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. 2010 Nov 15;123(Pt 22):3966-77.
doi: 10.1242/jcs.073908. Epub 2010 Oct 27.

The AP-1 Clathrin Adaptor Facilitates Cilium Formation and Functions With RAB-8 in C. Elegans Ciliary Membrane Transport

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Free PMC article

The AP-1 Clathrin Adaptor Facilitates Cilium Formation and Functions With RAB-8 in C. Elegans Ciliary Membrane Transport

Oktay I Kaplan et al. J Cell Sci. .
Free PMC article

Abstract

Clathrin adaptor (AP) complexes facilitate membrane trafficking between subcellular compartments. One such compartment is the cilium, whose dysfunction underlies disorders classified as ciliopathies. Although AP-1mu subunit (UNC-101) is linked to cilium formation and targeting of transmembrane proteins (ODR-10) to nematode sensory cilia at distal dendrite tips, these functions remain poorly understood. Here, using Caenorhabditis elegans sensory neurons and mammalian cell culture models, we find conservation of AP-1 function in facilitating cilium morphology, positioning and orientation, and microtubule stability and acetylation. These defects appear to be independent of IFT, because AP-1-depleted cells possess normal IFT protein localisation and motility. By contrast, disruption of chc-1 (clathrin) or rab-8 phenocopies unc-101 worms, preventing ODR-10 vesicle formation and causing misrouting of ODR-10 to all plasma membrane destinations. Finally, ODR-10 colocalises with RAB-8 in cell soma and they cotranslocate along dendrites, whereas ODR-10 and UNC-101 signals do not overlap. Together, these data implicate conserved roles for metazoan AP-1 in facilitating cilium structure and function, and suggest cooperation with RAB-8 to coordinate distinct early steps in neuronal ciliary membrane sorting and trafficking.

