Data supplements
Files in this Data Supplement:
- Supplemental Materials (PDF)
- Video 1
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Electron tomogram and 3D model of the ciliary base in L1-stage larva. Z scan through 130 nm of the ciliary base beginning in the transition zone and 3D reconstruction model highlighting ciliary membrane (gray) and doublet microtubules (green). Dual-axis tilt series was acquired on a Tecnai G2 20 microscope (FEI). Tomogram reconstruction was performed using IMOD. - Video 2
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Electron tomogram and 3D model of ciliary base in chemically fixed L4-stage larva. Z scan through 90 nm of the ciliary base beginning in the transition zone and 3D reconstruction model highlighting ciliary membrane (gray) and doublet microtubules (green). Dual-axis tilt series was acquired on a Tecnai G2 20 microscope (FEI). Tomogram reconstruction was performed using IMOD. - Video 3
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Neuronal development in late-stage Time-lapse sequence of an embryo expressing myristoylated GFP in amphid neurons beginning at the comma stage and continuing until the threefold stage. Single-plane GFP and transmitted light images were acquired every 5 min on a wide-field deconvolution microscope (DeltaVision; Applied Precision). The video playback is eight frames per second. Retrograde extension of dendrites begins at ∼30 min, with cell bodies moving posterior, whereas dendritic tips remain fixed in position. At ∼1 h 40 min, the worm begins to move inside the egg because of muscle contraction.C. elegans embryo. - Video 4
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Electron tomogram and 3D model of the centriole in a comma-stage embryo. Z scan through 85 nm of a centriole and 3D reconstruction model highlighting doublet microtubules (green). Dual-axis tilt series acquired on a Tecnai G2 20 microscope (FEI). Tomogram reconstruction was performed using IMOD. - Video 5
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Electron tomogram and 3D model of docked centriole in a twofold-stage embryo. Z scan through 150 nm of the amphid channel, zooming in on a docked centriole with a 3D reconstruction model highlighting ciliary membrane (gray), doublet microtubules (green), transition zone Y-links (red), and the central cylinder (brown). Parts of a second centriole, also composed of doublet microtubules and oriented orthogonally, are also captured in the section. Single-axis tilt series was acquired on a Tecnai G2 20 microscope (FEI). Tomogram reconstruction was performed using IMOD. - Video 6
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Electron tomogram and 3D model of cilium in L1-stage larva. Z scan through 110 nm of the base of the amphid channel, zooming in on cilium with a 3D reconstruction model highlighting the ciliary membrane (gray) and doublet microtubules (green). Dual-axis tilt series was acquired on a Tecnai G2 20 microscope (FEI). Tomogram reconstruction was performed using IMOD.
Article Figures & Data
Figures
- Figure 1.
Centrioles are not present at the base of mature C. elegans cilia. (A) Immunofluorescence micrograph of amphid cilia in L4-stage larva expressing GFP:CCEP-290 (transition zone) and stained for HYLS-1 (ciliary base) and glutamylated tubulin (axoneme). 10 cilia form a bundle that is in contact with the external environment via a channel formed by the sheath and socket glia cells. Note that cell bodies are located ∼100 µm from the ciliary base and, therefore, outside the field of view. (B) Transmission electron micrographs of L4-stage amphid cilium at the level of the proximal segment of the axoneme, transition zone, and ciliary base with explanatory schematics. Morphologically recognizable centriole/basal body structures are missing from the ciliary base. MTs, microtubules. (C) Schematic of centriole pair in C. elegans early embryo. SAS-6 is a component of the cartwheel/central tube, whereas SAS-4 is associated with the outer microtubule wall. (D) SAS-6 and SAS-4 are not present at the base of mature cilia. Panels show centrioles in two-cell and gastrula-stage embryos and ciliary base in L4-stage larvae of a strain coexpressing GFP:SAS-4/SAS-6:GFP and mCherry:HYLS-1 (mCh:HYLS-1). (E) Tomographic slices and 3D reconstruction model of the ciliary base of L1-stage amphid cilium. Axonemal microtubules continue proximally as cilium widens at the base. Note additional electron densities between doublet microtubules and cell membrane (arrowheads). Transition zone elements including Y-links (arrows) can be seen in more distal planes. See also Videos 1 and 2. Bars: (A) 5 µm; (B) 200 nm; (D, insets) 1 µm; (D) 10 µm; (E) 100 nm.
