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

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258497
Author(s):  
Tatsuro Noguchi ◽  
Kentaro Nakamura ◽  
Yuuki Satoda ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
Extracellular Vesicles ◽  
Protein Trafficking ◽  
Hedgehog Signaling ◽  
Intraflagellar Transport ◽  
Wild Type ◽  
Ciliary Membrane ◽  
Ciliary Protein ◽  
Exogenous Expression ◽  
Turnaround Process

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.

scholarly journals Interaction of heterotrimeric kinesin-II with IFT-B–connecting tetramer is crucial for ciliogenesis

2018 ◽  
Vol 217 (8) ◽  
pp. 2867-2876 ◽  
Author(s):  
Teruki Funabashi ◽  
Yohei Katoh ◽  
Misato Okazaki ◽  
Maho Sugawa ◽  
Kazuhisa Nakayama
Keyword(s):  
Protein Interaction ◽  
Protein Trafficking ◽  
Molecular Basis ◽  
Intraflagellar Transport ◽  
Wild Type ◽  
Immunoprecipitation Assay ◽  
Exogenous Expression

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.

scholarly journals Genetic interaction of mammalian IFT-A paralogs regulates cilia disassembly, ciliary protein trafficking, Hedgehog signaling and embryogenesis

2019 ◽  
Author(s):  
Wei Wang ◽  
Bailey A. Allard ◽  
Tana S. Pottorf ◽  
Jay L. Vivian ◽  
Pamela V. Tran
Keyword(s):  
Protein Trafficking ◽  
Hedgehog Signaling ◽  
Double Mutant ◽  
Primary Cilia ◽  
Genetic Interaction ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Ciliary Protein ◽  
Transport Defect

AbstractPrimary cilia are sensory organelles that are essential for eukaryotic development and health. These antenna-like structures are synthesized by intraflagellar transport protein complexes, IFT-B and IFT-A, which mediate bi-directional protein trafficking along the ciliary axoneme. Here using mouse embryonic fibroblasts (MEF), we investigate the ciliary roles of two mammalian orthologues of Chlamydomonas IFT-A gene, IFT139, namely Thm1 (also known as Ttc21b) and Thm2 (Ttc21a). Thm1 loss causes perinatal lethality, and Thm2 loss allows survival into adulthood. At E14.5, the number of Thm1;Thm2 double mutant embryos is lower than that for a Mendelian ratio, indicating deletion of Thm1 and Thm2 causes mid-gestational lethality. We examined the ciliary phenotypes of mutant MEF. Thm1-mutant MEF show decreased cilia assembly, shortened primary cilia, a retrograde IFT defect for IFT and BBS proteins, and reduced ciliary entry of membrane-associated proteins. Thm1-mutant cilia also show a retrograde transport defect for the Hedgehog transducer, Smoothened, and an impaired response to Smoothened agonist, SAG. Thm2-null MEF show normal ciliary dynamics and Hedgehog signaling, but additional loss of a Thm1 allele impairs response to SAG. Further, Thm1;Thm2 double mutant MEF show enhanced cilia disassembly, and relative to Thm1-null MEF, increased impairment of IFT81 retrograde transport and of INPP5E ciliary import. Thus, Thm1 and Thm2 have unique and redundant roles in MEF. Thm1 regulates cilia assembly, and together with Thm2, cilia disassembly. Moreover, Thm1 alone and together with Thm2, regulates ciliary protein trafficking, Hedgehog signaling, and embryogenesis. These findings shed light on mechanisms underlying Thm1-, Thm2- or IFT-A-mediated ciliopathies.

scholarly journals Interaction of WDR60 intermediate chain with TCTEX1D2 light chain of the dynein-2 complex is crucial for ciliary protein trafficking

2018 ◽  
Vol 29 (13) ◽  
pp. 1628-1639 ◽  
Author(s):  
Yuki Hamada ◽  
Yuta Tsurumi ◽  
Shohei Nozaki ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
Cell Lines ◽  
Light Chain ◽  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
Immunoprecipitation Assay ◽  
Established Cell Lines ◽  
Ciliary Protein ◽  
Established Cell ◽  
Exogenous Expression ◽  
Auxiliary Role

