Dataset Information

In situ Proteomics Unveils Specialized Domains for Extrasynaptic Signaling on Neuronal Cilia

ABSTRACT: Neuronal cilia have emerged as signaling hubs, but their molecular composition and integration with synaptic communication remain unexplored. Using an engineered Arl13b-TurboID mouse model, we achieved robust cilia-specific biotinylation and proteomic profiling across diverse cell types. Comparative analyses revealed that brain and kidney cilia share only half of their proteome, with neuronal cilia uniquely enriched in synaptic proteins, adhesion molecules, and neurotransmitter receptors. Surprisingly, several signaling and adhesion molecules localize to neuronal cilia in discrete patterns maintained by active retrieval mechanisms. 16-fold expansion microscopy of mouse cortex revealed that the NMDA receptor is non-randomly positioned on ciliary membranes near glutamatergic synapses, suggesting that cilia eavesdrop on synaptic activity. These findings highlight the diversity and specialization of mammalian ciliary proteomes and indicates that nanoscale organization may endow neuronal cilia with extrasynaptic functions.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Neuron

SUBMITTER: Chia-Hsiang Chang

LAB HEAD: Maxence V. Nachury

PROVIDER: PXD067892 | Pride | 2026-06-08

REPOSITORIES: Pride

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In situ proteomics unveils specialized domains for extrasynaptic signaling on neuronal cilia.

Chang Chia-Hsiang CH Trinh Van Ngu VN Novak Sammy Weiser SW Lokesh Nidhi Rani NR Montecinos Catalina Kretschmar CK Boassa Daniela D Pownall Mark E ME Kalocsay Marian M Nachury Maxence V MV

Science advances 20260603 23

Neuronal cilia have emerged as crucial signaling hubs, yet their molecular composition and integration with synaptic communication remain poorly understood. Using a newly developed <i>Arl13b-TurboID</i> mouse model, we achieved robust cilia-specific biotinylation and proteomic profiling across diverse tissues and cell types. Comparative proteomics revealed notable tissue-specific specialization, with neuronal cilia uniquely enriched in synaptic proteins, adhesion molecules, and neurotransmitter ...[more]

PMID: 42234764

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Project description:The outermost layer of eukaryotic cilia is a coat of membrane anchored, glycosylated proteins often referred to as glycocalyx. This highly heterogeneous layer provides functions from regulation of adhesion, force transduction and protection to signaling. We describe the structure of the ciliary coat of the green alga Chlamydomonas by cryo-electron tomography and proteomic approaches and present the high-resolution single particle structure of FMG1B via cryo-electron microscopy, the most abundant constituent of the ciliary coat. We report FMG1B to be a highly unusual mucin orthologue, which lacks the major O-glycosylation of mammalian mucins, but undergoes significant N-glycosylation. We find that an isoform of FMG1-B, FMG1-A, previously believed to be not expressed, is present in Chlamydomonas and differentially regulated with FMG1-B. By micro-flow-based adhesion assays, we observe increased surface adhesion in the glycocalyx deficient double-mutant fmg1a-fmg1b. We find this mutant to be fully capable of surface-gliding, suggesting that neither isoform is required for extracellular force transduction by intraflagellar transport. Our data provide in-depth structural details of the ciliary coat that functions as the primary contact to the environment and reveal that FMG1 acts primarily as a protective layer with adhesion-regulative properties. Data deposited here include label free quantification data on whole cells or isolated cilia confirming single knockout of FMG1B and high abundance of FMG1A in CC4533fmg1b (combined_protein_WC_CC4533fmg1b.tsv ) and double knockout of FMG1A and FMG1B in M32fmg1afmg1b (combined_proteinCilia_M32_M32fmg1afmg1b.tsv). The latter dataset was further analyzed for abundance changes in the cilia proteome (Log2_LFQ_ratios_M32_M32fmg1afmg1b.csv). Further, isolated cilia of wild type M32 were analyzed under non-reducing conditions to confirm structure predicted disulfide bridges via LC-MS/MS in FMG1B (FMG1B_intraprotein_disulfides.xlsx). Finally, isolated cilia of M32 were subjected to N-glycopeptide analyses using InSource-CID (FMG1B_HexNAc_modified_peptides.xlsx).

			Action	DRS
	NPER1.csv	Csv
	WGA.csv	Csv
	eb11353_tc-1217a_QIP.mzIdentML	Mzid
	eb11353_tc-1217a_QIP.raw	Raw
	eb11353_tc_1217a_QIP.mzXML	Mzxml