- Figure 2.
Centrioles are lost early in neuronal differentiation. (A) Stills from time-lapse video of embryo expressing myristoylated GFP (myrGFP) in amphid neurons undergoing retrograde migration. DIC and single-plane GFP images taken at the indicated time intervals corresponding to comma, 1.5-fold, twofold, and threefold stages. Note the position of dendritic tips (arrowheads) remains fixed as cell bodies (asterisks) move. At the threefold stage, the worm begins to move inside the egg because of muscle contraction. See also Video 3. (B) Tomographic slice and 3D reconstruction model of centriole in a myristoylated GFP–positive neuron of a comma-stage embryo. Note the centriole displays doublet microtubules. Electron densities at the center of the centriole may represent elements of the central tube/cartwheel (arrowhead). See also Video 4. (C and D) Immunofluorescence micrographs of comma-, 1.5-fold–, twofold-, and threefold-stage embryos expressing myristoylated GFP in amphid neurons and stained for HYLS-1 and SAS-6 (C) or SAS-4 (D). Insets show magnified view of ciliary base (1) and nonneuronal cells elsewhere in the head of the embryo (2). Centriolar signal is lost from the ciliary base, with loss of SAS-6 preceding that of SAS-4, while HYLS-1 remains. All three proteins are lost in nonneuronal cells coinciding with terminal differentiation. (E and F) Quantitation of centriolar signal in amphid neurons (percentage of HYLS-1 foci positive for SAS-6 or SAS-4; E) and nonneuronal cells (HYLS-1 foci per nucleus; F) from images as in C and D. Error bars are 95% confidence intervals. n = 5–9 animals per condition. Bars: (A, C, and D) 10 µm; (B) 50 nm; (C and D, insets)1 µm.
- Figure 3.
Transition zone assembly. (A and B) 3D reconstruction model (A) and molecular assembly hierarchies (B) for the C. elegans transition zone based on Schouteden et al. (2015) (A) and Huang et al. (2011), Williams et al. (2011), Jensen et al. (2015), Roberson et al. (2015), Schouteden et al. (2015), and Li et al. (2016) (B). CCEP-290 is an essential component of the central cylinder, whereas MKS and NPHP module components function in assembly of Y-links. MKSR-2 and NPHP-4 are upstream components in the MKS and NPHP assembly pathways, respectively. (C) Recruitment of transition zone components to the site of cilia assembly marked by HYLS-1. Panels show 1.5-fold–, twofold-, and threefold-stage embryos and L1-stage larvae coexpressing GFP:CCEP-290, MKSR-2, NPHP-4, and mCherry:HYLS-1. Initial recruitment of all three components occurs at the twofold stage, with signal reaching maximal levels at the threefold stage. (D) Tomographic slices and 3D reconstruction model of the ciliary base of twofold-stage amphid cilium. A pair of centrioles can be seen, one of which is decorated by a single array of Y-links and a discontinuous central cylinder and enveloped by the ciliary membrane. Note marked widening of the centriole toward the base. See also Video 5. Bars: (C) 10 µm; (C, insets) 1 µm; (D) 100 nm.
- Figure 4.
