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

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Learn more: PMC Disclaimer | PMC Copyright Notice Sci Adv . 2026 Jun 3;12(23):eaed5548. doi: 10.1126/sciadv.aed5548 In situ proteomics unveils specialized domains for extrasynaptic signaling on neuronal cilia Chia-Hsiang Chang 1, 2, † , Van Ngu Trinh 3 , Sammy Weiser Novak 4, ‡ , Nidhi Rani Lokesh 2, 5 , Catalina Kretschmar Montecinos 1, 2 , Daniela Boassa 4 , Mark E Pownall 2, 5 , Marian Kalocsay 3 , Maxence V Nachury 1, 2, * Author information Article notes Copyright and License information 1 Department of Ophthalmology, University of California, San Francisco, San Francisco, CA 94143, USA. 2 Smith Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA. 3 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 4 Waitt Advanced Biophotonics Core, Salk Institute, La Jolla, CA 92093, USA. 5 Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA. * Corresponding author. Email: maxence.nachury@ucsf.edu † Present address: Institute of Brain Science, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan. ‡ Present address: Department of Biology, University of Victoria, Victoria, BC V8P 5C2, Canada. Roles Chia-Hsiang Chang : Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing Van Ngu Trinh : Formal analysis, Investigation, Methodology, Resources, Validation, Writing - review & editing Sammy Weiser Novak : Data curation, Formal analysis, Methodology, Software, Validation, Visualization, Writing - review & editing Nidhi Rani Lokesh : Formal analysis, Investigation, Writing - review & editing Catalina Kretschmar Montecinos : Investigation, Methodology, Writing - review & editing Daniela Boassa : Resources, Supervision, Writing - review & editing Mark E Pownall : Formal analysis, Funding acquisition, Methodology, Resources, Software, Supervision, Writing - review & editing Marian Kalocsay : Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing - review & editing Maxence V Nachury : Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing Received 2025 Nov 3; Accepted 2026 Apr 23; Collection date 2026 Jun 5. Copyright © 2026 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited. PMC Copyright notice PMCID: PMC13232615  PMID: 42234764 Abstract Neuronal cilia have emerged as crucial signaling hubs, yet their molecular composition and integration with synaptic communication remain poorly understood. Using a newly developed Arl13b-TurboID 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 receptors. Unexpectedly, several signaling and adhesion molecules localize to neuronal cilia in discrete nanodomains maintained by active retrieval mechanisms. In the mouse cortex, expansion microscopy revealed that the NMDA receptor subunit GluN1 is organized in nanodomains on neuronal ciliary membranes, which are precisely positioned to sample neurotransmitter efflux from neighboring glutamatergic synapses. These findings establish neuronal cilia as specialized extrasynaptic signaling platforms, with nanoscale organization enabling them to integrate local synaptic cues and modulate neuronal connectivity. In situ proteomics and expansion microscopy reveal nanoscale organization of postsynaptic proteins on neuronal cilia. INTRODUCTION Primary cilia are micron-scale cellular projections that receive and transduce signals ( 1 , 2 ). In the central nervous system (CNS), most neurons and glial cells harbor a primary cilium, and human genetic studies have linked ciliary dysfunction to neurodevelopmental and cognitive disorders ( 3 ), including Bardet-Biedl syndrome (BBS) ( 4 ), Joubert syndrome ( 5 ), schizophrenia ( 3 , 6 – 8 ), bipolar disorder ( 3 , 6 ), autism spectrum disorders ( 9 – 13 ), and Parkinson’s disease ( 14 – 17 ). Despite their emerging role in neural signaling, the molecular mechanisms by which neuronal cilia contribute to brain connectivity and communication remain poorly understood. Neuronal communication can proceed via either synaptic (wiring) or extrasynaptic (signal diffusion in a local volume) transmission. Neuronal cilia are increasingly recognized as both synaptic and extrasynaptic devices that mediate communication between neurons and other cells within the brain ( 18 , 19 ), acting as cellular antennae that transduce signals ( 20 , 21 ). In specialized sensory neurons, such as photoreceptors and olfactory receptor neurons, cilia organize entire signaling cascades ( 22 , 23 ). By contrast, the function of cilia in most brain cells remains poorly understood. Recent studies suggest that neuronal cilia may influence synaptic connectivity ( 24 – 26 ). Volume electron microscopy (EM) has revealed that neuronal cilia are integrated into the neuronal circuitry in the mammalian brain ( 27 – 29 ): Mouse hippocampal cilia form intimate associations with axons named axociliary synapses ( 27 ), human cortical cilia frequently contact tripartite synapses involving astrocytes ( 29 ), and mouse visual cortex cilia are positioned to eavesdrop on neighboring synaptic signals ( 28 ). Despite these advances, the molecular underpinnings of cilia-synapse interactions remain elusive. Proteomic studies of sensory cilia have been facilitated by their biochemical isolation, enabling detailed characterization of their molecular makeup ( 30 – 33 ). However, most neuronal cilia cannot be purified, leaving their proteome largely unexplored. To