Studies from our group and others suggest that the importins, traditionally thought of as nucleocytoplasmic shuttles, may function as ciliary transporters as well. Our earlier work demonstrated that a small transmembrane protein Crb3b (also called Crumbs3-CLPI) binds to importin β1. In addition, Crb3b colocalizes with importin β1 in the primary cilia, which suggests that importin β1 may target Crb3b to this organelle (Fan et al., 2007
). In a separate report, we showed that importin β2 (also called transportin) directly interacts with RP2. Of note, knockdown of importin β2 blocked the ciliary localization of both endogenous RP2 and EGFP-RP2, implicating importin β2 as the ciliary targeting protein for RP2 (Hurd et al., 2011
). This is in agreement with concurrent studies from our group that identified two conserved NLS sequences encoded in KIF17, a kinesin-2 motor. It was discovered that the C-terminal NLS sequence serves as an authentic CLS. Mutation of the CLS barred KIF17 ciliary entry, and this observation led to the elucidation of a mechanism in which importin β2 binds the CLS of KIF17 and regulates its ciliary localization in a Ran GTP–dependent manner (Dishinger et al., 2010
). In collaboration with the Verhey laboratory, we also found that Ran GTP was enriched in the primary cilia of NIH 3T3 cells (Dishinger et al., 2010
In this study, we expand upon the existing paradigm and explore the functional role of Ran GTP in the centrosome/basal body and its relationship to ciliogenesis. To this end, we tested the Ran GTP distribution in several cell lines and tissues that display different types of cilia. We found that Ran GTP is remarkably enriched in both cilia and the basal bodies. We recognized a tight association between cell culture conditions that favor cilia formation—for instance, polarization of MDCK cells and serum starvation of TERT RPE cells—and a marked accumulation of Ran GTP at the centrosomes. In MDCK cells, ciliogenesis begins when the cells are fully polarized, and this process usually takes 5–7 d to complete. In fibroblasts and some epithelial cells, serum-free culture initiates primary cilia formation within 24–48 h. These findings prompted us to propose that up-regulation of Ran GTP may be a crucial effector for ciliogenesis.
If Ran GTP is indeed a required factor in ciliogenesis, then we imagined that adjusting intracellular Ran GTP levels should yield profound effects on cilia formation. Fortuitously, earlier work showed that Ran GTP regulatory proteins reside in the centrosomes, among them the well-described RanBP1 (Di Fiore et al., 2003
). RanBP1 is not a catalytic partner that regulates GTP/GDP turnover of Ran. Rather, RanBP1 binds to RanGAP1, and RanGAP1 promotes the hydrolysis of Ran GTP to Ran GDP (Bischoff et al., 1995
; Kuhlmann et al., 1997
; Lounsbury and Macara, 1997
). In addition, RanBP1 inhibits the activity of RCC1, a Ran GEF, and thereby suppresses the Ran GDP to Ran GTP conversion (Hayashi et al., 1995
). In short, the presence of RanBP1 promotes the formation of Ran GDP.
Thus we focused our attention on RanBP1 as a molecular tool to regulate Ran GTP. We demonstrated that endogenous and epitope-tagged RanBP1 localizes to cilia and/or basal bodies in several ciliated cell lines. Next, we reduced RanBP1 levels by shRNA-knockdown techniques and evaluated its effects on cilia formation and intracellular Ran GTP in TERT RPE cells. We found that RanBP1-knockdown RPE cells robustly initiated primary cilia formation independent of serum starvation, normally a required condition for ciliogenesis. We noted that Ran GTP is simultaneously increased in the centrosomes/basal bodies of RanBP1 RPE–knockdown cells. These experiments provide additional support for the concept that Ran GTP enrichment at the centrosome promotes cilia formation.
