Assembly and mother centriole recruitment of IFT-B subcomplexes to form IFT-B holocomplex

Abstract

For the biogenesis and maintenance of cilia, bidirectional protein trafficking within cilia is crucial, and is conducted by intraflagellar transport (IFT) trains containing the IFT-A and IFT-B complexes that are powered by dynein-2 and kinesin-II motors. We have recently shown that before the assembly of anterograde IFT trains, the IFT-A, IFT-B, and dynein-2 complexes are independently recruited to the mother centriole/basal body. The IFT-B complex, which consists of 16 subunits, can be divided into the IFT-B1 and IFT-B2 subcomplexes, and IFT-B1 can be further divided into the IFT-B1a and IFT-B1b subgroups. Here we investigated how the IFT-B complex is assembled and recruited to the mother centriole for ciliogenesis. Analyses using cells with knockouts of individual IFT-B subunits, and analyses of proteins coimmunoprecipitated with EGFP-fused IFT-B2, IFT-B1b, and IFT-B1a subunits expressed in these knockout cells demonstrated the following: (i) although IFT-B2 is dispensable for the linkage between IFT-B1b and IFT-B1a, it is essential for their localization to the mother centriole; (ii) IFT-B1b is essential both for bridging IFT-B2 and IFT-B1a, and for their localization to the mother centriole; (iii) IFT-B1a is not required for the linkage between IFT-B2 and IFT-B1b nor for their localization to the mother centriole; and (iv) all IFT-B components (IFT-B2, IFT-B1b, and IFT-B1a) are essential for ciliogenesis. Thus, although ciliogenesis is not a prerequisite for the recruitment of the IFT-B complex to the mother centriole, the linkage between IFT-B2 and IFT-B1b is crucial for the mother centriole localization of the IFT-B complex for ciliogenesis.

Key words: cilia, ciliogenesis, distal appendages, IFT-B complex, mother centriole

Graphical Abstract

Introduction

Primary cilia are antenna-like organelles that sense and transduce extracellular signals, such as the Hedgehog (Hh) family of morphogens, and play crucial roles in embryonic development and adult organ homeostasis (Anvarian et al., 2019; Hilgendorf et al., 2024; Kopinke et al., 2021). Their structure is based on axonemal microtubules that extend from the basal body derived from the mother centriole, which has distal appendages (DAPs) with nine-fold symmetry, and are surrounded by the ciliary membrane (Breslow and Holland, 2019; Kumar and Reiter, 2021). To perform their sensory functions, specific signaling proteins are present in the cilioplasm and on the ciliary membrane, such as G protein-coupled receptors, including Smoothened and GPR161, which regulate Hh signaling positively and negatively, respectively (Anvarian et al., 2019; Kopinke et al., 2021; Schou et al., 2015). The distinct composition of ciliary proteins is achieved by the transition zone (TZ) at the ciliary base, which functions as a barrier controlling the import and export of soluble and membrane proteins (Garcia-Gonzalo and Reiter, 2017; Park and Leroux, 2022).

The biogenesis of cilia and their maintenance rely on the intraflagellar transport (IFT) machinery, which was first discovered in Chlamydomonas flagella, and is often referred to as IFT trains or IFT particles (Cole et al., 1998; Kozminski et al., 1993, 1995; Piperno and Mead, 1997). The IFT machinery consists of two main multisubunit complexes (Nakayama and Katoh, 2020; Taschner and Lorentzen, 2016). The IFT-A complex is composed of six subunits, and participates in retrograde protein trafficking within cilia driven by the dynein-2 complex, and in the import of membrane proteins across the TZ together with the TULP3 adaptor protein. The IFT-B complex is composed of 16 subunits, and participates in anterograde trafficking driven by heterotrimeric kinesin-II, and in the export of membrane proteins across the TZ along with the BBSome complex, which is composed of eight Bardet-Biedl syndrome (BBS) proteins.

Recent studies of Chlamydomonas flagella using cryoelectron microscopy and cryoelectron tomography revealed that the anterograde IFT trains are composed of repetitive units of the IFT-B complex and the IFT-A complex that are located between the IFT-B repeats and the ciliary membrane; the IFT-B/IFT-A stoichiometry is approximately 2:1. The dynein-2 complex is then loaded onto the assembled trains as an anterograde IFT cargo (Jordan et al., 2018; Lacey et al., 2023; Toropova et al., 2019; van den Hoek et al., 2022).

The 16 subunits of the IFT-B complex can be grouped into two subcomplexes; namely, the IFT-B1 subcomplex (IFT22/IFT25/IFT27/IFT46/IFT52/IFT56/IFT70/IFT74/IFT81/IFT88) and the IFT-B2 subcomplex (IFT20/IFT38/IFT54/IFT57/IFT80/IFT172) (see Fig. 5A) (Boldt et al., 2016; Katoh et al., 2016; Lucker et al., 2005; Petriman et al., 2022; Richey and Qin, 2012; Taschner et al., 2011, 2016). The two subcomplexes are connected by a tetramer unit involving IFT52 and IFT88 from IFT-B1, and IFT38 and IFT57 from IFT-B2; this tetramer also constitutes the binding site for heterotrimeric kinesin-II (Funabashi et al., 2018). The IFT-B1 subcomplex can be further divided into two subgroups, i.e., IFT-B1a (IFT22/IFT25/IFT27/IFT74/IFT81) and IFT-B1b (IFT46/IFT52/IFT56/IFT70/IFT88), which are connected via an interaction between the IFT74–IFT81 dimer from IFT-B1a and the IFT46–IFT52 dimer from IFT-B1b (Katoh et al., 2016; Petriman et al., 2022; Taschner et al., 2014; Zhou et al., 2022). The IFT-B complex is connected to IFT-A by an interaction involving IFT122–IFT144 from IFT-A and IFT52–IFT88 from IFT-B (Hesketh et al., 2022; Ishida et al., 2021; Kobayashi et al., 2021; Lacey et al., 2023). Among the IFT-B subunits, IFT74 and IFT81 form a tight heterodimer via their long coiled-coil regions, and have been shown to play a crucial role in transporting tubulin dimers to support the extension and maintenance of axonemal microtubules (Bhogaraju et al., 2013; Kubo et al., 2016). Therefore, depletion or knockout (KO) of any of the IFT-B subunits, including IFT74 and IFT81, often results in a cell phenotype that lacks cilia (Nakayama and Katoh, 2018).