Figures

Fig. 1.
Fig. 1.
C. elegans AP-1 complex is required for cilium formation, morphology and positioning. (A) AP-1 subunit genes (unc-101; μ1A, aps-1; σ1) function in ciliated cells to maintain normal dye-filling. Shown are merged DIC-fluorescence images following a DiI (red fluorescence) incorporation assay. Also denoted in top-left wild-type image is the position of environmentally exposed cilia (cil; yellow) at the distal tips of dendrites (den; blue). unc-101(sy108) mutants and aps-1(RNAi) worms (all ciliated cells; using arl-13 promoter-driven sense/antisense genomic fragments) possess strong Dyf defects in amphids and phasmid neurons (brackets). Also, unc-101(RNAi) and aps-1(RNAi) animals [using sense and antisense genomic fragments expressed under the srb-6 promoter, active only in PHA/B/ADL/ASH ciliated cells. (Troemal et al., 1995)] show severely reduced dye uptake in PHA/B and in some amphid cells (presumably ADL/ASH; asterisk). Dye-filling is restored in unc-101(sy108) mutants expressing unc-101::gfp (under endogenous unc-101 promoter or ciliated cell-specific srb-6 promoter) or apm-1::gfp (under an unc-101 promoter). As expected, rescue using srb-6-driven construct is restricted to PHA/B/ADL/ASH ciliated cells. Scale bars: 10 μm. (B) Analysis of dye-filling data from A. Percentage of worms with a dye-uptake defect. Note that for unc-101 and apm-1 RNAi strains (knockdown in PHA/B/ADL/ASH), amphid dye-filling was only grossly scored; detailed analysis would probably also uncover Dyf defects in ADL/ASH. n>50 worms. (C) unc-101 worms possess defective cilium morphologies. Shown are fluorescence images of unc-101(sy108) worms expressing ciliated cell-specific transcriptional GFP markers; srb-6p::gfp (PHA/B), str-1p::gfp (AWB), gcy-5p::gfp (ASER), gpa-6p::gfp (AWA) and str-2p::gfp (AWC). Scale bars: 3 μm. (D) Cilium morphology analysis from N2, unc-101(sy108) and aps-1(RNAi) (all ciliated cells) animals. Cilium lengths shown as mean with s.d. *P<0.001; nd, not determined; n, number of cilia assessed. (E) Cilia are mispositioned in unc-101 worms. Fluorescence images of one amphid cilium bundle in N2, unc-101(sy108) and aps-1(RNAi) (all ciliated cells) worms expressing IFT transgenes. Arrows, mispositioned cilia; arrowhead, cilium extending backwards; bracket, amphid ciliary bundle. Scale bars: 2 μm.
Fig. 2.
Fig. 2.
TEM analysis of unc-101 mutants reveals abnormalities in amphid pore formation, axonemes and ciliary microtubules. Shown are low and high magnification images from TEM serial cross sections of amphid channel cilia from N2 and unc-101(sy108) animals. (A–E) Distal region of amphid pore. N2 worms possess ten-singlet microtubule (MT)-containing axonemes (A,B), whereas axonemes are missing or degenerate and lacking MT structure in unc-101 mutants (C,D). Occasionally, an axoneme adjacent to the amphid pore is observed in unc-101 animals (E). Also, the amphid pore appears poorly formed and filled with electron dense material (arrows). (F–L) Sections 2–4 μm proximal to A–E through middle segment (MS) region of pore. Instead of ~12 axonemes containing nine outer-doublet MTs (F,G,K,L), many axonemes are missing in unc-101 mutants (H). Axonemes that are present often exhibit missing or disorganised doublet MTs (I,J) and are surrounded by an additional membrane (I,J,N; arrowheads), which might belong to the supporting sheath cell and indicate abnormal structural arrangements between neuronal cilia and the sheath cell (see Z). (M–Q) Sections 6–7 μm proximal to A–E through MS, transition zone (TZ) and transition fiber (TF) regions. Although most N2 ciliary axonemes exhibit TZ and TF structure at this point (P,Q), many ciliary axonemes in unc-101 worms still display middle segment type structure (N,O), indicating posteriorly shifted axonemes. Some unc-101 axonemes possess nine doublet MTs (O); however, in others, MTs are missing (M). (R–Y) Sections 7–12 μm proximal to A–E through TF region (U,V). Unlike N2 worms, where cilia are not observed beyond +8 to +9 μm, axonemal ultrastructure (e.g. MS, TZ) is found beyond this point in unc-101 animals (W–Y). Although unc-101 TZs are typically normal (T), misplaced MT doublets and an additional enveloping membrane (Y, arrowhead) are also sometimes observed (Y, arrow). (Z) Schematics of amphid channel cilia (longitudinal and transverse views) from N2 and unc-101 worms showing the major ultrastructural defects observed (not to scale; e.g. some unc-101 cilia shifted more posteriorly than indicated). Scale bars: 200 nm.
Fig. 3.
Fig. 3.
AP-1-depleted mammalian cells possess primary cilium defects. RPE1 cells transfected with control siRNA (luciferase; siLUC) or siRNA targeting γ-adaptin (siAP-1). (A) AP-1-depleted cells form cilia. Percentage of ciliated cells normalised to siLUC-treated cells. Mean ± s.d.; n=700 cells, three independent experiments. (B–F) Loss of AP-1 function disrupts cilium morphology, position or orientation, and microtubule acetylation. Shown in B are 3D reconstructions stained with acetylated tubulin (AT) (cilium, red) and DAPI (nucleus, blue) from deconvoluted Z-stack images. Quantification of cilia phenotypes (cilia with discontinuous AT staining (Dis. AT), and curved or twisted cilia) are shown in C and cilium length (μm) is shown in D. Spread of angles formed by cilia with adhesion surface is shown in E. Shown in F is the centre of mass (CM) of each cilium normalised to that of its corresponding nucleus. For each assay, n=70; three independent experiments. Means ± s.d. are shown.
Fig. 4.
Fig. 4.
