30 research outputs found
Structural and Functional Recovery of Sensory Cilia in <i>C</i>. <i>elegans</i> IFT Mutants upon Aging
<div><p>The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT). Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in <i>C</i>. <i>elegans</i> transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies.</p></div
Cilia-dependent sensory behaviors are improved in aged IFT-B mutants.
<p><b>(A)</b> Chemotaxis responses of 1d and 7d old animals of the indicated genotypes to a point source of bacteria (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006325#sec012" target="_blank">Materials and Methods</a>). Positive chemotaxis indices indicate attraction. ** and *** indicate different from 1d within a genotype at <i>P</i><0.01 and 0.001, respectively (Kruskal-Wallis non parametric test). Error bars are SEM. n>200 animals each from 8 independent assays. <b>(B)</b> Fraction of animals of the indicated genotypes and ages that remain within a ring of 8M glycerol after 2 minutes. Error bars are SEM. *** indicate different from 1d at <i>P</i>< 0.001 within a genotype (Kruskal-Wallis non-parametric test). n>100 animals each from 10 independent assays. <b>(C)</b> Length of ASH cilia in 7d old wild-type, <i>osm-5(p813)</i> and <i>osm-6(p811)</i> animals that remained within (inside), or escaped (outside), a ring of 8M glycerol after 2 minutes. Horizontal lines indicate 25<sup>th</sup>, 50<sup>th</sup> and 75<sup>th</sup> percentiles. *** indicates different from 1d within a genotype at <i>P</i>< 0.001 (Wilcoxon Mann-Whitney U test). n≥15 animals for each condition.</p
IFT motor proteins are necessary for age-dependent cilia recovery in IFT mutants.
<p><b>(A)</b> Quantification of ASI cilia length in 1d and 7d old animals of the indicated genotypes. ASI cilia were visualized via expression of <i>str-3</i>p::<i>srg-36</i>::<i>gfp</i>. Alleles used in the double mutant strains were <i>osm-6(p811)</i>, <i>kap-1(ok676)</i> and <i>osm-3(p802)</i>. Horizontal lines indicate 25<sup>th</sup>, 50<sup>th</sup> and 75<sup>th</sup> percentiles; bars indicate 5<sup>th</sup> and 95<sup>th</sup> percentiles. *, ** and *** indicate different from 1d within a genotype at <i>P</i><0.05, 0.01 and 0.001, respectively (Wilcoxon Mann-Whitney U test). n>30 for each; ≥3 independent experiments. <b>(B)</b> Histograms of KAP-1::GFP and <b>(C)</b> OSM-3::GFP anterograde velocities in the ASH/ASI cilia of 1d and 7d old wild-type or <i>osm-6(p811)</i> mutants. <i>kap-1</i>:<i>gfp</i> and <i>osm-3</i>::<i>gfp</i> were expressed under the <i>sra-6</i> promoter. IFT could not be reliably quantified in short cilia in 1d old <i>osm-6</i> mutants. Anterograde velocities in the middle and distal segments are indicated by black and gray bars, respectively; average velocities are indicated at top right in each panel in corresponding colors. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006325#pgen.1006325.s008" target="_blank">S2 Table</a> for statistical analyses.</p
Cilia of multiple sensory neurons exhibit structural recovery in aged IFT mutants.