Recent studies using super-resolution microscopy and expansion microscopy demonstrated that the IFT-B and IFT-A complexes are localized at gaps between the DAPs of the basal body (Ishida et al., 2021; Katoh et al., 2020; Yang et al., 2015, 2018), demonstrating that the localization of IFT-B and IFT-A is dependent on DAP proteins (Bowie et al., 2018; Čajánek and Nigg, 2014; Kanie et al., 2025; Mori et al., 2025; Reed et al., 2022; Schmidt et al., 2012). On the other hand, analyses of cilia/flagella of various species using fluorescence recovery after photobleaching suggested that the IFT-A and IFT-B complexes are independently recruited to the basal body pool (Hibbard et al., 2021; Mitra et al., 2024; Wingfield et al., 2017). Furthermore, we recently showed that the IFT-A, IFT-B, and dynein-2 complexes are independently assembled at the mother centriole/basal body before their incorporation into IFT trains (Tasaki et al., 2025). In the course of this study, we noticed the possibility that assembly of the IFT-B complex and its recruitment to the mother centriole/basal body may be dependent on a specific subcomplex/subgroup of IFT-B and is independent of ciliogenesis (Tasaki et al., 2025). Namely, although human telomerase reverse transcriptase-immortalized retinal pigment epithelial 1 (hTERT-RPE1) cells that are knocked out of IFT81 (an IFT-B1a subunit) have impaired ciliogenesis, IFT88 (an IFT-B1b subunit) and IFT38 (an IFT-B2 subunit) were recruited to the mother centriole in these cells (see Fig. 1I and Fig. 2I). In the present study, we therefore extended our previous study and aimed to clarify the process of IFT-B complex assembly and recruitment to the mother centriole by investigating the localization of IFT-B2, IFT-B1b, and IFT-B1a subunits and their interactions under individual IFT-B subunit knockout conditions.

Materials and Methods

Plasmids, antibodies, chemicals, and KO cell lines

Plasmids for the preparation of lentiviral vectors for the expression of IFT-B subunits, and for production of the glutathione S-transferase (GST)-tagged anti-GFP nanobody (Nb) are listed in Table S1. The antibodies used in this study are also listed in Table S1. The KO cell lines used in this study are listed in Table S2; all of them were established from hTERT-RPE1 cells in our previous studies (Funabashi et al., 2018; Hiyamizu et al., 2023; Ishida et al., 2022; Katoh et al., 2017; Nozaki et al., 2019; Tasaki et al., 2023; Zhou et al., 2022).

Immunofluorescence analysis

Parental hTERT-RPE1 cells (American Type Culture Collection, CRL-4000) and KO cells were cultured in DMEM/Ham’s F-12 medium supplemented with 10% fetal bovine serum (FBS) and 0.348% sodium bicarbonate at 37°C in 5% CO₂. To induce ciliogenesis under serum-starved conditions, the cells were grown on coverslips to 100% confluence, and cultured for 24 h in Opti-MEM (Thermo Fisher Scientific) containing 0.2% bovine serum albumin. For immunostaining, the cells were fixed and permeabilized with 3% paraformaldehyde for 5 min at 37°C, and further by methanol for 5 min at –20°C for IFT38, IFT88, and IFT81. The immunostained cells were observed using an Axio Observer microscope (Carl Zeiss).

To quantify the fluorescence intensities, all images in a set of experiments were acquired under the same setting and analyzed using the Zeiss ZEN 3.1 software. A region of interest (ROI) was constructed around the CEP43 signal using a drawing tool, and the fluorescence intensity in the ROI was quantified. To correct for local background intensity, the fluorescence intensity of a nearby region was subtracted. Statistical analyses were performed using GraphPad Prism version 10.1.2 software (GraphPad Software).

Preparation of lentiviral vectors and establishment of stable cell lines

Lentiviral vectors for the stable expression of EGFP-fused IFT38, IFT52, and IFT74 were prepared as described previously (Takahashi et al., 2012). Briefly, HEK293T cells (RBC2202, RIKEN BioResource Research Center) were cultured in DMEM with high glucose supplemented with 5% FBS. An EGFP-fused IFT-B construct in the pRRLsinPPT vector was transfected into HEK293T cells together with the packaging plasmids (pRSV-REV, pMD2.g, and pMDLg/pRRE; kind gifts from Peter McPherson, McGill University) (Thomas et al., 2009) using Polyethylenimine Max (Polysciences). Eight hours after the transfection, the culture medium was replaced with fresh medium. The medium containing viral particles was then collected at 24, 36, and 48 h after transfection, passed through a 0.45-μm filter, and centrifuged at 32,000 × g at 4°C for 4 h. The precipitated lentiviral particles were resuspended in DMEM/Ham’s F-12 medium, and stored at –80°C until use. KO cells stably expressing an EGFP-fused construct were prepared by addition of the lentiviral suspension to the culture medium, followed by selection of the cells in culture medium containing blasticidin S (10 μg/mL; InvivoGen) or Zeocin (10 μg/mL; InvivoGen).

Coimmunoprecipitation analysis

Cells stably expressing either EGFP-fused IFT38, IFT52, or IFT74 were grown to 100% confluence on a 6-cm plate, and cultured for 24 h in Opti-MEM containing 0.2% bovine serum albumin. These cells were then lysed in 300 μL of lysis buffer (20 mM HEPES–KOH [pH 7.4], 100 mM KCl, 5 mM NaCl, 3 mM MgCl₂, 1 mM dithiothreitol, 10% glycerol and 0.1% Triton X-100) containing EDTA-free protease inhibitor cocktail (Nacalai Tesque) by placing on ice for 20 min. After centrifugation of the lysates at 16,100 × g at 4°C for 15 min, the supernatants were incubated with 5 μL of GST-tagged anti-GFP Nb prebound to glutathione-Sepharose 4B beads. After brief centrifugation in a microcentrifuge, the beads were washed three times with the lysis buffer and boiled in SDS-PAGE sample buffer. Proteins bound to the beads were separated by SDS-PAGE, and electroblotted onto an Immobilon-P membrane (Merck Millipore). The membrane was blocked in 5% skimmed milk and incubated sequentially with the primary antibody and the peroxidase-conjugated secondary antibody. Protein bands were detected with a Chemi Lumi one L kit or Chemi-Lumi super kit (Nacalai Tesque).

Results

Differential effects of the lack of the IFT-B1a, IFT-B1b, or IFT-B2 subunit on recruitment of the remaining sets of IFT-B subunits to the mother centriole

In the present study, we used hTERT-RPE1 cells knocked out of IFT74 and IFT81 (IFT-B1a subunits), of IFT52 and IFT88 (IFT-B1b subunits), and of IFT38 and IFT54 (IFT-B2 subunits) (Hiyamizu et al., 2023; Ishida et al., 2022; Katoh et al., 2017; Nozaki et al., 2019; Tasaki et al., 2023; Zhou et al., 2022) to investigate whether the absence of one of the IFT-B subunits affects recruitment of the other subunits to the mother centriole. In addition, we also used cells knocked out of KIF3B, a subunit of heterotrimeric kinesin II (Funabashi et al., 2018), as a control of cells lacking cilia but with normal IFT-B complex assembly (Tasaki et al., 2025).

