Biology subjects

Boulin, T.

Publications and source records attributed to Boulin, T..

10 recordsLinked to original sources

bioRxiv2026

PZLS-1/PDZRN controls muscle polarity downstream of Wnt-Ror-Dvl signalling in C. elegans muscles

Non-canonical Wnt pathways provide directional cues for cell polarity, but the mechanisms that translate these cues into asymmetric cellular organisation remain incompletely defined. We previously showed that a non-canonical EGL-20/Wnt-CAM-1/Ror-DSH-1/Dishevelled pathway establishes polarised membrane compartments in Caenorhabditis elegans body wall muscles. However, how EGL-20/Wnt signalling is translated into muscle polarity remains unknown. Here, we identify PZLS-1, the C. elegans ortholog of human PDZRN3, PDZRN4 and PDZD4, as a regulator of muscle polarity. Although PDZRN proteins are known targets of non-canonical Wnt-Ror signalling, how they are spatially organised within polarised cells and how their protein domains contribute to their function remain largely unknown. We reveal that PZLS-1 is itself asymmetrically localised by Wnt-Ror-Dvl signalling, and is closely associated with DSH-1. PZLS-1 and DSH-1 colocalise in intracellular puncta and are mutually required for their localisation. In vivo structure-function analyses reveal conserved protein motifs that differentially control PZLS-1 localisation, abundance and function. This includes a previously uncharacterized highly conserved sequence that regulates PZLS-1 abundance in a DSH-1-dependent manner. We further show that PZLS 1 and DSH-1 control the abundance and polarised localisation of VAB-8/KIF26b, another conserved target of Wnt-Ror signalling. Together, these findings indicate that Wnt-Ror-Dvl signalling can control the spatial organisation and abundance of PDZRN- and KIF26B-family proteins in vivo, providing a mechanism to translate extracellular polarity cues into intracellular asymmetry.

cell biology↗

bioRxiv2026

K2P Channels Regulate Presynaptic Organisation through a Membrane Potential-Independent Mechanism

Neuronal ion channels have well-established effects on synaptic plasticity, in many cases by influencing pathways that depend on membrane excitability. Here we find that a C. elegans two-pore domain potassium (K2P) channel, TWK-40, regulates presynaptic organisation through a membrane potential-independent mechanism. Instead, this mechanism depends on TWK-40's effects on intracellular potassium levels. Loss-of-function mutations in TWK-40 lead to excessive presynaptic protein accumulation, while gain-of-function mutations lead to depleted presynaptic components and cause synaptic transmission deficits. These abnormalities are phenocopied by transporter mutations that mimic TWK-40's effects on intracellular potassium concentration, but not by sodium channel mutations that mimic its effects on membrane excitability. This indicates that cytoplasmic potassium promotes presynaptic assembly. This process depends on the PYK-1 pyruvate kinase, a potassium-sensitive enzyme, and three transcription factors. These findings establish a new pathway linking neuronal potassium homeostasis to the control of presynaptic organisation and synaptic function.

molecular biology↗

bioRxiv2025

Functional validation of human SK channels variants causing NEDMAB and Zimmermann-Laband syndrome-3 in C. elegans

Small-conductance Ca2+-activated K+ channels (SK channels) are widely expressed in the central nervous system, where they play a crucial role in modulating neuronal excitability. Recent studies have identified missense variants in the genes encoding SK2 and SK3 channels as the cause of two rare neurodevelopmental disorders: NEDMAB and ZLS3, respectively. Here, we used C. elegans as an in vivo model to investigate the functional consequences of these patient variants. The C. elegans ortholog KCNL-1 regulates neuronal and muscle excitability in the egg-laying system, a well-characterized model circuit. To visualize KCNL-1 expression and localization, we generated a fluorescent translational reporter at the endogenous kcnl-1 locus. We then introduced eight point mutations corresponding to pathogenic variants reported in NEDMAB or ZLS3 patients. Our study confirmed the molecular pathogenicity of the ZLS3-associated mutations, revealing a gain-of-function effect that led to increased in utero egg retention, likely due to electrical silencing of the egg-laying circuitry. NEDMAB mutations exhibited more complex phenotypic effects. Most caused a loss-of-function phenotype, indistinguishable from null mutants, while one displayed a clear gain-of-function effect. Additionally, a subset of NEDMAB variants altered KCNL-1 localization, suggesting an impairment in channel biosynthesis, trafficking or stability. These findings provide new insights into the molecular mechanisms underlying NEDMAB and ZLS3 physiopathology, enhancing our understanding of SK channel dysfunction in human disease. Moreover, they establish C. elegans as a robust and cost-effective in vivo model for rapid functional validation of new SK channel mutations, paving the way for future investigations.

neuroscience↗

bioRxiv2024

Constitutive sodium permeability in a C. elegans two-pore domain potassium channel

