Quantitative analysis and modeling of katanin function in flagellar length control.
Bottom Line: 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.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.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.
Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158.Show MeSH
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Mentions: Although three short-flagella (shf) mutants have already been identified, they are difficult to clone because their mutations are not sequence tagged. We therefore screened a collection of 20 insertional short-flagella mutants that we had previously identified in a screen of 10,000 random insertional lines using a phototaxis assay (Feldman et al., 2007). We analyzed the length distributions of all 20 insertional shf mutants, with results listed in Table 1. Although different mutants show a range of different length distributions, analysis of the shape of the length distribution, as judged by skew and kurtosis, allowed us to define three categories: class I, positive kurtosis, positive skew; class II, positive kurtosis, negative skew; and class III, negative kurtosis. The first two classes have a sharply peaked distribution that is of a shorter mean length than wild type, but because the distributions are peaked around a single value, these mutations appear to retain an effective length control system. We note that although some of these show a statistically significant positive skew, this is likely to be a byproduct of the fact that flagellar lengths cannot be negative, hence length distributions whose mean is less than the SD will produce truncated distributions and yield a positive skew when fit with a Gaussian. We focused our analysis on class III, as the negative kurtosis (flat distribution) matched our expectations for mutants with impaired pool regeneration. Class III comprised five mutants, whose length distributions are plotted in Figure 1. As can be seen, in comparison to wild type, all of these mutants show a broader distribution of lengths and decreased average length.
Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158.