Biology subjects

Glauser, D. A.

Publications and source records attributed to Glauser, D. A..

8 recordsLinked to original sources

bioRxiv2025

Starvation transforms signal encoding in C. elegans thermoresponsive neurons and suppresses heat avoidance via bidirectional glutamatergic and peptidergic signaling

Animals must continuously adapt their behavioral outputs in response to changes in internal state, including nutritional state. Here, we show that starvation induces a profound and progressive suppression of thermonociceptive behavior in Caenorhabditis elegans. During early food deprivation (1-hr off food), the thermoresponsive sensory neurons AWCs mediate robust heat-evoked reversals over a broad range of stimulus intensities via glutamate and FLP-6 neuropeptide signaling, each covering distinct heat intensity ranges. After six hours of food deprivation (prolonged starvation), heat-evoked reversal responses are nearly abolished, independently of any external food odor cues. Starvation triggers a shift in the distribution of AWC heat-evoked calcium response polarity, transitioning from mostly excitatory responses to a more heterogeneous pattern combining both excitatory and inhibitory activities. This switch relies on ASI neurons, proposed to work as internal state-sensing neurons. INS-32 and NLP-18 neuropeptide signals from ASI switch from a reversal-promoting to a reversal-inhibiting effect. In addition, reversal-promoting glutamatergic transmission by AWC is antagonized by glutamatergic transmission from non-AWC neurons that suppresses FLP-6-dependent reversals. Our findings define a circuit logic by which nociceptive responsiveness gating by internal nutritional state is linked to dynamic modulation of sensory neuron activity patterns and orchestrated by bidirectional glutamatergic and neuropeptidergic signals. More broadly, this study illustrates how sensory systems integrate metabolic information to prioritize behavioral outputs under changing physiological conditions, providing mechanistic insight into the plastic coupling between sensation, internal state, and action selection.

neuroscience↗

bioRxiv2024

Antagonist actions of CMK-1/CaMKI and TAX-6/Calcineurin along the C. elegans thermal avoidance circuit orchestrate nociceptive habituation

Response decrease following repeated exposure to innocuous or noxious stimuli is a conserved adaptation phenomenon often referred to as habituation. Impaired nociceptive habituation is associated with several pain conditions in human, but the underpinning molecular mechanisms are only partially understood. In the nematode Caenorhabditis elegans, thermo-nociceptive adaptation to repeated stimuli was previously shown to be regulated by the Ca2+/Calmodulin-dependent protein kinase 1 (named CMK-1), but its downstream effectors were unknown. Here, using in vitro kinase assays coupled with mass-spectrometry-based phosphoproteomics, we empirically identified hundreds of CMK-1 phospho-substrates. Among them, we found that CMK-1 can phosphorylate the calcineurin A (CnA) protein TAX-6 in vitro in a highly conserved regulatory domain, which led us to hypothesize that TAX-6/CnA might be a downstream mediator of CMK-1 signaling in the control of thermo-nociceptive adaptation. Combined genetic and pharmacological manipulations revealed a network of antagonistic actions between CMK-1 and calcineurin pathways in the regulation of the responsiveness of naive worms and the response adaptation to repeated noxious heat stimuli. However, the results of cell-specific rescue and gain-of-function experiments suggested that CMK-1 acts in AFD and ASER thermosensory neurons and that TAX-6/CnA acts in FLP thermosensory neuron and a set of downstream interneurons to regulate noxious heat avoidance behaviors. Because CMK-1 and TAX-6/CnA act in non-overlapping cell types, the phosphorylation event identified in vitro might not be relevant for this phenotype and the complex interaction between the two pathways might rather originate from their action in separate parts of the nervous system. As a whole, our study has identified (i) CMK-1 substrate candidates, which will fuel further research on the intracellular actuation of CMK-1-dependent signaling on various processes, and (ii) a complex set of antagonistic interactions between CMK-1 and calcineurin signaling operating at distributed loci within a sensory-behavior circuit, acting to adjust baseline thermo-nociception and regulate thermo-nociceptive adaptation.

neuroscience↗

bioRxiv2024

Core Tri-fucosylation of Nematode N-glycans Requires Golgi α-mannosidase III Activity that Impacts Animal Growth and Behaviours

