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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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Double-knockout cells have altered patterns and levels of PTMs on tubulin. (A) Western blots of cilia, M+M, and axoneme fractions of wild-type and 13BC-KO cells. The lanes were loaded using the amount of material obtained from the same number of processed cells. Arrows represents a size marker of 50 kDa. The antibodies recognize either α-tubulin (12G10) or various modified tubulin isoforms. The Coomassie blue–stained gels loaded with the same samples are shown in Supplemental Figure S5. (B, C) Charts documenting the levels of α-tubulin (B) and posttranslational modifications on tubulin (C) in the 13BC-KO cilia relative to wild-type levels. The data represent an average of band signals on three blots (including blots shown in A). In B, the signal values of tubulin were corrected to account for slightly unequal loading on each gel by measuring the signal intensity of five non-tubulin bands on the image of a corresponding Coomassie blue–stained gel. The level of wild type equals 1. In C, the levels of modified tubulins are normalized to the amount of α-tubulin, and the normalized signal is expressed as a ratio to the normalized wild-type signal that equals 1.
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Figure 7: Double-knockout cells have altered patterns and levels of PTMs on tubulin. (A) Western blots of cilia, M+M, and axoneme fractions of wild-type and 13BC-KO cells. The lanes were loaded using the amount of material obtained from the same number of processed cells. Arrows represents a size marker of 50 kDa. The antibodies recognize either α-tubulin (12G10) or various modified tubulin isoforms. The Coomassie blue–stained gels loaded with the same samples are shown in Supplemental Figure S5. (B, C) Charts documenting the levels of α-tubulin (B) and posttranslational modifications on tubulin (C) in the 13BC-KO cilia relative to wild-type levels. The data represent an average of band signals on three blots (including blots shown in A). In B, the signal values of tubulin were corrected to account for slightly unequal loading on each gel by measuring the signal intensity of five non-tubulin bands on the image of a corresponding Coomassie blue–stained gel. The level of wild type equals 1. In C, the levels of modified tubulins are normalized to the amount of α-tubulin, and the normalized signal is expressed as a ratio to the normalized wild-type signal that equals 1.

Mentions: We used Western blotting to quantify the levels of modified and total tubulin in the wild-type and 13BC-KO cilia. The 13BC-KO and wild-type cilia showed a nearly normal level of total α-tubulin in the axoneme but had reduced levels of soluble (membrane plus matrix [M+M]) α-tubulin (Figure 7, A and B). For quantification of the PTM levels, we averaged data obtained from three independent preparations of cilia and normalized the signals to the signal of total tubulin. Although there is some variability in the patterns of PTM signals between the experiments, on average, 13BC-KO cilia had increased levels of tubulin PTMs, especially monoglycylation. The average levels of PTMs on axonemes were also mildly elevated except for polyglycylation (Figure 7C). Surprisingly, the tubulin in the soluble fraction of 13BC-KO cilia, while less abundant, contained 2- to 2.5-fold increased levels of K-40 acetylated, polyglycylated, and polyglutamylated isoforms (Figures 7, A, right, and C). Thus kinesin-13 affects the levels of tubulin PTMs, particularly in the soluble compartment of cilia.


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)

Double-knockout cells have altered patterns and levels of PTMs on tubulin. (A) Western blots of cilia, M+M, and axoneme fractions of wild-type and 13BC-KO cells. The lanes were loaded using the amount of material obtained from the same number of processed cells. Arrows represents a size marker of 50 kDa. The antibodies recognize either α-tubulin (12G10) or various modified tubulin isoforms. The Coomassie blue–stained gels loaded with the same samples are shown in Supplemental Figure S5. (B, C) Charts documenting the levels of α-tubulin (B) and posttranslational modifications on tubulin (C) in the 13BC-KO cilia relative to wild-type levels. The data represent an average of band signals on three blots (including blots shown in A). In B, the signal values of tubulin were corrected to account for slightly unequal loading on each gel by measuring the signal intensity of five non-tubulin bands on the image of a corresponding Coomassie blue–stained gel. The level of wild type equals 1. In C, the levels of modified tubulins are normalized to the amount of α-tubulin, and the normalized signal is expressed as a ratio to the normalized wild-type signal that equals 1.
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Related In: Results  -  Collection

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Figure 7: Double-knockout cells have altered patterns and levels of PTMs on tubulin. (A) Western blots of cilia, M+M, and axoneme fractions of wild-type and 13BC-KO cells. The lanes were loaded using the amount of material obtained from the same number of processed cells. Arrows represents a size marker of 50 kDa. The antibodies recognize either α-tubulin (12G10) or various modified tubulin isoforms. The Coomassie blue–stained gels loaded with the same samples are shown in Supplemental Figure S5. (B, C) Charts documenting the levels of α-tubulin (B) and posttranslational modifications on tubulin (C) in the 13BC-KO cilia relative to wild-type levels. The data represent an average of band signals on three blots (including blots shown in A). In B, the signal values of tubulin were corrected to account for slightly unequal loading on each gel by measuring the signal intensity of five non-tubulin bands on the image of a corresponding Coomassie blue–stained gel. The level of wild type equals 1. In C, the levels of modified tubulins are normalized to the amount of α-tubulin, and the normalized signal is expressed as a ratio to the normalized wild-type signal that equals 1.
Mentions: We used Western blotting to quantify the levels of modified and total tubulin in the wild-type and 13BC-KO cilia. The 13BC-KO and wild-type cilia showed a nearly normal level of total α-tubulin in the axoneme but had reduced levels of soluble (membrane plus matrix [M+M]) α-tubulin (Figure 7, A and B). For quantification of the PTM levels, we averaged data obtained from three independent preparations of cilia and normalized the signals to the signal of total tubulin. Although there is some variability in the patterns of PTM signals between the experiments, on average, 13BC-KO cilia had increased levels of tubulin PTMs, especially monoglycylation. The average levels of PTMs on axonemes were also mildly elevated except for polyglycylation (Figure 7C). Surprisingly, the tubulin in the soluble fraction of 13BC-KO cilia, while less abundant, contained 2- to 2.5-fold increased levels of K-40 acetylated, polyglycylated, and polyglutamylated isoforms (Figures 7, A, right, and C). Thus kinesin-13 affects the levels of tubulin PTMs, particularly in the soluble compartment of cilia.

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