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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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Overexpressed GFP-Kin13Bp induces shortening and complete loss of axonemes. (A) Confocal immunofluorescence images of wild-type and MTT1-GFP-Kin13Bp expressing cells induced with 2.5 μg/ml CdCl2 for 0–24 h. The cells show a direct GFP signal (green) and an immunofluorescence signal (red) obtained with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG). In A, the inset shows a higher magnification of a shortening cilium. Bar, 20 μm. (B) Confocal immunofluorescence images of MTT1-GFP-KIN13B cells (red) induced with 2.5 μg/ml CdCl2 for 4 and 6 h and labeled with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG). Subsets of confocal sections were combined to show the cell cortex. Note that the overexpressing cells, while having lost most of their cilia, have rows with apparently normal basal bodies, transverse microtubules, and longitudinal microtubules (B′ and B′′′), as well as contractile vacuole pores (B′′ and B′′′′). The inset shows a higher magnification of basal bodies, transverse microtubules, and longitudinal microtubules (B′ and B′′′) and contractile vacuole pores (B′′ and B′′′′). Bar, 20 μm. (C) TEM images of wild-type and MTT1-GFP-Kin13Bp cells induced for 4 h with 2.5 μg/ml CdCl2. bb, basal body; cvp, contractile vacuole pore; lm, longitudinal microtubule; oa, oral apparatus; tm, transverse microtubule. Bar, 0.2 μm.
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Figure 6: Overexpressed GFP-Kin13Bp induces shortening and complete loss of axonemes. (A) Confocal immunofluorescence images of wild-type and MTT1-GFP-Kin13Bp expressing cells induced with 2.5 μg/ml CdCl2 for 0–24 h. The cells show a direct GFP signal (green) and an immunofluorescence signal (red) obtained with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG). In A, the inset shows a higher magnification of a shortening cilium. Bar, 20 μm. (B) Confocal immunofluorescence images of MTT1-GFP-KIN13B cells (red) induced with 2.5 μg/ml CdCl2 for 4 and 6 h and labeled with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG). Subsets of confocal sections were combined to show the cell cortex. Note that the overexpressing cells, while having lost most of their cilia, have rows with apparently normal basal bodies, transverse microtubules, and longitudinal microtubules (B′ and B′′′), as well as contractile vacuole pores (B′′ and B′′′′). The inset shows a higher magnification of basal bodies, transverse microtubules, and longitudinal microtubules (B′ and B′′′) and contractile vacuole pores (B′′ and B′′′′). Bar, 20 μm. (C) TEM images of wild-type and MTT1-GFP-Kin13Bp cells induced for 4 h with 2.5 μg/ml CdCl2. bb, basal body; cvp, contractile vacuole pore; lm, longitudinal microtubule; oa, oral apparatus; tm, transverse microtubule. Bar, 0.2 μm.

Mentions: Our results so far opened a possibility that Kin13Bp and Kin13Cp act inside cilia as an axoneme assembly–promoting factor. These observations seem inconsistent with the only known biochemical activity of kinesin-13, a microtubule-end depolymerase. Thus we tested whether Kin13Bp and Kin13Cp promote microtubule depolymerization in vivo by overproduction of either GFP-Kin13Bp or GFP-Kin13Cp under the cadmium-inducible MTT1 promoter. No transgenic strains were obtained for GFP-Kin13Cp, possibly because the transgene's uninduced expression was lethal. Over­expressed GFP-Kin13Bp localized to the basal bodies and formed large clusters in the middle or at the tips of cilia (Figure 6A, bottom). Overproduction of GFP-Kin13Bp led to shortening and complete loss of cilia within 24 h (Figure 6A). Some Kin13Bp-overproducing cells also showed accumulation of microtubules inside the macronucleus (Figure 6A, 2 and 4 h), suggesting that the soluble tubulin released from depolymerization of the extranuclear microtubules enters the macronucleus. Strikingly, the GFP-Kin13Bp–overproducing cells that lacked most axonemes had mostly intact cortical microtubules (Figure 6B), indicating a degree of selectivity for axonemes in the GFP-Kin13Bp–mediated depolymerization activity. TEM of GFP-Kin13Bp cells induced for 4 h showed that most of the basal bodies lacked an axoneme entirely (56.5%). An additional 15.2% of cilia had partly depolymerized distal portions with electron-dense aggregates that likely contained overproduced GFP-Kin13Bp (compare the inset in Figure 6, A, 4 h, and C, right), indicating that the depolymerization occurs mainly at the axoneme plus ends. The ciliated and nonciliated basal bodies did not show signs of depolymerization at the proximal side corresponding to the minus ends of microtubules (Figure 6C). Thus the overproduced Kin13Bp has a strong axoneme plus end–depolymerizing activity in vivo.


