Striated Rootlet and Nonfilamentous Forms of Rootletin Maintain Ciliary Function

SummaryPrimary cilia are microtubule-based sensory organelles whose structures and functions must be actively maintained throughout animal lifespan to support signal transduction pathways essential for development and physiological processes such as vision and olfaction [1]. Remarkably, few cellular components aside from the intraflagellar transport (IFT) machinery are implicated in ciliary maintenance [2]. Rootletin, an evolutionarily conserved protein found as prominent striated rootlets or a nonfilamentous form, both of which are associated with cilium-anchoring basal bodies, represents a likely candidate given its well-known role in preventing ciliary photoreceptor degeneration in a mouse model [3, 4]. Whether rootletin is universally required for maintaining ciliary integrity, and if so, by what mechanism, remains unresolved. Here, we demonstrate that the gene disrupted in the previously isolated C. elegans chemosensory mutant che-10 encodes a rootletin ortholog that localizes proximally and distally to basal bodies of cilia harboring or lacking conspicuous rootlets. In vivo analyses reveal that CHE-10/rootletin maintains ciliary integrity partly by modulating the assembly, motility, and flux of IFT particles, which are critical for axoneme length control. Surprisingly, CHE-10/rootletin is also essential for stabilizing ciliary transition zones and basal bodies, roles not ascribed to IFT. Unifying these findings, we provide evidence that the underlying molecular defects in the che-10 mutant stem from disrupted organization/function of the periciliary membrane, affecting the efficient delivery of basal body-associated and ciliary components and resulting in cilium degeneration. Together, our cloning and functional analyses of C. elegans che-10 provide the first mechanistic insights into how filamentous and nonfilamentous forms of rootletin play essential roles in maintaining ciliary function in metazoans

EFHC1, Implicated in Juvenile Myoclonic Epilepsy, Functions at the Cilium and Synapse to Modulate Dopamine Signaling

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling. Yet, the roles of cilia in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC-1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC-1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy

Defensive flocculent emissions in a tiger moth, Homoeocera stictosoma (Arctiidae: Arctiinae)

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Data from: Insights into the roles of CMK-1 and OGT-1 in interstimulus interval-dependent habituation in Caenorhabditis elegans

Habituation is a ubiquitous form of non-associative learning observed as a decrement in responding to repeated stimulation that cannot be explained by sensory adaptation or motor fatigue. One of the defining characteristics of habituation is its sensitivity to the rate at which training stimuli are presented – animals habituate faster in response to more rapid stimulation. The molecular mechanisms underlying this interstimulus interval (ISI)-dependent characteristic of habituation remain unknown. In this article we use behavioral neurogenetic and bioinformatic analyses in the nematode Caenorhabiditis elegans to identify the first molecules that modulate habituation in an ISI-dependent manner. We show that the Caenorhabditis elegans orthologs of Ca2+/calmodulin-dependent kinases CaMK1/4, CMK-1, and O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, both function in primary sensory neurons to inhibit habituation at short ISIs and promote it at long ISIs. In addition, both cmk-1 and ogt-1 mutants display a rare mechanosensory hyper-responsive phenotype, i.e. larger mechanosensory responses than wild-type. Overall, our work identifies two conserved genes that function in sensory neurons to modulate habituation in an ISI-dependent manner, providing the first insights into the molecular mechanisms underlying the universally observed phenomenon that habituation has different properties when stimuli are delivered at different rates

Data from: EFHC1, implicated in juvenile myoclonic epilepsy, functions at the cilium and synapse to modulate dopamine signaling

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling, yet their roles in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy

Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport

<div><p>Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode <i>C</i>. <i>elegans</i>, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of <i>C</i>. <i>elegans</i> RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a <i>rab-28</i> null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for identifying ciliary proteins, and unveil RAB28, a GTPase most closely related to the BBS protein RABL4/IFT27, as an IFT-associated cargo with BBSome-dependent cell autonomous and non-autonomous functions at the ciliary base.</p></div

RAB-28 undergoes IFT.

<p><b>(A, B)</b> Representative images of phasmid cilia (left) from N2 wild type and <i>che-11(e1810)</i> worms expressing GFP::RAB-28, together with corresponding kymographs and kymograph schematics derived from time-lapse imaging. Distribution plot (and mean values) in B shows kymography-determined anterograde and retrograde GFP::RAB-28 velocities from wild type worms. MS; middle segment, DS; distal segment, PC; periciliary membrane compartment, DD; distal dendrite. Scale bars; 3 μm (phasmid image; and horizontal bar on kymographs); 3 seconds (vertical bar on kymographs). <b>(C)</b> Fluorescence recovery after photobleaching (FRAP) plots for GFP::RAB-28 in the phasmid neurons of N2 and <i>che-11(e1810)</i> mutant worms. GFP::RAB-28 also undergoes free diffusion in wild-type (N2) and <i>che-11</i> IFT mutant animals. Ciliary GFP signals bleached at time 0. Intensity measurements normalised to pre-bleach levels. Curves derived from 3 separate FRAP experiments. Error bars; SEM. Images taken from a representative FRAP experiment in N2 wild type worms. s; seconds. Scale bar; 2 μm.</p

BBSome-dependent recruitment of activated RAB-28 to the periciliary membrane.

<p><b>(A, B)</b> Representative images of amphid and phasmid cilia from N2 wild type (A) and <i>bbs-8(nx77)</i> mutant (B) worms expressing GFP-tagged RAB-28(WT), RAB-28(GDP) and RAB-28(GTP) reporters. All reporters are driven by the endogenous <i>rab-28</i> gene promoter. Kymographs and kymograph schematics derived from time-lapse imaging of GFP signals in phasmid cilia. Phenotypes summarised in cartoons. Large phasmid images are placed on black backgrounds. m; middle segment, d; distal segment. pcmc; periciliary membrane compartment (also denoted by arrow). Scale bars; 2 μm and 5 μm (low magnification phasmid images).</p

Candidate cilium-associated proteins localise to ciliary structures Representative images of amphid (head) and phasmid (tail) cilia from N2 wild type worms co-expressing GFP-tagged ‘translational’ reporters for candidate ciliary genes and tdTomato-tagged XBX-1 (IFT protein that localises to the ciliary base and axoneme).

<p>All reporters driven by the endogenous gene promoter, except for YAP-1 and RAB-28, where a <i>bbs-8</i> gene promoter sequence was used (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006469#pgen.1006469.g003" target="_blank">Fig 3A</a> for a GFP::RAB-28 reporter driven by the endogenous promoter). GFP tags are on the N- or C-terminus, as indicated above the panels. PCMC (periciliary membrane compartment); distal dendrite (den) swelling enriched for endocytosis-associated proteins and vesicles that regulate ciliary membrane homeostasis [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006469#pgen.1006469.ref019" target="_blank">19</a>]. The transition zone (TZ) extends from basal body (BB) and functions as a ciliary gate that regulates protein entry and exit to and from cilia [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006469#pgen.1006469.ref006" target="_blank">6</a>]. Middle (MS) and distal (DS) segments; characterised by a circular array of 9 doublet (A and B tubules) and 9 singlet (A tubules) microtubules, respectively (not shown). Note that for GFP::CCDC-149, signals are observed at the amphid/phasmid basal bodies (arrows) and as punctae (asterisks) along the dendrite of the OLQ ciliated neuron running parallel to the amphid neurons. Scale bars; 5 μm.</p