Molecular basis of tubulin transport within the cilium by IFT74 and IFT81

Abstract

Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.

Fig. 1. IFT81 and IFT74 form a tubulin-binding module

(A) Cartoon representation of the crystal structure of CrIFT81N domain, with conserved lysines and arginines implicated in tubulin binding shown as sticks. (B) Electrostatic surface potential of IFT81N displaying the positively charged patch with the residues labeled according to the HsIFT81 sequence. (C) Surface conservation of IFT81N demonstrates that the basic patch is well conserved among different species (also see fig. S2). (D) Tubulin binding evaluated by glutathione (GSH) affinity pull-down of bovine αβ-tubulin using glutathione S-transferase (GST)–HsIFT81N. Whereas tubulin does not bind the GSH beads and is not pulled down by GST alone, a substantial portion is pulled down by GST-HsIFT81N, demonstrating binding. Whereas the single-point mutation R87E does not strongly impair binding, the K73K75/EE double mutant (mut¹) results in reduced amounts of pulled-down tubulin, indicating reduced binding. (E) Quantification of tubulin binding to untagged HsIFT81N by microscale thermophoresis reveals a K_d of 16 μM. (F) The HsIFT81N mut¹ has drastically reduced binding with a K_d of 187 μM, showing that the basic patch is required for tubulin binding. (G) Microscale thermophoresis titration of tubulin with truncated HsIFT7481 complex reveals a K_d of 0.9 μM. The curves in (E), (F), and (G) are calculated for three independent experiments, and the error bars represent the mean ± SD. (H) The experiments shown in (D) to (G), along with the data in fig. S5, suggest a model in which IFT81N recognizes the globular domain of tubulin, providing specificity, and IFT74N binds the acidic tail of β-tubulin, providing increased affinity.

Fig. 2. Tubulin binding by IFT81 is required for ciliogenesis in human cells

(A) Transient expression of Flag-IFT81, but not the tubulin-binding–deficient IFT81 mutants (in green), rescues the ciliogenesis defect after IFT81 siRNA knockdown. Primary cilia formation was induced by 0.5 μM cytochalasin D and detected by antibody to Arl13b (in red). CAP350 (in blue, inset images only) was used to visualize centrosomes. Mut¹ and Mut² are K73K75/EE and K73K75K113K114R115/EEEEE tubulin-binding mutants, respectively. Scale bar, 5 μm. (B) Quantification of the rescue experiment shown in (A). n = 3 independent experiments; statistical analyses by one-way analysis of variance.

Fig. 3. IFT81N is not required for normal IFT

(A) Immunofluorescence analysis of methanol-fixed trypanosomes expressing the indicated YFP fusion proteins from the endogenous locus stained with an antibody to green fluorescent protein (GFP) (green) and with the antibody to PFR2 L8C4 to visualize the flagellum (red). The left panel corresponds to a control strain expressing YFP::IFT81 and the right panel to the mutant YFP::IFT81Dm, where the IFT81N CH domain is unfolded. Scale bar, 5 μm. (B) Kymograph generation and separation of anterograde and retrograde traces. Kymographs were extracted from videos of cells expressing YFP::IFT81 (movie S1) or YFP::IFT81Dm (movie S2). Panels show the complete kymograph, anterograde events, and retrograde events (from left to right). The x axis corresponds to the length of the flagellum (horizontal scale bar, 5 μm) and the y axis to the elapsed time (vertical scale bar, 5 s). (C) Quantitation of the kymograph analysis shown in (B). Anterograde (blue) and retrograde velocity (red) distribution of IFT particles are calculated from cells expressing YFP::IFT81 and YFP::IFT81Dm. The kymographic analysis reveals robust anterograde trafficking with a speed of 1.75 ± 0.55 μm/s for YFP::IFT81 (n = 294 tracks from 15 cells) and 1.68 ± 0.72 μm s⁻¹ for YFP::IFT81Dm (n = 244 tracks from 15 cells). These values are in line with those reported for anterograde movement of GFP-IFT52 (15). Curiously, retrograde transport was slowed down in the case of YFP::IFT81Dm, where a second population of relatively slow trains was detected.

Fig. 4. Model for tubulin transport and ciliary length control

(A) Fraction of IFT complex bound to tubulin at varying tubulin concentrations is plotted using the equation O_IFT = [Tub]/{K_d + [Tub]}. O_IFT is the fraction of IFT bound to tubulin, K_d is the binding constant that is experimentally determined in this study as 0.9 μM, and [Tub] is the local concentration of free tubulin at the base of the cilium. (B) From the point of initiation of flagellar regeneration, the relationship between the ciliary length, the concentration of anterograde IFT particles, and O_IFT is plotted.