Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia

Williams, Corey L.; McIntyre, Jeremy C.; Norris, Stephen R.; Jenkins, Paul M.; Zhang, Lian; Pei, Qinglin; Verhey, Kristen; Martens, Jeffrey R.

doi:10.1038/ncomms6813

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Published: 15 December 2014

Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia

Corey L. Williams¹,
Jeremy C. McIntyre¹,
Stephen R. Norris²,
Paul M. Jenkins³,
Lian Zhang¹,
Qinglin Pei⁴,
Kristen Verhey² &
…
Jeffrey R. Martens¹

Nature Communications volume 5, Article number: 5813 (2014) Cite this article

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Abstract

Cilia dysfunction underlies a class of human diseases with variable penetrance in different organ systems. Across eukaryotes, intraflagellar transport (IFT) facilitates cilia biogenesis and cargo trafficking, but our understanding of mammalian IFT is insufficient. Here we perform live analysis of cilia ultrastructure, composition and cargo transport in native mammalian tissue using olfactory sensory neurons. Proximal and distal axonemes of these neurons show no bias towards IFT kinesin-2 choice, and Kif17 homodimer is dispensable for distal segment IFT. We identify Bardet–Biedl syndrome proteins (BBSome) as bona fide constituents of IFT in olfactory sensory neurons, and show that they exist in 1:1 stoichiometry with IFT particles. Conversely, subpopulations of peripheral membrane proteins, as well as transmembrane olfactory signalling pathway components, are capable of IFT but with significantly less frequency and/or duration. Our results yield a model for IFT and cargo trafficking in native mammalian cilia and may explain the penetrance of specific ciliopathy phenotypes in olfactory neurons.

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Introduction

The cilium is a sensory organelle that serves specialized roles on diverse cell types throughout eukaryotes. Disruption of cilia in vertebrates gives rise to developmental patterning defects, progressive degenerative disorders and sensory deficits. Penetrance of congenic human ciliopathy disease phenotypes varies among different tissues and can be influenced by the gene that is disrupted, the nature of mutation and the genetic background of the individual¹. The precise mechanisms of this variable penetrance are unclear, in particular when mutations affect ubiquitously expressed cilia genes. Across eukaryotes, cilia and flagella are built and maintained by intraflagellar transport (IFT), a process in which macromolecular protein ‘trains’ comprising IFT-A and IFT-B subcomplexes bidirectionally traverse the ciliary microtubule axoneme via association with kinesin and dynein motors². Mutations in several human IFT genes are linked to a group of gestational skeletal disorders^3,4,5,6 and, recently, one gene encoding a core IFT complex B protein was revealed as a disease locus for Bardet–Biedl syndrome (BBS)⁷. BBS is a heterogeneous pleiotropic ciliopathy that shows penetrance in a number of organs and clinically manifests in obesity, polydactyly, renal cyst formation, retinal cell death, male infertility and anosmia. Of note, only these latter two conditions are due to the absence of cilia structures^8,9,10 and it is unclear why cilia in one cell type or tissue are lost but persist (or degenerate slowly) in other organs.

Work in lower eukaryotes, namely in Chlamydomonas reinhardtii and Caenorhabditis elegans, has demonstrated functional links between the IFT machinery and BBSome in cilia formation and maintenance and, moreover, currently accounts for the majority of our knowledge concerning IFT dynamics^11,12. In mammals, IFT has been described in only a small number of studies on cultured cells^{13,14,15,16,17,18} and the fundamental questions of how the mammalian IFT machinery operates in native cilia or whether it associates with BBS proteins have not been addressed. These questions are particularly relevant in the context of IFT gene mutations causing BBS⁷ and BBS disease penetrance within the olfactory system where BBS protein disruption is detrimental to ciliation and odour detection^9,10. Importantly, these unanswered questions can have significant implications for therapeutic strategies to treat ciliopathies in different organ systems.

IFT studies in vertebrates present a challenge, because most cilia of vertebrate tissues are inaccessible in the live animal. In contrast, olfactory cilia are uniquely amenable to live analysis due to their direct contact with the external environment via the nasal passage. This enables manipulation of live olfactory sensory neurons (OSNs) without disrupting synaptic connections. Here we exploited adenovirus (AV)-mediated protein expression and live-cell time-lapse imaging to capture IFT and cargo-trafficking events within native mammalian olfactory cilia. We find that the IFT machinery in OSN cilia employs two kinesin motors (heterotrimeric kinesin-2 and homodimeric Kif17), but Kif17 is not required for IFT or OSN cilia maintenance. OSN IFT particle velocities vary stochastically among neurons, but velocities in different cilia of individual neurons are similar to one another. We reveal constituents of the core BBS protein complex as participants in OSN IFT that exist in 1:1 stoichiometry with IFT particles. In addition, peripheral membrane Arl proteins and polytopic transmembrane signalling proteins traffic with IFT in OSN cilia, but the transmembrane proteins do so much less frequency and/or duration. Overall, our findings provide framework for the first model of IFT in a native mammalian setting and implicate the relationship between IFT and the BBSome as a likely culprit in the penetrance of BBS mutations on ciliation in the olfactory epithelium (OE).