Two-pore domain potassium (K2P) channels play a central role in modulating cellular excitability and neuronal function. The unique structure of the selectivity filter in K2P and other potassium channels determines their ability to allow the selective passage of potassium ions across cell membranes. The nematode C. elegans has one of the largest K2P families, with 47 subunit-coding genes. This remarkable expansion has been accompanied by the evolution of atypical selectivity filter sequences that diverge from the canonical TxGYG motif. Whether and how this sequence variation may impact the function of K2P channels has not been investigated so far. Here we show that the UNC-58 K2P channel is constitutively permeable to sodium ions and that a cysteine residue in its selectivity filter is responsible for this atypical behavior. Indeed, by performing in vivo electrophysiological recordings and Ca2+ imaging experiments, we demonstrate that UNC-58 has a depolarizing effect in muscles and sensory neurons. Consistently, unc-58 gain-of-function mutants are hypercontracted, unlike the relaxed phenotype observed in hyperactive mutants of many neuromuscular K2P channels. Finally, by combining molecular dynamics simulations with functional studies in Xenopus laevis oocytes, we show that the atypical cysteine residue plays a key role in the unconventional sodium permeability of UNC-58. As predicting the consequences of selectivity filter sequence variations in silico remains a major challenge, our study illustrates how functional experiments are essential to determine the contribution of such unusual potassium channels to the electrical profile of excitable cells. SIGNIFICANCEPotassium channels play a central role in modulating cellular excitability, particularly of neuronal cells. Their unique structure determines their ability to let ions pass selectively through cell membranes. The impact of pathological or evolutionary variations in this selectivity filter remains difficult to predict. Here, we reveal that UNC-58, a member of the two-pore domain potassium channel family of C. elegans, exhibits an unusual sodium permeability due to a unique cysteine residue in its selectivity filter. Our findings underscore the importance of functional studies to determine how sequence variation in potassium channel selectivity filters can shape the electrical profiles of excitable cells.

physiology↗

bioRxiv2023

Distinct dystrophin and Wnt/Ror-dependent pathways establish planar-polarized membrane compartments in C. elegans muscles

Cell polarity mechanisms allow the formation of specialized membrane domains with unique protein compositions, signalling properties, and functional characteristics. By analysing the localization of potassium channels and proteins belonging to the dystrophin-associated protein complex, we reveal the existence of distinct planar-polarized membrane compartments at the surface of C. elegans muscle cells. We find that muscle polarity is controlled by a non-canonical Wnt signalling cascade involving the ligand EGL-20/Wnt, the receptor CAM-1/Ror, and the intracellular effector DSH-1/Dishevelled. Interestingly, classical planar cell polarity proteins are not required for this process. Using time-resolved protein degradation, we demonstrate that -while it is essentially in place by the end of embryogenesis- muscle polarity is a dynamic state, requiring continued presence of DSH-1 throughout post-embryonic life. Our results reveal the unsuspected complexity of the C. elegans muscle membrane and establish a novel genetically tractable model system to study cellular polarity and membrane compartmentalization in vivo.

cell biology↗

bioRxiv2023

Natural variation in the Caenorhabditis elegans egg-laying circuit modulates an intergenerational fitness trade-off

Evolutionary shifts from egg-laying (oviparity) to live birth (viviparity) are common across diverse taxa, and some species exhibit variability in egg-laying modes or an intermediate mode with eggs containing embryos at various stages of development. How such natural quantitative variation in egg retention arises through differences in genetics, behaviour, and physiology - and how this variation ultimately connects to variation in specific fitness components - remains poorly understood. Here, we investigate intraspecific variation in egg retention in the nematode Caenorhabditis elegans using a panel of 316 wild strains. We observe highly variable intra-uterine egg retention, with some strains exhibiting strongly reduced or increased retention with internal hatching. We identify multiple evolutionary origins of these phenotypic extremes and pinpoint candidate loci responsible for the observed variation. Analysis of a subset of strains confirms that natural variation in egg-laying behaviour contributes to differences in egg retention. Using neuromodulatory agents and CRISPR-Cas9-mediated genetic manipulation, we demonstrate that this behavioural variation arises from an evolutionarily divergent neuromodulatory architecture in the egg-laying circuitry. In addressing the question of why natural variation in C. elegans egg retention is maintained, we find that strong egg retention can reduce maternal fertility and survival due to detrimental hatching in utero. However, genotypes with strong egg retention may benefit from improved offspring protection against environmental insults and gain a competitive advantage with offspring exhibiting a shortened development time to reproductive maturity outside the uterus. The observed natural variation in egg-laying behaviour in C. elegans could therefore reflect modifications of a trade-off between alternative fitness components across generations. Our study reveals previously underappreciated diversity in the C. elegans egg-laying circuit and provides insights into the fitness consequences of this behavioural variation. We propose that intraspecific variation in nematode egg-laying behaviour offers an ideal system to identify molecular changes underlying evolutionary transitions between oviparity and viviparity in invertebrates.