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)

Overexpressed GFP-Kin13Bp induces shortening and complete loss of axonemes. (A) Confocal immunofluorescence images of wild-type and MTT1-GFP-Kin13Bp expressing cells induced with 2.5 μg/ml CdCl2 for 0–24 h. The cells show a direct GFP signal (green) and an immunofluorescence signal (red) obtained with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG). In A, the inset shows a higher magnification of a shortening cilium. Bar, 20 μm. (B) Confocal immunofluorescence images of MTT1-GFP-KIN13B cells (red) induced with 2.5 μg/ml CdCl2 for 4 and 6 h and labeled with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG). Subsets of confocal sections were combined to show the cell cortex. Note that the overexpressing cells, while having lost most of their cilia, have rows with apparently normal basal bodies, transverse microtubules, and longitudinal microtubules (B′ and B′′′), as well as contractile vacuole pores (B′′ and B′′′′). The inset shows a higher magnification of basal bodies, transverse microtubules, and longitudinal microtubules (B′ and B′′′) and contractile vacuole pores (B′′ and B′′′′). Bar, 20 μm. (C) TEM images of wild-type and MTT1-GFP-Kin13Bp cells induced for 4 h with 2.5 μg/ml CdCl2. bb, basal body; cvp, contractile vacuole pore; lm, longitudinal microtubule; oa, oral apparatus; tm, transverse microtubule. Bar, 0.2 μm.
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Figure 6: Overexpressed GFP-Kin13Bp induces shortening and complete loss of axonemes. (A) Confocal immunofluorescence images of wild-type and MTT1-GFP-Kin13Bp expressing cells induced with 2.5 μg/ml CdCl2 for 0–24 h. The cells show a direct GFP signal (green) and an immunofluorescence signal (red) obtained with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG). In A, the inset shows a higher magnification of a shortening cilium. Bar, 20 μm. (B) Confocal immunofluorescence images of MTT1-GFP-KIN13B cells (red) induced with 2.5 μg/ml CdCl2 for 4 and 6 h and labeled with a mixture of anti–α-tubulin mAb (12G10) and anti-polyglycylation antibodies (polyG). Subsets of confocal sections were combined to show the cell cortex. Note that the overexpressing cells, while having lost most of their cilia, have rows with apparently normal basal bodies, transverse microtubules, and longitudinal microtubules (B′ and B′′′), as well as contractile vacuole pores (B′′ and B′′′′). The inset shows a higher magnification of basal bodies, transverse microtubules, and longitudinal microtubules (B′ and B′′′) and contractile vacuole pores (B′′ and B′′′′). Bar, 20 μm. (C) TEM images of wild-type and MTT1-GFP-Kin13Bp cells induced for 4 h with 2.5 μg/ml CdCl2. bb, basal body; cvp, contractile vacuole pore; lm, longitudinal microtubule; oa, oral apparatus; tm, transverse microtubule. Bar, 0.2 μm.
Mentions: Our results so far opened a possibility that Kin13Bp and Kin13Cp act inside cilia as an axoneme assembly–promoting factor. These observations seem inconsistent with the only known biochemical activity of kinesin-13, a microtubule-end depolymerase. Thus we tested whether Kin13Bp and Kin13Cp promote microtubule depolymerization in vivo by overproduction of either GFP-Kin13Bp or GFP-Kin13Cp under the cadmium-inducible MTT1 promoter. No transgenic strains were obtained for GFP-Kin13Cp, possibly because the transgene's uninduced expression was lethal. Over­expressed GFP-Kin13Bp localized to the basal bodies and formed large clusters in the middle or at the tips of cilia (Figure 6A, bottom). Overproduction of GFP-Kin13Bp led to shortening and complete loss of cilia within 24 h (Figure 6A). Some Kin13Bp-overproducing cells also showed accumulation of microtubules inside the macronucleus (Figure 6A, 2 and 4 h), suggesting that the soluble tubulin released from depolymerization of the extranuclear microtubules enters the macronucleus. Strikingly, the GFP-Kin13Bp–overproducing cells that lacked most axonemes had mostly intact cortical microtubules (Figure 6B), indicating a degree of selectivity for axonemes in the GFP-Kin13Bp–mediated depolymerization activity. TEM of GFP-Kin13Bp cells induced for 4 h showed that most of the basal bodies lacked an axoneme entirely (56.5%). An additional 15.2% of cilia had partly depolymerized distal portions with electron-dense aggregates that likely contained overproduced GFP-Kin13Bp (compare the inset in Figure 6, A, 4 h, and C, right), indicating that the depolymerization occurs mainly at the axoneme plus ends. The ciliated and nonciliated basal bodies did not show signs of depolymerization at the proximal side corresponding to the minus ends of microtubules (Figure 6C). Thus the overproduced Kin13Bp has a strong axoneme plus end–depolymerizing activity in vivo.

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