Results

Distinct properties of the OSN ciliary axoneme and membrane

To properly dissect ciliary trafficking dynamics in native mammalian olfactory cilia, we first delineated the organization of OSN cilia architecture via molecular profiling in living mouse tissue. In addition to allowing measurement of cilia dynamics, live analysis of ultrastructural features enables accurate appraisal of cilia properties that can be lost with fixation. We used AV-mediated ectopic expression of fluorescent protein (FP)-tagged proteins that comprise various structural elements of the OSN cilium (Fig. 1). En face microscopic investigation of red FP-tagged α-tubulin revealed the microtubule backbones of the many cilia emanating from the dendritic knob of each AV-transduced OSN (Fig. 1c). OSN ciliary basal bodies and transition zones were identified using transgenic Centrin2:GFP mice ectopically expressing Nphp4:mCherry (Fig. 1d–f). Based on Nphp4:mCherry fluorescence, OSN ciliary transition zones measure 0.95±0.12 μm in length (n=72 cilia, 9 OSNs, 3 mice), similar to frog and nematode^19,20. To visualize OSN axoneme microtubule organization, we used green FP (GFP)-tagged EF-hand domain containing 1 (GFP:Efhc1), which associates with interdoublet links connecting doublet microtubules²¹ and is highly enriched in OSNs²². GFP:Efhc1 was restricted specifically within the proximal-most region of OSN cilia, revealing the OSN doublet–singlet microtubule transition at 2.34±0.42 μm from the base of the transition zone (Fig. 1g–i, n=141 cilia, 11 OSNs, 3 mice). Finally, to differentiate the proximal versus distal segments of OSN cilia²³, we co-expressed GFP:Efhc1 with mCherry-tagged ADP-ribosylation factor-like 13b (Arl13b:mCherry), which localizes along the full length of OSN cilia⁶ (Fig. 1j). Collectively, our data showing highly restricted localization patterns of FPs with strong correlation to previous electron microscopy data in rodent²³ demonstrate that ectopic expression accurately delineates endogenous protein localization in cilia of live OSNs.

**Figure 1: Molecular architecture of OSN cilia.**

Specific lipid modifications are sufficient to sequester proteins in membrane microdomains of unique lipid composition or organization²⁴. This is particularly relevant to ciliary membrane organization, given that loss of lipid modifications is detrimental to ciliary localization of Arl13b³⁰, Retinitis Pigmentosa 2 (refs 25, 26), cystin²⁷ and fibrocystin²⁸. We compared the ability of myristoylation and palmitoylation (MyrPalm), dual palmitoylation (PalmPalm) and geranylgeranyl (GerGer) signals (which differentially partition into detergent-resistant membrane domains (Supplementary Fig. 1a,b, see Supplementary Methods)), to enrich GFP within the OSN ciliary membrane. MyrPalm-GFP and PalmPalm-GFP were present along the length of olfactory cilia (Fig. 2a); however, GFP-GerGer was constrained within the OSN ciliary proximal region (Fig. 2a). GFP-GerGer localization extended beyond mCherry:Efhc1 (Fig. 2b), indicating that the GerGer membrane microdomain is distinct from and more extensive than the membrane surrounding the OSN cilium proximal segment proper. In contrast to the differential lipid partitioning observed in OSN cilia, we found that OSN axons are equally permissive to each of the three lipid modifications as seen via GFP infiltration of olfactory bulbs (Fig. 2d). Although the axon initial segment²⁹ and ciliary base^{30,31,32,33,34,35,36} both act as a filter for specific transmembrane or membrane-associated proteins, our data show both are permissive to the lateral enrichment of lipid modified proteins. However, beyond the ciliary base, there exists a differential partitioning of lipid anchors between the OSN proximal and distal segments.

**Figure 2: Differential membrane enrichment of acyl and prenyl anchors in OSN ciliary subdomains.**

Finally, to measure OSN cilia number and length, we used MyrPalm-GFP as an inert marker. OSNs possessed 13±5 cilia (n=81 OSNs, 3 mice) with lengths ranging from 2.5 to 110 μm (22.0±12.8 μm average, n=1,083 cilia, 3 mice; Supplementary Fig. 2a). We saw no correlation between the number and length of cilia on a given cell (Supplementary Fig. 2b; Pearson’s coefficient R²=0.04829) and cilia lengths were highly variable within cells, indicating that OSN cilia length varies stochastically.

