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Quantitative analysis and modeling of katanin function in flagellar length control.

Kannegaard E, Rego EH, Schuck S, Feldman JL, Marshall WF - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158.

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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.
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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.

Mentions: It has been reported that depletion of a depolymerizing kinesin, kinesin-13, from Chlamydomonas leads to cells with regeneration defects and short flagella (Piao et al., 2009; Wang et al., 2013). Wang et al. (2013) proposed that these phenotypes could arise if kinesin-13 acts in the cytoplasm to help release tubulin from cytoplasmic microtubules for use in flagellar growth, a claim supported by the observation that cytoplasmic microtubules shorten less during flagellar regeneration when kinesin-13 is depleted by RNA interference (RNAi; Wang et al., 2013). Because the proposed role for kinesin-13 in flagellar growth is similar to our model for katanin function, we modified the stochastic simulation of Figure 4 to simulate the effects of kinesin-13. This was done by adding a variable Kremove, which determines the probability per unit time of kinesin-13 removing the final tubulin from a microtubule (regardless of GTP state). We assume that the probability of terminal tubulin removal by kinesin-13 is independent of microtubule length, in contrast to the length-dependent activity of kinesin-8 (Varga et al., 2006). With this additional term added to the model, we find that as the rate of kinesin-13 activity is increased, microtubules undergo higher frequency of catastrophe and gradually shorten during simulated flagellar regeneration (Figure 6A). When we plot steady-state flagellar length versus kinesin-13 activity (Figure 6B), we find that just as with katanin, increased activity leads to increased flagellar length. Decreased activity leads to short flagella. Our computational model thus recapitulates the key result of Wang et al. (2013) and further confirms that modulation of enzymes affecting cytoplasmic microtubule dynamics can alter flagellar length via a competition mechanism.


Quantitative analysis and modeling of katanin function in flagellar length control.

Kannegaard E, Rego EH, Schuck S, Feldman JL, Marshall WF - Mol. Biol. Cell (2014)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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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.
Mentions: It has been reported that depletion of a depolymerizing kinesin, kinesin-13, from Chlamydomonas leads to cells with regeneration defects and short flagella (Piao et al., 2009; Wang et al., 2013). Wang et al. (2013) proposed that these phenotypes could arise if kinesin-13 acts in the cytoplasm to help release tubulin from cytoplasmic microtubules for use in flagellar growth, a claim supported by the observation that cytoplasmic microtubules shorten less during flagellar regeneration when kinesin-13 is depleted by RNA interference (RNAi; Wang et al., 2013). Because the proposed role for kinesin-13 in flagellar growth is similar to our model for katanin function, we modified the stochastic simulation of Figure 4 to simulate the effects of kinesin-13. This was done by adding a variable Kremove, which determines the probability per unit time of kinesin-13 removing the final tubulin from a microtubule (regardless of GTP state). We assume that the probability of terminal tubulin removal by kinesin-13 is independent of microtubule length, in contrast to the length-dependent activity of kinesin-8 (Varga et al., 2006). With this additional term added to the model, we find that as the rate of kinesin-13 activity is increased, microtubules undergo higher frequency of catastrophe and gradually shorten during simulated flagellar regeneration (Figure 6A). When we plot steady-state flagellar length versus kinesin-13 activity (Figure 6B), we find that just as with katanin, increased activity leads to increased flagellar length. Decreased activity leads to short flagella. Our computational model thus recapitulates the key result of Wang et al. (2013) and further confirms that modulation of enzymes affecting cytoplasmic microtubule dynamics can alter flagellar length via a competition mechanism.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158.

Show MeSH
Related in: MedlinePlus