Biology subjects

de Bono, M.

Publications and source records attributed to de Bono, M..

9 recordsLinked to original sources

bioRxiv2023

Neuroendocrine control of synaptic transmission by PHAC-1 in C. elegans.

A dynamic interplay between synaptic and neuromodulatory signalling guarantees flexible but robust neuronal circuits. Presynaptic modulation plays a crucial role in controlling the excitatory-inhibitory balance within networks. Here, we designed a genetic screen to identify genes involved in the neuromodulation of the C. elegans neuromuscular junctions (NMJ) and identified the orthologs of the Protein Phosphatase 1 regulatory subunit PHACTR1 (phac-1) and the presynaptic phosphoproteins Synapsin (snn-1). Five de novo variants of human PHACTR1 are associated with severe early-onset epilepsies (DEE70). To understand the impact of these variants, we introduced the DEE70 mutations into phac-1. These mutations resulted in the formation of a constitutively active PP1-PHAC-1 holoenzyme that disrupts cholinergic signalling at the NMJ. By using quantitative fluorescence imaging, electron microscopy and electrophysiology, we found that the constitutive holoenzyme alters the synaptic vesicle cycle, reduces the synaptic vesicle reserve pool, and increases neuropeptide release by dense-core vesicles. Notably, while SNN-1 phosphoregulation contributes to NMJ signalling, genetic interactions suggest that SNN-1 is not the main effector of PP1-PHAC-1 holoenzyme signalling. Collectively, our results confirm the pathogenicity of DEE70 variants, clarify their dominant-positive effects, and provide evidence of a presynaptic mode of action for DEE70.

neuroscience↗

bioRxiv2022

An improved TurboID pipeline in C. elegans by biochemical depletion of endogenously biotinylated carboxylases

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for C. elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant, endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a co-factor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase and MCCC-1/methylcrotonyl-CoA carboxylase alpha. We developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry, by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography (IMAC). To demonstrate the methods efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, as well as previously uncharacterized potentially synaptic proteins. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin- dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.

biochemistry↗

bioRxiv2021

Interactome analysis of C. elegans synapses by TurboID-based proximity labeling

Proximity labelling provides a powerful in vivo tool to characterize the proteome of sub-cellular structures and the interactome of specific proteins. Using the highly active biotin ligase TurboID, we optimize a proximity labelling protocol for C. elegans. We use this protocol to characterise the proteomes of the worms gut, muscle, skin, and nervous system. We express TurboID exclusively in the pair of AFD neurons and show we can identify known and previously unknown proteins expressed selectively in AFD. We knock TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identify many known ELKS-1 interacting proteins as well as previously uncharacterised synaptic proteins. Versatile vectors, and the inherent advantage of C. elegans for biochemistry, make proximity labelling a valuable addition to the nematodes armory. TeaserWe optimize a TurboID proximity labeling protocol for C. elegans and use it to characterize tissue and synaptic proteomes

biochemistry↗

bioRxiv2021

Impairing one sensory modality enhances another by reprogramming peptidergic circuits in Caenorhabditis elegans

Animals that lose one sensory modality often show augmented responses to other sensory inputs. The mechanisms underpinning this cross-modal plasticity are poorly understood. To probe these mechanisms, we perform a forward genetic screen for mutants with enhanced O2 perception in C. elegans. Multiple mutants exhibiting increased responsiveness to O2 concomitantly show defects in other sensory responses. One mutant, qui-1, defective in a conserved NACHT/WD40 protein, abolishes pheromone-evoked Ca2+ responses in the ADL chemosensory neurons. We find that ADLs responsiveness to pre-synaptic input from O2- sensing neurons is heightened in qui-1 and other sensory defective mutants resulting in an enhanced neurosecretion. Expressing qui-1 selectively in ADL rescues both the qui-1 ADL neurosecretory phenotype and enhanced escape from 21% O2. Profiling of ADL neurons indicates its acquired O2-evoked neurosecretion is the result of a transcriptional reprogramming that up-regulates neuropeptide signalling. We show that the conserved neuropeptide receptor NPR-22 is necessary and sufficient in ADL to enhance its neurosecretion levels. Sensory loss can thus confer cross-modal plasticity by re-wiring peptidergic circuits.