<p><b>(A)</b> Representative images of the fan-shaped AWC, and rod-like ASE, cilia in 1d and 7d old wild-type and <i>osm-6(p811)</i> mutants. Cilia were visualized via expression of <i>ceh-36</i>p::<i>gfp</i> which drives expression in AWC and ASE [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006325#pgen.1006325.ref044" target="_blank">44</a>]. White and yellow arrowheads mark the bases of the AWC and ASE cilia, respectively. Arrows mark the AWC cilia membraneous expansions (‘fans’). Numbers indicate the percentage of animals exhibiting phenotypes similar to those shown; n≥12 for each. Anterior is at top. Scale bar: 5 μm. <b>(B)</b> Quantification of AWC cilia fan area (left) and ASE cilia length (right) in 1d and 7d old wild-type and <i>osm-6(p811)</i> mutants. *** indicates different from 1d within a genotype at <i>P</i><0.001 (Wilcoxon Mann-Whitney U test). n>30 each; 3 independent experiments. <b>(C)</b> Representative images of ASH cilia in 1d and 7d old adult wild-type (WT) and <i>osm-6(p811)</i> mutants. Arrowheads indicate the cilia base. ASH neuronal processes including cilia are marked via expression of GFP under the <i>sra-6</i> promoter. The cilium base is marked via localization of MKSR-2::TagRFP. Anterior is at top. Scale bar: 5 μm. <b>(D)</b> Quantification of ASH cilia length in wild-type and <i>osm-6(p811)</i> mutants at the indicated days of adulthood. *** indicates different from 1d within a genotype at <i>P</i><0.001 (Wilcoxon Mann-Whitney U test). n>30 for each; ≥3 independent experiments. <b>(E)</b> Transmission electron microscopy of amphid channel sensory pores in adult 1d and 7d animals. Images acquired from serial cross sections of wild-type and <i>osm-6(m533)</i> mutant worms; each image pair consists of a low magnification image of the entire nose tip (left) and a high magnification image of an amphid pore (right; boxed regions in images at left). Numbers (microns) denote proximal positioning of section relative to the distal-most first section in the series; section positions also indicated in schematic. The schematic is a longitudinal representation of a wild-type amphid neuronal pore, enveloped by supporting sheath and socket glial cells. Pores consist of 10 ciliary axonemes (only 3 are shown), each with distal segment (DS; singlet microtubules), middle segment (MS; doublet microtubules), transition zone (TZ) and periciliary membrane (PCMC) subcompartments. Bars; 2 μm (low magnification images), 200 nm (high magnification images).</p
Cilia of the ASI sensory neurons elongate in aged IFT mutants.
<p><b>(A)</b> (Left) Cartoon of a worm showing a representative sensory neuron in the worm head. Cilia are present at the dendritic ends at the nose (box). (Right) Diagrammatic representation of the structure of a typical cilium and IFT in <i>C</i>. <i>elegans</i>. Arrows indicated direction of IFT. TZ—transition zone (showing Y-link microtubule-to-membrane connectors). <b>(B)</b> Representative images of ASI cilia in 1d and 7d old adult wild-type (WT) and <i>osm-6(p811)</i> mutants. Arrowheads indicate the cilia base. Anterior is at top. ASI cilia were visualized via expression of a GFP-tagged SRG-36 GPCR protein expressed under the ASI-specific <i>str-3</i> promoter. Scale bar: 5 μm. <b>(C)</b> Quantification of ASI cilia length in animals of the indicated genetic backgrounds at different larval stages (L2, L4) or days of adulthood. Horizontal lines indicate 25<sup>th</sup>, 50<sup>th</sup> and 75<sup>th</sup> percentiles; bars indicate 5<sup>th</sup> and 95<sup>th</sup> percentiles. * and *** indicate different from 1d within a genotype at <i>P</i><0.05 and 0.001, respectively; <sup>#</sup> and <sup>###</sup> indicate different from L2 within a genotype at <i>P</i><0.05 and 0.001, respectively (Kruskal-Wallis test with post hoc paired comparisons). n>30 for each; ≥3 independent experiments. Animals were grown at either 20°C or 25°C for each set of experiments (indicated at top right). <b>(D)</b> Quantification of ASI cilia length in 1d and 7d old animals of the indicated genotypes grown at 20°C. ASI cilia were visualized via expression of <i>str-3</i>p::<i>srg-36</i>::<i>gfp</i>. Alleles used in the double mutant strains were <i>osm-6(p811)</i>, <i>osm-5(p813)</i>, and <i>daf-10(e1387)</i>. Horizontal lines indicate 25<sup>th</sup>, 50<sup>th</sup> and 75<sup>th</sup> percentiles; bars indicate 5<sup>th</sup> and 95<sup>th</sup> percentiles. * and *** indicate different from 1d within a genotype at <i>P</i><0.05 and 0.001, respectively (Wilcoxon Mann-Whitney U test). n>30 for each; ≥3 independent experiments.</p
Improved protein quality control mechanisms may underlie age-dependent cilia recovery in IFT mutants.