Multiple kinesin-2 subtypes traverse entire OSN axonemes

Our data highlight structural and membrane compositional aspects distinguishing specific subdomains of OSN cilia, but the mechanisms by which the proximal and distal segments are established remain unclear. In C. elegans amphid/phasmid channel sensory cilia, two kinesin motors function in coordination to build the proximal/middle and distal ciliary axoneme segments³⁷. To examine whether multiple kinesin motors are used in OSN cilia, we expressed FP-tagged homodimeric Kif17 and a component of the heterotrimeric kinesin-2 (Kap3a). Both Kap3a and Kif17 localized in puncta along the full length of OSN axonemes and showed accumulations at ciliary tips (Fig. 3a–d). Thus, heterotrimeric kinesin-2 is not restricted to the proximal segment of OSN cilia as seen in nematode amphid/phasmid channel cilia. FP-tagged Dync2li1, a component of the ciliary retrograde dynein motor complex, showed similar localization (Fig. 3e,f). To assess ciliary movement of the motor complex proteins, we employed high contrast, rapid acquisition, total internal reflection fluorescence microscopy (TIRFm) on OSNs expressing Kap3a:GFP, Kif17:mCitrine or Dync2li1:GFP. Strikingly, each protein showed robust, processive particle movement in both the anterograde and retrograde directions along the entire OSN cilia (Fig. 3g–k and Supplementary Movie 1). To our knowledge, this is the first capture of IFT in any native tissue of a vertebrate and/or mammalian system.

**Figure 3: Homodimeric and heterotrimeric kinesin-2 motors cooperatively traffic along the full length of OSN cilia.**

Our time-lapse data indicates that both heterotrimeric kinesin-2 and Kif17 participate in IFT along the entire OSN axonemes. We therefore reasoned that the presence of OSN cilia distal segments may not depend solely on the function of Kif17 as is seen with OSM-3 in C. elegans amphid/phasmid channel cilia³⁷. To test this hypothesis, we used a truncated dominant-negative version of Kif17 (Kif17DN) lacking the motor domain. Expression of Kif17DN was not detrimental to OSN cilia formation (Fig. 3l,m), and Kif17DN showed mobility along the entire axonemes via association with IFT particles containing Kap3a (Fig. 3n,o and Supplementary Movie 2). These data indicate that distal segment formation and/or maintenance in OSN cilia is not dependent on Kif17 alone, and that heterotrimeric kinesin-2 is capable of IFT trafficking along the entire length of OSN axonemes when Kif17 is disrupted. Thus, we conclude that OSN cilia use a unique and heretofore undescribed programme featuring at least two types of kinesin motors for trafficking along distal singlet microtubules.

Stochastic variation in IFT velocities among OSNs

The basic mechanistic principles by which IFT trains enable cargo transport within cilia are well established from work in homogeneous age-synchronous cell populations or groups of cells derived from an invariant lineage, but we have no understanding about IFT dynamics in the complex setting of a native mammalian tissue or in multiciliated cells. As individual OSNs differ from each other on many levels including odourant receptor expression, basal stimulation/activation state, age and spatial distribution, the OE is well suited for analysis of IFT dynamics in a heterogeneous context. We measured IFT velocities using FP-tagged components of the IFT-A and IFT-B subcomplexes, which undergo robust bidirectional motility along the full length of OSN cilia (Fig. 4a–h and Supplementary Movie 3). To differentiate anterograde versus retrograde trajectories, only IFT particles travelling in clear view of the ciliary tip were included in our analysis and are representative of IFT velocities specifically on the OSN distal segment. Velocities of both anterograde and retrograde IFT particles ranged from nearly stationary to approaching 0.6 μm s⁻¹ (Fig. 4i), with anterograde and retrograde means of 0.22±0.08 and −0.14±0.08 μm s⁻¹, respectively (n=2,316 IFT122:GFP-labelled particles, 1,941 IFT88:GFP-labelled particles, 300 cilia, 55 OSNs and 9 mice). In addition, the magnitude of anterograde and retrograde mean particle velocities within individual OSN cilia strongly correlate (Fig. 4j, Pearson’s coefficient R²=0.6064). Notably, we saw stochastic variation in anterograde and retrograde velocities among neurons and the velocities measured in multiple cilia within individual cells were more similar to each other than to cilia on other cells (Fig. 4k; permutation test with 20,000 iterations, P<0.0001 for both anterograde and retrograde velocities, see Methods) (n=1,941 IFT88:GFP-labelled particles, 160 cilia, 25 OSNs and 5 mice). Thus, differential regulation of IFT velocity occurs across the OSN population and the velocities of IFT particles in the many cilia projecting from a single OSN are controlled together within each cell.

Although the majority of IFT particles in OSN cilia moved with constant velocity, in some instances we observed non-processive particle trajectories (Supplementary Fig. 3). These included single-particle velocity changes (stalling/restarting and mid-cilium direction reversal), which may represent spatio-temporal events where IFT particles perform maintenance on the axoneme through delivery and/or pickup of cargo³⁸. We also observed unidirectional particle interactions (fusions and fissions) and rare anterograde and retrograde particle collision/fusion events. These observations imply that individual microtubules may be simultaneously used by both unidirectional and opposing particles, and that particle remodelling is not strictly limited to events at the ciliary base and microtubule tips³⁹. Finally, increased particle size was occasionally observed, which may reflect a form of IFT avalanching at the ciliary base or tip as described in C. reinhardtii⁴⁰ and/or snowballing from multiple fusion events along the axoneme.

