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

Funabashi, Teruki; Katoh, Yohei; Michisaka, Saki; Terada, Masaya; Sugawa, Maho; Nakayama, Kazuhisa

doi:10.1091/mbc.e16-09-0648

Cited by 52 publications

(80 citation statements)

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“…This is consistent with studies demonstrating that loss of function in KIF3A, KIF3B, or KAP results in a complete absence of cilia in vertebrate organisms [15, 42–44]. However, KIF17 binds to IFT-B particles and co-migrates with IFT trains in mammalian cilia [45, 46], suggesting that this motor could take over from KIF3A/KIF3B/KAP and function in cilia maintenance in a similar manner as its worm homolog OSM-3. We tested this hypothesis by co-expressing i3A/i3B and KIF17 in the Kif3a -/- ; Kif3b -/- cells.…”

Section: Resultssupporting

confidence: 89%

Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

Engelke

Waas

Kearns

et al. 2018

Preprint

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The trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in IFT. We used a chemicalgenetic approach to engineer an inhibitable KIF3A/KIF3B (i3A/i3B) kinesin-2 motor that is capable of rescuing WT motor function in Kif3a/Kif3b double-knockout cells. Inhibitor addition blocks ciliogenesis or, if added to ciliated cells, blocks IFT within two minutes, which leads to a complete loss of primary cilia within six hours. The kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells nor delay the disassembly of full-formed cilia upon i3A/i3B inhibition.These data suggest that KIF3A/KIF3B/KAP is the sole and essential motor for cilia assembly and function in mammalian cells, indicating a species-specific adaptation of kinesin-2 motors for IFT function. E n g e l k e e t a l . 2 Δ 11 ), 12 (i3A Δ 12 ), or 13 (i3A Δ 13 ) amino acids. In similar fashion, we fused the DmrB domain to the N-terminus of KIF3B truncated by five (i3B Δ 5 ), six (i3B Δ 6 ), or seven (i3B Δ 7) amino acids (Fig. 2b). We compared the ability of each i3A construct to pair with each i3B construct and generate primary cilia in the absence but not in the presence of B/B inhibitor. Fusion of the Δ 12 /i3B Δ 6 motor resulted in ~200-fold luciferase induction (Fig. 3c). Addition of B/B inhibitor resulted in a greater increase in luciferase activity induction only in cells expressing the i3A Δ 12 /i3B Δ 6 motor, reflecting pathway hyper-activation only in cells that lack a functional primary cilium (Fig. 3c).In summary, we find that expression of the i3A Δ 12 and i3B Δ 6 constructs in Kif3a -/-;Kif3b -/cells results in a bona fide inhibitable kinesin-2 motor. The engineered i3A Δ 12 /i3B Δ 6 motor is referred to as i3A/i3B throughout the rest of the manuscript. Inhibition of KIF3A/KIF3B results in the stalling of IFT trains and their exclusion from ciliaThe generation of an inhibitable kinesin-2 motor enables us, for the first time, to directly examine the role of KIF3A/KIF3B/KAP motors during IFT in fully-formed cilia. To investigate this, Kif3a -/-;Kif3b -/cells were transfected with plasmids for expressing the inhibitable i3A/i3 motor together with a fluorescently-tagged subunit of the IFT-B complex (IFT88-mNG; mNeonGreen) as in previous studies [34, 35]. Analysis of kymographs generated from live-cell imaging experiments revealed that IFT88-marked IFT trains moved processively towards the tip of the cilium, paused for variable durations, and then trafficked back towards the base (Fig. 4a), similar to what has been observed previously [34, 35]. Anterograde and retrograde speeds were on the order of 0.7 μ m/s, consistent with previously E n g e l k e e t a l . 5

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Section: Resultssupporting

confidence: 89%

Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

Engelke

Waas

Kearns

et al. 2018

Preprint

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“…It is reported that the NLS-like sequence in the C-terminal region of KIF17 is required for its ciliary targeting (Dishinger et al, 2010). This NLS-like sequence was further confirmed as a classical mono-partite NLS (Funabashi et al, 2017). However, we noticed that PL, the PY variant, is located in the immediate downstream region of this NLS.…”

Section: Discussionsupporting

confidence: 54%

“…(Dishinger et al, 2010;Hurd et al, 2001;Madugula et al, 2016;Torrado et al, 2016;Han et al, 2017). However, additional data has shown that importin α1 and α6, but not importin β2, are responsible for ciliary targeting of soluble KIF17 (Funabashi et al, 2017). In general, importin β1, alone or in cooperation with importin α, transports substrates with a conventional NLS (Lange et al, 2007), whereas importin β2 transports substrates that contain the nonconventional PY-NLS (Lee et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

