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Dynein-2–driven intraciliary retrograde trafficking indirectly requires multiple interactions of IFT54 in the IFT-B complex with the dynein-2 complex

Hiyamizu, Shunya Qiu, Hantian Tsurumi, Yuta Hamada, Yuki Katoh, Yohei Nakayama, Kazuhisa 京都大学 DOI:10.1242/bio.059976

2023.07

概要

Within cilia, the dynein-2 complex needs to be transported as an anterograde cargo to achieve its role as a motor to drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. We previously showed that interactions of WDR60 and the DYNC2H1-DYNC2LI1 dimer of dynein-2 with multiple IFT-B subunits, including IFT54, are required for the trafficking of dynein-2 as an IFT cargo. However, specific deletion of the IFT54-binding site from WDR60 demonstrated only a minor effect on dynein-2 trafficking and function. We here show that the C-terminal coiled-coil region of IFT54, which participates in its interaction with the DYNC2H1-DYNC2LI1 dimer of dynein-2 and with IFT20 of the IFT-B complex, is essential for IFT-B function, and suggest that the IFT54 middle linker region between the N-terminal WDR60-binding region and the C-terminal coiled-coil is required for ciliary retrograde trafficking, probably by mediating the effective binding of IFT-B to the dynein-2 complex, and thereby ensuring dynein-2 loading onto the anterograde IFT trains. The results presented here agree with the notion predicted from the previous structural models that the dynein-2 loading onto the anterograde IFT train relies on intricate, multivalent interactions between the dynein-2 and IFT-B complexes.

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Biology Open (2023): doi:10.1242/bio.BIO059976: Supplementary information

Fig. S1. Analyses of IFT54 genomic sequences of the IFT54-KO cell line

(A) Genomic DNA extracted from the IFT54-KO cell line #IFT54-4-3 was subjected to PCR using primer sets

(a, b, and c; Table S3) to detect alleles with forward (b) or reverse (c) integration of the donor knock-in vector

or no insertion/small indel (a). (B) The amplified DNAs with no insertion/small indel (allele 1) and reverse

integration of the donor vector (allele 2) were subjected to sequence analysis. Note that for allele 2, as an

unrelated long sequence from human chromosome 1 (LOC126805873), in addition to the knock-in vector

sequence, was inserted, we could not determine the exact insertion site of the knock-in vector. However, as the

abnormal cilia-lacking phenotype of the cell line #IFT54-4-3 was rescued by exogenous expression of

IFT54(WT) (see Fig. 1D, E), the abnormal phenotype is not likely to result from an off-target effect. (C, D)

Control RPE1 cells (C) and the #IFT54-4-3 cell line (D) were cultured under serum-starved conditions for 24 h

boxed regions are shown on the right. Scale bars, 5 µm. The #IFT54-4-3 cell line could not form cilia, consistent

with the disruption of both IFT54 alleles.

Biology Open • Supplementary information

to induce ciliogenesis, and doubly immunostained for FOP and ARL13B. Enlarged (2.5-fold) images of the

Biology Open (2023): doi:10.1242/bio.BIO059976: Supplementary information

Fig. S2: Uncropped images

Fig. 2: mChe-IFT54

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Fig. 2: IFT88

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Fig. S2: Uncropped images

Fig. 2: IFT52

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Fig. 2: IFT81

Biology Open (2023): doi:10.1242/bio.BIO059976: Supplementary information

Fig. S2: Uncropped images

Fig. 2: IFT25

Fig. 2: GAPDH

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Fig. S1A

Biology Open (2023): doi:10.1242/bio.BIO059976: Supplementary information

Table S1. Plasmids used in this study

Vector

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Reference