We first investigated whether IFT38 (an IFT-B2 subunit) is able to localize to the mother centriole/basal body in these KO cells. In control RPE1 cells, IFT38 was primarily localized at the ciliary base, with a minor proportion within cilia (Fig. 1B). In KIF3B-KO cells, which lack cilia, IFT38 was localized at one of the two CEP43-positive centrioles (Fig. 1C), confirming that the IFT-B complex is recruited to the mother centriole independently of ciliogenesis (Tasaki et al., 2025). In IFT54-KO cells as well as in IFT38-KO cells, IFT38 signals were undetectable at either of the centrioles (Fig. 1D, E; also see Fig. 1J), indicating that incorporation of IFT38 into the IFT-B complex and/or its recruitment to the mother centriole is dependent on IFT54, another IFT-B2 subunit (see Fig. 1A). In IFT52-KO cells and IFT88-KO cells, IFT38 was not detectable at the centrioles (Fig. 1F, G; also see Fig. 1J), indicating that mother centriole recruitment of IFT38 is dependent on the IFT-B1b subunits. By contrast, IFT38 signals were found at one of the two centrioles in IFT74-KO and IFT81-KO cells (Fig. 1H, I; also see Fig. 1J), although ciliogenesis was abrogated. Thus, recruitment of IFT38 to the mother centriole is independent of the IFT-B1a subunits, although the IFT74 and IFT81 subunits are essential for cilia formation owing to their role in tubulin transport (Bhogaraju et al., 2013; Kubo et al., 2016).

Fig. 1

Components of IFT-B2 and IFT-B1b, but not IFT-B1a, are required for mother centriole recruitment of IFT38

(A) Model diagram of assembly of the IFT-B complex with IFT38 highlighted in red. The beginning of the subunit name indicates the N-terminal side. (B–I) Control RPE1 (B), KIF3B-KO (C), IFT38-KO (D), IFT54-KO (E), IFT52-KO (F), IFT88-KO (G), IFT74-KO (H), and IFT81-KO (I) cells were cultured under serum-starved conditions for 24 h to induce ciliogenesis, and immunostained with antibodies against IFT38 and acetylated α-tubulin (Ac-tubulin) + CEP43 (CEP43 was previously referred to as FOP/FGFR1OP). Images shown on the right side are 2.5-times enlarged images of the boxed regions. Scale bars, 5 μm. (J) Staining intensity of IFT38 at the mother centriole of various cells was measured and shown as scatter plots. Dots indicate individual samples, and horizontal lines and error bars indicate means and SDs, respectively. Total numbers of analyzed cells from a single experiment are shown (n). The immunostaining experiment was repeated three times to confirm the reproducibility. Statistical significances among multiple groups were calculated using one-way ANOVA followed by Tukey’s multiple comparison test. NS, not significant.

We then investigated the localization of IFT88 (an IFT-B1b subunit) in IFT-B KO cells. Like IFT38, IFT88 was mainly localized at the ciliary base, with a minor proportion within cilia in control RPE1 cells (Fig. 2B), and at one of the two CEP43-positive centrioles in KIF3B-KO cells (Fig. 2C). IFT88 signals were not detected at either of the centrioles in either IFT52-KO or IFT88-KO cells (Fig. 2F, G; also see Fig. 2J). In addition, IFT88 signals were not detected in the absence of IFT38 or IFT54 (Fig. 2D, E; also see Fig. 2J). By contrast, IFT88 was found localized at one of the two centrioles in IFT74-KO and IFT81-KO cells (Fig. 2H, I; also see Fig. 2J). Taken together with the data shown in Fig. 1, these observations indicate that the IFT-B2 subcomplex and the IFT-B1b subgroup are recruited to the mother centriole in a mutually dependent manner, but independently of the IFT-B1a subgroup.

Fig. 2

Components of IFT-B2 and IFT-B1b, but not IFT-B1a, are required for mother centriole recruitment of IFT88

(A) Model diagram of assembly of the IFT-B complex with IFT88 highlighted in red. (B–I) Control RPE1 (B), KIF3B-KO (C), IFT38-KO (D), IFT54-KO (E), IFT52-KO (F), IFT88-KO (G), IFT74-KO (H), and IFT81-KO (I) cells were cultured under serum-starved conditions for 24 h, and immunostained with antibodies against IFT88 and Ac-tubulin + CEP43. Images shown on the right side are 2.5-times enlarged images of the boxed regions. Scale bars, 5 μm. (J) Staining intensity of IFT88 at the mother centriole of various cells was measured and shown as scatter plots. Dots indicate individual samples, and horizontal lines and error bars indicate means and SDs, respectively. Total numbers of analyzed cells from a single experiment are shown (n). The immunostaining experiment was repeated three times to confirm the reproducibility. Statistical significances among multiple groups were calculated using one-way ANOVA followed by Tukey’s multiple comparison test. NS, not significant.

We also investigated the localization of IFT81 (an IFT-B1a subunit) in IFT-B KO cells. Like IFT38 and IFT88, IFT81 was mainly localized at the ciliary base with a minor proportion within cilia in control RPE1 cells, and at one of the two centrioles in KIF3B-KO cells (Fig. 3B, C). In IFT38-KO, IFT54-KO, IFT52-KO, and IFT88-KO cells, IFT81 signals were not detectable at the mother or daughter centriole (Fig. 3D–G; also see Fig. 3J), indicating that the mother centriole localization of IFT-B1a is dependent not only on IFT-B1b but also on IFT-B2, which is not directly linked to IFT-B1a (see Fig. 5A). However, it should be mentioned that, in IFT74-KO and IFT81-KO cells, IFT81 signals at the centriole were substantially decreased (Fig. 3H, I; also see Fig. 3J), but slight IFT81 staining remained (Fig. 3J). By immunoblotting analysis of lysates from IFT81-KO cells with anti-IFT81 antibody, we detected residual non-specific bands (see Fig. 4B, fifth panel, lane 11; and Fig. 4C, sixth panel, lane 11). Thus, although we do not know the exact reason, the residual IFT81 staining in IFT74-KO and IFT81-KO cells is probably inherent to the used IFT81 antibody.

Fig. 3

Components of IFT-B2 and IFT-B1b as well as IFT-B1a are required for mother centriole recruitment of IFT81

(A) Model diagram of assembly of the IFT-B complex with IFT81 highlighted in red. (B–I) Control RPE1 (B), KIF3B-KO (C), IFT38-KO (D), IFT54-KO (E), IFT52-KO (F), IFT88-KO (G), IFT74-KO (H), and IFT81-KO (I) cells were cultured under serum-starved conditions for 24 h to induce ciliogenesis, and immunostained with antibodies against IFT81 and Ac-tubulin + CEP43. Images shown on the right side are 2.5-times enlarged images of the boxed regions. Scale bars, 5 μm. (J) Staining intensity of IFT81 at the mother centriole of various cells was measured and shown as scatter plots. Dots indicate individual samples, and horizontal lines and error bars indicate means and SDs, respectively. Total numbers of analyzed cells from a single experiment are shown (n). The immunostaining experiment was repeated twice to confirm the reproducibility. Statistical significances among multiple groups were calculated using one-way ANOVA followed by Tukey’s multiple comparison test. NS, not significant.