“…It was proposed that there are shared mechanisms between ciliary import and nuclear import (Dishinger et al, 2010;Takao et al, 2014;Del Viso et al, 2016;Takao et al, 2017;Endicott et al, 2018). Several results indicated that RanGTP/importinβ/NLS import system is required for ciliary targeting of either membrane or soluble proteins including importin β1 for Crumsb3 (Fan et al, 2007), RanGTP/importin β2 for KIF17 (Dishinger et al, 2010), importin β2 for RP2 , importin α1/α6/NLS for KIF17 (Funabashi et al, 2017), and importin β2/PY-NLS for GLI2/GLI3 (Han et al, 2017). It was also reported that importin β2/Rab8 forms a ternary complex with ciliary localization sequences to direct membrane protein trafficking to cilia (Madugula et al, 2016), suggesting that this process is independent of RanGTP and a NLS or PY-NLS.…”

Section: Introductionmentioning

confidence: 99%

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RanGTP regulates cilium formation and ciliary trafficking of a kinesin-II subunit independent of its nuclear functions

Huang

Avasthi

2019

Preprint

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9Kinesin is part of the microtubule (MT)-binding motor protein superfamily, which exerts crucial 10 functions in cell division and intracellular transport in different organelles. The heterotrimeric 11 kinesin-2, consisting of the kinesin like protein KIF3A/3B heterodimer and kinesin-associated 12 protein KAP3, is highly conserved across species from the green alga Chlamydomonas to 13 humans. It plays diverse roles in cargo transport including anterograde (base to tip) trafficking in 14 cilium. However, the molecular determinants mediating trafficking of heterotrimeric kinesin-2 15 itself is poorly understood. Using the unicellular eukaryote Chlamydomonas and mammalian 16 cells, we show that RanGTP regulates ciliary trafficking of KAP3. We found the armadillo repeat 17 region 6-9 (ARM6-9) of KAP3, required for its nuclear translocation, is sufficient for its targeting 18 to the ciliary base. Given that KAP3 is essential for cilia formation and the emerging roles of 19 RanGTP/importin β in ciliary protein targeting, we further investigate the effect of RanGTP in 20 cilium length regulation in these two different systems. We demonstrate that precise control of 21 RanGTP levels, revealed by different Ran mutants, is crucial for cilium formation and 22 maintenance. Most importantly, we were able to segregate RanGTP regulation of ciliary protein 23 incorporation from of its nuclear roles. Our work provides important support for the model that 24 nuclear import mechanisms have been coopted for independent roles in ciliary import. 25 26

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“…In the latter system, as in C. elegans amphid channel cilia, there is a 1 : 1 stoichiometry of the BBSome to IFT particles and both heterotrimeric (KIF3) and homodimeric (KIF17) kinesin-2 motors traffic along the OSN cilia, but unlike in C. elegans amphids, homodimeric kinesin-2 is not required to build the distal region of the axoneme [95]. Some evidence supports the hypothesis that KIF17 may be transported as passive cargo associated with IFT train subcomplex B to the distal tip of the cilium, where its function is unknown [137]. Other studies, on the other hand, provide evidence that KIF17 may actively cooperate with IFT-B to deliver cyclic nucleotide-gated channels and dopamine receptors to the membranes of primary cilia [133,134].…”

Section: Vertebratesmentioning

confidence: 92%

Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery

2017

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Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance and length-control of cilia, which play critical roles in motility, sensory reception and signal transduction in virtually all eukaryotic cells. During IFT, anterograde kinesin-2 and retrograde IFT-dynein motors drive the bidirectional transport of IFT trains that deliver cargo, for example axoneme precursors such as tubulins as well as molecules of the signal transduction machinery, to their site of assembly within the cilium. Following its discovery in Chlamydomonas, IFT has emerged as a powerful model system for studying general principles of motor-dependent cargo transport and we now appreciate the diversity that exists in the mechanism of IFT within cilia of different cell-types. The absence of heterotrimeric kinesin-2 function, for example, causes a complete loss of both IFT and cilia in Chlamydomonas but following its loss in C. elegans, where its primary function is loading the IFT machinery into cilia, homodimeric kinesin-2-driven IFT persists and assembles a full-length cilium. Generally, heterotrimeric kinesin-2 and IFT-dynein motors are thought to play widespread roles as core IFT-motors whereas homodimeric kinesin-2 motors are accessory motors that mediate different functions in a broad range of cilia, in some cases contributing to axoneme assembly or the delivery of signaling molecules but in many other cases their ciliary functions, if any, remain unknown. In this review, we focus on mechanisms of motor action, motor cooperation and motor-dependent cargo delivery during IFT.

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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

Abstract: The ciliary kinesin KIF17 is a cargo, but not a motor, of the IFT-B complex. The ciliary entry of KIF17 through the transition zone depends on its binding to IFT-B, as well as on its nuclear localization signal.

Cited by 52 publications

References 50 publications

Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

RanGTP regulates cilium formation and ciliary trafficking of a kinesin-II subunit independent of its nuclear functions

Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery

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