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

Abstract

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

Functional exploration of heterotrimeric kinesin-II in IFT and ciliary length control in Chlamydomonas

“…In contrast to most kinesins with two identical motor subunits, kinesin-II consists of two non-identical motor subunits and one non-motor subunit (KAP) ( Hirokawa et al, 2009 ; Verhey and Hammond, 2009 ). The heterotrimeric organization of kinesin-II is required for IFT because mutation in either subunit abolishes or impairs IFT in various organisms ( Engelke et al, 2019 ; Kozminski et al, 1995 ; Liang et al, 2014 ; Lin et al, 2003 ; Miller et al, 2005 ; Mueller et al, 2005 ; Nonaka et al, 1998 ; Snow et al, 2004 ). This may result if a homodimer of the motor subunits cannot be properly formed and/or a homodimer cannot associate with KAP or IFT complexes.…”

“…Though the assay conditions would slightly affect the measurements, the velocity of anterograde IFT is ~2.2 μm/s in Chlamydomonas and Trypanosome ( Bertiaux et al, 2018a ; Brown et al, 2015 ; Dentler, 2005 ; Engel et al, 2009 ; Liang et al, 2014 ; Wingfield et al, 2017 ). In contrast, mammalian cells and worms have a much slower velocity (~0.5 μm/s) ( Broekhuis et al, 2014 ; Engelke et al, 2019 ; Follit et al, 2006 ; Snow et al, 2004 ). Notably, Chlamydomonas and Trypanosoma have longer cilia whereas mammalian cells tend to have shorter cilia.…”

Intraflagellar transport trains and motors: Insights from structure

“…The principal motor driving anterograde movement of IFT trains is heterotrimeric kinesin-II (Kif3 in mammals) [ 12 , 35 , 36 , 88 , 89 ]. Within the heterotrimer, the Kif3A and Kif3B subunits each comprise an N-terminal motor domain, coiled-coil segments mediating heterodimerization, and putatively disordered tail ( Fig.…”

“…In vertebrates, a homodimeric kinesin-II (Kif17) also localizes to cilia, but does not function in conventional IFT [ 88 , 122 ]. Kif17 has been found to interact with a different site of the IFT train compared to C. elegans OSM-3 [ 81 ], and appears to be carried into cilia as a cargo; a process that is regulated by its nuclear localization signal [ 123 ].…”

Phosphosite T674A mutation in kinesin family member 3A fails to reproduce tissue and ciliary defects characteristic of CILK1 loss of function

“…18 Anterograde IFT mediated by KIF3A/KIF3B/KAP is essential for the assembly and maintenance of primary cilia. 19,20 The C-terminal cargo-binding domain of human KIF3A contains multiple phosphorylation sites, including PKA site Ser687, CaMKII sites Thr692 and Ser696, and CILK1 site Thr672. Phosphorylation of S687/T692/S696 enhances the cargo-binding and trafficking activities of KIF3A.…”