The active zone protein CLA-1 (Clarinet) bridges two subsynaptic domains to regulate presynaptic sorting of ATG-9

Autophagy, Год журнала: 2023, Номер 19(10), С. 2807 - 2808

Опубликована: Июнь 30, 2023

In neuronal synapses, autophagosome biogenesis is coupled with the activity-dependent synaptic vesicle cycle via ATG-9. How vesicles containing ATG-9 are sorted at presynapse unknown. We performed forward genetic screens single synapses of C. elegans neurons for mutants that disrupt presynaptic localization, and identified long isoform active zone protein CLA-1 (Clarinet; L). find disrupting L results in abnormal accumulation ATG-9-containing enriched clathrin. The adaptor complexes proteins periactive genetically interact sorting. Moreover, phenotype cla-1(L) was not observed integral proteins, suggesting distinct mechanisms regulate sorting vesicles. Our findings reveal novel roles macroautophagy/autophagy.

Язык: Английский

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Activity-driven trafficking of endogenous synaptic proteins through proximity labeling

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Апрель 23, 2024

Abstract To enable transmission of information in the brain, synaptic vesicles fuse to presynaptic membranes, liberating their content and exposing transiently a myriad vesicular transmembrane proteins. However, versatile methods for quantifying translocation endogenous proteins during neuronal activity remain unavailable, as fast dynamics vesicle cycling difficult specific isolation trafficking such transient surface exposure. Here we developed novel approach using cleft proximity labeling capture quantify activity-driven at synapse. We show that accelerating biotinylation times match exocytosis allows capturing exposed neural activity, enabling first time study nearly every protein. As proof-of-concept, further applied this technology obtain direct evidence non-canonical proteins, ATG9A NPTX1, which had been proposed traffic but proof not yet shown. The technological advancement presented here will facilitate future studies dissecting molecular identity exocytosed synapse helping define machinery sustains neurotransmission mammalian brain. Significance statement Synaptic is critical neurons communicate sustain brain function. Pascual-Caro de Juan-Sanz develop pioneering method any Coordinating clefts just few seconds, authors visualize This work provides framework uncover complex choreography events occurring within firing synapses, deeper control communication circuit physiology

Язык: Английский

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A modular system to label endogenous presynaptic proteins using split fluorophores inC. elegans

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июль 30, 2024

Abstract Visualizing the subcellular localization of presynaptic proteins with fluorescent is a powerful tool to dissect genetic and molecular mechanisms underlying synapse formation patterning in live animals. Here, we utilize split green red visualize endogenously expressed at single neuron resolution Caenorhabditis elegans. By using CRISPR/Cas9 genome editing, generated collection C. elegans strains which (RAB-3/Rab3, CLA-1/Piccolo, SYD-2/Liprin-α, UNC-10/RIM ELKS-1/ELKS) are tagged tandem repeats GFP 11 and/or wrmScarlet . We show that expression 1-10 under neuron-specific promoters can robustly label different types. believe combinations knock-in plasmids versatile modular system examine endogenous any type.

Язык: Английский

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Monitoring of activity-driven trafficking of endogenous synaptic proteins through proximity labeling

PLoS Biology, Год журнала: 2024, Номер 22(10), С. e3002860 - e3002860

Опубликована: Окт. 28, 2024

To enable transmission of information in the brain, synaptic vesicles fuse to presynaptic membranes, liberating their content and exposing transiently a myriad vesicular transmembrane proteins. However, versatile methods for quantifying translocation endogenous proteins during neuronal activity remain unavailable, as fast dynamics vesicle cycling difficult specific isolation trafficking such transient surface exposure. Here, we developed novel approach using cleft proximity labeling capture quantify activity-driven at synapse. We show that accelerating biotinylation times match exocytosis allows capturing exposed neural activity, enabling first time study nearly every protein. As proof-of-concept, further applied this technology obtain direct evidence noncanonical proteins, ATG9A NPTX1, which had been proposed traffic but proof not yet shown. The technological advancement presented here will facilitate future studies dissecting molecular identity exocytosed synapse helping define machinery sustains neurotransmission mammalian brain.

Язык: Английский

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A modular system to label endogenous presynaptic proteins using split fluorophores in C. elegans

Genetics, Год журнала: 2024, Номер unknown

Опубликована: Дек. 22, 2024

Abstract Visualizing the subcellular localization of presynaptic proteins with fluorescent is a powerful tool to dissect genetic and molecular mechanisms underlying synapse formation patterning in live animals. Here, we utilize split green red visualize endogenously expressed at single neuron resolution Caenorhabditis elegans. By using CRISPR/Cas9 genome editing, generated collection C. elegans strains which (RAB-3/Rab3, SNG-1/Synaptogyrin, CLA-1/Piccolo, SYD-2/Liprin-α, UNC-10/RIM, RIMB-1/RIM-BP, ELKS-1/ELKS) are tagged tandem repeats GFP11 and/or wrmScarlet11. We show that expression GFP1-10 wrmScarlet1-10 under neuron-specific promoters can robustly label different types. believe combination our knock-in plasmids versatile modular system useful for neuroscientists examine endogenous any type

Язык: Английский

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