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. 2018 Aug 6;217(8):2867-2876.
doi: 10.1083/jcb.201801039. Epub 2018 Jun 14.

Interaction of Heterotrimeric kinesin-II With IFT-B-connecting Tetramer Is Crucial for Ciliogenesis

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Free PMC article

Interaction of Heterotrimeric kinesin-II With IFT-B-connecting Tetramer Is Crucial for Ciliogenesis

Teruki Funabashi et al. J Cell Biol. .
Free PMC article

Abstract

Intraflagellar transport (IFT) is crucial for the assembly and maintenance of cilia and is mediated by IFT particles containing IFT-A and IFT-B complexes. IFT-B powered by heterotrimeric kinesin-II and IFT-A powered by the dynein-2 complex are responsible for anterograde and retrograde protein trafficking, respectively. However, little is known about the molecular basis of the trafficking of these IFT particles regulated by kinesin and dynein motors. Using the visible immunoprecipitation assay, we identified in this study a three-to-four protein interaction involving the kinesin-II trimer KIF3A-KIF3B-KAP3 and the IFT-B-connecting tetramer IFT38-IFT52-IFT57-IFT88; among the kinesin-II subunits, KIF3B contributed mainly to IFT-B binding. Furthermore, we showed that the ciliogenesis defect of KIF3B-knockout cells can be rescued by the exogenous expression of wild-type KIF3B but not by that of its mutant compromised with respect to IFT-B binding. Thus, interaction of heterotrimeric kinesin-II with the IFT-B-connecting tetramer is crucial for ciliogenesis via the powering of IFT particles to move in the anterograde direction.

Figures

Figure 1.
Figure 1.
KIF3B is an essential component of the kinesin-II trimer. (A and B) Lysates prepared from HEK293T cells coexpressing the indicated combination of EGFP-fused and mChe-fused kinesin-II subunits were processed for the VIP assay using GST–anti-GFP Nb prebound to glutathione–Sepharose beads. EGFP (A) and mChe (B) signals on the precipitated beads were observed under a microscope. Bars, 100 µm. (C) Kinesin-II models predicted from the binary interaction data. (D–I) Control RPE1 cells (D and G) and KIF3B-KO cell lines 3B-2-1 (E and H) and 3B-2-4 (F and I) were serum-starved to induce ciliogenesis and triple immunostained for either IFT88 (D–F) or IFT140 (G–I), together with Ac-α-tubulin (D′–I′) and γ-tubulin (D″–I″). Bars: (main images) 10 µm; (insets) 5 µm.
Figure 2.
Figure 2.
Interaction of heterotrimeric kinesin-II with the IFT-B–connecting tetramer. (A) A model for the interaction of IFT-B with homodimeric KIF17 and heterotrimeric kinesin-II. (B) Identification of an interaction between heterotrimeric kinesin-II and the IFT-B–connecting tetramer. Lysates from cells coexpressing EGFP-fused kinesin-II subunits and all IFT-B subunits, subunits of all core, core 1, core 2, or peripheral subcomplexes, or connecting tetramer fused to mChe/tRFP were processed for the VIP assay. (C) Lack of an interaction between the IFT-A complex and heterotrimeric kinesin-II. Lysates from cells coexpressing EGFP-fused kinesin-II subunits and mChe-fused IFT-A subunits (all, core, or peripheral) were subjected to the VIP assay. (D and E) Subtractive VIP assay and immunoblotting (IB) analysis to determine subunits of the IFT-B–connecting tetramer required for its interaction with kinesin-II. Lysates from cells coexpressing EGFP-fused kinesin-II subunits and all but one (as indicated) subunits of the IFT-B tetramer fused to mChe/tRFP were processed for the VIP assay (D) or immunoblotting analysis (E) using the following antibodies: an anti-RFP antibody (top), which reacts with mChe; an anti-tRFP antibody (middle), which reacts with tRFP and cross-reacts with the mChe portion of mChe-IFT52 (indicated by an asterisk); or an anti-GFP antibody (bottom). Note that the bands for tRFP-fused IFT38 and IFT57 were overlapped with each other. As a negative control, a mixture of mChe-fused and tRFP-fused IFT56 was used in place of mChe/tRFP-fused tetrameric subunits (labeled as mChe+tRFP). Bars, 100 µm.
Figure 3.
Figure 3.
KIF3B mainly contributes to the interaction with the IFT-B–connecting tetramer. (A and B) Subtractive VIP assay and immunoblotting (IB) analysis to determine subunits of kinesin-II required for its interaction with the IFT-B tetramer. (C and D) Subtractive VIP assay and immunoblotting analysis to determine subunits of the IFT-B–connecting tetramer required for its interaction with KIF3B. Details are essentially the same as described in the legend for Fig. 2 (C and D), except that exposure time for red fluorescence was 0.2 s. Bars, 100 µm. In B and D, asterisks indicate the position of mChe-IFT52, with which anti–tRFP antibody cross-reacted.
Figure 4.
Figure 4.
Regions of the KIF3B protein responsible for its interactions with KIF3A, KAP3, and the IFT-B–connecting tetramer. (A) Schematic representation of the KIF3B constructs used and their domain organizations. On the right side, interactions of these constructs with KIF3A, KAP3, and the IFT-B–connecting tetramer are summarized. +, strong interaction; ±, weak interaction; −, no interaction; ND, not determined. (B–F and H) VIP assay (B, D, and F) and immunoblotting (IB) analysis (C, E, and H) to determine the regions of KIF3B responsible for its interaction with KIF3A (B and C), KAP3 (D and E), or the IFT-B–connecting tetramer (F and H). Bars, 100 µm. In H, an asterisk indicates the position of mChe-IFT52, with which anti–tRFP antibody cross-reacted. (G) Red fluorescence intensities in the acquired images shown in F were measured using ImageJ, and relative fluorescence intensities are expressed as bar graphs. Values are means ± SD of three independent experiments. P-values were determined by one-way ANOVA followed by Tukey’s post hoc analysis.
Figure 5.
Figure 5.
KIF3B–IFT-B interaction is essential for ciliogenesis. (A–F) The KIF3B-KO cell line 3B-2-1 stably expressing EGFP-fused KIF3B(WT) (A), KIF3B(1–704) (B), KIF3B(1–662) (C), KIF3B(1–625) (D), KIF3B(1–579) (E), or EGFP (F) were triple immunostained for Ac-α-tubulin (A–F), γ-tubulin (A′–F′), and GFP (A″–F″). Bars: (main images) 10 µm; (insets) 5 µm. (G) Ciliated cells of the KIF3B-KO cell lines (3B-2-1 and 3B-2-4) stably expressing EGFP-fused KIF3B(WT), KIF3B(1–704), or KIF3B(1–662) were counted, and percentages of ciliated cells are represented as bar graphs. Values are shown as means ± SEM of three independent experiments. In each set of experiments, 31–50 cells with EGFP signals were analyzed, and the total number of cells analyzed (n) for each sample is shown.

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