neuroscience↗

bioRxiv2020

Transcriptional Control Of Calmodulin By CAMTA Regulates Neural Excitability

Calmodulin (CaM) is the major calcium ion (Ca2+) sensor in many biological processes, regulating for example the CaM kinases, calcineurin, and many ion channels. CaM levels are limiting in cells compared to its myriad targets, but how CaM levels are controlled is poorly understood. We find that CaM abundance in the C. elegans and Drosophila nervous systems is controlled by the CaM-binding transcription activator, CAMTA. C. elegans CAMTA (CAMT-1), like its fly and mammalian orthologues, is expressed widely in the nervous system. camt-1 mutants display pleiotropic behavioural defects and altered Ca2+ signaling in neurons. Using FACS-RNA Seq we profile multiple neural types in camt-1 mutants and find all exhibit reduced CaM mRNA compared to controls. Supplementing CaM levels using a transgene rescues camt-1 mutant phenotypes. Chromatin immunoprecipitation sequencing (ChIP-Seq) data show that CAMT-1 binds several sites in the calmodulin promoter. CRISPR-mediated deletion of these sites shows they redundantly regulate calmodulin expression. We also find that CaM can feed back to inhibit its own expression by a mechanism that depends on CaM binding sites on CAMT-1. This work uncovers a mechanism that can both activate and inhibit CaM expression in the nervous system, and controls Ca2+ homeostasis, neuronal excitability and behavior.

neuroscience↗

bioRxiv2019

Long-term activity drives dendritic branch elaboration of a C. elegans sensory neuron

Neuronal activity often leads to alterations in gene expression and cellular architecture. The nematode Caenorhabditis elegans, owing to its compact translucent nervous system, is a powerful system in which to study conserved aspects of the development and plasticity of neuronal morphology. Here we focus on one sensory neuron in the worm, termed URX, which senses oxygen and signals tonically proportional to environmental oxygen. Previous studies have reported that URX has variable branched endings at its dendritic sensory tip. By controlling oxygen levels and analyzing mutants, we found that these branched endings grow over time as a consequence of neuronal activity. Furthermore, we observed that the branches contain microtubules, but do not appear to harbor the guanylyl cyclase GCY-35, a central component of the oxygen sensory transduction pathway. Interestingly, we found that although URX dendritic tips grow branches in response to long-term activity, the degree of branch elaboration does not correlate with oxygen sensitivity at the cellular or the behavioral level. Given the strengths of C. elegans as a model organism, URX may serve as a potent system for uncovering genes and mechanisms involved in activity-dependent morphological changes in neurons.

neuroscience↗

bioRxiv2019

MALT1 mediates IL-17 Neural Signaling to regulate C. elegans behavior, immunity and longevity

Besides well-known immune roles, the evolutionarily ancient cytokine interleukin-17 (IL-17) modulates neural circuit function. We investigate how IL-17 signals in neurons, and the extent to which this signaling can alter organismal phenotypes. We combine immunoprecipitation and mass spectrometry to biochemically characterize endogenous signaling complexes that function downstream of IL-17 receptors in C. elegans (Ce) neurons. We identify the Ce ortholog of MALT1 as a critical output of the pathway. MALT1 was not previously implicated in IL-17 signaling or in nervous system function. MALT1 forms a complex with homologs of Act1 and IRAK and functions both as a scaffold for I{kappa}B recruitment, and as a protease. MALT1 is expressed broadly in the Ce nervous system, and neuronal IL-17-MALT1 signaling regulates many phenotypes, including escape behavior, associative learning, immunity and longevity. Our data suggest MALT1 has an ancient role modulating neural function downstream of IL-17 to remodel physiological and behavioral state.

