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Cited by 3Genetic and Kidney Cyst DiseasesCystic Fibrosis Research AdvancesMicrotubule and mitosis dynamics

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Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies

Open access
Dutta, Soumita, Avasthi, Prachee·2017·mSphere
DOI 10.1128/msphere.00003-17PMC PMC5343170

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Abstract

Cilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer. Chlamydomonas reinhardtii is an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of small changes in ciliary length by minimizing variability in the population. We find that this method alters the key relationship between cell size and the amount of protein accumulated for flagellar growth. This provides a rapid alternative to traditional methods of cell synchronization for uncovering novel regulators of cilia.

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Figures

FIG 1 F-L synchronization narrows the flagellar length distribution compared to other synchronization methods. (a) Distribution of steady-state flagellar lengths after the use of different synchronization methods. Nonsynchronous cells were used as a control, and steady-state flagellar lengths were measured after the use of each synchronization method as described in the text (n = 100/each). The F test was performed for comparing variance levels, and two-tailed P values were determined. Asterisks indicate significant differences (****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01). Standard deviations of each distribution are shown below the individually plotted values. (b) Wild-type flagellar length distribution at various time intervals during the regeneration after amputation. Predeflagellation nonsynchronous cells (pre) are shown in red. Regeneration was carried out for the indicated times after deflagellation by pH shock (green). Lighter green and darker green indicate before and after the time of F-L synchronization, respectively (n = 50/each). The F test was performed for comparing variance levels (control = predeflagellation nonsynchronous cells). Bonferroni corrected αaltered = 0.007. The asterisks indicate significant difference below αaltered (**, P = 0.004). Standard deviations are expressed as bar graphs in the bottom half of the panels. The filled standard deviation bar represents the condition with the smallest variance.
FIG 1 F-L synchronization narrows the flagellar length distribution compared to other synchronization methods. (a) Distribution of steady-state flagellar lengths after the use of different synchronization methods. Nonsynchronous cells were used as a control, and steady-state flagellar lengths were measured after the use of each synchronization method as described in the text (n = 100/each). The F test was performed for comparing variance levels, and two-tailed P values were determined. Asterisks indicate significant differences (****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01). Standard deviations of each distribution are shown below the individually plotted values. (b) Wild-type flagellar length distribution at various time intervals during the regeneration after amputation. Predeflagellation nonsynchronous cells (pre) are shown in red. Regeneration was carried out for the indicated times after deflagellation by pH shock (green). Lighter green and darker green indicate before and after the time of F-L synchronization, respectively (n = 50/each). The F test was performed for comparing variance levels (control = predeflagellation nonsynchronous cells). Bonferroni corrected αaltered = 0.007. The asterisks indicate significant difference below αaltered (**, P = 0.004). Standard deviations are expressed as bar graphs in the bottom half of the panels. The filled standard deviation bar represents the condition with the smallest variance.CC BY · PubMed Central
FIG 2 Effects of length-altering chemical treatments are more apparent after F-L synchronization. Flagella were measured from each cell after incubation with the appropriate concentration of different chemicals for 90 min. (a) 0.4 mM IBMX. (b) 10 µM LatB. (c) 10 mM NaPPi. (d) 25 mM LiCl. For each pair: red, nonsynchronized; blue, L-D synchronized; purple, M-N synchronized; green, F-L synchronized. The first and second distributions for each pair represent control and treated conditions, respectively. n = 100 cells (one flagellum per cell). Bars represent means and standard deviations (top half of each panel). In each case, percent change in mean flagellar length is shown below individual plotted values.
FIG 2 Effects of length-altering chemical treatments are more apparent after F-L synchronization. Flagella were measured from each cell after incubation with the appropriate concentration of different chemicals for 90 min. (a) 0.4 mM IBMX. (b) 10 µM LatB. (c) 10 mM NaPPi. (d) 25 mM LiCl. For each pair: red, nonsynchronized; blue, L-D synchronized; purple, M-N synchronized; green, F-L synchronized. The first and second distributions for each pair represent control and treated conditions, respectively. n = 100 cells (one flagellum per cell). Bars represent means and standard deviations (top half of each panel). In each case, percent change in mean flagellar length is shown below individual plotted values.CC BY · PubMed Central
