Interaction of heterotrimeric kinesin-II with IFT-B-connecting tetramer is crucial for ciliogenesis.

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.

Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system.

The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.

Ciliary entry of KIF17 is dependent on its binding to the IFT-B complex via IFT46-IFT56 as well as on its nuclear localization signal.

Cilia function as cellular antennae to sense and transduce extracellular signals. A number of proteins are specifically localized in cilia. Anterograde and retrograde ciliary protein trafficking are mediated by the IFT-B and IFT-A complexes in concert with kinesin-2 and dynein-2 motors, respectively. However, the role of KIF17, a homodimeric kinesin-2 protein, in protein trafficking has not been fully understood in vertebrate cilia. In this study, we demonstrated, by using the visible immunoprecipitation assay, that KIF17 interacts with the IFT46-IFT56 dimer in the IFT-B complex through its C-terminal sequence located immediately upstream of the nuclear localization signal (NLS). We then showed that KIF17 reaches the ciliary tip independently of its motor domain and requires IFT-B binding for its entry into cilia rather than for its intraciliary trafficking. We further showed that KIF17 ciliary entry depends not only on its binding to IFT-B but also on its NLS, to which importin α proteins bind. Taking the results together, we conclude that in mammalian cells, KIF17 is dispensable for ciliogenesis and IFT-B trafficking but requires IFT-B, as well as its NLS, for its ciliary entry across the permeability barrier located at the ciliary base.

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E.

ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46 -: IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E.

COPI-mediated retrieval of SCAP is crucial for regulating lipogenesis under basal and sterol-deficient conditions.

Retrograde trafficking from the Golgi complex to endoplasmic reticulum (ER) through COPI-coated vesicles has been implicated in lipid homeostasis. Here, we find that a block in COPI-dependent retrograde trafficking promotes processing and nuclear translocation of sterol regulatory element binding proteins (SREBPs), and upregulates the expression of downstream genes that are involved in lipid biosynthesis. This elevation in SREBP processing and activation is not caused by mislocalization of S1P or S2P (also known as MBTPS1 and MBTPS2, respectively), two Golgi-resident endoproteases that are involved in SREBP processing, but instead by increased Golgi residence of SREBPs, leading to their increased susceptibility to processing by the endoproteases. Analyses using a processing-defective SREBP mutant suggest that a fraction of SREBP molecules undergo basal cycling between the ER and Golgi in complex with SREBP cleavage-activating protein (SCAP). Furthermore, we show that SCAP alone is retrieved from the Golgi and moves to the ER after processing of SREBP under sterol-deficient conditions. Thus, our observations indicate that COPI-mediated retrograde trafficking is crucial for preventing unnecessary SREBP activation, by retrieving the small amounts of SCAP-SREBP complex that escape from the sterol-regulated ER retention machinery, as well as for the reuse of SCAP.