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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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GFP-Kin13Bp and GFP-Kin13Cp rescue the mutant phenotype of 13BC-KO cells and localize to assembling cilia. (A) TIRF images of 13BC-KO cells rescued with either GFP-Kin13Bp (A′) or GFP-Kin13C (A′′, A′′′). GFP-Kin13Bp and GFP-Kin13Cp are detected near the basal bodies, CVP, and in a subset of cilia, possibly enriched at their tips. (B) TIRF images of 13BC-KO cells rescued with GFP-Kin13Bp that were deciliated and allowed to regenerate their cilia for 5, 30, and 100 min. The background of faintly autofluorescent mitochondria is visible. Note the GFP-positive cilia on the cell surface at 30 min. (C) TIRF imaging of 13BC-KO cells rescued with GFP-Kin13Cp that were deciliated and allowed to regenerate cilia for 5, 60, and 90 min. Note short cilia at 60 min. Red boxes outline growing cilia. Arrowheads mark growing cilia where the pattern of fluorescence is consistent with the strong presence of kinesin-13 limited to the distal tips. bb, basal body; cvp, contractile vacuole pore. Bar, 20 μm.
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Figure 3: GFP-Kin13Bp and GFP-Kin13Cp rescue the mutant phenotype of 13BC-KO cells and localize to assembling cilia. (A) TIRF images of 13BC-KO cells rescued with either GFP-Kin13Bp (A′) or GFP-Kin13C (A′′, A′′′). GFP-Kin13Bp and GFP-Kin13Cp are detected near the basal bodies, CVP, and in a subset of cilia, possibly enriched at their tips. (B) TIRF images of 13BC-KO cells rescued with GFP-Kin13Bp that were deciliated and allowed to regenerate their cilia for 5, 30, and 100 min. The background of faintly autofluorescent mitochondria is visible. Note the GFP-positive cilia on the cell surface at 30 min. (C) TIRF imaging of 13BC-KO cells rescued with GFP-Kin13Cp that were deciliated and allowed to regenerate cilia for 5, 60, and 90 min. Note short cilia at 60 min. Red boxes outline growing cilia. Arrowheads mark growing cilia where the pattern of fluorescence is consistent with the strong presence of kinesin-13 limited to the distal tips. bb, basal body; cvp, contractile vacuole pore. Bar, 20 μm.

Mentions: The 13BC-KO cells died in the presence of a microtubule-stabilizing drug, paclitaxel, at the concentration in which wild-type cells multiply (see later discussion), indicating that the loss of kinesin-13 leads to abnormal microtubules. We took advantage of the paclitaxel sensitivity to confirm that the mutant phenotype of 13BC-KO cells is caused by the absence of Kin13Bp and Kin13Cp. Introduction of either GFP-Kin13Bp or GFP-Kin13Cp transgenes into the 13BC-KO cells resulted in paclitaxel-resistant rescue transformants that displayed nearly normal cell multiplication and motility rates, whereas no rescues were observed in a mock transformation experiment (107 cells). Based on TIRFM, the rescuing proteins had localization patterns that were similar but not identical to their natively tagged versions. Namely, the rescuing GFP-KIN13B was found at the CVP (Figure 3A and Supplemental Movies S6 and S7), whereas the natively tagged protein was not (Figure 1D). Likely, when both proteins are present, Kin13Cp has higher affinity for the CVP sites, but Kin13Bp can occupy the same sites in the absence of Kin13Cp. This can explain how Kin13Bp and Kin13Cp can be functionally redundant despite having nonoverlapping localizations when tagged in their native loci (Figure 1). Strikingly, in the live rescue cells observed in TIRFM, both GFP-Kin13Bp and GFP-Kin13Cp localized to a subset of cilia, which appeared short and likely were assembling (Figure 3A, arrowheads, and Supplemental Movies S6 and S7). To test further whether GFP-Kin13Bp and GFP-Kin13Cp localize preferentially to the assembling cilia, we observed the rescue cells as they regenerate cilia after deciliation. GFP-positive structures consistent with short assembling cilia were frequently seen on the surface of regenerating cells between 30 and 60 min after deciliation, when the majority of the regenerating cilia grow, and rarely at earlier and later time points (Figure 3, B and C, and Supplemental Movies S8 and S9). We conclude that Kin13Bp and Kin13Cp are partially functionally redundant and localize to the CVP, cortical microtubule bundles, basal bodies, and assembling 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)

GFP-Kin13Bp and GFP-Kin13Cp rescue the mutant phenotype of 13BC-KO cells and localize to assembling cilia. (A) TIRF images of 13BC-KO cells rescued with either GFP-Kin13Bp (A′) or GFP-Kin13C (A′′, A′′′). GFP-Kin13Bp and GFP-Kin13Cp are detected near the basal bodies, CVP, and in a subset of cilia, possibly enriched at their tips. (B) TIRF images of 13BC-KO cells rescued with GFP-Kin13Bp that were deciliated and allowed to regenerate their cilia for 5, 30, and 100 min. The background of faintly autofluorescent mitochondria is visible. Note the GFP-positive cilia on the cell surface at 30 min. (C) TIRF imaging of 13BC-KO cells rescued with GFP-Kin13Cp that were deciliated and allowed to regenerate cilia for 5, 60, and 90 min. Note short cilia at 60 min. Red boxes outline growing cilia. Arrowheads mark growing cilia where the pattern of fluorescence is consistent with the strong presence of kinesin-13 limited to the distal tips. bb, basal body; cvp, contractile vacuole pore. Bar, 20 μm.
