Quantitative analysis and modeling of katanin function in flagellar length control

Abstract

Flagellar length control in Chlamydomonas reinhardtii provides a simple model system in which to investigate the general question of how cells regulate organelle size. Previous work demonstrated that Chlamydomonas cytoplasm contains a pool of flagellar precursor proteins sufficient to assemble a half-length flagellum and that assembly of full-length flagella requires synthesis of additional precursors to augment the preexisting pool. The regulatory systems that control the synthesis and regeneration of this pool are not known, although transcriptional regulation clearly plays a role. We used quantitative analysis of length distributions to identify candidate genes controlling pool regeneration and found that a mutation in the p80 regulatory subunit of katanin, encoded by the PF15 gene in Chlamydomonas, alters flagellar length by changing the kinetics of precursor pool utilization. This finding suggests a model in which flagella compete with cytoplasmic microtubules for a fixed pool of tubulin, with katanin-mediated severing allowing easier access to this pool during flagellar assembly. We tested this model using a stochastic simulation that confirms that cytoplasmic microtubules can compete with flagella for a limited tubulin pool, showing that alteration of cytoplasmic microtubule severing could be sufficient to explain the effect of the pf15 mutations on flagellar length.

FIGURE 1:

Length distribution of the class III short flagella mutants, with wild type for comparison. (A) Wild-type strain cc-124, n = 165 flagella measured. (B) Mutant 1464, n = 385. (C) Mutant 784, n = 168. (D) Mutant 3584, n = 170. (E) Mutant 4580, n = 170. (F) Mutant 5899, n = 112.

FIGURE 2:

Identifying a short-flagella mutant with impaired precursor pool regeneration kinetics but normal transcriptional induction. (A) Regeneration kinetics in class III short-flagella mutants. Graph shows flagellar length vs. time after pH shock. Time 0 indicates length before pH shock. Error bars indicate SEM. Error bars smaller than the radius of the data-point marker are not visible. All data points are based on measurement of flagella from 60 cells. (B) Length vs. time after deflagellation normalized to predeflagellation length; red circles, wild type; orange squares, mutant 5899; blue squares, mutant 1464; green circles, mutant 4580; gray circles, mutant 3584; white circles, mutant 784. (C) Assay for flagellar pool regeneration as described in Lefebvre et al. (1978). Initial culture is subjected to pH shock (red arrow) and allowed to recover. At 10-min intervals, aliquots are removed, cycloheximide is added to the aliquot (vertical blue arrows), and then it is subjected to a second pH shock. Aliquots after the second aliquot are incubated for 2 h to reach steady-state length. (D) Result of pool regeneration assay. Time indicates the time at which the second pH shock was performed relative to when the initial pH shock was performed, that is, the time during which the cells were regenerating their pool before inhibition of protein synthesis. Length indicates the final steady-state length reached after regenerating from the second pH shock and is an indicator of the size of the protein pool at the time of cycloheximide addition; red circles, wild type; orange squares, mutant 5899; blue squares, mutant 1464; green circles, mutant 4580; gray circles, mutant 3584. (E) Induction of flagella-specific gene expression during regeneration in short-flagella mutants. Graph shows expression of RSP3 (normalized by expression of RBCS2 housekeeping gene) vs. time after pH shock measured by quantitative PCR, comparing wild-type cells, pf18 (chosen as a representative paralyzed mutation), and three previously described short-flagella mutants. Normal cells show up-regulation of flagella-specific genes at the 30- and 45-min time points, with expression dropping by 1 h. (F) Normalized expression of RSP3 in wild type and insertional short-flagella mutants. Error bars are SD among three separate experiments. Asterisks designate strains in which induction was significantly reduced (p < 0.001) compared with wild-type cells as determined by one-way analysis of variance, followed by Bonferroni's multiple comparison test.

FIGURE 3:

Mutation in p80 subunit of katanin results in short flagella with impaired precursor pool mobilization. (A) Position of pHyg insertion and accompanying deletion in genome of 1464 mutant as determined by RESDA mapping, followed by genomic PCR with flanking primers. (B) Region of PF15 gene that is deleted in the 1464 mutant, indicated by the solid bar below the gene map. (C) Electron microscopy showing loss of central pair microtubules in 1464 mutant. Top, wild-type cell showing clear central pair microtubules. Bottom, mutant 1464, showing that central-pair microtubules are missing and replaced with amorphous electron-dense material, similar to what is seen in the pf15a mutant. Scale bars, 50 nm. (D) Length distribution in wild type (red), mutant 1464 (green), and pf15a (blue). (E) Regeneration kinetics after pH shock in wild type (red), mutant 1464 (green), and pf15a (blue). Error bars show SE. (F) Comparison of effective pool regeneration kinetics for strain 1464 and pf15a measured by double-pH-shock procedure; red, wild type, green, 1464; blue, pf15a. (G) Comparison of flagellar gene induction in strain 1464 and pf15a measured by quantitative PCR. Plot shows ratio of RSP3 to RuBisCo message for wild type (red), 1464 (green), and pf15a (blue). Error bars show SD.

FIGURE 4:

Combined model of flagellar and cytoplasmic microtubule dynamics. (A) Diagram illustrating two populations of microtubules; red, cytoplasmic microtubules; green, flagellar microtubules. (B) Kymograph showing a single run of stochastic microtubule simulation using the method of Gregoretti et al. (2006), with addition of katanin-mediated cleavage. The plot shows a single microtubule, with subunits color coded to indicate nucleotide state. Arrows indicate examples of katanin-mediated severing events (red arrows) and a catastrophe induced by spontaneous loss of GTP cap (yellow arrow). These events are distinguishable in the kymograph because catastrophe is preceded by loss of the GTP cap. We note that microtubules regrow more rapidly after catastrophe than after severing in the simulation because shrinkage after catastrophe ends when a full cap is restored, leading to immediate growth, whereas severing often produces a microtubule that lacks a cap and does not start growing until a full cap is assembled. (C) Example of simulation showing length fluctuations of one cytoplasmic microtubule in parallel with length changes in one flagellum as the flagellum grows from an initial zero length, thus representing a regeneration experiment. We note that these data are plotted on a longer time scale than in B, so that variation in flagellar length, which takes place much more slowly than variation in individual microtubules, can be observed.

FIGURE 5:

Predicted effect of changes in katanin-mediated microtubule severing on flagellar length. Flagellar length (black) and ratio of polymerized to soluble tubulin (red) are plotted for different values of the katanin severing rate parameter. Results show that as severing rate is decreased, flagellar length decreases. Polymerized/soluble ratio values are reported using the numerical values indicated on the y-axis.

FIGURE 6:

Modeling effect of kinesin-13–mediated microtubule shortening on flagellar length. (A) Microtubule length fluctuation during simulated flagellar regeneration in the absence of kinesin-13 activity. (B) Microtubule length fluctuation during flagellar regeneration in the presence of kinesin-13 activity, showing higher catastrophe frequency and gradual shortening of microtubules as flagella grow. (C) Steady-state flagellar length vs. simulated kinesin-13 activity.