The BBSome is a constituent of the mammalian OSN IFT complex

Proteins homologous to those associated with human BBS participate in IFT in invertebrates^41,42,43. Although various lines of evidence have implicated mammalian BBS proteins as being resident within pericentriolar satellites⁴⁴, basal bodies⁴⁵ and cilia⁴⁶, no report has demonstrated that mammalian BBS proteins traffic with IFT. We examined constituents of the core BBSome⁴⁶ via expression of FP-tagged BBS1, BBS2 and BBS4 in OSNs. Confocal microscopy revealed strong enrichment of BBSome proteins within OSN knobs (Fig. 5a–f), consistent with basal body localization. In addition, BBS2:GFP was readily detectable in puncta along OSN axonemes (Fig. 5c inset), whereas BBS1:GFP was discretely present in the proximal-most regions of OSN axonemes (Fig. 5b) and BBS4:GFP was occasionally detected at ciliary tips (Fig. 5g). Using TIRFm, we observed that each of the three BBSome fusion proteins undergo bidirectional particle movement along the length of OSN cilia (Fig. 5j–l and Supplementary Movie 4) and do so with velocities similar to FP-tagged IFT proteins (Fig. 5m and Table 1; n=2,881 BBS4:GFP-labelled particles, 143 cilia, 48 OSNs and 7 mice). We next co-expressed BBS4:mCherry and IFT88:GFP, and found that BBS4:mCherry was present in all observed IFT88:GFP-labelled particles (Fig. 5n and Supplementary Movie 5), verifying that the BBSome is in fact a constituent of the IFT particle in OSN cilia.

**Figure 5: The BBSome is a constituent of OSN IFT particles.**

Table 1 Comparison of IFT velocities across species and cell types.

Full size table

Assembly of the BBSome depends up the activity of a BBS–chaperonin complex containing BBS6, BBS10 and BBS12 (ref. 47). As BBS10 and BBS12 are present at or near basal bodies⁴⁸ where the BBSome is most abundant in primary ciliated cells, we hypothesized that they may also associate with the BBSome during IFT in OSN cilia. Confocal analysis revealed BBS10:GFP localization almost exclusively in OSN knobs (Supplementary Fig. 4). Within the OSN knob, however, BBS10:GFP did not associate with all basal bodies as is typical for BBS proteins; instead, BBS10 accumulated as a single focus (Supplementary Fig. 4b–e). Remarkably, the BBS10:GFP focus was highly dynamic, displaying constant mobility (Supplementary Fig. 4f). It is enticing to speculate that BBS10 and other components of the BBS–chaperonin complex are resident within a single pericentriolar endosome that actively shuttles throughout the OSN knob to provide support to each olfactory cilium. Using TIRFm on OSNs co-expressing BBS10:GFP and BBS4:mCherry, we observed only rare, transient association of BBS10 with IFT particles (Supplementary Fig. 4g). Thus, we conclude that the major function of the BBS–chaperonin complex in BBSome assembly is performed outside of OSN cilia before incorporation of the BBSome into IFT particles.

Peripheral membrane Arl proteins are OSN IFT cargoes

We next examined peripheral ciliary membrane Arl proteins. Arl6 (hereafter referred to as BBS3) is required for BBSome recruitment onto membranes in vitro⁴⁹ and for localization of the BBSome within mammalian cilia on cultured cells⁵⁰. In C. elegans, BBS3 and Arl13b homologues move bidirectionally within cilia, suggesting they associate with IFT^41,51. Similar to Arl13b:mCherry, we observed BBS3:GFP throughout the full length of OSN axonemes (Fig. 6a,c). Remarkably, both BBS3:GFP and Arl13b:mCherry showed discrete bidirectional ciliary transport when imaged by TIRFm (Fig. 6d–g and Supplementary Movie 6). To assess the stoichiometry of BBS3 and Arl13b association with IFT particles, we co-expressed each protein with FP-tagged BBS4 and performed dual colour time-lapse TIRFm. Although BBS3:GFP appeared to co-localize with all BBS4:mCherry-labelled particles (Fig. 6f), Arl13b:mCherry was clearly absent from most (69.2%) BBS4:GFP particles (n=340) (Fig. 6g and Supplementary Movie 7), indicating that Arl13b is an intermittent IFT cargo and not a core constituent of the IFT machinery.

