Figure 2. Tubulin enters cilia by IFT and diffusion. (A) Gallery of kymograms depicting GFP–α-tubulin (or, in c, mNeonGreen-tubulin) moving inside cilia by anterograde IFT (open arrow in a), retrograde IFT (open arrow in b), and diffusion (arrowheads in c and d). Anterograde transport results in trajectories running from the bottom left (ciliary base) to the top right (ciliary tip; T); retrograde transport events result in top-left to bottom-right trajectories. (c) Tubulin diffusing inside the ciliary shaft; (d) reduced mobility of GFP–α-tubulin in the vicinity of the tip (filled arrows). Bars, 1 µm and 1 s. (B) Kymograms from simultaneous imaging of mNeonGreen–α-tubulin (a and b) and IFT20-mCherry (c and d) in growing cilia; IFT-like trajectories are marked in b and d. A merged kymogram is shown in e. A Western blot of this strain is shown in Fig. S3 B. Bars, 5 µm and 5 s. (C) Mean square displacement versus time for 64 sfGFP–α-tubulin particles diffusing inside the shaft and 58 particles diffusing near the tip of steady-state cilia. The standard error of the mean at each value is indicated. A linear fit to the data at the short time points, which is likely to represent pure diffusion, results in diffusion coefficients of ∼1.8 µm2/s and ∼0.2 µm2/s for 1D diffusion of GFP-tubulin along the ciliary shaft and at the tip, respectively. (D) Still images and kymograms of fla10-1 cells expressing either GFP–α-tubulin (a and b) or IFT20-mCherry (c and d) at the permissive temperature (22°C; a and c) and after >180 min at 32°C (b and d). IFT-like trajectories for tubulin and IFT20 were only observed at 22°C (open arrows in a and c). Diffusion of GFP–α-tubulin (arrowhead in b) into photobleached cilia continued at 32°C in the absence of detectable IFT (d). Bars, 1 µm and 1 s. (E) Frequency of anterograde GFP–α-tubulin transport by IFT in fla10-1 and control cells (FLA10 ift20-1 IFT20 mCherry). Steady-state cilia and cilia regenerated in the presence of CHX were compared; at 32°C, IFT-like tubulin transport was robust in control cells but not observed in fla10-1 cells. fla10-1 regenerates cilia only slowly at room temperature (∼22°C); most measurements are based on cells regenerating cilia at 16°C. Error bars indicate SEM.
Figure 2.

Tubulin enters cilia by IFT and diffusion. (A) Gallery of kymograms depicting GFP–α-tubulin (or, in c, mNeonGreen-tubulin) moving inside cilia by anterograde IFT (open arrow in a), retrograde IFT (open arrow in b), and diffusion (arrowheads in c and d). Anterograde transport results in trajectories running from the bottom left (ciliary base) to the top right (ciliary tip; T); retrograde transport events result in top-left to bottom-right trajectories. (c) Tubulin diffusing inside the ciliary shaft; (d) reduced mobility of GFP–α-tubulin in the vicinity of the tip (filled arrows). Bars, 1 µm and 1 s. (B) Kymograms from simultaneous imaging of mNeonGreen–α-tubulin (a and b) and IFT20-mCherry (c and d) in growing cilia; IFT-like trajectories are marked in b and d. A merged kymogram is shown in e. A Western blot of this strain is shown in Fig. S3 B. Bars, 5 µm and 5 s. (C) Mean square displacement versus time for 64 sfGFP–α-tubulin particles diffusing inside the shaft and 58 particles diffusing near the tip of steady-state cilia. The standard error of the mean at each value is indicated. A linear fit to the data at the short time points, which is likely to represent pure diffusion, results in diffusion coefficients of ∼1.8 µm2/s and ∼0.2 µm2/s for 1D diffusion of GFP-tubulin along the ciliary shaft and at the tip, respectively. (D) Still images and kymograms of fla10-1 cells expressing either GFP–α-tubulin (a and b) or IFT20-mCherry (c and d) at the permissive temperature (22°C; a and c) and after >180 min at 32°C (b and d). IFT-like trajectories for tubulin and IFT20 were only observed at 22°C (open arrows in a and c). Diffusion of GFP–α-tubulin (arrowhead in b) into photobleached cilia continued at 32°C in the absence of detectable IFT (d). Bars, 1 µm and 1 s. (E) Frequency of anterograde GFP–α-tubulin transport by IFT in fla10-1 and control cells (FLA10 ift20-1 IFT20 mCherry). Steady-state cilia and cilia regenerated in the presence of CHX were compared; at 32°C, IFT-like tubulin transport was robust in control cells but not observed in fla10-1 cells. fla10-1 regenerates cilia only slowly at room temperature (∼22°C); most measurements are based on cells regenerating cilia at 16°C. Error bars indicate SEM.

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