Figure 6. The concentration of soluble... | Rockefeller University Press

$Figure 6. The concentration of soluble tubulin is increased in growing cilia. (A) Western blot comparing the amounts of tubulin in the MM fractions of steady-state (ss), fully regenerated (fr), and regenerating (reg) cilia. MM fractions were loaded to represent matching volumes of MM. Toward this end, loading was adjusted for equal amounts of axonemal proteins in the corresponding axonemal fractions based on anti-IC2 staining. See Fig. S4 D for quantification of band intensities. (B–D) FRAP analysis of steady-state and growing cilia. (B) After local bleaching of cilia using a focused laser beam (brackets), partial recovery of GFP–α-tubulin fluorescence was observed. Shown are images before (pre), and immediately (after T0) and 55 s (after T55) after photobleaching. Bar, 1 µm. (C) Single measurements of the fluorescence recovery (in % of the pre-bleaching intensity) in the bleached areas of a steady-state and a regenerating cilium. (D) Mean FRAP of regrowing (reg; n = 18) versus steady-state and fully regenerated (ss & fr; n = 16) cilia. Error bars indicate SEM. (E and F) FRAP analysis of a long-short cell. (E) Still images showing a long-short cell before (pre1) and immediately (post1 T0) and 21 s (post1 T21) after spot bleaching of the short cilium (S); pre2, post2 T0, and post2 T19 indicate similar steps for the long cilium. Dashed circle: position of the bleaching laser. Arrow in h: incorporation of GFP-tubulin into the long cilium. Arrowheads with 2 indicate additional areas bleached between post2 T19s and post2–6 min. Note considerable elongation of the initially short cilium (after 6 min; indicated by brackets). Bar, 2 µm. (F) Kymograms showing FRAP in the long (L) and the short (S) cilium of a given cell. Open arrowhead: bleaching steps. Small arrows: GFP–α-tubulin trajectories. Note recovery of a diffuse background of GFP–α-tubulin in the bottom panel. Bar, 2 µm and 2 s. (G) Single measurements of the fluorescence recovery (in percentage of the pre-bleaching intensity) in the bleached areas of the short and long cilia shown in F. (H) Mean FRAP of short and long cilia of long-short cells. Newly assembled axonemal segments (see brackets in E, h) were excluded from FRAP analysis. Error bars indicate SEM.$

Figure 6.

The concentration of soluble tubulin is increased in growing cilia. (A) Western blot comparing the amounts of tubulin in the MM fractions of steady-state (ss), fully regenerated (fr), and regenerating (reg) cilia. MM fractions were loaded to represent matching volumes of MM. Toward this end, loading was adjusted for equal amounts of axonemal proteins in the corresponding axonemal fractions based on anti-IC2 staining. See Fig. S4 D for quantification of band intensities. (B–D) FRAP analysis of steady-state and growing cilia. (B) After local bleaching of cilia using a focused laser beam (brackets), partial recovery of GFP–α-tubulin fluorescence was observed. Shown are images before (pre), and immediately (after T0) and 55 s (after T55) after photobleaching. Bar, 1 µm. (C) Single measurements of the fluorescence recovery (in % of the pre-bleaching intensity) in the bleached areas of a steady-state and a regenerating cilium. (D) Mean FRAP of regrowing (reg; n = 18) versus steady-state and fully regenerated (ss & fr; n = 16) cilia. Error bars indicate SEM. (E and F) FRAP analysis of a long-short cell. (E) Still images showing a long-short cell before (pre1) and immediately (post1 T0) and 21 s (post1 T21) after spot bleaching of the short cilium (S); pre2, post2 T0, and post2 T19 indicate similar steps for the long cilium. Dashed circle: position of the bleaching laser. Arrow in h: incorporation of GFP-tubulin into the long cilium. Arrowheads with 2 indicate additional areas bleached between post2 T19s and post2–6 min. Note considerable elongation of the initially short cilium (after 6 min; indicated by brackets). Bar, 2 µm. (F) Kymograms showing FRAP in the long (L) and the short (S) cilium of a given cell. Open arrowhead: bleaching steps. Small arrows: GFP–α-tubulin trajectories. Note recovery of a diffuse background of GFP–α-tubulin in the bottom panel. Bar, 2 µm and 2 s. (G) Single measurements of the fluorescence recovery (in percentage of the pre-bleaching intensity) in the bleached areas of the short and long cilia shown in F. (H) Mean FRAP of short and long cilia of long-short cells. Newly assembled axonemal segments (see brackets in E, h) were excluded from FRAP analysis. Error bars indicate SEM.

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