Fig. 4

Differential effects of the lack of either the IFT-B1a, IFT-B1b, or IFT-B2 subunit on assembly of the remaining sets of IFT-B subunits

EGFP-fused IFT38 (A), IFT52 (B), or IFT74 (C) was stably expressed in control RPE1 cells, and in KIF3B-KO, IFT38-KO, IFT54-KO, IFT81-KO, and IFT88-KO cells as indicated. Lysates were prepared from the cells and processed for immunoprecipitation with GST-tagged anti-GFP Nb, followed by SDS-PAGE and immunoblotting analysis using antibodies against GFP, IFT38, IFT57, IFT52, IFT70, and IFT81, as indicated. γ-Adaptin (AP1G1) was used as a loading control. Asterisks indicate the positions of nonspecific bands. Blots shown are representative of two experiments.

Fig. 5

Model diagrams of the assembly and mother centriole recruitment of the IFT-B complex in cells lacking specific IFT-B subunits

Model diagrams of the assembly and recruitment to the mother centriole of the IFT-B2 subcomplex, and IFT-B1b and IFT-B1a subgroups in control RPE1 cells (A), cells lacking an IFT-B2 subunit (B; IFT38 or IFT54), cells lacking an IFT-B1b subunit (C; IFT88), and cells lacking an IFT-B1a subunit (D; IFT81). MC, mother centriole. Red triangles indicate DAPs.

Differential effects of the lack of the IFT-B1a, IFT-B1b, or IFT-B2 subunit on assembly of the remaining sets of IFT-B subunits

Next, we investigated whether the lack of one of the IFT-B subunits affects assembly of the remaining sets of the IFT-B subunits. To this end, we expressed one of the IFT-B subunits fused to EGFP in cells knocked out of one of the IFT-B subunits. Then, to determine if the EGFP-fused subunit can be incorporated into the IFT-B complex, lysates prepared from the cells were processed for immunoprecipitation with GST-tagged anti-GFP Nb prebound to glutathione-Sepharose beads (Katoh et al., 2015), followed by SDS-PAGE and immunoblotting analysis using antibodies against the various IFT-B subunits; however, it should be noted that the EGFP tag may interfere with the incorporation of the subunit into the complex.

To determine the assembly process of the IFT-B complex, we first expressed EGFP-fused IFT38 (an IFT-B2 subunit) in cells knocked out of one of the IFT-B subunits (Fig. 4A). When expressed in KIF3B-KO cells as the cilia-lacking control (Fig. 4A, lane 2), EGFP-IFT38 was able to coimmunoprecipitate IFT57 (an IFT-B2 subunit), IFT52 and IFT70 (IFT-B1b subunits), and IFT81 (an IFT-B1a subunit), indicating that EGFP-IFT38 was incorporated into the IFT-B holocomplex even in the absence of cilia formation. In cells knocked out of IFT54 (an IFT-B2 subunit) (Fig. 4A, lane 3), not only the amount of IFT57, but also the amounts of IFT52, IFT70, and IFT81 that were coimmunoprecipitated with EGFP-IFT38 were substantially reduced, suggesting that the IFT-B2 subcomplex was not normally formed in the absence of IFT54, and thereby EGFP-IFT38 was unable to coprecipitate the IFT-B1b and IFT-B1a subunits. In cells knocked out of IFT88 (an IFT-B1b subunit) (Fig. 4A, lane 5), EGFP-IFT38 was able to coprecipitate IFT57, indicating that the IFT-B2 subcomplex was normally formed even in the absence of the IFT-B1b subgroup. By contrast, EGFP-IFT38 was unable to coprecipitate IFT81 as well as IFT52 and IFT70, indicating that the IFT-B2 subcomplex and the IFT-B1a subgroup were not included in the same complex if the IFT-B1b subgroup was not present. In cells knocked out of IFT81 (an IFT-B1a subunit) (Fig. 4A, lane 4), EGFP-IFT38 was able to coprecipitate IFT57, IFT52, and IFT70, indicating that the IFT-B2 subcomplex and the IFT-B1b subgroup were linked to each other in the absence of the IFT-B1a subgroup. This is consistent with our previous studies showing that IFT38 and IFT57 from IFT-B2, and IFT52 and IFT88 from IFT-B1b are minimally required for the IFT-B2–IFT-B1b interaction (Katoh et al., 2016; Kobayashi et al., 2021).

We then analyzed the IFT-B KO cells expressing EGFP-fused IFT52 (an IFT-B1b subunit) (Fig. 4B). In KIF3B-KO cells (lane 2) as well as in control RPE1 cells (lane 1), all the analyzed IFT-B subunits were coprecipitated with EGFP-IFT52, indicating that EGFP-IFT52 is incorporated into the IFT-B holocomplex independently of cilia formation. In cells knocked out of IFT38 (lane 3) or IFT54 (lane 4) (IFT-B2 subunits), EGFP-IFT52 coprecipitated IFT70 (an IFT-B1b subunit) and IFT81 (an IFT-B1a subunit), indicating that the IFT-B1b subgroup is linked to the IFT-B1a subgroup even in the absence of the IFT-B2 subcomplex. In IFT81-KO cells (lane 5), EGFP-IFT52 was able to coprecipitate not only IFT70 but also IFT38 and IFT57, indicating that the IFT-B1b subgroup is linked to the IFT-B2 subcomplex in the absence of the IFT-B1a subgroup. In IFT88-KO cells (lane 6), neither IFT38 nor IFT57 was coprecipitated with EGFP-IFT52, in agreement with previous studies showing that the IFT52–IFT88 dimer of the IFT-B1b subgroup constitutes the interface with the IFT-B2 subcomplex (Katoh et al., 2016; Kobayashi et al., 2021; Petriman et al., 2022; Taschner et al., 2016).

We finally analyzed IFT-B KO cells expressing EGFP-fused IFT74 (an IFT-B1a subunit) (Fig. 4C). Again, all the analyzed IFT-B subunits were coprecipitated with EGFP-IFT74 in KIF3B-KO cells (lane 2) as well as in control RPE1 cells (lane 1), indicating that EGFP-IFT74 was incorporated into the IFT-B holocomplex in a ciliogenesis-independent manner. In cells knocked out of IFT38 (lane 3) or IFT54 (lane 4) (IFT-B2 subunits), IFT52 and IFT70 (IFT-B1b subunits) as well as IFT81 (an IFT-B1a subunit) were coprecipitated with EGFP-IFT74, confirming that the IFT-B1a and IFT-B1b subgroups are linked to each other even in the absence of the IFT-B2 subcomplex. By contrast, in IFT81-KO cells, the coimmunoprecipitation with EGFP-IFT74 of all the IFT-B subunits analyzed was virtually undetectable (lane 5). These results using IFT81-KO cells expressing EGFP-IFT74 are in good agreement with the fact that IFT74 forms a tight heterodimer with IFT81 via their long coiled-coil regions, and interacts with the IFT46–IFT52 dimer of the IFT-B1b subgroup (Petriman et al., 2022; Taschner et al., 2014; Zhou et al., 2022). In IFT88-KO cells (lane 6), IFT70 was coprecipitated with EGFP-IFT74. These results are consistent with the fact that the IFT74–IFT81 dimer interacts with the IFT46–IFT52 dimer of the IFT-B1b subgroup, to which IFT70 binds (Katoh et al., 2016; Petriman et al., 2022; Taschner et al., 2014). IFT38 or IFT57 was not coprecipitated with EGFP-IFT74 in IFT88-KO cells, consistent with the fact that the IFT52–IFT88 dimer interacts with IFT38 and IFT57 of the IFT-B2 subcomplex (Katoh et al., 2016; Kobayashi et al., 2021; Petriman et al., 2022; Taschner et al., 2016).

