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Cilia structure and intraflagellar transport differentially regulate sensory response dynamics within and between C. elegans chemosensory neurons [1]

['Alison Philbrook', 'Department Of Biology', 'Brandeis University', 'Waltham', 'Massachusetts', 'United States Of America', 'Michael P. O Donnell', 'Department Of Molecular', 'Cellular', 'Developmental Biology']

Date: 2024-12

Sensory neurons contain morphologically diverse primary cilia that are built by intraflagellar transport (IFT) and house sensory signaling molecules. Since both ciliary structural and signaling proteins are trafficked via IFT, it has been challenging to decouple the contributions of IFT and cilia structure to neuronal responses. By acutely inhibiting IFT without altering cilia structure and vice versa, here we describe the differential roles of ciliary trafficking and sensory ending morphology in shaping chemosensory responses in Caenorhabditis elegans. We show that a minimum cilium length but not continuous IFT is necessary for a subset of responses in the ASH nociceptive neurons. In contrast, neither cilia nor continuous IFT are necessary for odorant responses in the AWA olfactory neurons. Instead, continuous IFT differentially modulates response dynamics in AWA. Upon acute inhibition of IFT, cilia-destined odorant receptors are shunted to ectopic branches emanating from the AWA cilia base. Spatial segregation of receptors in these branches from a cilia-restricted regulatory kinase results in odorant desensitization defects, highlighting the importance of precise organization of signaling molecules at sensory endings in regulating response dynamics. We also find that adaptation of AWA responses upon repeated exposure to an odorant is mediated by IFT-driven removal of its cognate receptor, whereas adaptation to a second odorant is regulated via IFT-independent mechanisms. Our results reveal unexpected complexity in the contribution of IFT and cilia organization to the regulation of responses even within a single chemosensory neuron type and establish a critical role for these processes in the precise modulation of olfactory behaviors.

Here, we show that cilia architecture and IFT-mediated trafficking of signaling proteins differentially regulate response properties within and across 2 chemosensory neuron types in C. elegans. Via acute disruption of cilia structure but not IFT and vice versa, we show that cilia length but not continuous IFT is necessary for the responses to a subset of chemicals in the ASH nociceptive neurons. The roles of cilia shape and IFT are particularly complex in regulating odorant responses in the AWA olfactory neurons. We find that IFT is not required to maintain the complex AWA cilia architecture, but that prolonged IFT loss is associated with the formation of ectopic branches emanating from the periciliary membrane compartment (PCMC), regardless of the presence or absence of AWA cilia. Primary odorant responses are maintained under these conditions in part due to the shunting of odorant receptors and a subset of signaling molecules into these PCMC branches. However, spatial segregation of olfactory receptors from a cilia-localized GPCR kinase results in odorant desensitization defects in the absence of IFT. We also find that while IFT-mediated removal of the diacetyl receptor from AWA cilia is necessary for adaptation to this odorant upon repeated exposure, adaptation to the odorant pyrazine is mediated via IFT-independent mechanisms. Our results reveal unexpected complexity in the role of cilia and IFT in shaping responses to ecologically important chemical cues both within and between 2 different chemosensory neuron subtypes, and indicate that these processes play a critical role in diversifying and regulating stimulus-specific neuronal and organismal responses.

Caenorhabditis elegans sensory neurons provide an experimentally amenable system in which to correlate, and mechanistically describe, the role of cilia structure and IFT in regulating response properties. Only sensory neurons in C. elegans are ciliated, and as in other organisms, these cilia are built via IFT and house sensory transduction molecules [ 26 , 27 ]. Sensory cilia in C. elegans exhibit remarkably diverse morphologies which can be visualized and characterized on a neuron-by-neuron basis in single animals [ 27 – 29 ]. For instance, the 2 ASH nociceptive neurons in the bilateral amphid sense organs of the head each contain a single rod-like cilium, whereas the AWA olfactory neurons contain a highly branched cilium with a unique underlying cytoskeletal architecture [ 27 , 28 ]. Each sensory neuron pair responds to a range of stimuli within and across modalities, in part due to the expression of large numbers of sensory receptors in each neuron type [ 7 , 30 , 31 ]. Although loss of cilia in IFT mutant backgrounds is assumed to abolish all sensory responses in C. elegans [ 27 , 32 – 34 ], but see [ 35 ], the contribution of cilia and IFT to sensory transduction has not been analyzed systematically.

