Cultures of fla2 cells in TAP medium contain free flagella after incubation at the restrictive temperature of 33°C. Due to their small size, and tendency to stick to each other and to attached flagella, free flagella are difficult to accurately identify; however, as fla2 cells can regenerate and re-deflagellate at 33°C, the appearance of abundant free flagella is quite obvious. Our initial observations of the fla10 temperature-sensitive phenotype led us directly to the conclusion that this mutant also deflagellates, as many free flagella are seen in the presence of calcium at 33°C in both fla10 and fla2 cultures (Figure 1).

As fla10 has only been previously characterized as resorbing flagella at 33°C, and given our knowledge of the role played by calcium in the deflagellation pathway, we reasoned that deflagellation would be prevented if fla10 cells were incubated at 33°C in buffer lacking added calcium. We found this indeed to be the case; there is no indication of deflagellation by fla10 cells at 33°C in the absence of calcium, yet cultures are still mostly bald after ~6 hours (Figures 1, 2). We extended our observations by demonstrating that fla2 cells also do not deflagellate at 33°C in the absence of calcium (Figure 1). Adding calcium to minimal buffer to a concentration approximately that of TAP media (340 μM) 30 restored the deflagellation phenotype of both fla mutants. As a further control, blocking the deflagellation response by creating double mutants of either fla10 or fla2 with either of fa1 or fa2 also prevented the appearance of free flagella after six hours (Figure 1 and data not shown). Wild-type cells do not deflagellate under these conditions.

Having examined the culture media under various conditions, we turned our attention to the cells themselves. The average flagellar lengths of fla10 and fla2 cells, but not wild-type cells, decrease at 33°C (Figure 2). We have shown that the fla mutants deflagellate at 33°C in the presence of added calcium, yet the differences in the average flagellar length plots are difficult to discern whether or not calcium was added, especially for fla10 (diamonds in Figure 2A,2B). This similarity of the average flagellar length plots holds whether or not zero-length flagella are included in the average length calculations (Figure 2C,2D).

To better illustrate the effects of calcium at the restrictive temperature on the fla mutants, we have chosen a novel means of presenting flagellar length data. By plotting the length of the longer flagellum versus the length of the shorter flagellum for fla10, fla2, and wild-type cells (Figures 3, 4, and 5, respectively), we can more clearly observe the states of individual cells during timecourses at the restrictive temperature. Points representing bald cells fall at the origin, while points representing uniflagellate cells are found on the x-axis. Additionally, we have plotted the percent of biflagellate, uniflagellate, and bald cells in the population for each timepoint as insets in the scatter plots. This presentation allows one to quickly assess the flagellation state of a population, and changes in that state over time.

Figure 3 presents timecourses for fla10 flagellar lengths at 33°C, in the absence or presence of added calcium. In both populations, cells begin with flagella of approximately equal lengths, and the distribution of flagellar lengths falls from ~8–11 μm. After one hour at the restrictive temperature, flagellar lengths of most cells in calcium remain unchanged, although a few cells now have flagella of unequal lengths. In contrast, the flagella of cells in calcium-free buffer have shortened to 5–10 μm (average length of 7.5 μm, Figure 2C).

After three hours at 33°C, cells in each population have shorter flagella, and there is an increase in the numbers of uniflagellate and bald cells in both populations. At this time in nominally calcium-free buffer, several cells with flagella shorter than 4 μm are observed. These short flagella indicate that resorption is occurring. Conversely, in the presence of calcium, no flagella <4 μm are seen, suggesting that cells deflagellate before flagellar resorption is complete. fla10 cells incubated at 20°C did not undergo significant flagellar loss or shortening, and after 6 hours at 20°C, populations resembled zero-hour timepoints (bottom panels in Figure 3).