<p><b>(A-D)</b> Quantification of ASI cilia length in animals of the indicated genotypes and adult ages. Alleles used were <i>osm-6(p811)</i>, <i>daf-16(mu86)</i>, and <i>hsf-1(sy441ts)</i>. ASI cilia were visualized via expression of <i>str-3</i>p::<i>srg-36</i>::<i>gfp</i> (A,C,D) or <i>srg-47</i>p::<i>TagRFP</i> (B). An <i>hsf-1</i> cDNA tagged with <i>gfp</i>, and <i>hsf-1</i> and <i>daf-21</i> sense and antisense sequences were expressed in ASI under the <i>srg-47</i> promoter. Lines 1 and 2 represent independent transgenic lines. Animals were grown at 20°C (A,C,D) or 25°C (B). Horizontal lines indicate 25<sup>th</sup>, 50<sup>th</sup> and 75<sup>th</sup> percentiles; bars indicate 5<sup>th</sup> and 95<sup>th</sup> percentiles. * and *** indicate different from 1d within a genotype at <i>P</i><0.05 and 0.001, respectively (Wilcoxon Mann-Whitney U test). n>30 for each; ≥3 independent experiments. <b>(E)</b> Quantification of ASI cilia length in 1d and 4d old animals of the indicated genotypes, expressing <i>srg-47</i>p::<i>osm-5(p813)</i>::<i>gfp</i>. ASI cilia were visualized via expression of <i>srg-47</i>p::<i>TagRFP</i>. Horizontal lines indicate 25<sup>th</sup>, 50<sup>th</sup> and 75<sup>th</sup> percentiles; bars indicate 5<sup>th</sup> and 95<sup>th</sup> percentiles. *** indicates different from 1d of the same genotype at <i>P</i><0.001 (Wilcoxon Mann-Whitney U test). n>50 each; 3 independent experiments. <b>(F)</b> Quantification of ASI cilia length in 1d and 4d old animals of the indicated genotypes. ASI cilia were visualized via expression of <i>str-3</i>p::<i>srg-36</i>::<i>gfp</i>. Animals were grown on 10 μM Bortezomib (BTZ). Horizontal lines indicate 25<sup>th</sup>, 50<sup>th</sup> and 75<sup>th</sup> percentiles; bars indicate 5<sup>th</sup> and 95<sup>th</sup> percentiles. *** indicates different from 1d within a genotype at <i>P</i><0.001; <sup>###</sup> indicates different between the indicated conditions at <i>P</i><0.001 (Wilcoxon Mann-Whitney U test). n>50 each; 3 independent experiments.</p
The <i>C</i>. <i>elegans</i> ortholog of Tectonic1 is a transition zone component.
<p>(A) Schematic of the X-box sequence and <i>ok3021</i> allele of <i>C</i>. <i>elegans tctn-1</i>. (B) GFP-tagged TCTN-1 localizes specifically to the transition zones (TZs) in head (amphid) and tail (phasmid) cilia of <i>C</i>. <i>elegans</i>. Basal bodies (BBs) and ciliary axonemes are marked with tdTomato-tagged XBX-1, a component of the ciliary dynein. Schematics illustrate the position of TCTN-1 at the transition zone with respect to the basal body and axoneme. Scale bar, 5 μm.</p
TCTN-1 contributes to <i>C</i>. <i>elegans</i> ciliary gate function.