evolutionary biology↗

bioRxiv2022

K2P channel TWK-40 Regulates a Rhythmic Behavior in C. elegans

Leak potassium (K+) currents, conducted by two-pore domain K+ (K2P) channels, are critical for the stabilization of the membrane potential. The effect of K2P channels on motor rhythm remains enigmatic. We show here that the K2P TWK-40 regulates the rhythmic defecation motor program (DMP) in Caenorhabditis elegans. Disrupting TWK-40 suppresses the expulsion defects of nlp-40 and aex-2 mutants. By contrast, a gain-of-function (gf) mutant of twk-40 significantly reduces the expulsion frequency per DMP cycle. In situ whole-cell patch clamping demonstrates that TWK-40 forms an outward current that hyperpolarize the resting membrane potential of DVB neuron. In addition, TWK-40 substantially contributes to the rhythmic activity of DVB. Specifically, DVB Ca2+ oscillations exhibit obvious defects in twk-40 mutants. Expression of TWK-40(gf) in DVB recapitulates the expulsion deficiency of the twk-40(gf) mutant, and inhibits DVB Ca2+ oscillations in both wild-type and twk-40(lf) animals. Moreover, DVB innervated enteric muscles also exhibit rhythmic Ca2+ defects. Taken together, these results demonstrate that TWK-40 is an essential neuronal inhibitor of DMP, thus revealing a cellular mechanism linking leak K2P channels with rhythmic motor activity.

neuroscience↗

bioRxiv2022

A descending interneuron with depolarized resting membrane potential controls C. elegans motor states

Continuity of behaviors requires animals to make smooth transitions between successive and mutually exclusive behavioral states. Neural principles that govern these transitions are not well understood. C. elegans spontaneously switch between two opposite motor states, forward and backward movement, a phenomenon long thought to reflect the reciprocal inhibition between two interneurons that separately gate the forward and backward motor circuits, AVB and AVA. Combining experimental data and mathematical modeling, we report that spontaneous forward and backward locomotion and their corresponding motor circuits are not separately controlled. AVA and AVB are neither functionally equivalent nor strictly reciprocally inhibitory. Instead, while AVA phasically inhibits the forward promoting interneuron AVB at a fast timescale, it maintains a tonic, extrasynaptic excitation on AVB over the longer timescale. AVAs depolarized spontaneous membrane potential is necessary for this tonic excitation. We propose a new, master neuron model for locomotion. AVA, with tonic and phasic activity of opposite polarities on different time scales, acts as a master neuron to break the symmetry between the underlying forward and backward motor circuits. This offers a parsimonious solution for sustained locomotion consisted of mutually exclusive motor states. TeaserA tonically active C. elegans premotor interneuron functions as the master neuron that underlies continuous modulation of forward and backward movement to ensure smooth transitions between the two opposing motor states.

neuroscience↗

bioRxiv2020

A single nucleotide change underlies the genetic assimilation of a plastic trait

Genetic assimilation - the evolutionary process by which an ancestral environmentally sensitive phenotype is made constitutive - is a fundamental concept in biology. Its evolutionary relevance is debated, and our understanding of its prevalence, and underlying genetics and molecular mechanisms, is poor. Matricidal hatching is an extreme form of maternal provisioning induced by adverse conditions, which varies among Caenorhabditis elegans populations. We identified wild isolates, sampled from natural populations across multiple years and locations, that express a derived state of near-constitutive matricidal hatching. A single amino acid change in kcnl-1, encoding a small-conductance calcium-activated potassium channel subunit, explains most of this variation. A gain-of-function mutation altering the S6 transmembrane domain causes inappropriate activation of the K+ channel, leading to reduced vulval muscle excitability, and thus reduced expulsion of embryos, irrespective of environment. Using reciprocal allelic replacements, we show that this amino acid change is sufficient to induce constitutive matricidal hatching whilst re-establishing the ancestral protein abolishes matricidal hatching and restores egg-laying, thereby doubling lifetime reproductive fitness under benign conditions. While highly deleterious in the laboratory, experimental evolution showed that KNCL-1(V530L) is maintained under fluctuating resource availability. Selection on a single point mutation can therefore underlie the genetic assimilation of an ancestrally plastic trait with drastic life-history consequences.

evolutionary biology↗

bioRxiv2017

Reliable CRISPR/Cas9 genome engineering in Caenorhabiditis elegans using a single efficient sgRNA and an easily selectable phenotype

CRISPR/Cas9 genome engineering strategies allow the directed modification of the C. elegans genome to introduce point mutations, generate knock-out mutants and insert coding sequences for epitope or fluorescent tags. Three practical aspects however complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an 8-fold increase in fluorescence in a direct comparison.

genetics↗