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. 2014 Dec 15;5:5813.
doi: 10.1038/ncomms6813.

Direct Evidence for BBSome-associated Intraflagellar Transport Reveals Distinct Properties of Native Mammalian Cilia

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Free PMC article

Direct Evidence for BBSome-associated Intraflagellar Transport Reveals Distinct Properties of Native Mammalian Cilia

Corey L Williams et al. Nat Commun. .
Free PMC article

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.

Figures

Figure 1
Figure 1. Molecular architecture of OSN cilia.
(a) (Left) Illustration depicting a sagittal view of the mouse OE. (Middle) Scanning electron micrograph of the OE surface showing a dense mat of olfactory cilia. (Right) Illustration depicting multiple cilia on an OSN. (b) Representative confocal image of a coronal OE section from a Centrin2:GFP transgenic mouse stained for acetylated α-tubulin to reveal cilia. NC, nasal cavity. (Arrowheads) Side-by-side dendritic knobs revealed by Centrin2:GFP signal. (cj) Representative live en face confocal images of AV-transduced native OE expressing various cilia domain-specific marker proteins. (c) AV-α-tubulin:RFP expression reveals many individual OSN microtubule axonemes projecting from two OSN dendritic knobs (arrowheads) on the surface of the OE. (d) Centrin-2:GFP transgenic mouse transduced with AV-Nphp4:mCherry. From left: Centrin-2:GFP-labelled basal bodies (BBs) line the periphery of OSN knobs. Nphp-4:mCherry marks ciliary transition zones (TZ), distally associated with each BB as seen in e. (f) A single TZ/BB unit. Far right: representative line-scan intensity plot showing the fluorescence profile of a single BB/TZ. (g) Co-expression of AV-Nphp-4:mCherry and doublet microtubule marker AV-GFP:Efhc1. GFP:Efhc1 is restricted to the proximal segment (PS) of each OSN ciliary axoneme. (i) A single TZ/PS unit. Far right: representative line-scan intensity plot showing the fluorescence profile of a single TZ/PS. (j) Co-expression of AV-GFP:Efhc1 and cilia peripheral membrane marker AV-Arl13b:mCherry. Arl13b:mCherry reveals axonemes of variable length (compare arrow and arrowhead), each extending from proximal segments marked by GFP:Efhc1. (k) Illustration depicting the ultrastructure of an OSN cilium in which a basal body gives rise to a transition zone followed by a ~2.5 μm doublet microtubule proximal segment and finally a singlet microtubule distal segment (DS) of variable length, sometimes exceeding 100 μm. Scale bars, 5 μm (a); 10 μm (bd,g,j); 2.5 μm (e,h); 1.25 μm (f,i).
Figure 2
Figure 2. Differential membrane enrichment of acyl and prenyl anchors in OSN ciliary subdomains.
(a) Representative confocal en face images captured from whole OE transduced with AV- (left) MyrPalm-GFP, (middle) PalmPalm-GFP or (right) GFP-GerGer. Whereas MyrPalm and PalmPalm are present throughout OSN ciliary membranes, GerGer is restricted to within the proximal regions of cilia near the OSN knobs. (b) GerGer-GFP ciliary membrane localization can extend beyond the proximal doublet microtubule segments as revealed by AV-mCherry:Efhc1 expression. Arrows highlight a cilium where GFP-GerGer localization is coincident with mCherry:Efhc1. Arrowheads point to a ciliary segment where GFP-GerGer localizes beyond mCherry:Efhc1. (c) Illustration of an OSN depicting the location of the axon initial segment and of synaptic termini in a glomerulus of the olfactory bulb. (d) Lipid-anchored GFPs freely access OSN axons. Representative confocal images of fixed sections through glomeruli of the olfactory bulb from mice expressing (left) MyrPalm-GFP, (middle) PalmPalm-GFP and (right) GFP-GerGer are shown. Empty circular regions surrounded by DAPI (4',6-diamidino-2-phenylindole; blue)-labelled nuclei represent glomeruli where OSN axons terminate. Scale bars, 10 μm (a); 5 μm (b); 20 μm (d).
Figure 3
Figure 3. Homodimeric and heterotrimeric kinesin-2 motors cooperatively traffic along the full length of OSN cilia.
