Figure 2. Measuring turnover based on spatial distribution of incorporated HA-tagged tubulin versus time. Each bar represents average labeling of flagella at a given time point after cell fusion. Red segment gives average length of labeled portion, green segment gives average length of unlabeled portion. Over time, HA-tagged tubulin incorporates over an increasingly large distal portion of the flagellum. At the same time, flagella are not growing but are in fact shortening as a consequence of the mating developmental pathway. Therefore, incorporation does not simply reflect residual flagellar growth, but must entail turnover of tubulin already present in the flagellum. Average number of flagella measured per time point was 27. Error bars in all graphs represent standard error of the mean. of assembly and disassembly by measuring the changes in the lengths of the labeled and unlabeled segments. However, we note that the apparent rates of assembly and disassembly measured here apply strictly to mated cells, and are likely to be slightly different in vegetative cells. The time course of the experiment was limited to 120 min because after longer time intervals, all four flagella rapidly and synchro- nously resorb in preparation for meiosis. Consequently, we were not able to observe these cells long enough to see com- plete labeling of the flagella. Nevertheless, after 120 min, in some cases as much as a third of the flagellum was found to incorporate tagged tubulin. Fig. 2 shows that the labeled re- gion at the distal end of the flagellum grew at the expense of the unlabeled region, causing the unlabeled proximal region to appear to shorten throughout the course of the experi- ment. The continuous addition of new tubulin in the ab- sence of growth indicated that during the course of the ex- periment, old unlabeled tubulin subunits were continuously removed in order to accommodate the new tubulin assem- bly, i.e., turnover was occurring. Colchicine blocks both tubulin addition and removal Colchicine at a concentration sufficient to prevent flagellar regeneration does not cause flagellar shortening (Behnke and Forer, 1967; Tilney and Gibbins, 1968; Rosenbaum et al., 1969), and this is frequently taken as evidence that flagellar microtubules are not turning over (see, for example, Jensen et al., 1987; Tilney et al., 2000). The dikaryon assay of Fig. 1 was repeated in the presence of colchicine at a concentra- tion of 3 mg/ml, a concentration sufficient to completely block flagellar regeneration following deflagellation. As plot- ted in Fig. 3, colchicine did indeed completely block incor- poration of tagged tubulin into flagella, confirming that the incorporation of tubulin into the outer doublets seen in Fig. 1 represents bona fide microtubule polymerization. In addi- tion, colchicine also blocked the removal of unlabeled tubu- lin that we had observed in untreated cells, as indicated by the fact that the unlabeled proximal segment did not be- come shorter. Therefore, colchicine seems to arrest all mi- crotubule dynamics within the flagellum, and the fact that Flagellar length control | Marshall and Rosenbaum 407 Figure 3. Colchicine blocks flagellar tubulin turnover in wild-type cells. Compared to untreated cells, in cells treated with 3 mg/ml colchicine, a concentration that totally blocks flagellar regenera- tion, no assembly of HA-tubulin was observed at the distal ends of the flagella, confirming that the assembly observed in untreated cells represents bona fide microtubule polymerization. Colchicine also completely blocked disassembly of the outer doublets, thereby shutting down turnover. Average number of measurement per graph entry is 28. colchicine does not cause flagella to shorten is misleading and cannot be taken as evidence against microtubule turn- over. We do not know how colchicine blocks turnover and disassembly, but one possibility is that colchicine-tubulin dimers might cap the plus ends of the outer doublets and stabilize them against further depolymerization, as has been demonstrated with microtubules in vitro (Panda et al., 1995). Tubulin turnover restricted to plus end The data show that tubulin is continuously added onto the ends of flagella that are not growing, implying that tubulin is turning over at steady state. But from which end of the fla- gellum are tubulin subunits removed? Either (a) addition and removal could both occur at the plus end (Fig. 4 A), or (b) tubulin could assemble at the plus end and disassemble from the minus end (treadmilling) (Fig. 4 B). Of the two mechanisms, treadmilling seems less likely a priori because outer doublet microtubules are continuous with the basal bodies, so it is unclear where or how subunit removal could take place by this process. Regardless, we distinguished between these mechanisms using the strategy illustrated in Fig. 4 C. Unlabeled cells were deflagellated and allowed to regenerate flagella in the presence of cycloheximide. Under these conditions, flagella regenerate to half normal length because the pool of axone- mal proteins is depleted (Rosenbaum et al., 1969; Johnson and Rosenbaum, 1992). These cells were mated with cells expressing HA-tubulin that had not been deflagellated, and hence still contained a full pool of axonemal precursor pro- teins. After mating, the extra protein pool donated by the HA-tagged cells allowed the half-length flagella to grow rap- idly to the same length as the other two flagella (Johnson and Rosenbaum, 1992). Once all four flagella were of equal length, the experiment proceeded exactly as in Fig. 1, except that now the two flagella of the cell that regenerated its fla- gella in the presence of cycloheximide started out labeled over half their length. If turnover occurs entirely at the plus end, newly incorpo- rated, labeled tubulin would simply replace tubulin subunits that were already labeled, and would not affect unlabeled tubulin in the proximal half of the flagellum; thus, the unlabeled segment would maintain a constant length. In contrast, if turnover involved treadmilling, the unlabeled