**Figure 6: Peripheral ciliary membrane-associated Arl proteins participate in IFT.**

Polytopic signalling proteins rarely associate with OSN IFT

A major unanswered question is how olfactory receptors and other ciliary transmembrane proteins are trafficked within cilia. Evidence from invertebrates has demonstrated the capacity of transient receptor potential vanilloid channels to processively traffic with IFT^52,53. By contrast, Ye et al.¹⁶ recently reported that in mammalian digitonin-permeablized cells, the G protein-coupled receptor SSTR3 and Smo only associate with IFT over short intervals and more often move by diffusion. To address whether mammalian transmembrane signalling proteins use IFT for ciliary trafficking within native OSNs, we expressed an FP-tagged odourant receptor, adenylate cyclase and a subunit of the olfactory cyclic nucleotide-gated ion channel (Olfr78:GFP, ACIII:GFP and CNGA2:GFP, respectively). By confocal microscopy, each FP-tagged signalling protein showed distribution throughout OSN ciliary membranes (Fig. 7a–c). Through TIRFm, we discerned brief translocation events for each FP-tagged protein (Fig. 7d–f), reminiscent of disordered SSTR3 and Smo movement in cultured cell cilia¹⁶. To verify whether these movements correspond to IFT particles, we co-expressed ACIII:GFP and BBS4:mCherry, and found that ACIII:GFP movements were indeed coincident with IFT trafficking (Fig. 7h and Supplementary Movie 8). Interestingly, only a fraction of all IFT particles (14.4% of 479 particles captured from 60 cilia on 9 cells) were associated with ACIII and, moreover, only a fraction of all cilia (36.7%) showed any IFT-associated ACIII translocation during the given interval of data acquisition (Fig. 7g,h). Our data indicate that although transmembrane signalling proteins can use IFT for movement within OSN cilia, processive translocation events are rare at steady state, and we predict the function of these proteins is probably not dependent on constant association with IFT.

**Figure 7: Polytopic olfactory signalling proteins have the capacity to undergo IFT.**

Discussion

Genetic anomalies disrupting IFT or BBS proteins manifest a wide array of variably penetrant disease pathologies that arise from dysfunction of cilia found on cells of various tissues. In general, the genetic loci underlying clinically distinct ciliopathies are coincident with biochemical data showing interactions between their encoded proteins and cooperation as a functional unit⁵⁴. However, genetic and phenotypic overlap among ciliopathies predicts that some ciliary units functionally interact. In support of this notion, our data reveal for the first time the BBSome as a constituent of the mammalian olfactory IFT particle and implies that altered (but not ablated) IFT underlies BBS disease pathology. Interestingly, ciliation of the OE, but not ciliation in most other tissues, is dependent on normal BBSome function^8,9,10, suggesting the precise role of BBS proteins in IFT will vary by cell type, thereby influencing disease penetrance in different tissues. This is reflected in lower eukaryotes where loss of BBS proteins has species-specific consequences. In C. elegans the BBSome is essential for IFT particle integrity and motor coordination^37,55,56, but in C. reinhardtii BBS proteins are dispensable for IFT and flagellar biogenesis³⁴. C. reinhardtii BBSome proteins associate with only a subset of IFT particles, whereas IFT particles in OSN cilia show a 1:1 ratio of BBS protein labelling. This 1:1 stoichiometry may represent a critical requirement specifically in OSNs for BBS proteins to facilitate assembly and/or maintenance of cilia via modulation of the IFT machinery. The mammalian BBSome may function similar to its role in C. elegans where the coupling of IFT-A and IFT-B into a complete unit is dependent on the presence of the BBS proteins. The inability of the A and B subcomplexes to form a fully functional unit would probably have major ramifications on their ability to properly maintain the remarkably long axonemes of OSN cilia. Alternatively, the BBSome itself may provide docking sites for IFT cargoes that are necessary for assembly/maintenance of OSN cilia. Although biochemical data is lacking, genetic disruption of a BBS protein combined with reduced IFT88 expression exacerbates ciliopathy phenotypes in mouse⁵⁷ and IFT27 mutations give rise to BBS phenotypes in humans⁷, together demonstrating a potential functional link between the mammalian BBSome and IFT. Direct analysis of IFT assembly in the context of BBS mutant OSNs should provide insight into the functional relationship between the mammalian BBSome and IFT in the future. In addition to alterations in IFT function, the loss of OSN cilia seen in BBS mutant animals may also occur as a consequence of compromised ciliary membrane delivery, a role in which the BBSome is implicated⁴⁹. As OSN cilia require relatively large amounts of membrane to encapsulate their full length, ciliary membrane defects due to BBS mutations might have a greater adverse effect on the length of these cilia compared with cilia on other cell types.