(ac) Representative live en face confocal images of native OE ectopically expressing components of the IFT motor complexes. (a) Homodimeric kinesin-2 protein Kif17:mCitrine is present in cilia and at ciliary distal tips. (b) Accumulations of Kif17:mCitrine at distal tips (arrowheads) and in puncta along axonemes (arrow). (c) Heterotrimeric kinesin-2 component Kap3a:GFP is abundant along cilia axonemes. (d) Accumulations of Kap3a:GFP at ciliary distal tips (arrowheads) and distribution along axonemes (arrows). (e) Cytoplasmic dynein component Dync2li1:GFP is detectable along cilia axonemes. (f) Dync2li1:GFP is highly abundant along OSN cilia, detectable as discrete puncta (arrows), and is present at ciliary distal tips (arrowheads). Bracket indicates signal in a cilium from an OSN expressing only AV-Dync2li1:GFP. (g) TIRFm time-lapse capture of Kap3a:GFP particle movement in an OSN cilium. Exposures from different time points were overlayed onto an average intensity projection of the entire series to resolve individual particles in relation to the axoneme (ciliary tip is to the right, top). (Arrows) Anterograde particles. (Arrowheads) Retrograde particles. (hk) Line-scan kymograms generated from single cilia of OSNs ectopically expressing the indicated motor component. (h) Orientation of particle movement in relation to the ciliary tip. Axes represent distance (μm) over time (s). (i) Kif17:mCitrine-, (j) Kap3a:GFP- and (k) Dync2li1:GFP-labelled particles moving in anterograde and retrograde directions. (l) Disruption of Kif17 is not detrimental to the maintenance of OSN axonemes. Representative live en face confocal image of an OSN co-expressing AV-Kif17DN:GFP and AV-Arl13b:mCherry. (m) The presence of Kif17DN:GFP at distal tips (arrowheads) and in puncta along axonemes (arrows). (n,o) Kif17DN is transported to distal tips in IFT particles containing Kap3a. (n) Representative live en face confocal image of an OSN co-expressing AV-Kif17DN:mCherry and AV-Kap3a:GFP shows their co-localization in cilia and at ciliary distal tips (arrowheads). (o) Kymogram generated from a cilium of an OSN expressing (top) AV-Kif17DN:GFP and (middle) AV-Kap3a:mCherry. Scale bars, 10 μm (ae,l,n); 2.5 μm (zoomed panels); 2.5 μm (g); 10 μm × 30 s (hk,o).
Figure 4
Figure 4. Stochastic variation in IFT velocities among OSNs.
(a,c,e) Representative live en face confocal images of native OE ectopically expressing components of the IFT particle subcomplexes. Localization of (A) IFT-A subcomplex protein IFT122:GFP and (c) IFT-B subcomplex protein IFT88:GFP in the OSN knob and in puncta along the length of cilia as revealed by Arl13b:mCherry co-expression. Zoomed colour-shifted images show accumulation of (b) IFT122:GFP and (d) IFT88:GFP at the distal tips of cilia (arrow) and in puncta along axonemes (arrowheads). (e,g) Representative live en face TIRFm images captured from OSNs expressing IFT122:GFP or IFT88:GFP. Two-second exposures are shown. Insets show GFP puncta on OSN ciliary axonemes (arrowheads) and accumulations at ciliary distal tips (arrows). (f,h) Representative line-scan kymograms generated from single cilia of OSNs ectopically expressing the indicated IFT protein. (i) Histogram distribution of IFT122:GFP and IFT88:GFP particle velocities. IFT122:GFP, n=1,315 anterograde particles and 1,001 retrograde particles, 4 mice; IFT88:GFP, n=1,060 anterograde particles and 881 retrograde particles, 5 mice. (j) Plot of individual cilium mean anterograde versus retrograde velocities, showing a positive association between the magnitude of anterograde and retrograde velocities in a given cilium (Pearson’s coefficient R2=0.6064). Linear regression is shown. (k) Plot of individual cilium mean anterograde versus retrograde velocities, clustered by OSN (each group of points encapsulated by a coloured oval represents the cilia from a single cell). IFT88:GFP data set is shown. Scale bars, 10 μm (a,c); 2.5 μm (b,d); 10 μm, inset, 2.5 μm (e,g); 5 μm × 10 s (f,h).
Figure 5
Figure 5. The BBSome is a constituent of OSN IFT particles.