A defining morphological feature of many sensory neurons is the localization of sensory signal transduction molecules to microtubule-based primary cilia. Although primary cilia are now known to be present on nearly all cell types in mammals including on central neurons [ 15 – 17 ], sensory neuron cilia are unique in that they exhibit remarkably varied and complex cell type-specific morphologies and are directly or indirectly exposed to the external environment [ 17 , 18 ]. Most cilia are built by the highly conserved process of intraflagellar transport (IFT), a motor-driven process that traffics ciliary structural and signaling proteins into and out of the cilia [ 19 ]. The structural complexity of photoreceptor ciliary outer segments allows for dense packing and organization of phototransduction molecules, thereby accounting in part for the remarkable ability of these neurons to efficiently capture photons [ 20 , 21 ]. In the vertebrate olfactory epithelium, olfactory receptors and transduction channels are localized to multiple cilia of varying numbers and lengths present at the distal ends of sensory neuron dendrites [ 22 – 24 ]. Although overall cilia length may be correlated with odorant sensitivity of these neurons [ 25 ], whether and how structural diversity in cilia architecture shapes the response profiles of individual olfactory neurons is unknown. Moreover, given the requirement of IFT for building cilia, it has been challenging to distinguish between the contributions of cilia morphology, and continuous IFT-mediated trafficking of signaling proteins, to sensory neuron response properties.

Individual sensory neurons are highly specialized to respond to defined environmental stimuli. Although subsets of polymodal sensory neurons have been described across organisms [ 1 – 4 ], the majority of sensory neuron types are unimodal. Photoreceptors respond only to light, and olfactory neurons detect and respond only to volatile odorants. However, even unimodal sensory neurons exhibit extensive functional heterogeneity. Cone photoreceptor subtypes respond to distinct wavelengths of light [ 5 , 6 ], and individual olfactory and gustatory neuron types sense and drive behavioral responses to unique chemical subsets [ 7 – 11 ]. This functional diversity is largely mediated via the cell type-specific expression of sensory receptors and other signal transduction molecules within each neuron type [ 9 , 12 – 14 ]. Whether other features also contribute to diversification of response properties within and across sensory neuron subtypes remains to be fully described.

