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Kinesin-13 regulates the quantity and quality of tubulin inside cilia.

Vasudevan KK, Jiang YY, Lechtreck KF, Kushida Y, Alford LM, Sale WS, Hennessey T, Gaertig J - Mol. Biol. Cell (2014)

Bottom Line: Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase.The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding.Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA 30602;

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The 13BC-KO cells have overgrown cortical microtubules and shorter axonemes. (A) Confocal immunofluorescence images of mixed wild-type (labeled by loading the food vacuoles with India ink) and 13BC-KO cells labeled with anti–α-tubulin monoclonal antibody (mAb; 12G10), anti–acetyl K40 mAb (6-11B-1), or anti-polyglutamylation antibodies (polyE). Insets represent a threefold magnification of the selected areas. Bar, 20 μm. Arrows point at the central rings of CVPs. Arrowheads identify the ends of the CVP rootlet microtubules that are overgrown in the 13BC-KO cells. (B) Average length of the transverse microtubule bundles based on immunofluorescence using either an anti–α-tubulin 12G10 or anti–acetyl-K40 α-tubulin 6-11B-1 antibody. (C) Corrected total fluorescence signal of cilia, basal body, and cell body areas of wild-type and 13BC-KO cells labeled with the anti-polyglutamylation antibodies (polyE). The intensity of twenty-five 200-pixel circles (in five cells) was measured for each strain. Bars represent SE. *p = 0.00012 for basal body, and **p < 0.0001 for cell body. (D) Confocal immunofluorescence images of growing wild-type and 13BC-KO cells labeled by immunofluorescence with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG), which strongly label cilia. Bar, 20 μm. (E) Average length of cilia of growing or starved cells (20 cells for each strain). The values are 5.05 ± 0.04 μm for WT and 4.47 ± 0.04 μm for 13BC-KO growing and 4.92 ± 0.06 μm for WT and 4.54 ± 0.04 μm for 13BC-KO starved for 24 h. *p = 0.0028 for growing cells, and **p < 0.0001 for starved cells. (F) Average length of cilia in regenerating cells (taken from growing conditions and deciliated with a pH shock) in the presence or absence of 20 μg/ml cycloheximide (Chx; 10 cells for each sample). For each time point, there is a significant difference between the lengths of the wild-type and 13BC-KO cilia (p < 0.0001). tm, transverse microtubules.
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Figure 4: The 13BC-KO cells have overgrown cortical microtubules and shorter axonemes. (A) Confocal immunofluorescence images of mixed wild-type (labeled by loading the food vacuoles with India ink) and 13BC-KO cells labeled with anti–α-tubulin monoclonal antibody (mAb; 12G10), anti–acetyl K40 mAb (6-11B-1), or anti-polyglutamylation antibodies (polyE). Insets represent a threefold magnification of the selected areas. Bar, 20 μm. Arrows point at the central rings of CVPs. Arrowheads identify the ends of the CVP rootlet microtubules that are overgrown in the 13BC-KO cells. (B) Average length of the transverse microtubule bundles based on immunofluorescence using either an anti–α-tubulin 12G10 or anti–acetyl-K40 α-tubulin 6-11B-1 antibody. (C) Corrected total fluorescence signal of cilia, basal body, and cell body areas of wild-type and 13BC-KO cells labeled with the anti-polyglutamylation antibodies (polyE). The intensity of twenty-five 200-pixel circles (in five cells) was measured for each strain. Bars represent SE. *p = 0.00012 for basal body, and **p < 0.0001 for cell body. (D) Confocal immunofluorescence images of growing wild-type and 13BC-KO cells labeled by immunofluorescence with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG), which strongly label cilia. Bar, 20 μm. (E) Average length of cilia of growing or starved cells (20 cells for each strain). The values are 5.05 ± 0.04 μm for WT and 4.47 ± 0.04 μm for 13BC-KO growing and 4.92 ± 0.06 μm for WT and 4.54 ± 0.04 μm for 13BC-KO starved for 24 h. *p = 0.0028 for growing cells, and **p < 0.0001 for starved cells. (F) Average length of cilia in regenerating cells (taken from growing conditions and deciliated with a pH shock) in the presence or absence of 20 μg/ml cycloheximide (Chx; 10 cells for each sample). For each time point, there is a significant difference between the lengths of the wild-type and 13BC-KO cilia (p < 0.0001). tm, transverse microtubules.

Mentions: When the wild-type and 13BC-KO cells were analyzed side by side by immunofluorescence using anti–α-tubulin antibodies, the 13BC-KO cells showed an increased signal of tubulin in the cell bodies (Figure 4A, left). The organization of cortical microtubules seemed normal, except that some of the microtubule types were excessively long. Whereas in the wild-type cells, the CVP rootlets are limited to the vicinity of the CVP rings within two ciliary rows, in the 13BC-KO cells, the same microtubules were exceptionally long, covering almost the entire posterior region of the cell (Figure 4A, left and middle). The mutant transverse microtubule bundles were slightly but significantly longer (Figure 4, A, middle, and B). We conclude that Kin13Bp and Kin13Cp function in the shortening of subtypes of cortical microtubules. In the cell body, the consequences of the absence of kinesin-13 can be explained by its canonical activity as a microtubule-end depolymerizer.