Discussion

We previously showed that the IFT-B, IFT-A, and dynein-2 complexes are independently recruited to the mother centriole (Tasaki et al., 2025). In the present study, we set out to pursue the association between the assembly and localization to the mother centriole of the IFT-B complex, which can be divided into the IFT-B2 subcomplex and IFT-B1 subcomplex (IFT-B1a and IFT-B1b subgroups) (see Fig. 5A). Table 1 summarizes the results obtained in the present study, and Fig. 5 shows model diagrams of the assembly and mother centriole recruitment of the IFT-B complex in control RPE1 cells and in KO cells of each IFT subunit.

Table 1

Summary of the results of the present study

Subcomplex/subgroup	Cell	Cilia formation	Basal body/mother centriole localization			Assembly
			IFT-B2 (IFT38)	IFT-B1b (IFT88)	IFT-B1a (IFT81)	IFT-B2–IFT-B1b	IFT-B1b–IFT-B1a
	Control RPE1	+	+	+	+	+	+
	KIF3B-KO	–	+	+	+	+	+
IFT-B2	IFT38-KO	–	–	–	–	–	+
	IFT54-KO	–	–	–	–	–	+
IFT-B1b	IFT52-KO	–	–	–	–	NI	NI
	IFT88-KO	–	–	–	–	–	±*1
IFT-B1a	IFT74-KO	–	+	+	–	NI	NI
	IFT81-KO	–	+	+	–	+	–

NI: not investigated.

*¹ In IFT88-KO cells, there was partial interaction between IFT-B1a and the remaining IFT-B1b components (see Fig. 4C). We previously demonstrated that the IFT74–IFT81 dimer of the IFT-B1a subgroup can interact with the IFT46–IFT52 dimer of the IFT-B1b subgroup in the absence of IFT88 (Katoh et al., 2016; Zhou et al., 2022).

Immunofluorescence analysis of cells knocked out of individual IFT-B subunits (Fig. 1–3) and coimmunoprecipitation analysis of these KO cells expressing one of the IFT-B subunits fused to EGFP (Fig. 4) showed that, in the absence of an IFT-B1b subgroup subunit (IFT52 or IFT88), none of the analyzed IFT-B subunits could localize to the mother centriole, although the IFT-B2 subcomplex and the IFT-B1a subgroup appeared to be able to be assembled independently (Fig. 5C). Thus, given the central role of the IFT-B1b subgroup in bridging IFT-B2 and IFT-B1a (see Fig. 5A) (Petriman et al., 2022) and the potential role of a tetramer unit involving IFT52 and IFT88 from IFT-B1b and IFT38 and IFT57 from IFT-B2 in the localization of IFT-B to the mother centriole (see below), cilia do not form in the absence of IFT-B1b owing to the lack of assembly of the IFT-B holocomplex.

In the absence of the IFT-B2 subunit (IFT38 or IFT54), the IFT-B1b and IFT-B1a subgroups can be linked to each other (Fig. 4B, C; also see Fig. 5B); namely, the IFT-B1 subcomplex is formed. This is in line with early biochemical studies of Chlamydomonas IFT-B proteins showing that the peripheral (IFT-B2) subunits associate with the core (IFT-B1) of the IFT-B complex (the IFT-B1 subcomplex) (Lucker et al., 2005, 2010; Taschner et al., 2011), and with previous coimmunoprecipitation studies of EGFP-fused and mCherry-fused IFT-B proteins showing that the IFT46–IFT52 dimer from IFT-B1b and IFT81 from IFT-B1a are involved in the IFT-B1b–IFT-B1a linkage (Katoh et al., 2016; Tasaki et al., 2023; Zhou et al., 2022). However, neither the IFT-B1b (IFT88) subunit nor the IFT-B1a (IFT81) subunit can localize to the mother centriole, indicating that the IFT-B1b–IFT-B1a linkage (in other words, the IFT-B1 subcomplex) is not sufficient for localization of the IFT-B complex to the mother centriole (Fig. 5B). In the absence of the IFT-B2 subcomplex, the IFT-B complex cannot be recruited to the mother centriole, resulting in the absence of ciliogenesis.

By contrast, in the absence of the IFT-B1a subunit (IFT74 or IFT81), the IFT-B2 subcomplex and IFT-B1b subgroup can be linked to each other (Fig. 5D), which is consistent with previous studies showing that the tetramer unit composed of IFT38 and IFT57 from IFT-B2, and IFT52 and IFT88 from IFT-B1b is crucial for the linking of the IFT-B2 and IFT-B1 subcomplexes (Katoh et al., 2016; Kobayashi et al., 2021; Taschner et al., 2016). In addition, both IFT-B2 (IFT38) and IFT-B1b (IFT88) can localize to the mother centriole in the absence of IFT-B1a (Fig. 5D). Thus, it is likely that the IFT-B2–IFT-B1b linkage is essential for the mother centriole localization of the IFT-B complex; this was somewhat unexpected because early biochemical studies on Chlamydomonas IFT-B proteins indicated that the IFT-B peripheral (IFT-B2) proteins are associated with the core (IFT-B1) of the IFT-B complex (Lucker et al., 2005, 2010; Taschner et al., 2011). However, even with the recruitment of IFT-B2–IFT-B1b to the mother centriole, cilia do not form in the absence of IFT74 or IFT81. This is probably owing to the crucial role of the IFT74–IFT81 dimer in the transport of tubulin dimers for extension and maintenance of axonemal microtubules (Bhogaraju et al., 2013; Kubo et al., 2016).

Thus, although ciliogenesis is inhibited in the absence of the analyzed individual IFT-B subunits, the factors involved in the inhibition are likely to vary depending on the missing subunits, including impaired recruitment of the IFT-B complex to the mother centriole and impaired tubulin transport. A key remaining question is which subunits participate in the localization of IFT-B to the mother centriole. One possibility is the tetramer unit involved in the linkage between IFT-B2 and IFT-B1b (IFT38 and IFT57 from IFT-B2, and IFT52 and IFT88 from IFT-B1b), as IFT-B2 or IFT-B1 (IFT-B1b + IFT-B1a) alone is not sufficient for the mother centriole localization of the IFT-B complex. As the tetramer unit constitutes the binding site of heterotrimeric kinesin-II (Funabashi et al., 2018), one possible scenario is that the IFT-B complex is recruited to the mother centriole via the tetramer, and after leaving the mother centriole, kinesin-II occupies the tetramer and drives anterograde transport of the IFT trains.