Results

Mutations in IFT genes differentially affect chemosensory responses in the ASH nociceptive neurons Eight of the 12 sensory neuron pairs including ASH in the amphid sense organs contain 1 or 2 rod-like cilia at their distal dendritic ends that are enclosed within a glial channel and are directly exposed to the environment [27–29] (Fig 1A). To test how truncation of cilia in IFT mutants affects neuronal responses, we examined chemical-evoked intracellular calcium dynamics in ASH neurons expressing GCaMP3. ASH responds to multiple aqueous and volatile nociceptive chemicals including glycerol and quinine, as well as high concentrations of isoamyl alcohol and 1-heptanol [36–38]. Mutations in the osm-6/IFT52 component of the IFT-B complex severely truncate ASH cilia (Fig 1A) [27]. ASH failed to respond to examined glycerol and quinine concentrations in osm-6 mutants (Figs 1B, S1A and S1B). However, these neurons retained the ability to respond to higher concentrations of both isoamyl alcohol and 1-heptanol albeit with significantly decreased response amplitudes and altered response kinetics (Figs 1B, 1C, S1C and S1D). Adaptation of ASH to a subset of chemicals has previously been shown to be modulated by GABAergic signaling from surrounding glial cells [39,40]. We observed no changes in glycerol responses in ASH in either wild-type or osm-6 mutants in the presence of exogenous GABA or the GABA A receptor antagonist bicuculline (S1E Fig), suggesting that mutations in IFT genes do not alter ASH responses via modulation of glial GABAergic signaling. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Cilia length but not continuous IFT regulates glycerol responses in ASH. (A) (Left) Cartoons (top) and representative images (bottom) of ASH cilia expressing gfp in wild-type and osm-6(p811) mutants under the sra-6 promoter. Yellow/white arrowheads: cilia base/cilia tip; white asterisk: neighboring ASI cilium. Scale bar: 1 μm. (Right): Quantification of ASH cilia length in wild-type and osm-6(p811) adult hermaphrodites. ***: different from wild type at P < 0.001 (t test). (B) Mean GCaMP3 fluorescence changes in ASH to a 30 s pulse of the indicated odorants in wild-type and osm-6(p811) mutants. Shaded regions indicate SEM. n ≥ 17 neurons each. For glycerol responses, data are reported from the second stimulus pulse (see Methods); the 0 time point represents 90 s following initiation of imaging. (C) Quantification of peak fluorescence intensity changes in ASH expressing GCaMP3 to 1 M glycerol (left) or 10−2 dilution of IAA (right) in animals of the indicated genotypes. * and ***: different from wild type at P < 0.05 and P < 0.001, respectively (glycerol: one-way ANOVA with Tukey’s multiple comparisons test; IAA: t test). Numbers above indicate approximate ASH cilia lengths. (D) Quantification of ASH cilia lengths in the indicated conditions and genotypes. ***: different from corresponding wild type at P < 0.001 (t test). (E) (Left) Representative kymographs of OSM-6::split-GFP movement in ASH cilia for the indicated conditions in kap-1(ok676 lf); osm-3(oy156ts) mutants. (Right) Percentage of cilia exhibiting OSM-6::split-GFP movement in 30 s (see S1 Table). ***: different at P < 0.001 from kap-1; osm-3(ts) prior to temperature upshift (Fisher’s exact test). (F, J) Mean GCaMP3 fluorescence changes in ASH to a 30 s pulse of 1 M glycerol for the indicated genotypes and conditions. The che-3(nx159ts) allele was used in J. Shaded regions indicate SEM. n ≥ 15 neurons each. For glycerol responses, the 0 time point represents 90 s following initiation of imaging (see Methods). (G, K) Quantification of avoidance to 8 M Glycerol for the indicated genotypes and conditions; 0 and 1 indicate 0% and 100% avoidance, respectively. ***: different at P < 0.001 between indicated values (one-way ANOVA with Tukey’s multiple comparisons test). (H) Quantification of ASH cilia lengths in the indicated genotypes and conditions. ***: different at P < 0.001 between indicated values (one-way ANOVA with Tukey’s multiple comparisons test). (I) Percentage of cilia exhibiting OSM-6::split-GFP movement in the indicated genotypes and conditions. ***: different at P < 0.001 from che-3(ts) prior to temperature upshift (Fisher’s exact test). Each dot in the scatter plots is the measurement from a single ASH neuron except in G and K where each dot indicates a single assay of 10 animals each. Horizontal lines in all plots indicate the mean. Errors are SEM. Data shown are from a minimum of 2 to 3 independent experiments. ns: not significant. Underlying data are provided in https://doi.org/10.5281/zenodo.13748735. IAA, isoamyl alcohol; IFT, intraflagellar transport. https://doi.org/10.1371/journal.pbio.3002892.g001 To determine whether there is a minimum cilium length above which responses to aqueous chemicals are maintained, we examined glycerol-evoked calcium dynamics in IFT mutants with different ASH cilia lengths. Mutations in the daf-10/IFT122 IFT-A complex protein also significantly truncate ASH cilia, whereas only the distal ciliary segments are lost in osm-3 homodimeric kinesin-2 anterograde IFT motor mutants (Fig 1C) [27,41]. ASH cilia length is unaltered in animals mutant for the kap-1 component of the heterotrimeric anterograde kinesin-II motor (Fig 1C). kap-1 but not daf-10 mutants retained the ability to robustly respond to glycerol, whereas responses in osm-3 mutants were variable (Fig 1C). We infer that a minimum cilium length may contribute to robust responses to aqueous but not volatile chemicals in ASH. Alternatively, IFT may differentially regulate responses to these chemicals in ASH.