The dynein-2 complex mediates trafficking of ciliary proteins by powering the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Although 11 subunits are known to constitute the dynein-2 complex, with several light-chain subunits shared by the dynein-1 complex, the overall architecture of the dynein-2 complex has not been fully clarified. Utilizing the visible immunoprecipitation assay, we demonstrated the interaction modes among the dynein-2 subunits, including previously undefined interactions, such as that between WDR60 and the TCTEX1D2–DYNLT1/DYNLT3 dimer. The dynein-2 complex can be divided into three subcomplexes, namely DYNC2H1–DYNC2LI1, WDR34–DYNLL1/DYNLL2–DYNLRB1/DYNLRB2, and WDR60–TCTEX1D2–DYNLT1/DYNLT3. We established cell lines lacking WDR60 or TCTEX1D2, both of which are dynein-2–specific subunits encoded by ciliopathy-causing genes, and found that both WDR60-knockout (KO) and TCTEX1D2-KO cells show defects in retrograde ciliary protein trafficking, with WDR60-KO cells demonstrating more severe defects probably due to failed assembly of the dynein-2 complex. The exogenous expression of a WDR60 mutant lacking TCTEX1D2 binding partially restored retrograde trafficking to a level comparable to that of TCTEX1D2-KO cells. Thus, our results demonstrated that WDR60 plays a major role and TCTEX1D2 plays an auxiliary role in the dynein-2 complex to mediate retrograde ciliary protein trafficking.

scholarly journals Anterograde trafficking of ciliary MAP kinase–like ICK/CILK1 by the intraflagellar transport machinery is required for intraciliary retrograde protein trafficking

2020 ◽  
Vol 295 (38) ◽  
pp. 13363-13376 ◽  
Author(s):  
Kentaro Nakamura ◽  
Tatsuro Noguchi ◽  
Mariko Takahara ◽  
Yoshihiro Omori ◽  
Takahisa Furukawa ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
Extracellular Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Ciliary Protein ◽  
Mitogen Activated Protein ◽  
Exogenous Expression ◽  
Bidirectional Movement ◽  
Dependent Transport ◽  
G Protein Coupled

ICK (also known as CILK1) is a mitogen-activated protein kinase–like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)–B complex of the IFT machinery and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of ICK knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary G protein–coupled receptors. In addition, we found that in ICK knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and released into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in ICK knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.

scholarly journals ARL13B regulates Sonic Hedgehog signaling from outside primary cilia

2019 ◽  
Author(s):  
Eduardo D. Gigante ◽  
Megan R. Taylor ◽  
Anna A. Ivanova ◽  
Richard A. Kahn ◽  
Tamara Caspary
Keyword(s):  
Signal Transduction ◽  
Sonic Hedgehog ◽  
Protein Trafficking ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Complete Loss ◽  
Wild Type ◽  
Sonic Hedgehog Signaling ◽  
Biochemical Function

AbstractARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358A protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13bV358A/V358A mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13bV358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B’s role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.

scholarly journals ARL13B regulates Sonic hedgehog signaling from outside primary cilia

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Eduardo D Gigante ◽  
Megan R Taylor ◽  
Anna A Ivanova ◽  
Richard A Kahn ◽  
Tamara Caspary
Keyword(s):  
Signal Transduction ◽  
Sonic Hedgehog ◽  
Protein Trafficking ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Complete Loss ◽  
Wild Type ◽  
Sonic Hedgehog Signaling ◽  
Biochemical Function

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358A protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13bV358A/V358A mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13bV358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B’s role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.

scholarly journals Interactions of the dynein-2 intermediate chain WDR34 with the light chains are required for ciliary retrograde protein trafficking

2019 ◽  
Vol 30 (5) ◽  
pp. 658-670 ◽  
Author(s):  
Yuta Tsurumi ◽  
Yuki Hamada ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
Protein Trafficking ◽  
Binding Sites ◽  
Intraflagellar Transport ◽  
Dominant Negative ◽  
Light Chains ◽  
Wd40 Repeat ◽  
Phenotypic Differences ◽  
Repeat Domain ◽  
Ciliary Protein ◽  
Intermediate Chain

