Molecular connections between nuclear and ciliary import processes
© Kee and Verhey; licensee BioMed Central Ltd. 2013
Received: 22 May 2013
Accepted: 30 July 2013
Published: 28 August 2013
As an organelle, the cilium contains a unique complement of protein and lipid. Recent work has begun to shed light on the mechanisms that regulate entry of ciliary proteins into the compartment. Here, we focus on the mechanisms that regulate ciliary entry of cytosolic molecules. Studies have revealed a size exclusion mechanism for ciliary entry that is similar to the barrier to nuclear entry. Active import into the ciliary compartment involves nuclear trafficking components including importins, a Ran-guanosine triphosphate gradient, and nucleoporins. Together, this work indicates that nuclei and cilia share molecular, structural and mechanistic components that regulate import into the compartments.
KeywordsCilia Ciliary pore complex Flagella Nuclear import Nuclear pore complex Nucleoporin Ran Size exclusion
Eukaryotic cells have evolved to maintain specialized functions and morphologies by compartmentalizing cellular activities within topologically distinct organelles such as the nucleus, mitochondrion and endoplasmic reticulum. Recent work has suggested that the cilium is also a specialized organelle. Cilia and flagella are microtubule-based organelles that protrude from the cell surface and function in cellular motility and extracellular sensing. For example, motile cilia (or flagella) beat to move mucus up the respiratory tract, establish left-right asymmetry in the embryonic node, and propel sperm. Non-motile cilia, also called primary or sensory cilia, were once believed to be vestigial organelles without complex function. They are now known to act as cellular ‘signaling antennas’ responsible for a variety of functions including olfaction in olfactory neurons, photoreception in photoreceptor cells, mechanosensing of fluid flow in kidney epithelial cells, and responding to extracellular signals like Hedgehog, Wnt and platelet-derived growth factor ligands (reviewed in [1, 2]). The modern view of primary cilia as sensory antennae has been driven by recent findings that defects in ciliary formation, function and/or signaling underlie a group of phenotypically diverse disorders now known as ciliopathies [3, 4].
An important characteristic of the cilium or flagellum is that the organelle protrudes from the cell surface such that the ciliary membrane is continuous with the plasma membrane and the intraciliary space is exposed to the cytosolic space. This raises the important question of how ciliary components are targeted to and/or retained in the organelle. For example, structural components such as the outer dynein arm and radial spoke complexes of motile cilia are assembled in the cytosol and trafficked specifically to the cilium [5, 6]. In addition, the enrichment of many membrane and soluble signaling factors in the ciliary compartment is required for proper motile and sensory function. For example, in the Hedgehog pathway, trafficking of soluble Gli transcription factors through the ciliary compartment is required for proper Gli proteolysis and subsequent transcriptional output [7, 8].
Two pathways for ciliary trafficking need to be considered - entry and exit of membrane proteins, and entry and exit of cytosolic proteins. Several lines of evidence support the idea that ciliopathy gene products and septins play important roles in regulating the entry and exit of membrane proteins [13–17]. In this review, we will focus on the trafficking of cytosolic proteins into the ciliary compartment.
Is there a barrier for entry of soluble proteins into the ciliary compartment?
As the intraciliary space appears to be continuous with that of the cytosolic space, whether entry of cytosolic components into the ciliary compartment is restricted is an important question. Using soluble GFP (approximately 27 kDa, 4.2 nm × 2.4 nm barrel) as a model protein in Xenopus photoreceptor cells, Calvert et al. showed that the connecting cilium (the transition zone equivalent) provides only a modest barrier to diffusion between the inner and outer segments . Further work showed that tandem GFP proteins, 2xGFP (approximately 54 kDa) and 3xGFP (approximately 81 kDa), freely entered the outer segment compartment, albeit to a lesser extent than single GFP . This work concluded that no diffusion barrier exists to regulate the entry of cytosolic proteins into the ciliary compartment, at least for proteins of up to approximately 80 kDa. Rather, size-restricted flux into photoreceptor outer segments was postulated to be due to steric volume exclusion within this compartment . In this model, the membranous discs and high protein concentration in the outer segment reduce the aqueous volume available to soluble molecules such that larger molecules will be less abundant in this environment than smaller proteins.