Cilia length but not continuous IFT regulates glycerol responses in ASH Mutations in IFT genes disrupt not only cilia structure but also IFT-mediated trafficking of signaling proteins, a subset of which is localized to the distal ciliary tip [42–46]. To decouple the contributions of IFT and cilia length to sensory signaling, we engineered a temperature-sensitive (ts) mutation in osm-3. The fla8-2 mutation (F55S) in the motor domain of the highly conserved FLA8 ciliary kinesin motor protein of Chlamydomonas reinhardtii results in rapid loss of flagella upon a shift from permissive to the restrictive temperature [47–49]. An osm-3(oy156) mutant engineered to carry the corresponding F55S mutation (S2A Fig) was defective in its ability to uptake a lipophilic dye within 4 h of a shift from 20°C to 30°C similar to the dye uptake defects of osm-3(p802) loss-of-function (lf) mutants (S2B Fig). Since dye-filling defects are associated with structural alterations in a subset of amphid sense organ sensory cilia [27,50], these observations suggest that osm-3(oy156) may be a ts allele. We next examined ASH cilia length and IFT in osm-3(oy156) mutants upon shifting to the restrictive temperature for different periods of time. Since OSM-3 acts redundantly with the heterotrimeric kinesin-II motor to build the middle segments of channel cilia [51], we examined ASH cilia in kap-1(ok676 lf); osm-3(oy156) double mutants. ASH cilia were significantly truncated within approximately 1 h of shift to either 27°C or 30°C in osm-3(oy156) mutants and were truncated to the same extent as in kap-1(ok676); osm-3(p802) lf double mutants after 4 h at 30°C (Fig 1D). To visualize movement of IFT proteins at endogenous levels in ASH cilia, we tagged the osm-6 IFT-B gene with the split-GFP reporter GFP 11 (oy166 allele) via gene editing. ASH cilia length and dye-filling was unaltered in these animals (S2C Fig) indicating that the tag did not disrupt osm-6 function. We reconstituted GFP via expression of the GFP 1-10 fragment in ASH and monitored OSM-6 movement. OSM-6::split-GFP moved at speeds similar to those of overexpressed OSM-6::GFP reported previously (Figs 1E and S2D and S1 Table). Both anterograde and retrograde movement were markedly reduced upon a 1-h shift to 30°C, but continued, albeit at a reduced frequency after shifting to 27°C for 1 h (Fig 1E and S1 Table). These observations confirm that osm-3(oy156) represents a bona fide ts allele (henceforth referred to as osm-3(ts)). Shifting these double mutants to the semi-permissive temperature of 27°C provides a time window within which to assess the effects of cilia length truncation on glycerol responses in ASH in the presence of continued IFT. While ASH responded robustly to glycerol in kap-1(lf); osm-3(ts) animals at 20°C, these responses were eliminated upon an acute shift to 27°C for 1 h (Fig 1F). Moreover, while these animals avoided a ring of glycerol at the permissive temperature similar to wild-type animals, they escaped the ring upon exposure to 27°C for 1 h (Fig 1G). To determine how the converse conditions in which cilia length is maintained but IFT is acutely blocked affect glycerol responses, we examined che-3(nx159ts) mutants [52]. che-3 encodes the ciliary retrograde IFT dynein-2 motor, and the ts allele has previously been shown to disrupt both anterograde and retrograde IFT within 3 h of a temperature upshift without significant effects on cilia length [52]. Consistently, while ASH cilia length was slightly but significantly shorter in che-3(ts) mutants at the permissive temperature of 15°C, length was not further affected even after growth at 25°C for 6 h (Fig 1H). However, movement of OSM-6::split-GFP was significantly reduced within 3 h following a temperature upshift (Fig 1I and S1 Table). Neither glycerol-evoked calcium responses nor behavioral avoidance of glycerol were affected in che-3(ts) mutants under these conditions (Fig 1J and 1K). Together, these results indicate that glycerol responses in ASH require a minimum cilium length but not continuous IFT.