Intraflagellar transport is not affected in AP-1-depleted cells. (A) Fluorescence images of amphid or phasmid cilia in N2 and unc-101(sy108) worms overexpressing an osm-3::gfp transgene. OSM-3 localises normally to ciliary axonemes (c) and at the base of cilia (asterisk) in unc-101 worms. Note anteriorly shifted phasmid cilium in unc-101 animals (bottom right; arrow). Kymographs and kymograph schematics, derived from time-lapse movies (3 frames/second), show OSM-3::GFP particles moving anterogradely along unc-101 cilia. Scale bars: 2 μm. (B) Time-lapse fluorescence images of C. elegans amphid dendrites showing that similarly to N2 worms, CHE-11::GFP-associated particles (arrow) translocate bidirectionally along unc-101 dendrites. (C) AP-1-depleted mammalian cells possess normal IFT protein ciliary localisations. RPE1 cells treated with siRNA to knock down γ-adaptin (siAP-1) or luciferase (siLUC; control) and stained for acetylated tubulin (AT, cilia, green), AP-1 (γ-adaptin, green), IFT46 (red) and nuclei (DAPI). Magnifications correspond to ciliary regions. Scale bars: 5 μm.
Fig. 5.
Fig. 5.
Disruption of C. elegans rab-8 and chc-1 function phenocopies the cilium formation and ciliary membrane protein transport defects of unc-101 mutants. (A) chc-1 mutants and N2 animals expressing an arl-13p::rab-8(wt)::gfp transgene possess dye-filling defects; rab-8 and clic-1 loss of function (LOF) mutants are dye-filling normal. Shown is the percentage of animals incorporating DiI in amphid and phasmid neurons. (B) AWB cilium (brackets) morphologies are disrupted in chc-1(b1025ts) mutants and N2 worms expressing an srb-6p::rab-8(Q67L) transgene [encodes GTP-locked RAB-8 expressed in AWB cells (Mukhopadhyay et al., 2008)], but not in rab-8(tm2526) LOF mutants. Images are from worms of indicated genotype expressing the AWB cilium marker, str-1p::gfp. Cilium length is indicated as mean/s.d. n>50. Scale bars: 3 μm. (C) ODR-10::GFP is abnormally sorted to all plasma membrane destinations and ODR-10 dendritic vesicles fail to form in AP-1 subunit and chc-1 mutants, and srb-6p::rab-8(Q67L)-expressing worms. Fluorescence images of AWB neurons from worms expressing an odr-10::gfp transgene. Presented for each strain is a low magnification AWB image [left, showing entire cell with cilium (c), dendrite (d) and cell body (cb) denoted] and two smaller high-magnification images, showing AWB cilium (top image; asterisk denotes cilium base) and cell body (bottom image). Also shown are three images of AWB dendrite (third from left) from a time-lapse movie (s, seconds), as well as corresponding kymographs (second from right) and kymograph schematics (right). Arrowhead denotes ODR-10 at plasma membrane. Scale bars: 10 μm (left panels) and 3 μm (all other panels). (D) Expansion of AWB ciliary membrane in grk-2(268) mutants requires unc-101. Fluorescence images of worms expressing an str-1p::gfp transgene. Arrows denote large membrane fans at tips of AWB cilium forks. Scale bars: 2 μm.
Fig. 6.
Fig. 6.
Subcellular localisation and motility behaviours of AP-1 subunit, RAB-8 and ODR-10 in C. elegans sensory neurons. (A) ODR-10 colocalises with RAB-8 in cell soma, whereas ODR-10 and UNC-101/TGN signals are juxtaposed and do not overlap. Shown are green, red and merged fluorescence images of AWB or PHA/B cell soma from worms expressing the indicated transgenes. SYN-16::dsRed marks TGN. Arrows, UNC-101::GFP; arrowheads (closed), ODR-10::dsRed; arrowhead (open), colocalised ODR-10::GFP and RAB-8::dsRed. Scale bars: 2 μm. (B,C) RAB-8 and ODR-10 translocate in cell soma and dendrites. Individual frames from fluorescence time-lapse recordings in worms expressing srb-6p::gfp::rab-8 (PHA/B cells) or str-1p::odr-10::gfp (AWB cells). s, seconds. Arrows, moving particles; asterisk, non-moving reference point. C shows kymographs and kymograph schematics from dendritic time-lapse movies in B, with particles moving in both directions. Scale bars: 2 μm. (D) Anterograde (towards cilium) and retrograde (towards cell soma) velocities of ODR-10::GFP (AWB), GFP::RAB-8 (PHA/B) and RAB-8::GFP (AWB) moving along dendrites. Velocities derived from multiple kymographs (N). n, number of particles measured. *, pairwise comparisons (using t-test analysis) of anterograde ODR-10 rates with anterograde GFP::RAB-8 (P=0.041) and RAB-8::GFP (P=0.384) rates. (E–G) Moving dendritic vesicles containing ODR-10 also contain RAB-8. Shown in E and F are red, green and merged images from time-lapse movies (time shown in seconds) of PHA/B dendrites in worms expressing srb-6p::odr-10::dsRed and arl-13p::gfp::rab-8 transgenes. Green and red images simultaneously acquired using an image beam splitter. Arrow, anterograde (towards cilium)-moving particle containing ODR-10 and RAB-8. Shown in F is a kymograph derived from a dendritic time-lapse movie, focusing on a specific section of one of the PHA/B dendrites. Note perfect overlap of a retrograde (towards cell body) moving ODR-10 and RAB-8 marked vesicle (bracket). cb, cell soma; d, dendrites. G is a merged kymograph analysis showing % ODR-10::dsRed lines colocalising with GFP::RAB-8 lines (ODR-10 + RAB-8) or not (ODR-10 alone). Also shown is % GFP::RAB-8 lines colocalising with ODR-10::dsRed lines (RAB-8 + ODR-10) or not (RAB-8 alone). Data for anterograde (Ant) and retrograde (Ret) moving particles is shown. Number of lines counted for each category is shown in parentheses. Scale bars: 3 μm (E), 5 μm (F).
Fig. 7.
Fig. 7.
Model of AP-1/RAB-8 function in sorting ciliary membrane proteins in C. elegans sensory neurons. In wt neurons, default or constitutive secretory pathways are proposed to target membrane proteins to all plasma membrane destinations. Ciliary transmembrane proteins are rescued from these pathways by AP-1 and RAB-8 activities functioning at distinct cell soma compartments. Initial budding of cilium-destined vesicles probably occurs at TGN/AP-1-positive endosomes, in a clathrin-dependent manner, followed by rapid uncoating of vesicles, and subsequent fusion with RAB-8-positive compartments. RAB-8 then facilitates formation of vesicles destined for dendrites and cilia. RAB-8 probably serves several roles in the delivery of ciliary proteins and membrane, both in the cell soma and in dendrites. Although ciliary membrane is likely to flow from TGN/AP-1-positive endosomes to RAB-8-positive endosomes, the involvement of a reverse retrograde trafficking route (question mark) is also possible.

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