neuroscience↗

bioRxiv2018

A neuronal MAP kinase constrains growth of a C. elegans sensory dendrite throughout the life of the organism

Neurons develop elaborate morphologies that provide a model for understanding cellular architecture. By studying C. elegans sensory dendrites, we previously identified genes that act to promote the extension of ciliated sensory dendrites during embryogenesis. Interestingly, the nonciliated dendrite of the oxygen-sensing neuron URX is not affected by these genes, suggesting it develops through a distinct mechanism. Here, we use a visual forward genetic screen to identify mutants that affect URX dendrite morphogenesis. We find that disruption of the MAP kinase MAPK-15 or the {beta}H-spectrin SMA-1 causes a phenotype opposite to what we had seen before: dendrites extend normally during embryogenesis but begin to overgrow as the animals reach adulthood, ultimately extending up to 150% of their normal length. SMA-1 is broadly expressed and acts non-cell-autonomously, while MAPK-15 is expressed in many sensory neurons including URX and acts cell-autonomously. MAPK-15 acts at the time of overgrowth, localizes at the dendrite ending, and requires its kinase activity, suggesting it acts locally in time and space to constrain dendrite growth. Finally, we find that the oxygen-sensing guanylate cyclase GCY-35, which normally localizes at the dendrite ending, is localized throughout the overgrown region, and that overgrowth can be suppressed by overexpressing GCY-35 or by genetically mimicking elevated cGMP signaling. These results suggest that overgrowth may correspond to expansion of a sensory compartment at the dendrite ending, reminiscent of the remodeling of sensory cilia or dendritic spines. Thus, in contrast to established pathways that promote dendrite growth during early development, our results reveal a distinct mechanism that constrains dendrite growth throughout the life of the animal, possibly by controlling the size of a sensory compartment at the dendrite ending.\n\nAUTHOR SUMMARYLewis Carrolls Alice told the Caterpillar, \"Being so many different sizes in a day is very confusing.\" Like Alice, the cells of our bodies face a problem in size control - they must become the right size and remain that way throughout the life of the organism. This problem is especially relevant for nerve cells (neurons), as the lengths of their elaborate dendrites determine the connections they can make. To learn how neurons control their size, we turned not to a Caterpillar but to a worm: the microscopic nematode C. elegans, in which single neurons can be easily visualized and the length of each dendrite is highly predictable across individuals. We focused on a single dendrite, that of the oxygen-sensing neuron URX, and we identified two genes that control its length. When these genes are disrupted, the dendrite develops correctly in embryos but then, like Alice, grows too much, eventually extending up to 1.5x its normal length. Thus, in contrast to known pathways that promote the initial growth of a dendrite early in development, our results help to explain how a neuron maintains its dendrite at a consistent length throughout the animals life.

developmental biology↗

bioRxiv2017

A memory of recent oxygen experience switches pheromone valence in C. elegans

Animals adjust their behavioral priorities according to momentary needs and prior experience. We show that C. elegans changes how it processes sensory information according to the oxygen environment it experienced recently. C.elegans acclimated to 7% O2 are aroused by CO2 and repelled by pheromones that attract animals acclimated to 21% O2. This behavioral plasticity arises from prolonged activity differences in a circuit that continuously signals O2 levels. A sustained change in the activity of O2 sensing neurons reprograms the properties of their post-synaptic partners, the RMG hub interneurons. RMG is gap-junctionally coupled to the ASK and ADL pheromone sensors that respectively drive pheromone attraction and repulsion. Prior O2 experience has opposite effects on the pheromone responsiveness of these neurons. These circuit changes provide a physiological correlate of altered pheromone valence. Our results suggest C. elegans stores a memory of recent O2 experience in the RMG circuit and illustrate how a circuit is flexibly sculpted to guide behavioral decisions in a context-dependent manner.

neuroscience↗