The current model of proximal–distal kinesin-based axoneme assembly, generated from studies in C. elegans amphid/phasmid channel sensory cilia, states that two motor complexes cooperate to build distinct compartments of the cilium; the Kif17 homologue OSM-3 is solely responsible for distal singlet microtubule biogenesis and heterotrimeric kinesin-2 mediates IFT in cooperation with OSM-3 only along the proximal segment^20,37. This model is incompatible with our data showing that Kap3a travels throughout OSN cilia. Interestingly, in C. elegans dyf-5 MAP kinase loss-of-function mutants, the heterotrimeric kinesin-2 motor abnormally gains access to the distal segment, suggesting that the underlying microtubule architecture itself does not dictate kinesin usage⁵⁸. Thus, a divergent phosphorylation programme may be responsible for the differential localization of heterotrimeric kinesin-2 in OSN cilia versus C. elegans amphid/phasmid channel cilia. We also find that Kif17 is dispensable for both OSN distal segment formation/maintenance (Fig. 3) and for sustaining anterograde velocity (Table 1). Although this data is in contrast with the ciliogenic role of OSM-3 in C. elegans amphid/phasmid channel cilia distal segments, it is consistent with the dispensability of OSM-3 in amphid wing `C' cell ciliogenesis⁵⁹ and in the formation of amphid wing `B' (AWB) cell distal segments⁶⁰. However, in AWB cilia the heterotrimeric kinesin-2 is nevertheless restricted from distal segments, highlighting a divergence between AWB and OSN cilia as well⁶⁰. In support of our findings, disruption of Kif17 in cultured cells affects ciliary cargo trafficking without having an essential function in ciliogenesis⁶¹, and genetic ablation of Kif17 is apparently not inhibitory to ciliation in mice⁶². Instead, formation of OSN ciliary distal segments is probably facilitated by one or more other kinesins and our data points to heterotrimeric kinesin-2, which distinctively participates in IFT along the full length of OSN axonemes. Indeed, heterotrimeric kinesin-2 is solely responsible for proximal and distal segment formation in the cilia of Drosophila olfactory receptor neurons⁶³. Overall, our data uncovers a unique collaboration between the heterotrimeric and homodimeric kinesin-2 motors in OSN cilia and further demonstrates how kinesin usage is diversified in different organisms and cell types.

IFT velocities significantly vary among neighbouring OSNs. This is not surprising, considering the extent of heterogeneity that defines the OE. Each OSN within the olfactory system predominantly expresses one of ~1,000 odourant receptors^64,65; the OE therefore comprises subpopulations of cells that are differentially activated by various stimuli. As changing intracellular cyclic AMP and calcium influences IFT velocity¹⁷, we hypothesize that differential basal stimulation through environmental odours could have downstream effects to produce the cell-by-cell variation in IFT dynamics we observed. Similarly, the stochastic variation may be influenced by spatial distribution of individual OSNs within the OE or their age/maturation state.

Delineating the mechanisms of ciliary cargo trafficking, and membrane protein trafficking in particular, remains a key area of ongoing research interest. Recent data from single-molecule imaging in cultured cells suggests that individual ciliary membrane signalling receptors dynamically switch between short bursts of active transport by IFT and longer intervals of passive diffusion¹⁶. Likewise, clusters of flagellar membrane glycoproteins transiently associate with moving IFT particles in C. reinhardtii⁶⁶. However, previous ensemble imaging has shown that populations of certain membrane proteins can associate with IFT particles over comparatively longer distances^52,53. Our ensemble data showing peripheral membrane Arl proteins and transmembrane olfactory signalling proteins undergoing bouts of processive translocation in OSN cilia clearly demonstrates a capacity for these cargoes to use IFT in their trafficking in a native mammalian setting. However, the associations between IFT and the cargo proteins analysed here are intermittent and infrequent among OSN cilia and particles within cilia, suggesting that continuous association between cargo and particle is not essential to the normal function of these proteins.

Overall, from this work we have deconstructed mammalian OSN cilia axoneme and membrane organization, the composition and dynamic properties of OSN IFT and the degree to which IFT contributes to peripheral and transmembrane protein trafficking. Our data establish for the first time BBS protein participation in vertebrate IFT and provide novel insight into how kinesin motors are used in OSN cilia (Fig. 7i). Although invertebrate studies have informed our understanding of the players involved in ciliary transport, our findings highlight divergent qualities of mammalian IFT. In addition, the unique aspects of OSN IFT versus other mammalian cell types (Table 1) underscores the necessity to ascertain how cellular specification of the IFT machinery affects tissue-specific ciliopathy disease penetrance.

Methods

Mice

CD1 and GFP-CETN2 (Jackson Laboratory) mice were housed at the University of Florida. All procedures were approved by the University Committee for the Use and Care of Animals. Unless otherwise stated, two to three mice were used in each experimental condition.

Scanning electron microscopy

After deep anaesthesia and transcardial perfusion with 2% glutaraldehyde, 0.15 M cacodylate in water, olfactory tissue was processed using the OTOTO protocol. Analysis was performed on an Amray (Drogheda, Ireland) 1910FE field emission scanning electron microscope at 5 kV and recorded digitally with Semicaps software.