Figure 4 is a timecourse for the fla2 mutant, which was previously reported to deflagellate at the restrictive temperature 21214. After 6 hours in HEPES at 33°C, but not at 20°C, over one-quarter of the fla2 cells are bald. Fewer cells with flagella >9 μm are seen after 6 hours at 33°C compared with earlier timepoints and 20°C controls. At the three hour timepoint in the presence of calcium, there is a dramatic increase in bald cells in the fla2 population (Figure 4, inset). In contrast to fla10, fla2 cells are able to regenerate flagella at the restrictive temperature [1214, and data not shown]. This regeneration accounts for the increase in the number of cells with short flagella in the fla2 population at later timepoints. After overnight incubation at 33°C, fla2 cultures are often entirely bald.

Figure 5 shows the flagellar lengths of wild-type cells after a shift to 33°C. In contrast to the fla mutants, wild-type flagella do not undergo appreciable changes in length at 33°C. In principle, the resorption phenotype of fla10 at 33°C could be solely due to constitutive disassembly in the absence of anterograde IFT, and the deflagellation phenotype a secondary consequence due to the stress of elevated temperature. However, wild-type cells do not deflagellate at 33°C (Figures 1, 5), although they will deflagellate at 42°C (data not shown).

The deflagellation phenotype of fla10 and fla2 at 33°C in calcium is blocked by either fa mutation (Figure 1 and data not shown). We wished to examine double mutant cell populations, and because fla2 is able to regenerate flagella at the restrictive temperature, we focused on fla10-fa mutants. We find that both fla10-fa1 and fla10-fa2 double mutants resorb flagella more slowly than fla10 at 33°C (Figure 6). This held true whether or not calcium was present in the medium, and, as for the fla10 single mutant, similar flagellar loss kinetics were observed regardless of calcium (Figure 3 and data not shown). This result is reminiscent of the partial block of fla10 flagellar resorption by six different extragenic suppressors 31; it is unknown whether any of these suppressors are fa genes.

fla10 mutant populations are >90% bald after 6 hours at 33°C (Figures 3, 6), whereas only ~1/3 of fla10-fa cells are bald at this time (Figure 6). Moreover, at later timepoints when fla10 cells are all bald, many double mutant cells still retain flagella. Strikingly, at all timepoints, the double mutant populations are enriched in cells with unequal length flagella as well as uniflagellate cells. Resorbing populations of fla10 cells also have a few uniflagellate cells, but cells with unequal length flagella are not often observed (Figure 3). Thus, the appearance of cells with flagella of unequal length in double mutant populations correlates with an overall decrease in the resorption rate.

The prevalence of uniflagellate cells, as well as flagella of unequal lengths, is at odds with the classical picture of Chlamydomonas long-zero flagellar dynamics 832. This model predicts that cells with flagella of unequal length should be rarely seen: the combination of length-control kinetics, and the fact that the two flagella share a common pool of precursors, means that any length imbalance between the flagella of a single cell will be corrected 8. However, long-zero experiments are typically carried out in conditions permissive for flagellar regeneration, which is not the case for fla10 mutants at the restrictive temperature. In agreement with this, fla2 cultures contain fewer uniflagellate cells than fla10 cultures at the restrictive temperature, and this is likely due to the ability of fla2, but not fla10, to reassemble flagella at the restrictive temperature.

The observation of unequal shortening of flagella in a resorbing population (Figure 6) parallels the observation that one flagellum is prone to deflagellate before the other (Figures 3, 4, 6 and data not shown). Loss of one flagellum at a time is not observed in response to a strong deflagellation signal, but is observed after treatment with weaker stimuli such as a mild acid shock (data not shown) or threshold concentrations of alcian blue 33. The two flagella of Chlamydomonas are not equivalent; the cis flagellum is defined as the one closest to the eyespot. Rather than the random loss of either flagellum, we hypothesize that the cis and trans flagella are differentially sensitive to disassembly signals, as they are to motility-related calcium signals 34. This hypothesis predicts that disassembly will be initiated earlier and/or proceed more rapidly in one flagellum, which could explain the abundance of unequal length flagella observed in Figure 6.