One possible explanation for the differences between the work of Najafi et al.  and Kee et al.  is the transport substrate, in that the former study used proteins linked as beads on a string and the later study used globular proteins of different sizes. To directly compare entry into the primary cilium to that of photoreceptors, we created fusion proteins containing tandem fluorescent proteins (FPs). Like single GFP, proteins consisting of two FPs (approximately 54 kDa) or three FPs (approximately 81 kDa) were able to enter into primary ciliary (Figure 2). Although fusing FPs in tandem increases the molecular weight and the length of the molecule in a linear fashion, the width of the single and tandem FPs are the same and they are therefore able to cross the diffusion barrier and enter the outer segment of photoreceptor cells  and primary cilia of hTERT-RPE cells (Figure 2). Collectively, this work indicates that a ciliary barrier restricts the free entry of soluble proteins into the compartment and that a variety of features, including molecular weight and the overall structural conformation of a transport substrate, impact a molecule’s ability to cross this barrier.
A recent study approached the issue of access of soluble proteins to the ciliary compartment by using a high-affinity interaction induced by the drug rapamycin to trap soluble proteins that diffuse into primary cilia . This technique allowed the authors to specifically measure the kinetics of ciliary accumulation of proteins of various sizes. The authors found that steric volume exclusion is not likely to be a defining feature of the barrier in primary cilia. Rather, the ciliary barrier was found to behave like a molecular sieve in that the entry of proteins into primary cilia was restricted in a size-dependent manner. The major discrepancy with the work of Kee et al.  appears to be in the size for restricted entry; Lin et al.  found that large multimeric complexes up to 8 nm in radius and 650 kDa in size could become trapped in the cilium.
Two parameters must be kept in mind when evaluating the differences between these studies. The first is experimental. Each of the experimental setups (microinjection and dimerization-induced trapping) has its drawbacks. Whereas the trapping of FPs in the ciliary compartment enables better visualization of the ciliary proteins over the cytosolic pool (a major limitation in the microinjection system), the use of a membrane protein as an anchor for the ‘trap’ may cause aberrant entry of large cytosolic proteins into the ciliary compartment. Clearly, more work is needed to define the physical properties of the ciliary barrier. The second parameter that must be considered is that factors in addition to molecular weight are likely to influence protein mobility and movement through the pore.
Collectively, these experiments demonstrate that entry of soluble proteins into the ciliary compartment is restricted by a size-based exclusion mechanism. This is reminiscent of entry into the nucleus, which has mechanisms in place to prevent entry of cytosolic molecules. Protein gateways, the NPCs, span the nuclear envelope and create pores that function to control the exchange of molecules between the cytoplasm and nucleoplasm. The NPC forms a permeability barrier and allows the diffusional entry of small molecules (<40 kDa) but hinders the passage of larger molecules, thus maintaining the nucleus as a privileged domain with unique composition [23–25]. This protects the eukaryotic cell’s genetic material and transcriptional machinery, and ensures proper functioning of nuclear activities.
Nucleoporins constitute a ciliary pore complex at the base of the cilium
What are the molecular components of the diffusion barrier at the base of cilia? Nucleoporin proteins make up the NPCs that are embedded in the nuclear envelope and regulate entry into this compartment [26–28]. Recent work has shown that endogenous and expressed nucleoporins also localize to the base of primary and motile cilia in mammalian cells  to form a CPC. Furthermore, nucleoporin function is required for the gated entry of the cytosolic kinesin-2 motor KIF17 into the ciliary compartment . Although further work is needed to verify and extend these results in other ciliated cells, this work demonstrates that the nuclear and ciliary barriers share molecular components that regulate organelle composition. These results raise many interesting questions about the molecular, structural and evolutionary relationships between the NPC and CPC.