It is known that knockdown of RanBP1 can have small effects on the progression of the cell cycle and induce apoptosis (Tedeschi et al., 2007
). In our system, we saw a slight, 4–6% decrease of the mitotic index in RanBP1-knockdown TERT RPE cells by fluorescence-activated cell sorting (FACS) analysis (Supplemental Figure S3, A and B); however, this could not explain the nearly 20- to 30-fold increase we saw in ciliogenesis. Similarly, in IMCD3 cells RanBP1 knockdown resulted in specific defects in cilia trafficking, independent of ciliogenesis. In addition, overexpression of RanBP1 has been reported to impair S phase entry and the mitotic cycle and to induce multipolar spindles (Battistoni et al., 1997
; Guarguaglini et al., 1997
; Di Fiore et al., 2003
). We did not detect any spindle or centrosome cohesion defects in cells overexpressing EGFP-RanBP1 in TERT RPE cells. Our FACS results showed that overexpression of EGFP-RanBP1 did not block cell cycle exit from G1 to G0 after serum starvation (Supplemental Figure S4, C and D). In the report by Di Fiore et al. (2003
), the authors observed an approximately fourfold increase of overexpressed RanBP1 in their experiment, but only the cells with the highest levels showed mitotic centrosomal abnormalities. This suggests that the loss of cilia formation in the presence of EGFP-RanBP1 is not a secondary effect of cell cycle defects. Thus our findings suggest that ciliogenesis correlates best with levels of Ran GTP in the cytoplasm and centrosome and not with cell cycle effects.
Although we do find Ran GTP and RanBP1 concentrated at the ciliary tips in our study, we do not yet understand their function in this specialized location. It is interesting to note that the specific placement of importin β and RanBP1 mRNAs within the neuronal axon results in their localized protein synthesis when the axon is injured. Subsequently, importin α and the dynein motor are released from their association with Ran GTP. The proteins reshuffle to form an importin α/importin β/dynein cassette, and this complex transports specific cargoes to the cell body to signal axon injury (Lai et al., 2008
; Yudin et al., 2008
). It is tempting to speculate that the loss of KIF17-mCit from ciliary tips in RanBP1-knockdown cells may be explained by a universal Ran/importin/RanBP1–regulated anterograde/retrograde transport system in polarized cellular extensions such as axons or cilia.
Because Ran GTP accrues in the basal bodies, we attempted to identify the specific Ran GEF responsible for the GDP/GTP turnover in this organelle. The Ran GEF RCC1 exists exclusively in the nuclei, and we did not detect RCC1 in the centrosome by immunocytochemical or immunoblot analysis (S. Fan and B. L. Margolis, unpublished results). Recently, it was reported that RanBP10 might act as a cytosolic Ran GEF (Schulze et al., 2008
). However, we did not observe any effects on ciliogenesis when we overexpressed RanBP10 in RPE cells (S. Fan and B. L. Margolis, unpublished results). There is also the possibility that Ran GTP is created in the nucleus and then enters the cytoplasm to concentrate at the basal body.
In summary, our studies suggest that the accumulation of Ran GTP at the centrosome might function as a local transit center: releasing ciliary cargoes from importin α/β complexes, allowing these cargo proteins to reshuffle binding partners, enabling the cargoes to dock with ciliary elements such as IFT complexes, and ultimately initiating and maintaining ciliogenesis. In RPE and similar nonpolarized cells such as fibroblasts, interfering with the formation of Ran GTP by RanBP1 overexpression was sufficient to block ciliogenesis. In contrast, increasing the level of Ran GTP by RanBP1 knockdown appears to promote ciliogenesis in RPE cells. In a polarized cell such as IMCD3, modulating Ran GTP levels did not appear to affect ciliogenesis but did affect trafficking of the Kif17 motor. Thus careful control of Ran GTP levels may be necessary for optimal protein trafficking into and within the cilia. Perturbation of trafficking induced by lowering Ran GTP levels may be sufficient to prevent ciliogenesis, especially in nonpolarized cells. However, other explanations are possible for Ran-mediated modulation of ciliogenesis. For example, in spindle formation, the Ran importin system releases spindle assembly factors such as HURP and TPX2 that promote formation of the spindles (Kalab and Heald, 2008
). In a similar manner Ran and importins could regulate ciliary microtubular dynamics during cilia formation in addition to having effects on ciliary transport. Regardless of mechanism, it is clear from our studies that there is a coordinated regulation of Ran GTP levels at the centrosome and cilia that is essential for proper cilia formation and function.