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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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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.
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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.

Mentions: To determine the regions flanking the inserted sequence in our collection of insertional mutants, we used restriction enzyme site-directed amplification PCR (RESDA-PCR; Gonzalez-Ballester et al., 2005). Our initial RESDA-PCR results identified flanking sequence in four mutants: 1464, 3584, 4580, and 9111. However, only in line 1464 did the insertion occur in an annotated gene (Figure 3A), namely PF15, which encodes the p80 subunit of katanin (Hartman et al., 1998; McNally et al., 2000; Dymek et al., 2004), and we therefore chose this insertional line for further analysis. Genomic PCR revealed that the insertional mutagenic event deleted all but the first three predicted exons of the PF15 gene (Figure 3B). Mutations in the PF15 gene were previously identified in screens for paralyzed flagellar motility (Dymek et al., 2004), and consistent with this result, we observed that in addition to its length defect, the mutant 1464 flagella were paralyzed. Because the original pf15a strain was previously shown to lack the central pair of microtubules (Adams et al., 1981; Dymek et al., 2004), we examined the structure of axonemes in mutant 1464 by electron microscopy (Figure 3C). For the wild type, 38 flagella were analyzed, all of which had a central pair. For the mutant 1464, 32 flagella were analyzed, of which 30 lacked a central pair and two contained electron-dense material in the center of the axoneme that could not be clearly recognized as a central pair. If these two are conservatively classified as positive, the fraction of flagella with a central pair is 100% for the wild type and 6% for the mutant. The central-pair defect seen in mutant 1464 is thus comparable to that seen in pf15a mutants.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: To determine the regions flanking the inserted sequence in our collection of insertional mutants, we used restriction enzyme site-directed amplification PCR (RESDA-PCR; Gonzalez-Ballester et al., 2005). Our initial RESDA-PCR results identified flanking sequence in four mutants: 1464, 3584, 4580, and 9111. However, only in line 1464 did the insertion occur in an annotated gene (Figure 3A), namely PF15, which encodes the p80 subunit of katanin (Hartman et al., 1998; McNally et al., 2000; Dymek et al., 2004), and we therefore chose this insertional line for further analysis. Genomic PCR revealed that the insertional mutagenic event deleted all but the first three predicted exons of the PF15 gene (Figure 3B). Mutations in the PF15 gene were previously identified in screens for paralyzed flagellar motility (Dymek et al., 2004), and consistent with this result, we observed that in addition to its length defect, the mutant 1464 flagella were paralyzed. Because the original pf15a strain was previously shown to lack the central pair of microtubules (Adams et al., 1981; Dymek et al., 2004), we examined the structure of axonemes in mutant 1464 by electron microscopy (Figure 3C). For the wild type, 38 flagella were analyzed, all of which had a central pair. For the mutant 1464, 32 flagella were analyzed, of which 30 lacked a central pair and two contained electron-dense material in the center of the axoneme that could not be clearly recognized as a central pair. If these two are conservatively classified as positive, the fraction of flagella with a central pair is 100% for the wild type and 6% for the mutant. The central-pair defect seen in mutant 1464 is thus comparable to that seen in pf15a mutants.

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