Another important question concerning the relationship between the NPC and the CPC concerns the overall structure of the CPC. Each NPC has typically an eight-fold rotational symmetry [31, 32], although pores with nine- or ten-fold symmetry have been noted [33, 34]. By contrast, the cilium is characterized by nine-fold symmetry due to the core microtubule doublets of the axoneme (Figure 1). It is not clear whether the difference between the eight-fold symmetry of the NPC and the nine-fold symmetry of the cilium is important, as we do not have any information about how the nucleoporin subunits are arranged at the base of the cilium to form an actual pore. One possibility is that there is one large pore at the base of the cilium with the axoneme protruding through the middle of the pore (Figure 3A). Such a pore would presumably have a nine-fold symmetry based on that of the axoneme. An alternative possibility is that there are nine pores positioned between the Y-links at the base of the cilium (Figure 3B). In this scenario, each CPC would retain the characteristic eight-fold symmetry of the NPC. In support of this possibility, recent electron cryotomography analysis of isolated basal body structures from the protist Tetrahymena pyriformis demonstrated the presence of an electron-dense ‘terminal plate’ structure that spans the ciliary base and contains nine pore structures, one adjacent to each microtubule doublet of the axoneme (Figure 3C) . Are these Tetrahymena CPCs of the terminal plate the same barriers as the nucleoporin-containing CPCs found in mammalian primary and motile cilia? One striking finding in support of this is that the CPCs in the Tetrahymena terminal plate have a diameter of approximately 53 nm, similar to the pore diameter of mammalian NPCs . In addition, proteomic analysis of the isolated Tetrahymena basal bodies identified proteins involved in nuclear transport including Ran and the transmembrane nucleoporin NDC-1 . Further proteomic and structural analysis will reveal the exact molecular composition of the CPC and its organization at the ciliary base.
The shared gating mechanism of nuclei and cilia has evolutionary implications as well. Cilia are found in a wide range of eukaryotic taxa and were already present in the last eukaryotic common ancestor . Unlike nuclei, cilia were then independently lost from multiple eukaryotic lineages (for example, fungi, amoebae and some plants) [38, 39]. Recent work has uncovered structural and sequence similarities between outer ring nucleoporins, intraflagellar transport (IFT) proteins, and vesicle coat proteins (COPs and clathrins) [40–44]. These findings have led to the hypothesis that a ‘protocoatamer’ gave rise to membrane-coating components during eukaryotic evolution [45, 46]. It thus appears that the evolutionary appearance of both nuclei and cilia involved the adaptation of an ancestral protocoatamer component into both gating (NPC and CPC) and trafficking (IFT, coatamer) components.
Active transport of soluble proteins into the ciliary compartment
Gated entry into the nuclear and ciliary compartments has shared mechanisms beyond the size-exclusion barrier and nucleoporin-containing pore complexes. Entry of proteins above the size barrier into the nuclear compartment requires an active transport mechanism involving cytosolic recognition of nuclear localization sequences (NLS) by transport receptors called importins (or karyopherins), shuttling across the NPC, and release of NLS-containing proteins in the nuclear compartment by the small G protein Ran. Interestingly, entry of cytosolic proteins into the ciliary compartment has also been shown to utilize an NLS-like signal, importins and Ran.
Two classes of NLS have been described. First, the classical NLS consists of one or two stretches of basic residues that bind directly to an importin-α adaptor protein and thereby indirectly to importin-β1 in order to traverse the NPC. The best-studied NLSs of this class are the monopartite sequence of the SV40 large T antigen and the bipartite sequence of nucleophosmin . Second, nonclassical NLSs have diverse amino acid sequences that bind directly and specifically to other members of the importin-β family. Best-studied in this class is the M9 sequence from the heterogeneous nuclear ribonucleoprotein A1 protein, which binds directly to importin-β2 (transportin-1) .
Further work demonstrated that an NLS and importin-β2 are required for ciliary entry of retinitis pigmentosa 2 (RP2), a lipid-anchored peripheral membrane protein . In this case, both classical and nonclassical NLS sequences were identified in the retinitis pigmentosa 2 primary sequence and mutational analysis determined that the nonclassical sequence is critical for mediating ciliary entry of retinitis pigmentosa 2 . That a nonclassical NLS binds to importin-β2 and mediates transport across the CPC parallels what has been observed for nuclear import. The fact that KIF17 appears to use a classical NLS to interact with importin-β2 and traverse the CPC is puzzling. Further mutational analysis of the KIF17 NLS is required to define the sequence parameters that mediate the interaction with importin-β2 and ciliary entry.
Importin-β1 has been shown to bind to the ciliary transmembrane proteins Crumbs  but whether this interaction regulates ciliary entry is unknown. Expression of dominant negative importin-β1 or knockdown of the endogenous protein resulted in defects in ciliogenesis , suggesting that importins and their cargoes play important roles in ciliary processes in addition to regulating ciliary entry.
