406 The Journal of Cell Biology | Volume 155, Number 3, 2001 al., 1977) prevent flagellar regeneration by arresting antero- grade IFT at the nonpermissive temperature (Kozminski et al., 1995). IFT is required to transport inner dynein arms (Piperno et al., 1996) and presumably other axonemal sub- units to the end of the growing flagellum, which is the site of new flagellar protein assembly during regeneration (Rosen- baum and Child, 1967; Johnson and Rosenbaum, 1992). Remarkably, IFT continues to be required even after flagellar assembly is completed. When fla10 mutants with intact flagella are shifted to the nonpermissive temperature, IFT stops (Kozminski et al., 1995) and the flagella begin to shorten and eventually disappear (Huang et al., 1977). This shortening would not occur if flagella were static structures. It suggests that flagella require a constant input of subunits to balance the loss during turnover, and emphasizes the dy- namic nature of the flagellar axoneme. These turnover re- sults raise several interesting questions, the first of which is, where along the flagellar length does the turnover occur? How much of the flagellar axoneme is turning over? Is the rate and extent of turnover sufficient to account for the rate of the complete flagellar resorption that occurs in fla10 mu- tants at the nonpermissive temperature when IFT is inhib- ited? Finally, one might also ask how such a turnover process fits in with mechanisms for flagellar length control. In order to determine the cytological localization and spa- tial extent of turnover, we have developed an assay to visual- ize turnover of tubulin in Chlamydomonas flagella. Our results show that tubulin assembles and disassembles contin- uously at the distal end of the flagella, indicating that flagella are dynamic structures. We have also found that IFT is re- quired for the steady-state assembly of outer doublet micro- tubules that occurs during turnover. On the other hand, the continuous disassembly of outer doublet microtubules was found to continue in the absence of IFT. These results sug- gested that length regulation may involve a balance between continuous assembly of tubulin at the tip, mediated by IFT, balanced by continuous disassembly. Consistent with this idea, we find that partial reduction in IFT leads to an shf phenotype, and conversely we find that an lf mutant shows a decreased rate of turnover. Results Visualizing flagellar microtubule turnover A method was developed to visualize flagellar microtubule turnover in situ in Chlamydomonas. As illustrated in Fig. 1 A, cells expressing hemaglutinin (HA) epitope–tagged -tubulin were mated to wild-type cells that lacked the tagged tubulin construct. Upon mating, the biflagellate Chla- mydomonas gametic cells fused to form a quadriflagellate dikaryon. At this point, only two of the four flagella con- tained HA-tagged tubulin. If flagellar tubulin turns over, HA-tubulin should begin to incorporate into the other two unlabeled flagella of the cell that did not initially contain tagged tubulin. As indicated by the arrows in Fig. 1, B and C, tagged tubulin was indeed found to incorporate into the distal regions of these flagella. To localize this incorporation more precisely, cells were washed with detergent to remove the flagellar membrane and then treated with ATP, allowing the dynein arms to rupture Figure 1. Visualizing flagellar microtubule dynamics. (A) Experi- mental strategy. Cells expressing HA-tubulin are mated with nonexpressing cells to create a quadriflagellated dikaryon in which two flagella contain HA-tubulin and two do not. Cells are fixed at successive timepoints after mating, and stained with antibodies recognizing HA-tubulin, to detect incorporation of tagged tubulin into the previously unlabeled flagella. (B and C) Typical dikaryon images. Cell fixed 90 min after mating. (Green) FITC immunofluo- rescence with antibodies to Fla10 kinesin, used as a marker for flagella and basal bodies. (Red) Texas red immunofluorescence with antibodies recognizing the HA epitope–tagged tubulin. Yellow results from overlap of HA-tubulin staining with Fla10 kinesin staining, indicating incorporation of tagged tubulin into flagella. Two flagella (originally belonging to the tagged tubulin expressing cell) are fully labeled and two are partially labeled (arrows), indicating incorporation of tagged tubulin following mating. (D–F) Flagella splayed open with detergent plus ATP following mating of HA-tagged tubulin expressing and nonexpressing cells. (D) Splayed flagella detected by FITC immunofluorescence using antibodies against tubulin. Central pair microtubules (arrow) have extruded and are clearly separated from the main mass of outer doublets. (E) Texas red immunofluorescence using antibodies to HA epitope- tagged tubulin. Tagged tubulin clearly has incorporated into the distal end of the outer doublets, and possible also the splayed-out central pair microtubules. (F) Overlay image showing that majority of incorporation is in the outer doublet microtubules. Bars: (B and C) 2 m; (D–F) 3 m. the flagella. Under these conditions, the set of nine outer doublets splits into two groups and the central pair microtu- bules splay out through the gaps (Johnson, 1998). As seen in Fig. 1, D–F, when partially labeled flagella in the course of these dikaryon experiments were splayed in this way, HA- tubulin had clearly incorporated into the outer doublets. Having demonstrated that tubulin incorporates into the outer doublets at the distal end, we next measured the spatial extent of this incorporation over time. Dikaryons were fixed at 20-min intervals after mating. At each time point, the av- erage length of the distal labeled region and the proximal unlabeled region were measured, with results plotted in Fig. 2. As time elapsed after cell fusion, tagged tubulin incorpo- ration could be seen over an increasingly larger distal region of the flagella. During this time, the flagella were not grow- ing. Flagella shorten gradually following mating, as seen in Fig. 2. In spite of this, we are still able to measure the rates