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. 2018 Aug 6;217(8):2831–2849. doi: 10.1083/jcb.201711104

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© 2018 Lee et al.

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Figure 1. — Superresolution tracking of single Qdot-labeled GPCRs reveals 2D transport on ciliary membranes that is distinct from IFT-based motor transport. (A) Schematic of the GPCR constructs used for single-molecule tracking and ensemble FRAP experiments. (B) Image of a cilium taken with the GFP channel shows uniform distribution of myc-Rhoi3S-EGFP. (C) Overlay of GFP and Qdot channels shown with tracking results from a single Qdot625-labeled myc-Rhoi3S-EGFP. Axial and circumferential movements (abaxial) are apparent (see Videos 1–4). (D) Axial position time course (kymograph) obtained from Qdot tracking. (E) Time course of movements perpendicular to the cilium axis. Zero in the ordinate indicates the center of mass of the cilium. (F) Instantaneous axial velocity histogram. Anterograde velocities are positive; retrograde are negative. Note the symmetry of the histogram, indicating that the distribution of velocities is the same in the anterograde and retrograde directions. (G) Representative IFT20-EGFP kymograph obtained under identical conditions (Video 5). Note smooth contiguous movement along the full length of the cilium. (H) Anterograde and retrograde IFT velocity histograms pooled from the slopes of 122 anterograde and 84 retrograde tracks from 14 cilia obtained from six independent experiments. Red lines, Gaussian fittings of the histograms. Anterograde, mean = 0.594, SD = 0.173 µm/s; retrograde, mean = 0.340, SD = 0.204 µm/s. (I and J) MSD analysis reveals significant subdiffusion of GPCRs within ciliary membranes. Black circles represent time-averaged MSDs of an individual Qdot-labeled GPCR plotted as a function of the time step size (τ). Error bars (SEM) are on the order of the size of the symbols; n > 1,000 for each τ. Red lines, linear fittings to MSD(τ = 0.3 and 0.6 s) from which apparent D_ax was estimated; dashed lines, predicted MSD(τ) based on estimated D_ax and Eq. 1. (J) MSD(τ) plot on expanded scale. (K) Axial position histogram for the individual GPCR tracking experiment. The length of a given cilium was divided into 20 bins into which axial positions were distributed. Zero on the abscissa represents the base of the cilium. In D–F and I–K, results are representative from one tracking experiment. Total number of Qdot-labeled myc-Rhoi3S-EGFP tracked = 9, on eight individual cilia, taken from four independent experiments (Table 1).