The dynein-2 complex drives retrograde ciliary protein trafficking by associating with the intraflagellar transport (IFT) machinery, containing IFT-A and IFT-B complexes. We recently showed that the dynein-2 complex, which comprises 11 subunits, can be divided into three subcomplexes: DYNC2H1–DYNC2LI1, WDR34–DYNLL1/DYNLL2–DYNLRB1/DYNLRB2, and WDR60–TCTEX1D2–DYNLT1/DYNLT3. In this study, we demonstrated that the WDR34 intermediate chain interacts with the two light chains, DYNLL1/DYNLL2 and DYNLRB1/DYNLRB2, via its distinct sites. Phenotypic analyses of WDR34-knockout cells exogenously expressing various WDR34 constructs showed that the interactions of the WDR34 intermediate chain with the light chains are crucial for ciliary retrograde protein trafficking. Furthermore, we found that expression of the WDR34 N-terminal construct encompassing the light chain–binding sites but lacking the WD40 repeat domain inhibits ciliary biogenesis and retrograde trafficking in a dominant-negative manner, probably by sequestering WDR60 or the light chains. Taken together with phenotypic differences of several WDR34-knockout cell lines, these results indicate that incorporation of DYNLL1/DYNLL2 and DYNLRB1/DYNLRB2 into the dynein-2 complex via interactions with the WDR34 intermediate chain is crucial for dynein-2 function in retrograde ciliary protein trafficking.

scholarly journals Loss of dynein-2 intermediate chain Wdr34 results in defects in retrograde ciliary protein trafficking and Hedgehog signaling in the mouse

2017 ◽  
Vol 26 (13) ◽  
pp. 2386-2397 ◽  
Author(s):  
Chuanqing Wu ◽  
Jia Li ◽  
Andrew Peterson ◽  
Kaixiong Tao ◽  
Baolin Wang
Keyword(s):  
Protein Trafficking ◽  
Hedgehog Signaling ◽  
Ciliary Protein ◽  
Intermediate Chain

scholarly journals Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein–coupled receptors

2017 ◽  
Vol 28 (3) ◽  
pp. 429-439 ◽  
Author(s):  
Tomoaki Hirano ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
G Protein Coupled Receptors ◽  
Protein Protein Interactions ◽  
Retrograde Trafficking ◽  
The Core ◽  
Ciliary Protein ◽  
G Protein Coupled

Cilia serve as cellular antennae where proteins involved in sensory and developmental signaling, including G protein–coupled receptors (GPCRs), are specifically localized. Intraflagellar transport (IFT)-A and -B complexes mediate retrograde and anterograde ciliary protein trafficking, respectively. Using a visible immunoprecipitation assay to detect protein–protein interactions, we show that the IFT-A complex is divided into a core subcomplex, composed of IFT122/IFT140/IFT144, which is associated with TULP3, and a peripheral subcomplex, composed of IFT43/IFT121/IFT139, where IFT139 is most distally located. IFT139-knockout (KO) and IFT144-KO cells demonstrated distinct phenotypes: IFT139-KO cells showed the accumulation of IFT-A, IFT-B, and GPCRs, including Smoothened and GPR161, at the bulged ciliary tips; IFT144-KO cells showed failed ciliary entry of IFT-A and GPCRs and IFT-B accumulation at the bulged tips. These observations demonstrate the distinct roles of the core and peripheral IFT-A subunits: IFT139 is dispensable for IFT-A assembly but essential for retrograde trafficking of IFT-A, IFT-B, and GPCRs; in contrast, IFT144 is essential for functional IFT-A assembly and ciliary entry of GPCRs but dispensable for anterograde IFT-B trafficking. Thus the data presented here demonstrate that the IFT-A complex mediates not only retrograde trafficking but also entry into cilia of GPCRs.

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

2021 ◽  
Vol 32 (1) ◽  
pp. 45-56
Author(s):  
Takuya Kobayashi ◽  
Yamato Ishida ◽  
Tomoaki Hirano ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
G Protein ◽  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
G Protein Coupled Receptors ◽  
Retrograde Trafficking ◽  
Ciliary Protein ◽  
G Protein Coupled

Little is known about how the IFT-A and IFT-B complexes of the intraflagellar transport machinery cooperate to mediate ciliary protein trafficking. We show that IFT144-IFT122 and IFT88-IFT52-IFT46 contribute to the IFT-A-IFT-B interface and that the interaction of IFT-A with IFT-B is required for retrograde trafficking of the IFT machinery and ciliary entry of G protein-coupled receptors.

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