Another question is what in the mother centriole determines the recruitment of the IFT-B complex. Previous studies showed that DAP proteins, including CEP164 and Tau-tubulin kinase 2 (TTBK2), are essential for recruitment of the IFT-A, IFT-B, and dynein-2 complexes to the mother centriole (Bowie et al., 2018; Čajánek and Nigg, 2014; Goetz et al., 2012; Kanie et al., 2025; Mori et al., 2025; Reed et al., 2022; Schmidt et al., 2012). A recent proximity labeling study on TTBK2 identified many ciliary, centrosomal, and pericentriolar material, as well as centriolar satellite proteins, suggesting direct or indirect interactions of these proteins with TTBK2; IFT46, IFT52, IFT74, and IFT81 were included in the TTBK2 proximity list (Nguyen and Goetz, 2023). However, our attempts to show direct interactions of TTBK2 with these IFT-B proteins have been unsuccessful to date (Mori et al., 2025). On the other hand, we showed that the kinase activity and CEP164-dependent recruitment of TTBK2 are required for the localization of the IFT-A, IFT-B, and dynein-2 complexes to the mother centriole (Mori et al., 2025). Recently, Kanie et al. reported that KO of TTBK2 in RPE1 cells impaired the localization of several DAP proteins, including CEP83, FBF1, ANKRD26, PIDD1, and NCS1 (Kanie et al., 2025). These proteins, which are phosphorylated by TTBK2, may hence be involved in the recruitment of the IFT-B complex to the mother centriole.

Thus, future issues to be addressed include which subunit(s) of the IFT-B complex determine its mother centriole localization and which protein(s) of the mother centriole interact with the IFT-B complex. The analyses we have employed to date, based on protein–protein interactions and the expression of various proteins in KO cells, will help to address these issues.

Data availability

All data in this study are included in the article or the supplementary material.

Author contributions

K.T. designed and performed the experiments and prepared the manuscript, and Y.K., H.-W.S., and K.N. designed the experiments and prepared the manuscript.

Conflict of Interest

The authors declare that they have no competing interests associated with this study.

Acknowledgments

We thank Peter McPherson for providing the plasmids for lentivirus production, Hiroshi Hamada and Takanobu A. Katoh for providing the anti-IFT38 antibody, and Helena Akiko Popiel for critical reading of the manuscript.

This study was partially supported by grants from the Japan Society for the Promotion of Science (grant numbers 22H05539 to Y.K. and 24K02022 to K.N.), and JST SPRING (grant number JPMJSP2110 to K.T.).