A Ran gradient for directional transport
An important question is how the ciliary RanGTP/GDP gradient is generated. Cytosolic RanGDP is generated, at least in part, by Ran GTPase activating protein and its cofactor RanBP1 (reviewed in ). Recent work suggests that RanBP1 also plays a role in regulating the ciliary RanGTP/GDP gradient as altering the levels of Ran binding protein 1 had distinct consequences for ciliogenesis . Nuclear RanGTP is generated by the guanine nucleotide exchange factor (GEF) RCC1. As a chromatin-bound protein, RCC1 is localized to the nucleus. Whether RCC1 also functions as a ciliary GEF for Ran or whether a cilia-specific GEF exists is unknown. Ciliary proteomes contain both RCC1 and the related protein RCC2 as well as several proteins with tandem RCC1 repeats, including X-linked retinitis pigmentosa GTPase regulator and Secretion-regulating guanine nucleotide exchange factor [55, 56]. Therefore, identifying the ciliary RanGEF is one of the next key experiments.
In addition to regulating trafficking across the ciliary-cytoplasmic barrier, recent work has shown that Ran regulates ciliogenesis in specific cell types. Ran has been localized to the centrosomes of elongating rat spermatids . In cultured hTERT-RPE cells, modulating RanGTP levels through knockdown or overexpression of Ran binding protein 1 either promoted or abolished ciliogenesis, respectively . As RanGTP regulates microtubule assembly during mitosis , it may also play a critical role in regulating microtubule assembly during axoneme formation. However, manipulating RanGTP levels in polarized MDCK cells had no effect on ciliogenesis but did significantly impair the ciliary trafficking of the kinesin-2 KIF17 motor . Clearly, more work is needed to understand the role of Ran during ciliogenesis and ciliary trafficking.
Conclusions and future directions
The work described above indicates that import into the nuclear and ciliary compartments share molecular, structural and mechanistic components. These findings raise the possibility that other regulators of nuclear-cytoplasmic trafficking may function to regulate ciliary protein localization and/or function. For example, small, ubiquitin-related modifiers (SUMOs) are approximately 100-amino-acid proteins that are covalently yet reversibly attached to substrate proteins during a variety of cellular processes including nuclear-cytoplasmic transport [59, 60]. Recent work has shown that SUMOylation of the small GTPase ARL-13, the worm ortholog of Arl13B that is mutated in the ciliopathy Joubert syndrome, regulates the proper ciliary targeting of various sensory receptors and the corresponding sensory functions . In addition, it seems likely that the nuclear export machinery could play a role in ciliary export processes. A recent paper suggests that phosphorylation of a potential nuclear export sequence regulates the localization of huntingtin protein to the ciliary shaft or the basal body .
The commonalities of nuclear and ciliary import processes raise the intriguing possibility that proteins can play functional roles in both compartments. For example, the IFT motor heterotrimeric kinesin-2 (KIF3A/KIF3B/KAP in mammals) has been found to shuttle between the nuclear and ciliary compartments in sea urchin embryos , although a nuclear function for kinesin-2 is not known. More established is the ciliary to nuclear shuttling of Gli transcription factors in response to extracellular Hedgehog ligand [7, 8]. Furthermore, centriolar proteins such as centrins have been found to play a role in mRNA and protein transport through the NPC [64, 65] and centrosomal and transition zone proteins have been found to localize to both the ciliary and nuclear compartments and have been implicated in the DNA damage response [66–69].
Both nuclear-cytoplasmic and ciliary-cytoplasmic transport events are restricted to interphase in metazoans. However, recent work has suggested that nuclear and ciliary components have important roles in the mitotic phase of the cell cycle. During mitosis, chromatin-bound RCC1 generates a spindle RanGTP gradient that activates spindle assembly factors and organizes spindle microtubules . Nucleoporins such as the NUP107/160 complex relocalize to the kinetochore during prophase, where they regulate spindle assembly and establishment of microtubule/kinetochore attachments [70, 71]. IFT components such as IFT88 support the formation of astral microtubules and thereby orientation of the mitotic spindle in dividing cells . Other IFT proteins, including IFT27, IFT46, IFT72 and IFT139, accumulate at the cleavage furrow of dividing Chlamydomonas cells , hinting for a role of IFT proteins in cytokinesis. These and other findings that ciliary proteins have important non-ciliary functions (for example, see ) have broad implications in understanding the disease mechanisms for ciliopathies.
Ciliary pore complex
Guanine nucleotide exchange factor
Green fluorescent protein
Nuclear localization sequence
Nuclear pore complex
Small ubiquitin-related modifiers.
Work in the laboratory of KJV is supported by the National Institute of General Medical Sciences (RO1-070862) and by the University of Michigan Center for Organogenesis. We thank John Dishinger, Lynne Blasius and Jay Pieczynski for discussions and support.
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