that the predominant modes of GPCR movement within the ciliary compartment are diffusion with transient confinement to membrane sub-regions. More study is needed to better understand the mechanisms of this confinement. One possibility involves GPCR diffusion between lipid rafts from which they are excluded.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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