FIG 3 F-L synchronization times differ in mutants of different lengths. For each mutant, distributions of flagellar length during regeneration are shown. (a) Long-flagella mutant lf4-7. (b) Short-flagella mutant shf1-253. (c) Mild phenotype long-flagella mutant cnk2-1. “Pre” represents the steady-state length of the mutant predeflagellation. Bars represent means and standard deviations (top half of each panel). Standard deviations are represented by bar graphs in the lower half of each panel, and the filled bar corresponds to the synchronization time for each mutant on the basis of minimal standard deviation. n = 50/each. The F test was performed for comparing variance levels with predeflagellation nonsynchronous controls. (a and c) Bonferroni corrected αaltered = 0.007; asterisks indicate significant differences below αaltered (****, P = 0.00005; ***, P = 0.0002; **, P = 0.004). (b) Bonferroni corrected αaltered = 0.0055; asterisks indicate significant differences below αaltered (**, P = 0.0008).
FIG 3 F-L synchronization times differ in mutants of different lengths. For each mutant, distributions of flagellar length during regeneration are shown. (a) Long-flagella mutant lf4-7. (b) Short-flagella mutant shf1-253. (c) Mild phenotype long-flagella mutant cnk2-1. “Pre” represents the steady-state length of the mutant predeflagellation. Bars represent means and standard deviations (top half of each panel). Standard deviations are represented by bar graphs in the lower half of each panel, and the filled bar corresponds to the synchronization time for each mutant on the basis of minimal standard deviation. n = 50/each. The F test was performed for comparing variance levels with predeflagellation nonsynchronous controls. (a and c) Bonferroni corrected αaltered = 0.007; asterisks indicate significant differences below αaltered (****, P = 0.00005; ***, P = 0.0002; **, P = 0.004). (b) Bonferroni corrected αaltered = 0.0055; asterisks indicate significant differences below αaltered (**, P = 0.0008).CC BY · PubMed Central
FIG 4 Distributions of flagellar length in long-flagella mutants before and after F-L synchronization. (a) lf3-2 mutant. (b) lf2-5 mutant. The upper parts of panels a and b represent the distributions of flagellar lengths in nonsynchronous cells. Cells were then deflagellated by acidic shock and regenerated for 4 h for F-L synchronization. The lower parts of panels a and b correspond to the distributions of flagellar lengths after F-L synchronization. n = 50/each (zeros excluded). The mean flagellar length of each mutant is marked by an asterisk. The green asterisks indicate the mean flagellar length observed in wild-type controls. (c) The mean flagellar lengths of wild-type and mutant cells were compared using Bonferroni’s post hoc test, and the results are represented. The first and second bars within each pair in panel c represent the mean flagellar lengths of nonsynchronized and synchronized cells, respectively. The Mann-Whitney U test was performed for comparing two means in a pair, and Bonferroni’s post hoc test was performed for multiple comparisons. Asterisks indicate significant differences (****, P ≤ 0.0001; **, P ≤ 0.01; ns, P > 0.05).
FIG 4 Distributions of flagellar length in long-flagella mutants before and after F-L synchronization. (a) lf3-2 mutant. (b) lf2-5 mutant. The upper parts of panels a and b represent the distributions of flagellar lengths in nonsynchronous cells. Cells were then deflagellated by acidic shock and regenerated for 4 h for F-L synchronization. The lower parts of panels a and b correspond to the distributions of flagellar lengths after F-L synchronization. n = 50/each (zeros excluded). The mean flagellar length of each mutant is marked by an asterisk. The green asterisks indicate the mean flagellar length observed in wild-type controls. (c) The mean flagellar lengths of wild-type and mutant cells were compared using Bonferroni’s post hoc test, and the results are represented. The first and second bars within each pair in panel c represent the mean flagellar lengths of nonsynchronized and synchronized cells, respectively. The Mann-Whitney U test was performed for comparing two means in a pair, and Bonferroni’s post hoc test was performed for multiple comparisons. Asterisks indicate significant differences (****, P ≤ 0.0001; **, P ≤ 0.01; ns, P > 0.05).CC BY · PubMed Central
FIG 5 Relationship between flagellar length and precursor pool distribution. (a) Flagellar precursor pool distributions (as determined by regeneration in cycloheximide after the use of different synchronization methods). Red, nonsynchronous; blue, L-D synchronized; purple, M-N synchronized; light green, F-L synchronized for 2 h; medium green, F-L synchronized for 3 h; dark green, F-L synchronized for 5 h. Results of F-L synchronization performed for 3 h show minimal variability among F-L synchronized cells. n = 100/each. The F test was performed for comparing variance levels (control = nonsynchronous cells). Asterisks indicate significant differences (****, P ≤ 0.0001; ***, P ≤ 0.001). Bars represent means and standard deviations. (b) Postsynchronization standard deviations (top panel) and corresponding precursor pool standard deviations (bottom panel) of each distribution are represented by bars with matching colors. Cell cycle synchronized cells (L-D synchronized) show the smallest variability in precursor pool distribution (blue, bottom panel). Of the F-L synchronized cells (green), 3 h of synchronization showed the smallest variability in precursor pool distribution (filled bar, bottom panel).