Kinesin-13 regulates the quantity and quality of tubulin inside cilia.

Vasudevan KK, Jiang YY, Lechtreck KF, Kushida Y, Alford LM, Sale WS, Hennessey T, Gaertig J - Mol. Biol. Cell (2014)

The 13BC-KO cells have overgrown cortical microtubules and shorter axonemes. (A) Confocal immunofluorescence images of mixed wild-type (labeled by loading the food vacuoles with India ink) and 13BC-KO cells labeled with anti–α-tubulin monoclonal antibody (mAb; 12G10), anti–acetyl K40 mAb (6-11B-1), or anti-polyglutamylation antibodies (polyE). Insets represent a threefold magnification of the selected areas. Bar, 20 μm. Arrows point at the central rings of CVPs. Arrowheads identify the ends of the CVP rootlet microtubules that are overgrown in the 13BC-KO cells. (B) Average length of the transverse microtubule bundles based on immunofluorescence using either an anti–α-tubulin 12G10 or anti–acetyl-K40 α-tubulin 6-11B-1 antibody. (C) Corrected total fluorescence signal of cilia, basal body, and cell body areas of wild-type and 13BC-KO cells labeled with the anti-polyglutamylation antibodies (polyE). The intensity of twenty-five 200-pixel circles (in five cells) was measured for each strain. Bars represent SE. *p = 0.00012 for basal body, and **p < 0.0001 for cell body. (D) Confocal immunofluorescence images of growing wild-type and 13BC-KO cells labeled by immunofluorescence with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG), which strongly label cilia. Bar, 20 μm. (E) Average length of cilia of growing or starved cells (20 cells for each strain). The values are 5.05 ± 0.04 μm for WT and 4.47 ± 0.04 μm for 13BC-KO growing and 4.92 ± 0.06 μm for WT and 4.54 ± 0.04 μm for 13BC-KO starved for 24 h. *p = 0.0028 for growing cells, and **p < 0.0001 for starved cells. (F) Average length of cilia in regenerating cells (taken from growing conditions and deciliated with a pH shock) in the presence or absence of 20 μg/ml cycloheximide (Chx; 10 cells for each sample). For each time point, there is a significant difference between the lengths of the wild-type and 13BC-KO cilia (p < 0.0001). tm, transverse microtubules.
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Figure 4: The 13BC-KO cells have overgrown cortical microtubules and shorter axonemes. (A) Confocal immunofluorescence images of mixed wild-type (labeled by loading the food vacuoles with India ink) and 13BC-KO cells labeled with anti–α-tubulin monoclonal antibody (mAb; 12G10), anti–acetyl K40 mAb (6-11B-1), or anti-polyglutamylation antibodies (polyE). Insets represent a threefold magnification of the selected areas. Bar, 20 μm. Arrows point at the central rings of CVPs. Arrowheads identify the ends of the CVP rootlet microtubules that are overgrown in the 13BC-KO cells. (B) Average length of the transverse microtubule bundles based on immunofluorescence using either an anti–α-tubulin 12G10 or anti–acetyl-K40 α-tubulin 6-11B-1 antibody. (C) Corrected total fluorescence signal of cilia, basal body, and cell body areas of wild-type and 13BC-KO cells labeled with the anti-polyglutamylation antibodies (polyE). The intensity of twenty-five 200-pixel circles (in five cells) was measured for each strain. Bars represent SE. *p = 0.00012 for basal body, and **p < 0.0001 for cell body. (D) Confocal immunofluorescence images of growing wild-type and 13BC-KO cells labeled by immunofluorescence with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG), which strongly label cilia. Bar, 20 μm. (E) Average length of cilia of growing or starved cells (20 cells for each strain). The values are 5.05 ± 0.04 μm for WT and 4.47 ± 0.04 μm for 13BC-KO growing and 4.92 ± 0.06 μm for WT and 4.54 ± 0.04 μm for 13BC-KO starved for 24 h. *p = 0.0028 for growing cells, and **p < 0.0001 for starved cells. (F) Average length of cilia in regenerating cells (taken from growing conditions and deciliated with a pH shock) in the presence or absence of 20 μg/ml cycloheximide (Chx; 10 cells for each sample). For each time point, there is a significant difference between the lengths of the wild-type and 13BC-KO cilia (p < 0.0001). tm, transverse microtubules.
Mentions: When the wild-type and 13BC-KO cells were analyzed side by side by immunofluorescence using anti–α-tubulin antibodies, the 13BC-KO cells showed an increased signal of tubulin in the cell bodies (Figure 4A, left). The organization of cortical microtubules seemed normal, except that some of the microtubule types were excessively long. Whereas in the wild-type cells, the CVP rootlets are limited to the vicinity of the CVP rings within two ciliary rows, in the 13BC-KO cells, the same microtubules were exceptionally long, covering almost the entire posterior region of the cell (Figure 4A, left and middle). The mutant transverse microtubule bundles were slightly but significantly longer (Figure 4, A, middle, and B). We conclude that Kin13Bp and Kin13Cp function in the shortening of subtypes of cortical microtubules. In the cell body, the consequences of the absence of kinesin-13 can be explained by its canonical activity as a microtubule-end depolymerizer.

Bottom Line: Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase.The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding.Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA 30602;

Show MeSH
Related in: MedlinePlus