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Figure 3: GFP-Kin13Bp and GFP-Kin13Cp rescue the mutant phenotype of 13BC-KO cells and localize to assembling cilia. (A) TIRF images of 13BC-KO cells rescued with either GFP-Kin13Bp (A′) or GFP-Kin13C (A′′, A′′′). GFP-Kin13Bp and GFP-Kin13Cp are detected near the basal bodies, CVP, and in a subset of cilia, possibly enriched at their tips. (B) TIRF images of 13BC-KO cells rescued with GFP-Kin13Bp that were deciliated and allowed to regenerate their cilia for 5, 30, and 100 min. The background of faintly autofluorescent mitochondria is visible. Note the GFP-positive cilia on the cell surface at 30 min. (C) TIRF imaging of 13BC-KO cells rescued with GFP-Kin13Cp that were deciliated and allowed to regenerate cilia for 5, 60, and 90 min. Note short cilia at 60 min. Red boxes outline growing cilia. Arrowheads mark growing cilia where the pattern of fluorescence is consistent with the strong presence of kinesin-13 limited to the distal tips. bb, basal body; cvp, contractile vacuole pore. Bar, 20 μm.
Mentions: The 13BC-KO cells died in the presence of a microtubule-stabilizing drug, paclitaxel, at the concentration in which wild-type cells multiply (see later discussion), indicating that the loss of kinesin-13 leads to abnormal microtubules. We took advantage of the paclitaxel sensitivity to confirm that the mutant phenotype of 13BC-KO cells is caused by the absence of Kin13Bp and Kin13Cp. Introduction of either GFP-Kin13Bp or GFP-Kin13Cp transgenes into the 13BC-KO cells resulted in paclitaxel-resistant rescue transformants that displayed nearly normal cell multiplication and motility rates, whereas no rescues were observed in a mock transformation experiment (107 cells). Based on TIRFM, the rescuing proteins had localization patterns that were similar but not identical to their natively tagged versions. Namely, the rescuing GFP-KIN13B was found at the CVP (Figure 3A and Supplemental Movies S6 and S7), whereas the natively tagged protein was not (Figure 1D). Likely, when both proteins are present, Kin13Cp has higher affinity for the CVP sites, but Kin13Bp can occupy the same sites in the absence of Kin13Cp. This can explain how Kin13Bp and Kin13Cp can be functionally redundant despite having nonoverlapping localizations when tagged in their native loci (Figure 1). Strikingly, in the live rescue cells observed in TIRFM, both GFP-Kin13Bp and GFP-Kin13Cp localized to a subset of cilia, which appeared short and likely were assembling (Figure 3A, arrowheads, and Supplemental Movies S6 and S7). To test further whether GFP-Kin13Bp and GFP-Kin13Cp localize preferentially to the assembling cilia, we observed the rescue cells as they regenerate cilia after deciliation. GFP-positive structures consistent with short assembling cilia were frequently seen on the surface of regenerating cells between 30 and 60 min after deciliation, when the majority of the regenerating cilia grow, and rarely at earlier and later time points (Figure 3, B and C, and Supplemental Movies S8 and S9). We conclude that Kin13Bp and Kin13Cp are partially functionally redundant and localize to the CVP, cortical microtubule bundles, basal bodies, and assembling 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