(af) Representative live en face confocal images of native OE ectopically expressing components of the BBSome. (a) Image of OE from animal transduced with AV-BBS1:GFP and AV-Arl13b:mCherry. BBS1:GFP concentrates strongly within the OSN knob and is distributed uniformly along the proximal-most segments (arrow) of OSN cilia as shown in the zoomed image (a). (c,d) Image of OE from animal transduced with AV-BBS2:GFP and AV-Nphp4:mCherry. (c) BBS2:GFP concentrates strongly within the OSN knob and is visible along ciliary axonemes in puncta (inset, arrowheads). (d) BBS2:GFP is enriched in basal bodies in the OSN knob. Merged panel shows BBS2:GFP basal body localization in close association with transition zones (arrows) as revealed by Nphp4:mCherry labelling. (e) Image of OE from animal transduced with AV-BBS4:GFP and AV-Arl13b:mCherry. BBS4:GFP concentrates strongly within the OSN knob. (f) Zoomed panel showing a transduced OSN knob where BBS4:GFP appears to localize at basal bodies. (g) Merged and zoomed panel shows accumulation of BBS4:GFP at the tip of an OSN cilium as revealed by Arl13b:mCherry labelling. (hn) TIRFm uncovers BBSome protein association with IFT particles in OSN cilia. (h,i) Collapsed maximum intensity projection of TIRFm time-series images captured from OSNs ectopically expressing (h) AV-BBS1:GFP or (i) AV-BBS4:GFP. The collapsed time series show that both BBS1:GFP and BBS4:GFP are present in OSN cilia. (jl) BBSome proteins show bidirectional particle movement in OSN cilia. Line-scan kymograms of cilia on OSNs ectopically expressing (j) AV-BBS1:GFP, (k) AV-BBS2:GFP or (l) AV-BBS4:GFP were generated from TIRF time-series acquisitions from native OE. (m) Histogram distribution of BBS4:GFP particle velocities. n=1,735 anterograde particles and 1,146 retrograde particles. (n) BBS4:mCherry associates with IFT particles. Representative kymogram was generated from a cilium of an OSN co-expressing AV-BBS4:mCherry and AV-IFT88:GFP. Scale bars, 10 μm (a,c,e); 2.5 μm (inset of c); 2.5 μm (b,d,f,g); 10 μm (h,i); 5 μm × 10 s (jl,n).
Figure 6
Figure 6. Peripheral ciliary membrane-associated Arl proteins participate in IFT.
(ac) Representative live en face confocal images of native OE ectopically co-expressing AV-BBS3:GFP and AV-Arl13b:mCherry. (a) BBS3:GFP is present along OSN ciliary axonemes. Inset shows zoomed image of two axonemes decorated with BBS3:GFP. (b) Zoomed image of the OSN knob showing BBS3:GFP accumulation. (c) Zoomed and colour-shifted image of an OSN ciliary distal tip showing co-localization of BBS3:GFP and Arl13b:mCherry. (d) Line-scan kymogram generated from a TIRFm time series of a BBS3:GFP AV-transduced OSN showing bidirectional movement of BBS3:GFP in an OSN cilium. (e) Line-scan kymogram generated from a TIRFm time series of an Arl13b:mCherry AV-transduced OSN showing particle movement of Arl13b:mCherry in an OSN cilium. (f) Line-scan kymogram of an OSN cilium co-expressing AV-BBS3:GFP and AV-BBS4:mCherry. (g) Line-scan kymogram of an OSN cilium co-expressing AV-Arl13b:mCherry and AV-BBS4:GFP. White arrows highlight a particle possessing both Arl13b:mCherry and BBS4:GFP. Black arrow indicates a group of particles that are labelled by BBS4:GFP but not Arl13b:mCherry. Scale bars, 10 μm (a); 2.5 μm (a (inset), b,c); 5 μm × 10 s (dg).
Figure 7
Figure 7. Polytopic olfactory signalling proteins have the capacity to undergo IFT.
(ac) Representative confocal images of AV-transduced OSNs expressing (a) Olfr78:GFP, (b) ACIII:GFP or (c) Cnga2:GFP. Each FP-tagged olfactory signalling protein was distributed along the full length of OSN cilia. (df) Representative time-lapse kymograms generated from cilia on AV-transduced OSNs expressing (d) Olfr78:GFP, (e) ACIII:GFP or (f) Cnga2:GFP. In each panel, occasional IFT-like translocation events are seen (arrowheads). (g,h) Representative time-lapse kymograms generated from two cilia on a single AV-transduced OSN co-expressing ACIII:GFP and BBS4:mCherry. In g, no association of ACIII with BBS4 is seen. In h, several BBS4-labelled IFT particles carry ACIII. Scale bars, 10 μm (ac); 5 μm × 10 s (dh). (i) Model of ciliary organization and IFT in OSNs. OSN cilia feature short (~2.5 μm) doublet proximal microtubules that transition to singlets that can span more than 100 μm. The OSN ciliary membrane gate located at or near the basal body (BB)/transition zone (TZ) complex is permissive to the ciliary entry of prenylated and acylated proteins, which differentially partition once within the OSN ciliary membrane. The mammalian OSN IFT particle comprises IFT-A, IFT-B and BBSome particle subcomplexes, and associates with both homodimeric Kif17 and heterotrimeric kinesin-2 along the full axoneme length. OSN IFT particles intermittently carry both peripheral membrane-associated proteins and polytopic olfactory signalling proteins as cargoes.

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