References

•  Anvarian,  Z.,  Mykytyn,  K.,  Mukhopadhyay,  S.,  Pedersen,  L.B., and  Christensen,  S.T. 2019. Cellular signaling by primary cilia in development, organ function and disease. Nat. Rev. Nephrol., 15: 199–219.
•  Bhogaraju,  S.,  Cajánek,  L.,  Fort,  C.,  Blisnick,  T.,  Weber,  K.,  Taschner,  M.,  Mizuno,  N.,  Lamla,  S.,  Bastin,  P.,  Nigg,  E.A., and  Lorentzen,  E. 2013. Molecular basis of tubulin transport within the cilium by IFT74 and IFT81. Science, 341: 1009–1012.
•  Boldt,  K.,  van Reeuwijk,  J.,  Lu,  Q.,  Koutroumpas,  K.,  Nguyen,  T.M.,  Texier,  Y.,  van Beersum,  S.E.C.,  Horn,  N.,  Willer,  J.R.,  Mans,  D.,  Dougherty,  G.,  Lamers,  I.J.,  Coene,  K.L.,  Arts,  H.H.,  Betts,  M.J.,  Beyer,  T.,  Bolat,  E.,  Gloeckner,  C.J.,  Haidari,  K.,  Hetterschijt,  L.,  Iaconis,  D.,  Jenkins,  D.,  Klose,  F.,  Knapp,  B.,  Latour,  B.,  Letteboer,  S.J.,  Marcelis,  C.L.,  Mitic,  D.,  Morleo,  M.,  Oud,  M.M.,  Riemersma,  M.,  Rix,  S.,  Terhal,  P.A.,  Toedt,  G.,  van Dam,  T.J.,  de Vrieze,  E.,  Wissinger,  Y.,  Wu,  K.M.,  Group,  U.K.R.D.,  Apic,  G.,  Beales,  P.L.,  Blacque,  O.E.,  Gibson,  T.J.,  Huynen,  M.A.,  Katsanis,  N.,  Kremer,  H.,  Omran,  H.,  van Wijk,  E.,  Wolfrum,  U.,  Kepes,  F.,  Davis,  E.E.,  Franco,  B.,  Giles,  R.H.,  Ueffing,  M.,  Russell,  R.B., and  Roepman,  R. 2016. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms. Nat. Commun., 7: 11491.
•  Bowie,  E.,  Norris,  R.,  Anderson,  K.V., and  Goetz,  S.C. 2018. Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability. PLoS Genet., 14: e1007844.
•  Breslow,  D.K. and  Holland,  A.J. 2019. Mechanism and regulation of centriole and cilium biogenesis. Annu. Rev. Biochem., 88: 691–724.
•  Čajánek,  L. and  Nigg,  E.A. 2014. Cep164 triggers ciliogenesis by recruiting Tau tubulin kinase 2 to the mother centriole. Proc. Natl. Acad. Sci. USA, 111: E2841–E2850.
•  Cole,  D.G.,  Diener,  D.R.,  Himelblau,  A.L.,  Beech,  P.L.,  Fuster,  J.C., and  Rosenbaum,  J.L. 1998. Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J. Cell Biol., 144: 993–1008.
•  Funabashi,  T.,  Katoh,  Y.,  Okazaki,  M.,  Sugawa,  M., and  Nakayama,  K. 2018. Interaction of heterotrimeric kinesin-II with IFT-B-connecting tetramer is crucial for ciliogenesis. J. Cell Biol., 217: 2867–2876.
•  Garcia-Gonzalo,  F.R. and  Reiter,  J.F. 2017. Open sesame: How transition fibers and the transition zone control ciliary composition. Cold Spring Harb. Perspect. Biol., 9: a028134.
•  Goetz,  S.C.,  Liem,  K.F., Jr., and  Anderson,  K.V. 2012. The spinocerebellar ataxia-associated gene Tau Tubulin Kinase 2 controls the initiation of ciliogenesis. Cell, 151: 847–858.
•  Hesketh,  S.J.,  Mukhopadhyay,  A.G.,  Nakamura,  D.,  Toropova,  K., and  Roberts,  A.J. 2022. IFT-A structure reveals carriages for membrane protein transport into cilia. Cell, 185: 4971–4985.
•  Hibbard,  J.V.K.,  Vazquez,  N.,  Satija,  R., and  Wallingford,  J.B. 2021. Protein turnover dynamics suggest a diffusion-to-capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins. Mol. Biol. Cell, 32: 1171–1180.
•  Hilgendorf,  K.I.,  Myers,  B.R., and  Reiter,  J.F. 2024. Emerging mechanistic understanding of cilia function in cellular signalling. Nat. Rev. Mol. Cell Biol., 25: 555–573.
•  Hiyamizu,  S.,  Qiu,  H.,  Tsurumi,  Y.,  Hamada,  Y.,  Katoh,  Y., and  Nakayama,  K. 2023. Dynein-2–driven intraciliary retrograde trafficking indirectly requires multiple interactions of IFT54 in the IFT-B complex with the dynein-2 complex. Biol. Open, 12: bio059976.
•  Ishida,  Y.,  Kobayashi,  T.,  Chiba,  S.,  Katoh,  Y., and  Nakayama,  K. 2021. Molecular basis of ciliary defects caused by compound heterozygous IFT144/WDR19 mutations found in cranioectodermal dysplasia. Hum. Mol. Genet., 30: 213–225.
•  Ishida,  Y.,  Tasaki,  K.,  Katoh,  Y., and  Nakayama,  K. 2022. Molecular basis underlying the ciliary defects caused by IFT52 variations found in skeletal ciliopathies. Mol. Biol. Cell, 33: ar83.
•  Jordan,  M.A.,  Diener,  D.R.,  Stepanek,  L., and  Pigino,  G. 2018. The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterogarde movement in cilia. Nat. Cell Biol., 20: 1250–1255.
•  Kanie,  T.,  Liu,  B.,  Love,  J.F.,  Fisher,  S.D.,  Gustavsson,  A.K., and  Jackson,  P.K. 2025. A hierarchical pathway for assembly of the distal appendages that organize primary cilia. eLife, 14: e85999.
•  Katoh,  Y.,  Chiba,  S., and  Nakayama,  K. 2020. Practical method for superresolution imaging of primary cilia and centrioles by expansion microscopy using an amplibody for fluorescence signal amplification. Mol. Biol. Cell, 31: 2195–2206.
•  Katoh,  Y.,  Michisaka,  S.,  Nozaki,  S.,  Funabashi,  T.,  Hirano,  T.,  Takei,  R., and  Nakayama,  K. 2017. Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated homology-independent knock-in system. Mol. Biol. Cell, 28: 898–906.
•  Katoh,  Y.,  Nozaki,  S.,  Hartanto,  D.,  Miyano,  R., and  Nakayama,  K. 2015. Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. J. Cell Sci., 128: 2351–2362.
•  Katoh,  Y.,  Terada,  M.,  Nishijima,  Y.,  Takei,  R.,  Nozaki,  S.,  Hamada,  H., and  Nakayama,  K. 2016. Overall architecture of the intraflagellar transport (IFT)-B complex containing Cluap1/IFT38 as an essential component of the IFT-B peripheral subcomplex. J. Biol. Chem., 291: 10962–10975.
•  Kobayashi,  T.,  Ishida,  Y.,  Hirano,  T.,  Katoh,  Y., and  Nakayama,  K. 2021. Cooperation of the IFT-A complex with the IFT-B complex is required for ciliary retrograde protein trafficking and GPCR import. Mol. Biol. Cell, 32: 45–56.
•  Kopinke,  D.,  Norris,  A.M., and  Mukhopadhyay,  S. 2021. Developmental and regenerative paradigms of cilia regulated hedgehog signaling. Sem. Cell Dev. Biol., 110: 89–103.
•  Kozminski,  K.G.,  Beech,  P.L., and  Rosenbaum,  J.L. 1995. The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane. J. Cell Biol., 131: 1517–1527.
•  Kozminski,  K.G.,  Johnson,  K.A.,  Forscher,  P., and  Rosenbaum,  J.L. 1993. A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc. Natl. Acad. Sci. USA, 90: 5519–5523.
•  Kubo,  T.,  Brown,  J.M.,  Bellve,  K.,  Craige,  B.,  Craft,  J.M.,  Fogarty,  K.,  Lechtreck,  K.-F., and  Witman,  G.B. 2016. The IFT81 and IFT74 N-termini together form the major module for intraflagellar transport of tubulin. J. Cell Sci., 129: 2106–2119.
•  Kumar,  D. and  Reiter,  J.F. 2021. How the centriole builds its cilium: Of mothers, daughters, and the acquisition of appendages. Curr. Opin. Struct. Biol., 66: 41–48.
•  Lacey,  S.E.,  Foster,  H.E., and  Pigino,  G. 2023. The molecular structure of IFT-A and IFT-B in anterograde intraflagellar transport trains. Nat. Struct. Mol. Biol., 30: 584–593.