<p>(A-D) Localization of different transition zone proteins in phasmid cilia of wild type, <i>tctn-1</i>, <i>nphp-4</i>, and <i>tctn-1; nphp-4</i> mutants. Each GFP, CFP, YFP, or tdTomato-tagged transition zone protein is co-localized with a ciliary marker (CHE-13, DYF-11 or XBX-1), as indicated. Abnormal localization of MKS-5 in the <i>nphp-4</i> single and <i>tctn-1; nphp-4</i> double mutants is indicated by asterisks. (E) ARL-13::GFP localizes ectopically to the PCMC in <i>tctn-1</i>, <i>nphp-4</i>, and <i>tctn-1; nphp-4</i> mutants as indicated by asterisks, but localizes normally to the middle segment (MS) of cilia. (F) ODR-10::GFP localizes to the cilia of AWA neurons in wild type, <i>tctn-1</i>, <i>nphp-4</i>, and <i>tctn-1; nphp-4</i> mutants. Anterior is to the right. (G) TRAM-1a::tdTomato localizes only to the PCMC in wild type animals, but enters phasmid cilia in <i>tctn-1</i> mutants, as indicated by asterisks. MKS-2::GFP marks the transition zone. Scale bars, 5 μm.</p
<i>C</i>. <i>elegans tctn-1</i> interacts with NPHP genes, but not MKS genes to affect ciliary structure.
<p>(A) Dye filling of amphid neurons in L4 nematodes in the indicated single transition zone mutants (left column) and <i>tctn-1</i> double mutants (right column). Lateral views, with anterior to the left. Genotypes including an allele affecting a previously recognized MKS complex component are indicated in green. Genotypes including an allele affecting an NPHP complex component are indicated in red. Scale bar, 20 μm. (B and C) Fluorescence intensity of DiI filled amphid neurons in single mutants of the MKS complex or NPHP complex, and double mutants with <i>tctn-1</i> relative to wild type. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s <i>t</i>-tests (* <i>p</i><0.001; ** <i>p</i><0.001 compared to <i>tctn-1</i>). (D) Low and high magnification TEM cross-sections of the distal segment, middle segment, transition zone (TZ), and distal dendritic periciliary membrane compartment (PCMC) of amphid cilia with schematics below (lateral and transverse views). Green arrowheads indicate intact Y-links in wild type and <i>tctn-1</i> transition zones whereas red arrowheads indicate reduced or missing Y-links, observed in <i>nphp-4</i> and <i>tctn-1; nphp-4</i> transition zones. Yellow arrowheads indicate open B-tubules and purple arrows indicate vesicle accumulation in the PCMCs of <i>nphp-4</i> and <i>tctn-1; nphp-4</i> mutants. <i>tctn-1; nphp-4</i> mutants display several truncated axonemes and axonemes with fewer microtubule doublets (blue arrows) compared to wild type, <i>tctn-1</i> and <i>nphp-4</i> mutants. Boxed numbers indicate distances (μm) from the distal ciliary tips. Scale bars,100 nm.</p
Mouse <i>Tctn1</i> genetically interacts with <i>Bbs1</i>.
<p>(A) Lateral views of wild type, <i>Tctn1</i><sup>-/-</sup>, <i>Bbs1</i><sup>-/-</sup> and <i>Tctn1</i><sup>-/-</sup><i>Bbs1</i><sup>-/-</sup> embryos at E14.5. Exencephaly is apparent in the <i>Tctn1</i><sup>-/-</sup><i>Bbs1</i><sup>-/-</sup> double mutant. Alcian blue staining of the corresponding genotype’s forelimb (top) and hindlimb (bottom) are included, with asterisks indicating extra digits. Scale bars, 1 mm. (B-D) Number of digits in the (B) forelimbs and (C) hindlimbs, and (D) the incidence of exencephaly for the indicated genotypes. Numbers of animals analyzed for polydactyly and exencephaly are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005627#pgen.1005627.s007" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005627#pgen.1005627.s008" target="_blank">S4</a> Tables. (E) Limb bud fibroblasts of the indicated genotypes immunostained for Tub<sup>Ac</sup> (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). Scale bar, 10 μm. (F) Quantification of proportion of ciliated limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s <i>t</i>-tests (* <i>p</i><0.0001). (G) Limb bud fibroblasts immunostained for Nphp1 (red), Tub<sup>Ac</sup> (green), and γ-tubulin (blue). Scale bar, 1 μm.</p