Many nematodes possess N-glycans with complex core chitobiose modifications, which is a feature observed in various free-living and parasitic nematodes but is absent in mammals. Using Caenorhabditis elegans as a model to study N-glycan biosynthesis, we demonstrated that the core N-acetylglucosamine (GlcNAc) residues can be modified by three fucosyltransferases in the Golgi, namely FUT-1, FUT-6 and FUT-8. While the asparagine-linked GlcNAc is modified with a 1,3- and 1,6-linked fucose by FUT-1 and FUT-8 respectively, the distal GlcNAc residue is 1,3-fucosylated solely by FUT-6. Interestingly, FUT-6 can only fucosylate N-glycan structures lacking the 1,6-mannose upper arm, indicating that a specific -mannosidase is required to generate substrates for subsequent FUT-6 activity. By analysing the N-glycomes of aman-3 mutants (tm5400 and a CRISPR/Cas9 knockout, hex-2;hex-3;aman-3) using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of the aman-3 gene abolishes 1,3-fucosylation of the distal GlcNAc of N-glycans, which suggests that AMAN-3 is the relevant mannosidase on whose action FUT-6 depends. To further investigate it, we recombinantly expressed AMAN-3 in insect cells and characterised its enzymatic activity in vitro. In contrast to the classical Golgi -mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent 1,6-mannosidase activity towards N-glycans. Using AMAN-3 and other recombinant C. elegans glycoenzymes, we remodelled a fluorescein conjugated-Man5GlcNAc2 structure; we were able to mimic N-glycan biosynthesis in the Golgi and generate a tri-fucosylated glycan in vitro. We performed confocal microscopy studies using a knock-in strain (aman-3::eGFP) and could show the Golgi localisation of AMAN-3. In addition, using a high-content computer-assisted C. elegans analysis platform, we observed that AMAN-3 deficient worms display significant developmental delays, morphological and behavioural alterations in comparison to the wild type. Therefore, our data suggested that AMAN-3 participates in nematode N-glycan biosynthesis in the Golgi and generates substrates for FUT-6; thereby, this enzyme is essential for the formation of the unusual tri-fucosylated chitobiose cores of nematode N-glycans, which may play important roles in nematode development and behaviour. BackgroundTri-fucosylation of N-glycan core is a conserved feature seen in the N-glycomes of several nematode species. However, beyond the three core fucosyltransferases, we know very little about the biosynthesis and biological function of these core modifications. ResultsComparative glycomics data revealed that aman-3 mutants possess underfucosylated N-glycomes. Biochemical characterisation of AMAN-3 clarified its optimal reaction conditions and substrate specificity and, we demonstrated a Golgi localisation. Thereafter in vitro reconstruction of biosynthesis of a core tri-fucosylated N-glycan was achieved using 8 recombinant C. elegans glycoenzymes. Notably, aman-3 deficient worms exhibited significant developmental and behavioural changes. ConclusionAMAN-3 is a Golgi -mannosidase required for core fucosylation of the distal N-acetylglucosamine of N-glycoproteins. SignificanceThis study elucidates the key role of a novel Golgi -mannosidase in the biosynthesis of the unusual N-glycans of C. elegans and related nematodes, thereby setting the stage for new approaches to study the roles of glycan in the biology and immunology of nematode glycoproteins.

biochemistry↗

bioRxiv2024

Cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and gates rapid phase shifts of the circadian clock

The circadian clock enables organisms to synchronize biochemical and physiological processes over a 24-hour period. Natural changes in lighting conditions, as well as artificial disruptions like jet lag or shift work, can advance or delay the clock phase to align physiology with the environment. Within the suprachiasmatic nucleus (SCN) of the hypothalamus, circadian timekeeping and resetting rely on both membrane depolarization and intracellular second-messenger signaling. Voltage-gated calcium channels (VGCCs) facilitate calcium influx in both processes, activating intracellular signaling pathways that trigger Period (Per) gene expression. However, the precise mechanism by which these processes are concertedly gated remains unknown. Our study demonstrates that cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and regulates phase shifts of the circadian clock. We observed that knocking down Cdk5 in the SCN of mice affects phase delays but not phase advances. This is linked to uncontrolled calcium influx into SCN neurons and an unregulated protein kinase A (PKA) - calcium/calmodulin-dependent kinase (CaMK) - cAMP response element-binding protein (CREB) signaling pathway. Consequently, genes such as Per1 are not induced by light in the SCN of Cdk5 knock-down mice. Our experiments identified Cdk5 as a crucial light-modulated kinase that influences rapid clock phase adaptation. This finding elucidates how light responsiveness and clock phase coordination adapt activity onset to seasonal changes, jet lag, and shift work.