IFT-dependent axoneme extension. (A) Schematic of anterograde IFT machinery in C. elegans. Assembly of the proximal segment depends on the coordinated transport of IFT trains by kinesin-II and OSM-3 together, while assembly of the distal segment depends on OSM-3 alone. (B) Recruitment of fluorescently tagged IFT components (green in merge) to the site of cilia assembly marked by the transition zone components MKS-6 and MKSR-2 (red). Panels show 1.5-fold–, twofold-, and threefold-stage embryos and L1- and L3-stage larvae coexpressing CHE-11:mCherry, KAP-1:GFP, OSM-3:mCherry, or OSM-6:GFP and GFP:MKSR2 or MKS-6:mCherry as indicated. Initial recruitment of KAP-1 and OSM-6 occurs at the twofold stage of CHE-11 and OSM-3 at the threefold stage. IFT trafficking is only observed at L1. (C) Tomographic slice through base of the amphid channel in L1 larva, with magnified view and 3D reconstruction model of a single cilium at the level of the transition zone and proximal axoneme. Note cilium appears to terminate close to the transition zone. See also Video 6. Bars: (B) 10 µm; (B, insets) 1 µm; (C) 200 nm; (C, insets) 100 nm.
- Figure 5.
HYLS-1 is dispensable for cilia maintenance. (A) Schematic of the method used to degrade HYLS-1 specifically in ciliated neurons. (B) Amphid cilia structural integrity as assessed by dye filling. Dye-fill phenotype expressed as percentage of wild-type complement is shown. hyls-1 mutants display strong defects because of compromised axoneme assembly. These are rescued by expression of GFP:HYLS-1 (Student’s t test; P < 0.0001). Degron-mediated degradation of GFP:HYLS-1 after onset of ciliogenesis does not impair dye filling (Student’s t test; P = 0.57). Error bars are 95% confidence interval. n > 50 animals per condition. (C) Loss of GFP:HYLS-1 because degron-mediated degradation occurs from the threefold stage. Control animals not expressing degron are shown for comparison. Some HYLS-1 foci remain in L4, presumably because of lack of expression of the degron in those neurons or inaccessibility of the GFP epitope. Number of foci/amphid bundle ± 95% confidence intervals (n = 5–15 amphids per condition) is shown. (D) hyls-1 mutants display defects in targeting mKate2:DYF-19 to the ciliary base in amphid neurons. DYF-19 targeting is rescued in animals expressing GFP:HYLS-1 and the degron, despite late loss of HYLS-1. Transition zone marker mNeon:CCEP-290 is shown as a point of reference. (E) hyls-1 mutants display defects in IFT trafficking, shown using CHE-11:mKate2. IFT trafficking is restored in animals expressing GFP:HYLS-1 and the degron. (F) Tomographic slice through the amphid channel in L4-stage larvae of wild type, hyls-1 mutants, and hyls-1 mutants expressing both GFP:HYLS-1 and the degron. The marked axoneme extension defects in hyls-1 mutants are not observed in animals expressing both GFP:HYLS-1 and the degron. Bars: (C–E) 10 µm; (C, insets) 2 µm; (D and E, insets) 3 µm; (F) 100 nm.
- Figure 6.
A timeline for C. elegans ciliogenesis. Centriole and ciliary architecture at key stages in the C. elegans life cycle. In contrast to the singlet centrioles in the early embryo (Wolf et al., 1978; Pelletier et al., 2006), centrioles in sensory neurons possess doublet microtubules (MTs). Within 1 h after terminal cell division (twofold stage; 490 min after fertilization), centrioles have docked to the plasma membrane and have begun assembling transition zone structures (Y-links and central cylinder). Expansion of transition zone structures requires another hour (threefold stage; 550 min; not depicted). IFT-dependent axoneme extension continues through the larval stages. Centriole degeneration begins with loss of the cartwheel/central tube (by twofold stage), followed by loss of the centriolar microtubule wall component SAS-4 (threefold stage). Centrioles are therefore not directly involved in later events including transition zone expansion and axoneme elongation. HYLS-1 remains on the centriolar remnant and is required for the initial DYF-19/Fbf1-dependent recruitment of IFT components. However, HYLS-1 is dispensable for continued IFT trafficking.
Current Issue
June 5, 2017
Volume 216, No. 6