•  Lucker,  B.F.,  Behal,  R.H.,  Qin,  H.,  Siron,  L.C.,  Taggart,  W.D.,  Rosenbaum,  J.L., and  Cole,  D.G. 2005. Characterization of the intraflagellar transport complex B core: Direct interaction of the IFT81 and IFT74/72 subunits. J. Biol. Chem., 280: 27688–27696.
•  Lucker,  B.F.,  Miller,  M.S.,  Dziedzic,  S.A.,  Blackmarr,  P.T., and  Cole,  D.G. 2010. Direct interactions of intraflagellar transport complex B proteins IFT88, IFT52, and IFT46. J. Biol. Chem., 285: 21508–21518.
•  Mitra,  A.,  Loseva,  E., and  Peterman,  E.J.G. 2024. IFT cargo and motors associate sequentially with IFT trans to enter cilia of C. elegans. Nat. Commun., 15: 3456.
•  Mori,  K.,  Yamazaki,  S.,  Yoshida,  K.,  Shirota,  R.,  Chiba,  S.,  Shin,  H.-W.,  Katoh,  Y., and  Nakayama,  K. 2025. Coordinated roles of the CEP164 homodimer and TTBK2 are required for recruitment of the IFT machinery to the mother centriole for ciliogenesis. Mol. Biol. Cell, 36: ar79.
•  Nakayama,  K. and  Katoh,  Y. 2018. Ciliary protein trafficking mediated by IFT and BBSome complexes with the aid of kinesin-2 and dynein-2 motors. J. Biochem., 163: 155–164.
•  Nakayama,  K. and  Katoh,  Y. 2020. Architecture of the IFT ciliary trafficking machinery and interplay between its components. Crit. Rev. Biochem. Mol. Biol., 55: 179–196.
•  Nguyen,  A. and  Goetz,  S.C. 2023. TTBK2 controls cilium stability by regulating distinct modules of centrosomal proteins. Mol. Biol. Cell, 34: ar8.
•  Nozaki,  S.,  Castro Araya,  R.F.,  Katoh,  Y., and  Nakayama,  K. 2019. Requirement of IFT-B–BBSome complex interaction in export of GPR161 from cilia. Biol. Open, 8: bio043786.
•  Park,  K. and  Leroux,  M.R. 2022. Composition, organization and mechanisms of the transition zone, a gate for the cilium. EMBO Rep., 23: e55420.
•  Petriman,  N.A.,  Loureiro-López,  M.,  Taschner,  M.,  Zacharia,  N.K.,  Georgieva,  M.M.,  Boegholm,  N.,  Wang,  J.,  Mourão,  A.,  Russell,  R.B.,  Andersen,  J.S., and  Lorentzen,  E. 2022. Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B. EMBO J., 41: e112440.
•  Piperno,  G. and  Mead,  K. 1997. Transport of a novel complex in the cytoplasmic matrix of Chlamydomonas flagella. Proc. Natl. Acad. Sci. USA, 94: 4457–4462.
•  Reed,  M.,  Takemaru,  K.,  Ying,  G.,  Frederick,  J.M., and  Baehr,  W. 2022. Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT). PLoS Genet., 18: e1010154.
•  Richey,  E.A. and  Qin,  H. 2012. Dissecting the sequential assembly and localization of intraflagellar transport particle complex B in Chlamydomonas. Plos One, 7: e43118.
•  Schmidt,  K.N.,  Kuhns,  S.,  Neuner,  A.,  Hub,  B.,  Zentgraf,  H., and  Pereira,  G. 2012. Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis. J. Cell Biol., 199: 1083–1101.
•  Schou,  K.B.,  Pedersen,  L.B., and  Christensen,  S.T. 2015. Ins and outs of GPCR signaling in primary cilia. EMBO Rep., 16: 1099–1113.
•  Takahashi,  S.,  Kubo,  K.,  Waguri,  S.,  Yabashi,  A.,  Shin,  H.-W.,  Katoh,  Y., and  Nakayama,  K. 2012. Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. J. Cell Sci., 125: 4049–4057.
•  Tasaki,  K.,  Satoda,  Y.,  Chiba,  S.,  Shin,  H.-W.,  Katoh,  Y., and  Nakayama,  K. 2025. Mutually independent and cilia-independent assembly of IFT-A and IFT-B complexes at mother centriole. Mol. Biol. Cell, 36: ar48.
•  Tasaki,  K.,  Zhou,  Z.,  Ishida,  Y.,  Katoh,  Y., and  Nakayama,  K. 2023. Compound heterozygous IFT81 variations in a skeletal ciliopathy patient cause Bardet-Biedl syndrome-like ciliary defects. Hum. Mol. Genet., 32: 2887–2900.
•  Taschner,  M.,  Bhogaraju,  S.,  Vetter,  M.,  Morawetz,  M., and  Lorentzen,  E. 2011. Biochemical mapping of interactions within the intraflagellar transport (IFT) B core complex: IFT52 binds directly to four other IFT-B subunits. J. Biol. Chem., 286: 26344–26352.
•  Taschner,  M.,  Kotsis,  F.,  Braeuer,  P.,  Kuehn,  E.W., and  Lorentzen,  E. 2014. Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly. J. Cell Biol., 207: 269–282.
•  Taschner,  M. and  Lorentzen,  E. 2016. The intraflagellar transport machinery. Cold Spring Harb. Perspect. Biol., 8: a028092.
•  Taschner,  M.,  Weber,  K.,  Mourão,  A.,  Vetter,  M.,  Awasthi,  M.,  Stiegler,  M.,  Bhogaraju,  S., and  Lorentzen,  E. 2016. Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin-binding IFT-B2 complex. EMBO J., 35: 773–790.
•  Thomas,  S.,  Ritter,  B.,  Verbich,  D.,  Sanson,  C.,  Bourbonnière,  L.,  McKinney,  R.A., and  McPherson,  P.S. 2009. Intersectin regulates dendritic spine development and somatodendritic endocytosis but not synaptic vesicle recycling in hippocampal neurons. J. Biol. Chem., 284: 12410–12419.
•  Toropova,  K.,  Zalyte,  R.,  Mukhopadhyay,  A.G.,  Mladenov,  M.,  Carter,  A.P., and  Roberts,  A.J. 2019. Structure of the dynein-2 complex and its assembly with intraflagellar transport trains. Nat. Struct. Mol. Biol., 26: 823–829.
•  van den Hoek,  H.,  Klena,  N.,  Jordan,  M.A.,  Viar,  G.A.,  Righetto,  R.D.,  Schaffer,  M.,  Erdmann,  P.S.,  Wan,  W.,  Geimer,  S.,  Plitzko,  J.M.,  Baumeister,  W.,  Pigino,  G.,  Hamel,  V.,  Guichard,  P., and  Engel,  B.D. 2022. In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains. Science, 377: 543–548.
•  Wingfield,  J.L.,  Mengoni,  I.,  Bomberger,  H.,  Jiang,  Y.Y.,  Walsh,  J.D.,  Brown,  J.M.,  Picariello,  T.,  Cochran,  D.A.,  Zhu,  B.,  Pan,  J.,  Eggenschwiler,  J.,  Gaertig,  J.,  Witman,  G.B.,  Kner,  P., and  Lechtreck,  K. 2017. IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium. eLife, 6: e26609.
•  Yang,  T.T.,  Chong,  W.M.,  Wang,  W.-J.,  Mazo,  G.,  Tanos,  B.,  Chen,  Z.,  Tran,  T.M.N.,  Chen,  Y.-D.,  Weng,  R.R.,  Huang,  C.-E.,  Jane,  W.-N.,  Tsou,  M.-F.B., and  Liao,  J.-C. 2018. Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components. Nat. Commun., 9: 2023.
•  Yang,  T.T.,  Su,  J.,  Wang,  W.-J.,  Craige,  B.,  Witman,  G.B.,  Tsou,  M.-F.B., and  Liao,  J.-C. 2015. Superresolution pattern recognition reveals the architectural map of the ciliary transition zone. Sci. Rep., 5: 14096.
•  Zhou,  Z.,  Qiu,  H.,  Castro-Araya,  R.-F.,  Takei,  R.,  Nakayama,  K., and  Katoh,  Y. 2022. Impaired cooperation between IFT74/BBS22–IFT81 and IFT25–IFT27/BBS19 in the IFT-B complex causes ciliary defects in Bardet-Biedl syndrome. Hum. Mol. Genet., 31: 1681–1693.

Abbreviations

BBS

Bardet-Biedl syndrome

DAP

distal appendage

FBS

fetal bovine serum

GST

glutathione S-transferase

Hh

Hedgehog

hTERT-RPE1

human telomerase reverse transcriptase-immortalized retinal pigment epithelial 1

IFT

intraflagellar transport

KO

knockout

Nb

nanobody

ROI

region of interest

SMO

Smoothened

TTBK2

Tau-tubulin kinase 2

TZ

transition zone