Constructs

Plasmids containing complementary DNA inserts used in this study were generously provided as follows: Nphp4:mCherry and IFT88 (B.K.Y., UAB); GFP:Efhc1 (K.Y., RIKEN); Arl13b (T.C., Emory); IFT122:GFP (J.E., UGA); Kap3a (T.S., Johns Hopkins); Olfr78 (J.P., Johns Hopkins); ACIII (R.R., Johns Hopkins); BBS1 (V.C.S., Iowa); and BBS2, BBS3, BBS4 and BBS10 (K.M., Ohio State). Dync2li1 was amplified from cDNA generated from mouse whole eye RNA. Lipid-anchored GFP constructs were generated by annealing oligonucleotides encoding the 13 amino-terminal residues from Lyn kinase (MyrPalm), the 20 N-terminal residues from GAP43 (PalmPalm) and the 9 carboxy-terminal residues from the guanine triphosphatase Rho (GerGer) into the KpnI and AgeI sites of pEGFP-N1 (Clontech). All cloning primer sequences are available on request.

Analysis of ectopic expression in mouse OSNs

For expression in native tissue, recombinant GFP-, mCherry- or mCitrine-flanked cDNAs were inserted into the adenoviral vector pAD/V5/−dest and virus was propagated using the ViraPower protocol (Invitrogen). AV particles were isolated with the Virapur Adenovirus mini purification Virakit (Virapur, San Diego, CA) and dialysed in 2.5% glycerol, 25 mM NaCl and 20 mM Tris-HCl, pH 8.0. Twenty-microlitre volumes of AV-containing constructs of interest were intranasally administered to the OE of pups at P7, P8 and P9. Transduced animals were killed for analysis on or after day P19.

For immunostaining, mice were deeply anaesthetized and then cardiac perfused with 4% paraformaldehyde. The lower jaw, teeth and hair were removed from snouts before postfixation in 4% paraformaldehyde for 2 h at 4 °C. Snouts were then decalcified in 0.5 M EDTA/ × PBS overnight, cryoprotected in 10%, 20% and 30% sucrose for 1 h, 1 h and overnight, respectively, at 4 °C, and embedded in optimal cutting temperature compound (Tissue-Tek). Coronal cryosections were sliced at a thickness of 10–12 μm and mounted onto Superfrost Plus slides (Fisher Scientific). For immunostaining, cryosections were permeabilized and blocked with 0.3% Triton X-100 and 2% goat serum in PBS for 30 min. Samples were incubated with acetylated α-tubulin antibody (clone 6–11 B-1, Sigma T6793) diluted 1:1,000 in 2% goat serum in PBS for 1 h at room temperature and washed three times with PBS. Cryosections were incubated in fluorescent-conjugated secondary antibodies for 1 h, washed three times with PBS and mounted using Prolong Gold (Invitrogen). Fixed tissue imaging was performed on an Olympus Fluoview 500 confocal microscope equipped with a 405-nm laser diode with a 430- to 460-nm band-pass filter, a 488-nm laser with a 505- to 525-nm band-pass filter, a 543-nm laser with a 560-nm long-pass filter and a 633-nm laser with a 660-nm long-pass filter; original magnification × 60, 1.40 numerical aperture (NA) or × 100,1.35 NA oil objectives were used.

For confocal en face imaging of transduced OSNs, animals were anaesthetized with CO₂, rapidly decapitated, split along the cranial midline and the OE was dissected out in PBS. Turbinates were placed in a tissue chamber and imaged on a Nikon TiE-PFS-A1R confocal microscope equipped with a 488-nm laser diode with a 510- to 560-nm band-pass filter and 561 laser with a 575- to 625-nm band-pass filter. A CFI Apo Lamda S × 60, 1.4 NA objective was used.

For en face TIRFm imaging of transduced OSNs, animals were anaesthetized with CO₂, rapidly decapitated, split along the cranial midline and further prepared in artificial cerebrospinal fluid (124 mM NaCl, 3 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 25 mM glucose) bubbled with 5% CO₂/95% O₂ for 10 min before use. The rostral- and caudal-most portions of one hemisphere were removed, leaving the OE and olfactory bulb intact in the skull. The tissue was placed face down in the imaging chamber, bathed in 35 °C artificial cerebrospinal fluid and held in place with mesh netting. TIRFm time series were captured at 200 ms exposure for 2–3 min on a Nikon Eclipse Ti-E/B inverted microscope equipped with a × 100 CFI APO TIRF 1.49 NA, × 1.5 tube lens, ZT488/561rpc dichroic, ZET488/561 × excitation filter, ZET488/561m-TRF emission filter (Chroma Technology) and an electron-multiplying charge-coupled device camera (iXon X3 DU897, Andor Technology). The 488-nm line of a fibre-coupled diode laser at incident power of 2 mw was used to illuminate a circular region of ~60 μm in diameter for capturing video sequences at 5–10 Hz. ImageJ was used to generate line-scan kymograms for measuring particle velocities from imported time series. Velocity was calculated with the following formula: tan(α*π/180)*b/c where α is angle, b is calibration in μm per pixel and c is exposure time per frame.

