Figure 6. Relationship between IFT, microtubule turnover, and flagellar length. (A) Partial reduction of IFT causes shf phenotype. Bars depict length of flagella in wild-type and fla10 cells grown at different temperatures. Typical shf and lf mutants are shown for comparison. In contrast to wild-type cells that show only a slight change in length at elevated temperatures, or temperature-sensitive fla10 cells grown at the nonpermissive temperature in which flagella are completely resorbed, when fla10 cells are grown at an intermediate temperature to partially reduce IFT, their flagella reach half length, comparable to bona fide shf mutants. (B) Length versus temperature in fla10 mutant cells grown at a range of temperatures spanning completely permissive and completely nonpermissive, resulting in intermediate steady state length. Average number of measurements per data point is 25. (C) Reduction in microtubule turnover rate in lf mutant. Compared to wild-type cells measured over an equal time interval, lf mutant lf2-5 shows less incorporation of new tubulin (reflected by the shorter length of incorporated tubulin at the plus end, indicated in red), as well as less removal of unlabeled tubulin (reflected by a smaller reduction in the length of the unlabeled proximal segment, indicated in green). Average number of measurements per entry in graph was 23. sult. This may potentially reflect a coupling of assembly and disassembly at the plus end; however, this coupling cannot be absolute because the unlabeled region does in fact shorten in fla10 cells, albeit not as rapidly as in wild-type cells. To further investigate the relation between IFT and turn- over, we noted that although colchicine has no effect on IFT itself (Kozminski et al., 1993b), it does block flagellar microtu- bule turnover (Fig. 3), raising the counterintuitive prediction that colchicine should block flagellar shortening in fla10 mu- tants at the restrictive temperature. As shown in Fig. 5 C, this was indeed the case. Treatment of fla10-1 cells with colchicine blocked flagellar shortening following a shift to the nonper- missive temperature. This result further supports the model that flagella shorten in fla10 mutants because IFT is normally required to balance flagellar disassembly during turnover. Microtubule turnover and flagellar length mutants The results of Fig. 5 suggest that intraflagellar transport is required continuously to maintain full-length flagella. This Flagellar length control | Marshall and Rosenbaum 409 raises the possibility that if IFT were to be partially reduced, but not completely eliminated, it might cause a partial, but not complete, reduction in flagellar length. fla10ts mutants were grown at an intermediate temperature between the fully permissive temperature of 21 C and the fully nonper- missive temperature of 34 C, so that some partial fla10 function would remain. We found that in fla10 cells grown at 28 C, the flagella shortened and equilibrated at roughly one half their wildtype length (Fig. 6 A), comparable to the lengths seen in previously identified shf mutants that can range in size from 5 to 8 m, depending on the particular shf allele (Kuchka and Jarvik, 1987). This new length was maintained even after 3 d of growth at the intermediate tem- perature. Half-length flagella were only obtained when fla10 cells were grown at exactly 28 C. Growing cells at a range of intermediate temperatures between fully permissive and fully nonpermissive produces a corresponding range of inter- mediate steady-state lengths (Fig. 6 B). When the tempera- ture is increased to 29 C or higher, the flagella disappear completely, confirming that IFT in Chlamydomonas is strictly required to maintain even very short flagella. If IFT maintains flagellar length by balancing turnover of microtubules at the plus end, then just as alterations in IFT can lead to changes in flagellar length, alterations in turn- over rates should also lead to changes in flagellar length. Thus, some of the known Chlamydomonas flagellar length mutants (McVittie, 1972; Jarvik et al., 1976, 1984; Barsel et al., 1988; Kuchka and Jarvik, 1987; Asleson and Lefebvre, 1998) might show changes in outer doublet microtubule turnover rate. We tested this possibility by analyzing micro- tubule turnover in the lf mutant lf2-5 (Barsel et al., 1988). As shown in Fig. 6 C, when the turnover assay was carried out in cells carrying the lf2-5 mutation, the extent of turn- over was greatly reduced compared with wild-type cells as re- flected in a decrease in the growth of the labeled region (re- flecting net assembly of labeled tubulin), as well as a decrease in the shortening of the unlabeled segment (reflecting net disassembly of unlabeled tubulin). This suggests that lf2-5 may have lf because of a decrease in the disassembly rate. IFT particles and flagellar length Because IFT appears to play an important role in maintain- ing flagellar length, it is of interest to determine how the density of IFT particles may change as a function of flagellar length. Fig. 7 A shows three cells from a flagellar regenera- tion time course, stained with an antibody that detects the IFT protein IFT52 (Cole et al., 1998; Deane et al., 2001). Shorter flagella clearly show a more intense staining of IFT52 protein than longer flagella. Because it can be diffi- cult to assess differences in fluorescence intensity by visual inspection, the average fluorescence intensity per unit length was quantified in cells at various stages of flagellar regenera- tion, and plotted in Fig. 7 B. This quantification confirms the visual impression that IFT52 is present at a higher con- centration per unit length in shorter flagella, and becomes relatively less intense as the flagella become longer. Fig. 7 C plots the total IFT52 intensity in the flagellum, calculated by multiplying the intensity per unit length by the total length. This data indicates that the total amount of IFT52 in the flagellum is constant and does not vary significantly