Separable roles for RanGTP in nuclear and ciliary trafficking of a kinesin-2 subunit

Abstract

Kinesin is part of the microtubule (MT)-binding motor protein superfamily, which serves important roles in cell division and intraorganellar transport. The heterotrimeric kinesin-2, consisting of the heterodimeric motor subunits KIF3A/3B and kinesin-associated protein KAP3, is highly conserved across species from the unicellular eukaryote Chlamydomonas to humans. It plays diverse roles in cargo transport including anterograde (base to tip) trafficking in cilia. However, the molecular determinants mediating trafficking of heterotrimeric kinesin-2 itself are poorly understood. It has been previously suggested that ciliary transport is analogous to nuclear transport mechanisms.Using Chlamydomonas and hTERT-immortalized retinal pigment epithelial cell line, we show that RanGTP, a small GTPase that dictates nuclear transport, regulates ciliary trafficking of KAP3, a key component for functional kinesin-2. We found that the armadillo repeat region 6-9 (ARM6-9) of KAP3, required for its nuclear translocation, is also necessary and sufficient for its targeting to the ciliary base. Given that KAP3 is essential for cilium formation and there are the emerging roles for RanGTP/importin β in ciliary protein targeting, we further investigated the effect of RanGTP in cilium formation and maintenance. We found that precise control of RanGTP levels, revealed by different Ran mutants, is crucial for cilium formation and maintenance. Most importantly, we were able to provide orthogonal support in an algal model system that segregates RanGTP regulation of ciliary protein trafficking from its nuclear roles. Our work provides important support for the model that nuclear import mechanisms have been co-opted for independent roles in ciliary import.

Motor Cooperation During Mitosis and Ciliogenesis

“…The base and tip of the cilia are important zones for switching between anterograde and retrograde IFT. The RanGTP nuclear trafficking machinery controls the import of some kinesins into cilia (Dishinger et al 2010, Huang et al 2020. Moreover, to enter or exit the cilium, the IFT machinery must cross the TZ, a dense gating structure that resists the passage of both antero-and retrograde IFT trains, so fully active kinesin-2 and dynein-2 are required to transport the IFT machinery across it (De-Castro et al 2022, Prevo et al 2015.…”

Primary Cilia: A Cellular Regulator of Articular Cartilage Degeneration

“…Intraflagellar transport (IFT) is a two-way cargo transport system, and all signaling within the primary cilia must be IFT-dependent to complete. Generally, it is divided into anterograde IFT (moves from the base of a cilium to the tip) and retrograde IFT (moves from the tip back to the base), which are mediated by heterotrimeric kinesin-2 and cytoplasmic dynein 2 motor (some call cytoplasmic dynein 1b motor), respectively [ 36 , 37 ]. In this IFT train, when the cargo needs to be transported in the basal body, the IFT-B complex aggregates and activates kinesin-2 and moves the cargo to the tip of axoneme and they dissociate, releasing the substance; then, the new cargo comes in and kinesin-2 deactivates whereas dynein 2 activates in bonding with the IFT-A complex and transports the new cargo to the basal body [ 38 ].…”

Non-transport roles of nuclear import receptors: In need of the right balance

“…KAP3 has armadillo-repeats with which importin beta-1 interacts and required both for KAP3 nuclear import and ciliary targeting. Chlamydomonas, a model system with cilia regenerating capacity, proved informative to disentangle these processes, due to the evolutionary conservation of KAP3: in that system, assays combining importazole (an inhibitor of importin beta1), and cycloheximide (inhibiting ciliogenesis during regeneration), demonstrated the functional separation of ciliary transport and nuclear import of KAP3 ( Huang et al, 2021 ).…”