Statistical analysis

As our IFT velocity data set significantly violated the constant variance assumption of analysis of variance, we employed a non-parametric permutation test to assess the variability of IFT rates on the cell and population level using the following formula, to obtain test statistics:

The numerator reflects the variance between the cells and the denominator reflects the variance within the cells (that is, between the cilia); i is the cell index, j is the cilia index. The designating observations are denoted by y_ijk where k is the IFT particle index. The notations y_ij., y_i.. and y... denote the average values of observations in a cilium, in a cell or in the entire population, respectively. Number of observations in a cilium were given as n_ij and number of observations in a cell were given as m_i. Calculation of anterograde and retrograde observations gave statistics of 14.23 and 10.12, respectively. Next, the anterograde and retrograde observations were permuted and randomly shuffled across cells 20,000 times and the test statistics were calculated for each permutation. The P-values were calculated to be the percentages of the test statistics obtained from permutated data sets falling below the test statistics obtained from the observed data. For both the anterograde and retrograde data, the P-values were <0.0001, demonstrating that variations in IFT velocities among cells are significantly greater than variations among cilia in each cell.

Additional information

How to cite this article: Williams, C. L. et al. Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia. Nat. Commun. 5:5813 doi: 10.1038/ncomms6813 (2014).

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Acknowledgements

We thank L. Beyer and members of the University of Michigan Microscopy and Image Analysis Laboratory for assistance with scanning electron microscopy. We thank H.L. Kee, J. Pieczynski and additional members of the Martens and Verhey labs for valuable discussions and technical assistance. This work was supported by NIH grants R01DC009606 (to J.R.M.), RO1070862 (to K.V.), T32DC00011 (to C.L.W.) and K99DC013555 (to J.C.M.).

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Authors and Affiliations

Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, 1200 Newell Drive, PO Box 100267, Gainesville, 32610, Florida, USA
Corey L. Williams, Jeremy C. McIntyre, Lian Zhang & Jeffrey R. Martens
Department of Cell and Developmental Biology, University of Michigan, 109 Zina Pitcher Place, 3041 Biomedical Science Research Building (BSRB), Ann Arbor, 48109, Michigan, USA
Stephen R. Norris & Kristen Verhey
Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, 48109-5632, Michigan, USA
Paul M. Jenkins
Department of Biostatistics, University of Florida, RM5225, 2004 Mowry Road, Gainesville, 32611, Florida, USA
Qinglin Pei

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Corey L. Williams
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Jeremy C. McIntyre
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Stephen R. Norris
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Lian Zhang
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Contributions

C.L.W, J.C.M., P.M.J. and J.R.M. designed experiments. C.L.W., J.C.M., S.R.N. and P.M.J. performed experiments. C.L.W., J.C.M., P.M.J. and L.Z. generated reagents. Q.P. performed biostatistical analysis. K.V. provided equipment and reagents. C.L.W. and J.R.M. wrote the manuscript, with all authors providing input. C.L.W. made illustrations and generated figures. J.R.M. directed the project.

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Correspondence to Jeffrey R. Martens.

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Supplementary information

Supplementary Figures and Supplementary Methods

Supplementary Figures 1-4 and Supplementary Methods (PDF 663 kb)

Supplementary Movie 1

Processive bidirectional trafficking of kinesin and dynein complex proteins in olfactory sensory cilia. (AVI 30008 kb)

Supplementary Movie 2

Disruption of Kif17 does not affect kinesin-2 trafficking to distal tips of olfactory sensory cilia. (AVI 28251 kb)

Supplementary Movie 3

Processive bidirectional trafficking of IFT-A and IFT-B subcomplex proteins in olfactory sensory cilia. (AVI 17044 kb)

Supplementary Movie 4

Processive bidirectional trafficking of BBSome proteins in olfactory sensory cilia. (AVI 29136 kb)

Supplementary Movie 5

Co-movement of BBS4 and IFT88 in olfactory sensory cilia. (AVI 27758 kb)

Supplementary Movie 6

Processive bidirectional trafficking of peripheral membrane-associated Arl proteins in olfactory sensory cilia (AVI 29244 kb)

Supplementary Movie 7

Co-movement of Arl13b and BBS4 in olfactory sensory cilia. (AVI 29633 kb)

Supplementary Movie 8

Co-movement of ACIII and BBS4 in olfactory sensory cilia (AVI 26024 kb)

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Williams, C., McIntyre, J., Norris, S. et al. Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia. Nat Commun 5, 5813 (2014). https://doi.org/10.1038/ncomms6813

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Received: 01 October 2014
Accepted: 07 November 2014
Published: 15 December 2014
DOI: https://doi.org/10.1038/ncomms6813

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