410 The Journal of Cell Biology | Volume 155, Number 3, 2001 Figure 7. Total quantity of intraflagellar transport particles is independent of flagellar length. (A) IFT52 immunofluorescence staining during flagellar regeneration. Three cells are shown at three different time points during regeneration. As regeneration progresses and flagella elongate, the density of IFT52 decreases visibly. Bar, 5 m. (B) Average IFT52 staining intensity, per unit length, plotted versus flagellar length. Quantification by digital imaging confirms the visual impression that IFT particle staining is more intense in short flagella. (C) Total IFT52 fluorescence intensity, a measure of IFT particle protein content, found by multiplying intensity per unit length by flagellar length. Graph indicates that the IFT particle content of a flagellum is independent of flagellar length. with flagellar length. This result suggests that from the be- ginning of flagellar assembly, each flagellum is allocated a fixed number of active IFT particles, regardless of flagellar length. The consequences of this result for the mechanism of length control will be discussed below. Discussion A function of IFT in flagellar maintenance IFT is required for flagellar and ciliary assembly and mainte- nance in a wide range of species (Rosenbaum et al., 1999). Given that flagellar assembly always takes place at the distal end of growing flagella (Johnson and Rosenbaum, 1992), the need for IFT during assembly is clear. However, the contin- ued need for IFT to maintain flagella after flagellar growth has been completed has never been explained. If, as is often as- sumed, cilia and flagella are static structures, then it is hard to understand why IFT would be needed after flagellar assembly is completed. In this case, one would have to propose that IFT plays some additional function besides simply bringing flagel- lar proteins out to the distal tip assembly site. But the fact that flagellar proteins turn over (Stephens, 1997, 2000), together with the results presented here, that flagella are constantly re- moving and incorporating tubulin subunits at the distal ends of the outer doublets, leads to a much simpler explanation for the continuous requirement for IFT. Because flagellar microtubules are constantly turning over, transport of fresh subunits is needed to support continuous assembly at the distal end. We have shown that when IFT is shut down, thus inhibiting assembly (Fig. 5 B), disassembly continues unabated, causing the flagella to shorten. We have also found that when turnover is blocked (i.e., by colchicine treatment), IFT is no longer required to maintain the flagella (Fig. 5 C). Therefore, we conclude that the primary role of IFT in maintaining cilia and flagella is to balance the ongo- ing disassembly of outer doublet microtubules by providing a constant supply of new material to assemble at the distal end. However, it is important to note that at present, we do not know exactly how IFT acts to bring material to the flagellar tip. Whether the IFT particles directly bind precursor pro- teins or instead facilitate their motion more indirectly, re- mains an open question that our data do not address. Although shutting off IFT in Chlamydomonas by the fla10 mutation results in complete loss of flagella at the nonper- missive temperature, antibody injection experiments in sea urchin embryos (Morris and Scholey, 1997) found that in- hibiting kinesin-II (the homologue of fla10) resulted in short, paralyzed, but still visible cilia. This could either re- flect a difference between sea urchins and Chlamydomonas, or differences in efficacy between antibody blocking ap- proaches and direct mutations in the kinesin itself. We also note that whereas this model accounts for the re- sorption of flagella when IFT is shut off, this may not be the only mechanism for resorbing a flagellum. In Chlamydomo- nas, recently mated cells retain their flagella for 2 h and then rapidly resorb them prior to meiosis. This resorption is completed within just 20 min, and is thus much faster than the flagellar resorption seen when IFT is shut down, which takes several hours before resorbtion is complete. Moreover, whereas colchicine blocks the slow resorption of flagella in fla10 mutants (Fig. 5 C) by arresting turnover, it has no ef- fect on the rapid premeiotic flagellar resorption during mat- ing (unpublished data). These differences suggest that the two types of resorption may occur via different mechanisms. A model for flagellar length control The preceding results yield three main facts that, taken to- gether, suggest a simple model for flagellar length control: (a) flagellar microtubules turn over continuously at the plus end; (b) IFT is required for assembly at the plus end and must, therefore, be able to provide flagellar precursor pro- teins fast enough to balance the ongoing disassembly; and (c) the number of IFT particle proteins per flagellum is inde- pendent of flagellar length. We propose that flagellar length may be determined by an equilibrium between assembly and disassembly at the plus end. In order to see how such an equilibrium can yield a unique steady-state length, we must consider the length de- pendence of the assembly and disassembly rates. When IFT is blocked in fla10 mutants, flagella shorten at a constant rate, indicating that the disassembly rate at the plus end is independent of flagellar length. In contrast, when flagella re- generate, the rate of flagellar elongation decreases as the fla- gellum grows (Rosenbaum and Child, 1967; Tamm, 1967), indicating that the assembly rate is length dependent and de- creases as the flagellum becomes longer.