Mutations in IFT genes differentially affect responses to volatile odorants in the AWA olfactory neurons We next examined the contributions of cilia morphology and IFT to odorant responses in the AWA olfactory neurons which contain highly elaborate arborized cilia whose distal ends are embedded within glial processes [27–29]. These neurons mediate responses to a panel of bacterial food-related attractive volatile odorants including diacetyl and pyrazine [53]. It has previously been reported that truncation of AWA cilia in IFT mutants does not result in loss of the primary response to diacetyl but instead leads to defects in response desensitization (defined here as the decay of the response) and adaptation (defined here as progressively decreasing responses upon repeated stimulation; previously referred to as habituation [35]). Responses to other AWA-sensed odorants in IFT mutants were not examined. We investigated the effects of IFT mutations on responses to diacetyl and pyrazine as well as structurally related odorants in AWA. AWA cilia as visualized via expression of a myr-GFP membrane-associated reporter were severely truncated in both osm-6(p811) and kap-1(ok676); osm-3(p802) lf double mutants (Fig 2A). We also noted a large number of ectopic processes emanating from the PCMC at the base of the AWA cilia (henceforth referred to as PCMC branches) (Fig 2A); these are discussed further below. As reported previously [35], these mutants retained primary responses over a broad range of diacetyl concentrations but exhibit desensitization defects (Figs 2B and S3A). Similarly, osm-6 mutants continued to respond robustly to 2-heptanone [54] at different concentrations but exhibited altered desensitization rates (S3B Fig). In contrast, response amplitudes in AWA to different concentrations of both pyrazine and 2-methylpyrazine [55] were markedly decreased in IFT mutants (Figs 2C, S3C and S3D). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Chronic disruption of IFT differentially affects the expression of odorant receptors in AWA to drive distinct odorant responses. (A) Representative images of AWA cilia and PCMC branches (white arrows) in the indicated strains. AWA was visualized via expression of AWAp::myr-GFP. Numbers at top right indicate the percentage of neurons exhibiting the shown phenotype; n ≥ 20. (B) (Top) Mean GCaMP fluorescence changes in AWA in response to a 30 s pulse of diacetyl. Shading indicates SEM. (Bottom) Quantification of peak fluorescence intensities (left) and desensitization rates (right) for the diacetyl response in AWA in animals of the indicated genotypes. >95% of traces fit t 1/2 . *: different from wild type at P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). (C) (Top) Mean GCaMP fluorescence changes in AWA in response to a 10 s pulse of pyrazine at the indicated concentrations. Shading indicates SEM. (Bottom) Quantification of peak fluorescence intensities for the pyrazine responses in AWA in animals of the indicated genotypes. ***: different from wild type at P < 0.001 (left: one-way ANOVA with Tukey’s multiple comparisons test, right: t test). (D) (Left) Schematic of a behavioral microfluidics device (from [38]). (Right) Average histograms showing mean relative x-y residence of animals of the indicated genotypes in the behavioral device with a central stripe of the indicated odorants. (E) Chemotaxis indices calculated from the behavioral assays shown in D. *,**, ***: different from wild type at P < 0.05, P < 0.01, P < 0.001 (t test; each mutant strain was assayed in parallel with wild-type controls in the same device in each assay). (F) Representative images of ODR-10::split-GFP, SRX-64::split-GFP, or odr-10p::SRX-64::GFP localization in AWA in the indicated strains expressing the shown reporters. Numbers at bottom right indicate the percentage of animals showing the phenotype. n ≥ 20 for each. (G) (Left) Mean GCaMP fluorescence changes in AWA in response to a 30 s pulse of pyrazine. Shading indicates SEM. (Middle) Quantification of peak fluorescence intensities and (right) desensitization rates for the diacetyl response in AWA in animals of the indicated genotypes. 100% of traces fit t 1/2 . * and ***: different from indicated genotype at P < 0.05 and P < 0.001 (middle: one-way ANOVA with Tukey’s multiple comparisons test, right: t test). In all images, yellow/white arrowheads indicate cilia base/cilia tip, arrows indicate PCMC branches. Scale bars: 5 μm. Each dot in the scatter plots in B, C, and G is the measurement from a single AWA neuron; each dot in the plots in E is the chemotaxis index from a single behavioral assay. Horizontal lines in all plots indicate the mean. Errors are SEM. Data shown are from a minimum of 2 to 3 independent experiments. ns: not significant. Alleles used were: osm-6(p811), kap-1(ok676), and osm-3(p302). All calcium imaging was initiated 30 s prior to stimulus onset; responses from 5 s prior to stimulus onset are shown. Underlying data are provided in https://doi.org/10.5281/zenodo.13748735. IFT, intraflagellar transport; PCMC, periciliary membrane compartment. https://doi.org/10.1371/journal.pbio.3002892.g002 To determine whether the observed response phenotypes result in defects in attraction to these odorants, we examined behavioral responses to diacetyl and pyrazine in microfluidics behavioral arenas [38,56] that allow assessment of attraction to the same chemical concentrations used in calcium imaging experiments. Wild-type animals were robustly attracted to both odorants and accumulated within the central odorant stripe over the 20 min assay period (Fig 2D and 2E). While osm-6 mutants retained the ability to be attracted to a range of diacetyl concentrations, kap-1(ok676); osm-3(p802) double mutants retained partial attraction to high but not low diacetyl concentrations (Figs 2D, 2E and S3E) [35]. Consistent with reduced pyrazine response amplitudes, osm-6(p811) and kap-1(ok676); osm-3(p802) mutants were indifferent to all tested concentrations of pyrazine (Figs 2D, 2E and S3E). We conclude that truncation of cilia and/or loss of IFT differentially affects AWA responses to distinct volatile odorants.