On the basis of a survey of the modes of flagellar loss in unicellular algae and fungi, Bloodgood divided flagellar loss into several categories 23. Examples were cited of the same organism (or even the same cell) undergoing flagellar loss sometimes by resorption and sometimes by deflagellation 23. Rather than supposing that organisms undergo flagellar loss by different mechanisms at different stages of their life cycles, we contend that the mechanism of flagellar loss is conserved, and that subtle variations in signaling leads to the appearance of either deflagellation or various forms of resorption. This conserved mechanism would involve severing of the axoneme at the base of the flagellum.

We now summarize specific experimental data suggesting that resorption is mediated at the transition zone, the site of deflagellation, rather than by constitutive disassembly at flagellar tips. First, fla10 and fla2 mutant cells disassemble their flagella at the restrictive temperature via deflagellation in the presence of calcium, and via resorption in the absence of added calcium. Second, fa mutants, which are unable to deflagellate, are slow to resorb; this effect would not be predicted if resorption were entirely due to dynamics at flagellar tips. The slow resorption phenotype of fa1 is especially informative, as this mutant is slow to assemble flagella (JDKP, Ben Montpetit, LMQ, unpublished observations), in which case the length-control theory predicts that fa1 should be fast to resorb. Third, the slow resorption of fla10-fa double mutant cells produces uniflagellate cells in the absence of deflagellation, a consequence not predicted by the dynamic length control model but in accordance with the results of mild deflagellation treatments. Fourth, while Chlamydomonas cells resorb their flagella prior to mitosis 24, EM studies have provided evidence that flagella are detached from basal bodies, but not lost from cells, during pre-mitotic resorption 29. This seeming contradiction may be explained by our proposal that resorption requires microtubule severing activity at the flagellar transition zone.

There are also some theoretical considerations. Not only the tips of the outer doublet microtubules, but rather the whole axoneme, has been shown to undergo turnover in sea urchin embryos 35. If this turnover occurs in all eukaryotic flagella, then flagellar tip dynamics alone 8 or lattice translocation models 36 could potentially account for this turnover. However, we believe a regulated ratcheting of the axoneme at the transition zone, coupled with dynamic activity at the tip, provides the most satisfying explanation for the occurrence of axonemal turnover even while a flagellum remains functional. Additionally, there is the consideration that flagellar length in Chlamydomonas is cell-cycle dependent, and flagella undergo a period of slow resorption prior to a period of fast resorption preceding cell division 8. We hypothesize that the slow phase of resorption is mediated by the length-control mechanism, while the fast phase of resorption is mediated by severing of axonemal microtubules in the transition zone.

The deflagellation phenotype of fla10 is significant, as it implies an active signalling event is at work. We predicted that if the deflagellation severing complex is responsible for resorption, then providing a deflagellation stimulus to a cell unable to deflagellate should induce resorption. Indeed, this is consistent with temperature-sensitive resorption of fla10 in the absence of added calcium, and with temperature-induced resorption of fla10-fa double mutants with or without added calcium. To investigate whether this effect is more general, or only due to some special nature of the fla phenotype, we examined the effect of pH shock on mutants unable to respond by deflagellation. As previously shown, acid treatment of fa1, fa2, or adf1 mutants does not lead to deflagellation as it does for wild-type cells 3738. However, as predicted by our model, these mutants do indeed resorb their flagella within one hour after a 30 s acid shock (Figure 7). This resorption is not necessarily complete, and is complicated by the tendency of the acid-treated fa cells to coil their flagella starting from the distal ends. These coiled flagella get smaller over time, and before disappearing entirely they become dense, stumpy structures. Sanders and Salisbury noted that the Chlamydomonas centrin mutant, vfl-2, will sometimes resorb rather than deflagellate in response to the deflagellation agent, dibucaine 39. We have further extended these results by performing deflagellation experiments on wild-type cells in low-calcium buffer. Acid shock, dibucaine treatment, and 42°C heat shock all induce flagellar resorption of wild-type cells in low calcium buffer (data not shown).