FIG 5 Relationship between flagellar length and precursor pool distribution. (a) Flagellar precursor pool distributions (as determined by regeneration in cycloheximide after the use of different synchronization methods). Red, nonsynchronous; blue, L-D synchronized; purple, M-N synchronized; light green, F-L synchronized for 2 h; medium green, F-L synchronized for 3 h; dark green, F-L synchronized for 5 h. Results of F-L synchronization performed for 3 h show minimal variability among F-L synchronized cells. n = 100/each. The F test was performed for comparing variance levels (control = nonsynchronous cells). Asterisks indicate significant differences (****, P ≤ 0.0001; ***, P ≤ 0.001). Bars represent means and standard deviations. (b) Postsynchronization standard deviations (top panel) and corresponding precursor pool standard deviations (bottom panel) of each distribution are represented by bars with matching colors. Cell cycle synchronized cells (L-D synchronized) show the smallest variability in precursor pool distribution (blue, bottom panel). Of the F-L synchronized cells (green), 3 h of synchronization showed the smallest variability in precursor pool distribution (filled bar, bottom panel).CC BY · PubMed Central
FIG 6 F-L synchronization changes the relationship between the precursor pool size and cell size. Flagellar length in cycloheximide, which corresponds to the precursor pool, is plotted along the y axis. Matching cell volume is plotted along the x axis. Values are plotted for (a) nonsynchronized (red), (b) L-D synchronized (blue), (c) M-N synchronized (purple), and (d) F-L synchronized (green) populations. n = 100. Nonsynchronized cells showed a significant correlation between cell size and precursor pool size (r = 0.73). L-D and M-N synchronized cells had narrow cell volume and precursor pool ranges. F-L synchronized cells showed a narrow precursor pool range without limiting cell volume. (e) Relation between nonsynchronous and F-L synchronized cells. The straight lines represent the best-fitted lines through the data point and were drawn after linear regression (red, nonsynchronous cells; green, F-L synchronized cells). SE = standard error of the slopes. The slope became smaller in the case of F-L synchronized cells, a result which is significantly different from that seen with nonsynchronous cells (P = 0.0012).
FIG 6 F-L synchronization changes the relationship between the precursor pool size and cell size. Flagellar length in cycloheximide, which corresponds to the precursor pool, is plotted along the y axis. Matching cell volume is plotted along the x axis. Values are plotted for (a) nonsynchronized (red), (b) L-D synchronized (blue), (c) M-N synchronized (purple), and (d) F-L synchronized (green) populations. n = 100. Nonsynchronized cells showed a significant correlation between cell size and precursor pool size (r = 0.73). L-D and M-N synchronized cells had narrow cell volume and precursor pool ranges. F-L synchronized cells showed a narrow precursor pool range without limiting cell volume. (e) Relation between nonsynchronous and F-L synchronized cells. The straight lines represent the best-fitted lines through the data point and were drawn after linear regression (red, nonsynchronous cells; green, F-L synchronized cells). SE = standard error of the slopes. The slope became smaller in the case of F-L synchronized cells, a result which is significantly different from that seen with nonsynchronous cells (P = 0.0012).CC BY · PubMed Central
FIG 7 Proposed model of flagellar length synchronization. Thick light green and medium green lines represent slow-growing and fast-growing flagella, respectively. The red dotted line denotes the 3 h postdeflagellation time at which flagellar length variability was minimized. Regeneration is initially the dominant factor. With time, other listed factors may contribute, resulting in higher levels of flagellar length variability.
FIG 7 Proposed model of flagellar length synchronization. Thick light green and medium green lines represent slow-growing and fast-growing flagella, respectively. The red dotted line denotes the 3 h postdeflagellation time at which flagellar length variability was minimized. Regeneration is initially the dominant factor. With time, other listed factors may contribute, resulting in higher levels of flagellar length variability.CC BY · PubMed Central

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Metadata

Authors
Dutta, S., & Avasthi, P.
Affiliations
University of Kansas Medical Center (US)
Corresponding author
Avasthi, Prachee
ORCID
Venue
mSphere
Publisher
American Society for Microbiology
Published
2017-04-26
Volume
2
Issue
2
ISSN
2379-5042 (ISSN-L)
DOI
10.1128/msphere.00003-17
PMC
PMC5343170
OpenAlex
W2951402366
Cited by
3
References
73
Citations by year
2022: 1 · 2019: 1 · 2018: 1
FWCI
0.36
Type
journal article
Language
en
Open access
Licence
Locations
  • mSphere (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • KU ScholarWorks (University of Kansas) (submittedVersion, cc-by)
  • KU ScholarWorks (The University of Kansas) (submittedVersion, cc-by)
Funders
HHS | NIH | National Institute of General Medical Sciences (P20 GM104936, P20 GM103418)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic and Kidney Cyst Diseases
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Cystic Fibrosis Research Advances
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
Keywords
flagellar synchronization, cell cycle synchronization, cilia, flagella, Chlamydomonas reinhardtii, ciliary length, cell size, ciliary assembly, ciliary motility

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