Differential expression of odorant receptors upon chronic loss of IFT may drive distinct olfactory responses in AWA To begin to explore how mutations in IFT genes result in odorant-specific response phenotypes in AWA, we hypothesized that lf mutations in IFT genes may alter the localization and/or levels of the odr-10 and srx-64 diacetyl and pyrazine receptors, respectively, in AWA [57,58]. Endogenously tagged and functional ODR-10 [59] and SRX-64 proteins (S4A Fig) localized specifically to the AWA cilia in wild-type animals (Fig 2F). We detected expression of ODR-10::split-GFP both in the truncated cilium and the PCMC branches in osm-6 and kap-1; osm-3 mutants (Fig 2F). However, levels of SRX-64::split-GFP were markedly down-regulated, and this fusion protein was barely detectable in either the ciliary stub or PCMC branches in IFT mutants (Figs 2F and S4B) likely accounting for the marked decrease in their pyrazine responses. This down-regulation appears to be mediated via transcriptional mechanisms since expression of an endogenous SRX-64::SL2::split-GFP transcriptional reporter was also significantly decreased in an IFT mutant (S4C Fig). Consistent with this notion, expression of srx-64::gfp under the odr-10 promoter restored SRX-64 protein levels in osm-6 mutants, with localization in both the ciliary stub and the PCMC branches (Fig 2F). Pyrazine responses were also restored in these animals, and as in the case of diacetyl, these responses exhibited desensitization defects (Fig 2G). These results indicate that reduced expression of SRX-64 likely accounts for the pyrazine response defect in IFT lf mutant backgrounds. These observations further imply that upon localization of both receptors to the cilia stub and the PCMC branches, lf mutations in IFT genes primarily affect odorant desensitization in AWA.

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