neuroscience↗

bioRxiv2023

Cohesin forms fountains at active enhancers in C. elegans

Transcriptional enhancers must locate their target genes with both precision and efficiency. In mammals, this specificity is facilitated by topologically associated domains (TADs), which restrict the enhancer search space through three-dimensional genome organization. In contrast, the nematode genome lacks such TAD-based segmentation despite harboring over 30000 sequences with chromatin signature characteristic of enhancers, thereby raising the question of how enhancer-promoter specificity is achieved. Using high-resolution Hi-C in C. elegans, we identify distinct 3D chromatin structures surrounding active enhancers, which we term fountains. These structures span 38 kb in average, are unique to active enhancers, and are enriched for the major somatic cohesin complex. Fountains collapse upon in vivo cohesin cleavage, indicating their cohesin dependency. Notably, fountains accumulate topological stress, as evidenced by the enrichment of topoisomerases and the psoralen-binding signature of negatively-supercoiled DNA. Functionally, fountain disassembly correlates with transcriptional upregulation of active enhancer-proximal genes, suggesting that fountains act as spatial repressors of enhancer activity. This repression is particularly pronounced for neuronal genes, including the skn-1/Nrf gene, which becomes upregulated, switches isoform and transcription start site upon cohesin loss in a pair of head neurons. Behaviorally, cohesin cleavage alters nematode movement and foraging behavior, linking enhancer-driven transcriptional changes to neural circuit function and organismal phenotypes, reminiscent of pathologies caused by cohesin mutations in humans. Together, our findings uncover fountains as a novel 3D chromatin feature that modulates enhancer activity in a TAD-less genome, establishing a mechanistic link between genome architecture, gene regulation and behavior.

genomics↗

bioRxiv2022

Multiple antagonist calcium-dependent mechanisms control CaM Kinase-1 subcellular localization in a C. elegans thermal nociceptor

Nociceptive habituation is a conserved process through which pain-sensitivity threshold is adjusted based on past sensory experience and which may be dysregulated in human chronic pain conditions. Noxious heat habituation in C. elegans involves the nuclear translocation of CaM kinase-1 (CMK-1) in the FLP thermo-nociceptors neurons, causing reduced animal heat sensitivity and avoidance responses. The phosphorylation of CMK-1 on T179 by CaM kinase kinase-1 (CKK-1) is required for nuclear entry. Recently, we identified a specific Nuclear Export Sequence (NES) required to maintain CMK-1 in the cytoplasm at rest (20 {degrees}C) and showed that Ca2+/CaM binding is sufficient to enhance CMK-1 affinity for IMA-3 via a specific Nuclear Localization Signal (NLS) in order to promote nuclear entry after persistent heat stimulation (90 min at 28{degrees}C) (Ippolito et al., 2021). Here, we identified additional functional NES and NLS on CMK-1, whose activity can counteract previously identified elements. Furthermore, we clarify the relationship between the CaM-binding-dependent and T179-dependent effects. T179 phosphorylation can promotes nuclear entry both downstream of CaM-binding and as part of an independent/parallel pathway. Moreover, T179 phosphorylation can also produce the opposite effect by promoting nuclear export. Taken together, our studies show that multiple calcium-dependent regulatory mechanisms converge to bias the activity pattern across a network of NES/NLS elements, in order to activate CMK-1 nucleo-cytoplasmic shuttling, change its localization equilibrium and actuate stimulation-dependent nociceptive plasticity.

neuroscience↗

bioRxiv2022

Multisite gating in tonic sensory circuits integrates multimodal context to control persistent behavioral states

Maintaining or shifting between behavioral states according to context is essential for animals to implement fitness-promoting strategies. How integration of internal state, past experience and sensory inputs orchestrate persistent multidimensional behavior changes remains poorly understood. Here, we show that C. elegans integrates food availability and environment temperature over different timescales to engage in persistent dwelling, scanning, global or glocal search strategies matching thermoregulatory and feeding needs. Transition between states, in each case, requires lifting multiple regulatory gates including AFD or FLP tonic sensory neurons activity, neuropeptide expression and downstream circuit responsiveness. State-specific FLP-6 or FLP-5 neuropeptide signaling acts on a distributed set of inhibitory receptors to promote scanning or glocal search, respectively, bypassing dopamine and glutamate-dependent behavioral state control. Multisite gating-dependent behavioral switch by GPCRs in tonic sensory circuits might represent a conserved regulatory logic for persistent behavioral state transitions enabling a flexible prioritization on the valance of multiple inputs.

neuroscience↗

bioRxiv2020

Tissue-specific transcription footprinting using RNA PoI DamID (RAPID) in C. elegans

Differential gene expression across cell types underlies the development and cell physiology in multicellular organisms. C. elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates, however, to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the RNA PoI DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit in order to create transcriptional footprints via methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, sorted embryonic blastomeres and in different tissues from intact young adults by driving Dam fusion expression tissue-specifically. We obtained meaningful transcriptional footprints in line with RNA-seq studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 2362 candidate genes with putatively active transcription in XXX cells, among which the few known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were expressed in XXX and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging. Article summaryGene expression is a major determinant of cell fate and physiology, yet it is notoriously difficult to characterize in individual cell types for the widely used model system C. elegans. Here, we introduce a method based on the in vivo covalent modification of DNA by transcribing RNA polymerases to determine genome-wide transcription patterns in single tissues of embryos or young adult animals. We show that the method is able to identify actively transcribed genes in tissues representing down to 0.2% of the somatic cells in adult animals. Additionally, this method can be fully performed in a single laboratory by using third generation sequencing methods (ONT).

genetics↗