408 The Journal of Cell Biology | Volume 155, Number 3, 2001 Figure 4. Mechanism of flagellar microtubule turnover: plus end turnover versus treadmilling. (A) Plus end only turnover model. Individual outer doublet microtubules alternate between cycles of growth and shortening, with all dynamics restricted to the plus end. Plus and minus ends of flagellar doublets are indicated by ( ) and ( ). (B) Treadmilling model. New subunits are assembled at the plus end of the outer doublets, while simultaneously, subunits are disassembled from the minus end. (C) Experimental strategy to distinguish plus end turnover from treadmilling. Cells with regener- ated half-length flagella were mated to cells expressing HA-tubulin. Right after mating, the half-length flagella rapidly elongate to full length, incorporating HA-tubulin along their distal half. (D) Result of partial regeneration experiment. The unlabeled proximal segment did not shorten, either during the rapid regeneration phase or during the subsequent steady-state phase. This result is inconsistent with a treadmilling mechanism, but matches the predictions of the plus end turnover model. Average number of flagella measured per time point was 10. proximal segment should shorten because tubulin is con- stantly removed from the base of the flagellum. As illustrated in Fig. 4 D, the results of this experiment are inconsistent with treadmilling: the unlabeled proximal seg- ment did not shorten, either during the regeneration phase (which was completed within the first 20 min) or during the subsequent steady state phase that occurred once the flagella reached full length. Statistical analysis shows that slight dif- ferences in the unlabeled segment length at these time points are not significant. Even after 140 min, when the flagella have begun to shorten, the unlabeled segment length does not change, suggesting that not only assembly, but also dis- assembly, is restricted to the plus end. We conclude that the labeling of outer doublet microtubules seen in Fig. 1 occurs strictly by plus end turnover. Intraflagellar transport balances plus end turnover The continuous turnover of tubulin suggests that IFT is re- quired to support the constant assembly at the distal end of the flagellum (Fig. 5 A). Presumably, if IFT is stopped, dis- assembly will continue and lead to flagellar resorption. To test the model that IFT is required to support continu- ous tubulin assembly at the distal end in order to balance continuous tubulin removal, we measured tubulin incorpo- ration in flagella of cells in which IFT has been stopped by Figure 5. Role of IFT in flagellar maintenance. (A) Model: IFT particles carry structural proteins, including tubulin, to the plus end of the flagellum where they are incorporated in order to compensate for subunits removed during the disassembly portion of the turnover process. Plus and minus ends of the flagellar outer doublet microtubules are indicated by ( ) and ( ). (B) fla10 mutants at the nonpermissive temperature block the assembly portion of turnover, as judged by the lack of tagged tubulin incorporation at the tip, but do not block disassembly, as judged by shortening of the unlabeled region of the flagella. This confirms that IFT is needed for ongoing incorporation of new tubulin at the distal end. Average number of measurements per graph entry was 19. (C) Colchicine is predicted to rescue the fla10 phenotype by blocking outer doublet disassem- bly. As shown in graph, colchicine blocks flagellar resorption in fla10 cells shifted to the nonpermissive temperature. (Dotted line) fla10 cells treated with 3 mg/ml colchicine. (Solid line) Untreated fla10 cells. Length ratio plots ratio of length at non- permissive temperature to length at permissive temperature. the temperature-sensitive fla10 mutation. The fla10 muta- tion is known to cause a reduction in both the number and velocity (Kozminski et al., 1995; Iomini et al., 2001) of IFT particles in flagella at elevated temperatures. As seen in Fig. 5 B, wild-type cells and fla10 mutant cells at the permissive temperature show similar levels of new tubulin incorpora- tion, as judged by growth of the distal labeled flagellar seg- ment, and they show similar levels of tubulin disassembly, as judged by shortening of the proximal unlabeled segment. In contrast, fla10 mutant cells at the nonpermissive tempera- ture showed almost no new tubulin incorporation (as judged by the almost complete lack of a distal labeled segment after 90 min), but showed the same rate of disassembly (as judged by shortening of the unlabeled proximal segment) as seen at the permissive temperature. Thus, IFT is needed to support the ongoing assembly of new tubulin at the distal ends of the outer doublets. We note that wild- type cells at 34 C appear to show a greater net loss of unlabeled tubulin compared with fla10 cells at the same temperature, as judged by appar- ent shortening of the unlabeled proximal region. Our data do not suggest an obvious explanation for this interesting re-