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

Kinesin is part of the microtubule-binding motor protein superfamily, which serves important roles in cell division and intraorganellar transport. The heterotrimeric kinesin-2, consisting of the heterodimeric motor subunits, kinesin family member 3A/3B (KIF3A/3B), and kinesin-associated protein 3 (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 human telomerase reverse transcriptase-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 to 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.


Introduction 30
Cilia are microtubule-based protrusions with sensory and/or motile functions. In mammals, 31 defects in assembly and maintenance of cilia results in a series of diseases called "ciliopathies"

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Cilia of Chlamydomonas can be regenerated to full length in two hours, and unlike 104 mammalian cells, ciliary assembly does not need to be induced (Rosenbaum et al., 1969) to 105 result in heterogeneous population of ciliated and non-ciliated cells. The molecular mechanism 106 of the nucleo-cytoplasmic trafficking is likely conserved between the Chlamydomonas and 107 humans (Li et al., 2018). However, there are fewer constituent nucleoporins in the 108 Chlamydomonas NPC compared to that of humans (Neumann et al., 2006). By using both 109 mammalian and Chlamydomonas cells, we have found that ciliary trafficking of kinesin 110 associated protein KAP3 is regulated by RanGTP. We demonstrated that precise manipulation 111 of RanGTP level is crucial for regulating cilium formation. Importantly, we were able to clearly 112 show that RanGTP plays a direct role in incorporation of ciliary proteins that is independent of 113 its nuclear roles. These results provide potential insights for the molecular mechanism 114 orchestrating multi-compartment trafficking of the heterotrimeric kinesin-2 motor complex.

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Further, they answer a long-standing open question in the field about whether nuclear import 116 mechanisms have been coopted for direct ciliary import.

Results 119
Ciliary protein KAP3 can localize to the nucleus 120 The heterotrimeric kinesin-2 motor complex consists of the heterodimeric motor proteins 121 KIF3A/3B and the adaptor protein KAP3. In contrast, the homodimeric kinesin-2 KIF17 motor 122 does not need an adaptor protein to exert its function. Although it was suggested that KAP3 123 functions as a linker between KIF3A/3B and the specific cargoes to facilitate intracellular 124 transport, the function of KAP3 is still not well characterized. To explore this, we firstly 125 investigated the intracellular localization of KAP3 in ciliated and non-ciliated cells. HA-tagged 5 distributed in the cytoplasm. We also showed that EGFP-tagged KAP3A and KAP3B could 135 localize in the nucleus of MDCK cells ( Figure 1C). Nuclear localization of KAP3A was 136 consistent with that of EGFP tagged KAP3A in a previous report (Tenny et al., 2016).

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To dissect critical regions within KAP3A responsible for its nuclear localization, as depicted 138 in Figure 1D, EGFP-fused KAP3 truncations were constructed (henceforth, KAP3 refers to the 139 long isoform KAP3A). First, the expression of appropriately-sized truncations in MDCK cells was 140 detected by western blot analysis ( Figure 1E). Second, the subcellular distributions of these 141 KAP3 truncations in MDCK cells were analyzed via fluorescence microscopy. As shown in  6-9, were predominantly localized in the nucleus, which is similar to full-length KAP3. These 146 data indicate that the region between amino acids 461 and 660 is crucial for nuclear localization 147 of KAP3. Taken together, our data demonstrate that ciliary protein KAP3 can localize to the 148 nucleus under the control of armadillo repeats 6-9.

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As shown in Figure 1, KAP3 is distributed to the nucleus in different cells. To determine the 152 molecular mechanism of KAP3 nuclear translocation, we tested a well-studied pathway for 153 protein nuclear import, RanGTP mediated nuclear import, which requires a high concentration of 154 RanGTP in the nucleus for the disassembly of the imported complexes. To determine whether 155 RanGTP drives nuclear import of KAP3, the dominant negative mutant RanQ69L which cannot 156 hydrolyze GTP, was used in this study. As shown in Figure 2A, Ectopic expression of RanQ69L 157 blocked nuclear localization of KAP3 in COS-7 cells, resulting in a more cytoplasmic distribution 158 of KAP3 relative to wild-type controls. This data suggests that nuclear translocation of KAP3 is 159 mediated by a RanGTP-dependent nuclear import pathway.

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We mapped the region responsible for nuclear localization of KAP3 and found that KAP3

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It was reported that the import receptor importin β2 plays critical roles in both nuclear import

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KAP3 can localize in both the cilium and nucleus. We dissected the regions required for 182 nuclear translocation of KAP3. Next, we mapped the regions required for ciliary targeting of 183 KAP3 in hTERT-RPE cells. As depicted in Figure 3A, full-length KAP3 contains three regions: a 184 non-conserved N-terminal domain, nine armadillo repeats, and a C-terminal conserved domain 185 (Jimbo et al., 2002;Shimuzu et al., 1996). Based on this, a series of truncations of KAP3 were 186 generated and intracellular localization of these truncations was examined after cilium induction.

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As shown in Figure 3B

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These data indicate that the RanGTP level in hTERT-RPE cells is a determinant of initiation of 222 cilium formation. Taken together, the ability to bind and hydrolyze GTP by Ran in vivo, revealed 223 by different dominant Ran mutants, regulates its essential functions on the generation of cilia.

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To determine why there is reduced cilium formation in RanQ69L-expressing RPE cells, we

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To exclude that the phenotype was caused by the off-target effect of the inhibitor, the GTP-248 locked Ran1 mutant Ran1Q73L, corresponding to human RanQ69L, was transformed into

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Chlamydomonas. As shown in Figure 5C, there is strong Ran1Q73L expression as expected in

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These results demonstrated that RanGTP plays pivotal roles in ciliary length regulation in 257 Chlamydomonas.

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We showed that KAP3, but not IFT81, couldn't be targeted to ciliary base in hTERT-RPE

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To tease apart nuclear and non-nuclear effects, we were able to use the small molecular 285 inhibitor cycloheximide (CHX) to inhibit new protein synthesis during cilia regeneration. As 286 shown in Figure 6A, in wild-type Chlamydomonas cells, this typically results in growth of cilia to  Figure 6B). To confirm that the lack of ciliary 299 growth wasn't due to cell toxicity and that IPZ only impacts the ability of existing proteins to 300 enter cilia, we washed out IPZ but still continued CHX treatment to inhibit new protein synthesis.

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There is increasing data that nuclear import and ciliary import shares similar mechanisms,

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CA). The unpaired student's t-test was used to assess statistical significance of two groups. A 501 value of p < 0.05 was considered statistically significant.

Competing interests 504
The authors declare no competing or financial interests 505 506 Acknowledgements 507 16 We would like to thank Dr. Yuh Min Chook (University of Texas Southwestern Medical Center), 508 Drs. Kristen Verhey and Benjamin Allen (University of Michigan) for providing DNA plasmids.

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We also thank our colleague Dr. Pamela Tran for sharing some reagents. We are grateful to the