Cooperation of the IFT-A complex with the IFT-B complex is required for ciliary retrograde protein trafficking and GPCR import

Abstract

Cilia sense and transduce extracellular signals via specific receptors. The intraflagellar transport (IFT) machinery mediates not only bidirectional protein trafficking within cilia but also the import/export of ciliary proteins across the ciliary gate. The IFT machinery is known to comprise two multisubunit complexes, namely, IFT-A and IFT-B; however, little is known about how the two complexes cooperate to mediate ciliary protein trafficking. We here show that IFT144–IFT122 from IFT-A and IFT88–IFT52 from IFT-B make major contributions to the interface between the two complexes. Exogenous expression of the IFT88(Δα) mutant, which has decreased binding to IFT-A, partially restores the ciliogenesis defect of IFT88-knockout (KO) cells. However, IFT88(Δα)-expressing IFT88-KO cells demonstrate a defect in IFT-A entry into cilia, aberrant accumulation of IFT-B proteins at the bulged ciliary tips, and impaired import of ciliary GPCRs. Furthermore, overaccumulated IFT proteins at the bulged tips appeared to be released as extracellular vesicles. These phenotypes of IFT88(Δα)-expressing IFT88-KO cells resembled those of IFT144-KO cells. These observations together indicate that the IFT-A complex cooperates with the IFT-B complex to mediate the ciliary entry of GPCRs as well as retrograde trafficking of the IFT machinery from the ciliary tip. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

CCRK/CDK20 regulates ciliary retrograde protein trafficking via interacting with BROMI/TBC1D32

“…As shown in S1 and S2 Videos and in Fig 2S and 2T, the release of ECVs from ciliary tips was often observed by live-cell imaging of CCRK-KO cells expressing ARL13B(ΔGD)-EGFP. These observations, in conjunction with our previous studies [30,71], indicate that the IFT turnaround process at the tip is impaired in the absence of CCRK, and indicate that overaccumulated proteins at the ciliary tips are packaged within ECVs and eliminated to relieve cilia from excessive stress. As bulbous ciliary tips were observed in Ccrk-KO mice [44,77], it is likely that ECV release also occurs in vivo in the absence of CCRK.…”

“…In control RPE1 cells, IFT88 (an IFT-B subunit) was detected mainly at the base of cilia, with a small proportion detected at the tip (Fig 2E). By contrast, the proportion of IFT88 found at the ciliary tip was significantly increased in CCRK-KO cells (Fig 2F We previously showed that, in ICK-KO cells and IFT144-KO cells, as well as in cells with a compromised interaction between the IFT-A and IFT-B complexes, ciliary proteins that are excessively accumulated at the ciliary tips owing to an impairment in the turnaround process are packaged into ECVs and eliminated to relieve cilia of the stress of protein overaccumulation [30,71]. This was also the case in CCRK-KO cells.…”

“…Live-cell imaging to observe the release of ECVs from cilia was performed as described previously [30,71,72]. Briefly, CCRK-KO cells expressing EGFP-fused ARL13B(ΔGD) were serum-starved for 24 h on a glass-bottom culture dish (MatTek) and observed under an A1R-MP confocal laser-scanning microscope (Nikon).…”

Interactions between TULP3 tubby domain cargo site and ARL13B amphipathic helix promote lipidated protein transport to cilia

“…The IFT-A core comprises of IFT144, IFT122 and IFT140, whereas IFT43, IFT139 and IFT121 are peripheral subunits (Behal et al, 2012;Hirano et al, 2017;Mukhopadhyay et al, 2010). ARL13B levels in cilia are reduced in both IFT122 (Takahara et al, 2018) and IFT144 (Kobayashi et al, 2021) knockout cell lines. A recent proteomics study showed that the flagellar levels of multiple ARF family GTPases such as ARL13B, and other myristoylated and farnesylated proteins were reduced in an IFT-140 null Chlamydomonas mutant (Picariello et al, 2019).…”

Intraflagellar Transport Proteins as Regulators of Primary Cilia Length

“…Loss of the IFT74 residues required to bind IFT-A caused stunted cilia, thus revealing a role for the interdependence between IFT-B and IFT-A in ciliogenesis ( Brown et al, 2015 ). Additionally, in mammalian retinal pigment epithelial (RPE) cells, expression of a truncated form of IFT88 on a CRISPR/Cas9-mediated IFT88 knockout background produced a ciliary phenotype similar to IFT-A knockout cells ( Kobayashi et al, 2021 ). In C. reinhardtii and in mice, Ift54 -null mutants lack cilia ( Berbari et al, 2011 ; Zhu et al, 2017b ), and a recent study showed that in C. reinhardtii and mammalian cells, IFT54 interacts with both the kinesin-2 and dynein motors ( Zhu et al, 2020 ).…”