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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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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.
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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.

Mentions: We next examined flagellar regeneration kinetics directly in this subclass of short-flagella mutants (Figure 2A). In all five of the candidate mutants, the regeneration curves plateau abruptly at a short length relative to wild type and in this sense are reminiscent of wild-type cells treated with protein synthesis inhibitors. Such might be the expected result for a mutant with defects in pool regeneration. However, the initial growth rate is slightly reduced compared with wild-type cells. This could indicate some pool-independent effect of the mutation, or it could indicate the presence of a reduced pool from the very beginning of the time course, unlike in wild-type cells, where the pool starts out at full capacity.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: We next examined flagellar regeneration kinetics directly in this subclass of short-flagella mutants (Figure 2A). In all five of the candidate mutants, the regeneration curves plateau abruptly at a short length relative to wild type and in this sense are reminiscent of wild-type cells treated with protein synthesis inhibitors. Such might be the expected result for a mutant with defects in pool regeneration. However, the initial growth rate is slightly reduced compared with wild-type cells. This could indicate some pool-independent effect of the mutation, or it could indicate the presence of a reduced pool from the very beginning of the time course, unlike in wild-type cells, where the pool starts out at full capacity.

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