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Han Yen Tan, Ivayla Geneva, Peter D Calvert; Single molecule tracking of rhodopsin-like GPCRs and chimeras reveal diverse mechanisms of transport to, and within sensory cilia. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5518.
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© ARVO (1962-2015); The Authors (2016-present)
Cilia act as antennae probing the physical environment of cells. Given the diversity of ciliated cells and signalling cascades that are delivered to these exclusive compartments, we sought to understand whether cascade components from cilia of one cell type would be localized to the cilia of a foreign cell, and if not, what molecular cues drive specificity. We thus examined the localization of two GPCRs, rhodopsin (Rho) and the rhodopsin-like somatostatin receptor type 3 (SSTR3), as well as their chimeras, in two ciliated cells - Xenopus rod photoreceptors and mouse IMCD3 cells.
myc-Rho-EGFP-VXPX, myc-Rhoi3sstr3-EGFP-VXPX, IFT20-EGFP, myc-SSTR3-EGFP or SSTR3-mKate2 were expressed in transfected IMCD3 cells. Cells were starved for 48 hours to induce cilia. Rho-EGFP or SSTR3-EGFP expression in Xenopus rods was achieved by REMI. Cilia localization was quantified with confocal microscopy. Single molecule tracking of Qdot labelled GPCRs was accomplished with an epifluorescence microscope.
Rho-EGFP appears to be excluded from IMCD3 cilia (𝐹𝑐𝑖𝑙/𝐹𝑎𝑚 = 1.050.34 n=14 cells; 𝐹𝑐𝑖𝑙/𝐹𝑎𝑚 > 3.5 indicates ciliary localization). SSTR3-GFP does localize to IMCD3 cilia (𝐹𝑐𝑖𝑙/𝐹𝑎𝑚 = 7.113.49 n=65 cells). Replacing the third intracellular loop of Rho-EGFP with the third intracellular loop of the SSTR3 (Rhoi3sstr3-EGFP) results in its IMCD3 ciliary localization (𝐹𝑐𝑖𝑙/𝐹𝑎𝑚 = 4.572.02 n=36 cells). Kymographs showed that IFT20-GFP moves continuously from one end of the cilium to the other. In contrast, individual Qdot tagged myc-SSTR3-EGFP and myc-RHOi3sstr3-EGFP mostly exhibit random, Brownian motion. Occasionally, GPCRs moved in a vectoral fashion, but rarely over the entire ciliary length. At times GPCR motion stopped at ciliary tip or base, as if docking, before resuming largely random motion.<br />
Rho-EGFP is localized to the rod outer segment of photoreceptors, whereas it is excluded from the primary cilia of IMCD3 cells. Conversely, SSTR3-EGFP is localized to the primary cilia of IMCD3 cells. The i3 loop of SSTR3 appears to be sufficient for Rho ciliary localization in IMCD3 cells, and thus appears to be an important cue for GPCR ciliary localization/exclusion. GPCR transport within the cilium is primarily via simple diffusion, with infrequent, weak coupling to IFT.
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