We conclude that resorption and deflagellation both may result from a "flagellar disassembly" signal. If the fast phase of flagellar resorption results from disassembly mediated by the severing complex at the transition zone, as we have argued, then a new picture of deflagellation emerges: calcium-mediated hyperactivation of the severing complex following a disassembly signal leads to deflagellation. This model exposes our lack of understanding of the endogenous signaling pathways that lead to flagellar disassembly. Flagellar disassembly occurring prior to mitosis or meiosis in Chlamydomonas must occur via resorption, even in medium containing calcium, as cultures typically contain few free flagella. If agents such as intracellular acidification, dibucaine, and heat shock induce deflagellation by hyperactivation of an endogenous disassembly pathway by causing an increase in intracellular calcium, then why should similar treatments in low calcium cause resorption? Two nonexclusive possibilities present themselves: either the severing apparatus has progressively greater activity at higher calcium thresholds, and has partial activity sufficient to mediate resorption without full calcium activation; or there is a calcium-independent signaling pathway which is induced by treatments such as intracellular acidification.

A candidate for providing such a signal is the IFT pathway. Recent work has suggested that IFT has signaling functions 40, and it has been suggested that these signalling functions are perhaps more important for flagellar assembly than the physical transport of flagellar components 5. Halting IFT in the presence of calcium induces deflagellation, as we have shown with the IFT mutant fla10, in which IFT halts quickly after shift to the restrictive temperature 41. fla10 and fla2 are not the only IFT mutants which deflagellate; we have observed deflagellation at the restrictive temperature for several other fla mutants (data not shown). As most fla mutants have defects at some point in the IFT cycle at the permissive temperature 16, it is likely that IFT is disrupted in these mutants at the restrictive temperature. As first mentioned anecdotally by Kozminski et al. 41, halting IFT seems to lead to deflagellation.

As IFT is so intimately involved in flagellar assembly, it should not come as a surprise that it may play a role in flagellar disassembly. It is intuitive that a cell should not attempt to continue building a flagellum at a time when it is actively disassembling a flagellum, and this logic suggests that signalling events that regulate IFT should be related to flagellar disassembly signals. Deflagellation of fla10 at the restrictive temperature, when anterograde IFT is halted, raises the possibility that functional IFT counteracts a constitutive disassembly signal. This could be adaptive. For example, for cells with damaged flagella, deflagellation would be the favored disassembly response, due to its speed and ability of Chlamydomonas to upregulate genes required for flagellar assembly post-deflagellation. If deflagellation is blocked, flagellar disassembly will instead occur via resorption.

Chlamydomonas reinhardtii mutants fla2 (CC-1390) and fla10-1 (CC-1919) were provided by the Chlamydomonas Genetics Center. Wild-type strain B214 was provided by Dr. G. Pazour (University of Massachusetts). The fa1-1 mutant was isolated by R.A. Lewin 42; the fa2-1 and adf1-5 mutants were isolated in our lab 38. Genetic crosses to obtain the double mutants fla10-fa1-1, fla10-fa2-1, fla2-fa1-1, and fla2-fa2-1 were performed by standard techniques 30. All cells were maintained on TAP (Tris-Acetate-Phosphate) media 30 under constant illumination at 20°C.

Since Chlamydomonas cells transiently resorb their flagella after resuspension in HEPES buffer (10 mM HEPES-KOH, pH 7.2) (with or without added calcium), cultures were incubated overnight in HEPES prior to HEPES experiments. No chelators were used in the preparation of low-calcium HEPES buffer as trace amounts of calcium are required for cell viability over the length of the experiments.

Cells were resuspended at 3 × 10⁶ cells/mL in buffer or media, pre-warmed to the temperature of the incubation, at time zero. We find that the flagellar loss phenotypes of fla10 are very temperature-sensitive, and a 1°C temperature difference can have a large effect on the kinetics of the experiment; this likely accounts for varying reports on the time required for fla10 cultures to become bald. Thus, the same culture of cells was used for both +/- calcium at each temperature. Samples were fixed in 2% gluteraldehyde for flagellar length measurements and free flagella determinations. Cells were examined under DIC on an Olympus IX70 microscope (Carsen Group Inc., Ontario, Canada) and lengths of both flagella on at least 70 cells were measured using software provided with the DeltaVision system (Applied Precision, Seattle, Washington). All experiments were repeated on at least three independent cultures.