Kinesin-13 regulates the quantity and quality of tubulin inside cilia.
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.
Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA 30602;
- Gene Knockout Techniques
- Morphogenesis/drug effects/genetics
- Protein Processing, Post-Translational
- Tetrahymena thermophila/genetics/physiology
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Figure 1: Tetrahymena expresses three homologues of kinesin-13, each with a distinct pattern of localization. (A) A comparison of predicted domain organizations of the well-studied human kinesin-13 (MCAK) and homologues of T. thermophila. CT, C-terminal domain; NT, N-terminal domain; NLS, nuclear localization signal (predicted using cNLS mapper). (B, C) Confocal immunofluorescence images of cells in which either Kin13Ap or Kin13Cp is tagged with a C-terminal GFP expressed in the native locus. The cells show a direct kinesin-13–GFP signal (green) and nuclear DNA stained with propidium iodide (red). (B) Kin13Ap localizes to the nuclei when they divide. The cells on the left and right are in an advanced (left) or early (right) stage of cell division, respectively, whereas the middle bottom cell is in interphase. In the cell on the left, the macronucleus undergoes amitosis, whereas the micronucleus is in the telophase of mitosis. The insets show a higher magnification of the micronucleus (white circle) and the macronucleus (red box) in the boxed area. In the cell on the right, the micronucleus is in early anaphase. The white circles and oval in B′ mark the micronuclei in mitosis. The two dividing cells have weak green dots in the cell cortex, which are likely the somatic and oral basal bodies. Bar, 50 μm. (C) Kin13Cp associates with cortical microtubules and cilia. The images show a dividing cell that is surrounded by three interphase cells. All cells show weak dots of cortical labeling consistent with basal bodies. Both dividing and two of the three nondividing cells show a strong CVP signal (red box). The dividing cell shows a very strong signal in the growing cilia of oral apparatuses (the anterior one is magnified in the white box) in both the anterior and posterior daughter cells. Bar, 50 μm. (D) TIRF image of a cell with a natively tagged Kin13Bp-GFP that is detected near the basal bodies and cortical microtubules (transverse and longitudinal). The structures are identified based on their shape and relative locations. The schematic organization of the cell cortex microtubules viewed from the ventral side is shown in the right bottom corner (modified from Sharma et al., 2007). Bar, 20 μm. bb, basal body; cvp, contractile vacuole pore; lm, longitudinal microtubule; mac, macronucleus; mic, micronucleus; noa, new oral apparatus; oa, oral apparatus; tm, transverse microtubule.
The genome of T. thermophila contains three genes encoding kinesin-13 homologues, KIN13A (TTHERM_00790940), KIN13B (TTHERM_00429870), and KIN13C (THERM_00648540) (Wickstead et al., 2010). Each of the predicted gene products encodes a protein with an organization typical of kinesin-13 (Figure 1A and Supplemental Figure S1), including a catalytic domain with KEC/KVD motifs in the loop 2 that are important for the microtubule- depolymerization activity (Ogawa et al., 2004; Shipley et al., 2004). Kin13Bp and Kin13Cp (but not Kin13Ap) have a positively charged neck, an ∼70-residue extension N-terminal to the catalytic domain, that contributes to the recruitment of kinesin-13 to microtubules (Moores et al., 2006; Cooper et al., 2010; Wang et al., 2012).