address this gap, we developed an in situ proximity labeling approach in mice and systematically profiled the brain cilia proteome. We found that neuronal cilia harbor a diverse array of signaling proteins, adhesion molecules, and neurotransmitter receptors, distinct from renal cilia. Using expansion microscopy, we reveal the nanoscale spatial organization of ciliary proteins and demonstrate that neuronal cilia are closely positioned to glutamatergic synapses. Notably, the N -methyl- d -aspartate (NMDA) receptor subunit GluN1 is nonrandomly distributed on ciliary membranes near synaptic structures, suggesting that cilia may “eavesdrop” on synaptic communication. RESULTS ARL13B-TurboID enables efficient and cilium-selective biotinylation in mice To determine the most efficient proximity labeling strategy in neuronal cilia, we screened three ciliary targeting proteins and two proximity labeling enzymes via lentiviral transduction of cultured cerebellar granule neuron progenitors (GNPs) (fig. S1A). While well-validated in other systems, the ciliary targeting sequence of NPHP3 ( 34 ), and the ciliary G protein–coupled receptor (GPCR) somatostatin receptor 3 (SSTR3) were only partially enriched in GNP cilia (fig. S1, B and C). In contrast, ARL13B (ADP-ribosylation factor-like protein 13B) efficiently and specifically targeted GNP cilia (fig. S1, B and C). Unexpectedly, despite achieving robust biotinylation of the cilia proteome in kidney cells ( 34 , 35 ), APEX2 (ascorbate peroxidase 2) fused to ARL13B failed to biotinylate the ciliary content of GNPs, even under elevated biotin-phenol concentrations (fig. S1, D and E). Instead, the engineered biotin ligase TurboID ( 36 ) carried out efficient and selective biotinylation in GNP cilia when fused to ARL13B (fig. S1, D and E). Similarly, ARL13B-TurboID efficiently biotinylated ciliary contents in multiciliated ependymal cells (fig. S1, F and G) and in the human cerebellar Daoy cell line (fig. S1, H and I). The V359A mutation of the ciliary targeting motif of human ARL13B ( 37 ) abolished ciliary biotinylation, confirming the specificity of the system (fig. S1, H and I). Collectively, these findings demonstrate that ARL13B-TurboID achieves robust, cilium-specific biotinylation across various CNS cell types. To implement cilia-specific biotinylation in vivo while avoiding overexpression-related perturbations ( 38 – 40 ), we inserted a GFP-TurboID cassette at the endogenous Arl13b locus to express ARL13B-GFP-TurboID (referred to as ARL13B-TurboID) ( Fig. 1A ). The Arl13b-GFP-TurboID allele did not affect development, body weight, or cilia length in heterozygous and homozygous state [see figs. S2F and S5 (A and B)], indicating that the fusion protein is fully functional. We next introduced a point mutation in the ciliary targeting signal of mouse ARL13B ( 37 ) to generate the Arl13b[V358A]-TurboID allele ( Fig. 1A ). Fig. 1. ARL13B-TurboID robustly and specifically biotinylates ciliary proteins. Open in a new tab ( A ) Diagram of the targeting strategy. The GFP-TurboID (G-T) cassette was inserted into exon 10 of Arl13b in the mouse genome immediately before the stop codon to generate the Arl13b-GFP-TurboID allele. The stop codon appears 77 bp from the beginning of exon 10, and the remaining 1947 bp of exon 10 encode the 3′ untranslated region. Mutagenesis at exon 8 generated the Arl13b[V358A]-GFP-TurboID allele. Solid lines represent the sites for genome editing. *: Stop codon. ( B to G ) Cilium-specific biotinylation by ARL13B-TurboID. Cortical neurons (B), GNPs (D), and ependymal cells (F) were cultured from embryonic day 15.5 (E15.5) cerebral cortex, P6 cerebellum, and P0 cerebral cortex of mice with the indicated genotypes. Cultured cells were stained for GFP (green), cilia markers (ACIII or ARL13B, red), biotin (fluorophore-conjugated streptavidin, white), and DNA (blue). Individual channels from the boxed regions are displayed to the right of each image in the following order (top to bottom): GFP, cilia marker, biotin, and merge of the three channels. Scale bars, 5 μm (main) and 2.5 μm (insets). Scatterplots depict the fluorescence intensity of the ciliary biotin signal [relative to WT cilia; relative fluorescence unit (RFU)] for cortical neurons (C), GNPs (E), and ependymal cells (G). The thick and dotted lines indicate the median and interquartile range, respectively. Number of cilia analyzed N = 110 to 159 from three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with post hoc Bonferroni test (**** P < 0.0001). Primary cultures from diverse CNS cell types—cortical neurons, GNPs, and ependymal cells—revealed robust cilia-specific biotinylation only in cells from Arl13b +/TurboID mice ( Fig. 1, B to G ). In vivo, prominent biotin signals were detected in cilia in the cerebral cortex and hippocampus of Arl13b +/TurboID mice at 1 and 3 months of age ( Fig. 2A and fig. S2, A to C). Given that ciliary ARL13B levels are much lower in adult neurons than in glial cells in vivo, we expected ciliary biotinylation to be relatively weak in neuronal cilia in the brain of adult mice. Instead, the intensity of ciliary biotin signals was comparable between cilia marked by ARL13B (glial cells) or adenylate cyclase III (ACIII; neurons) in adult mouse cortex ( Fig. 2, A and B ). As the levels of ciliary biotin were similar in cortical neurons from heterozygous and homozygous Arl13b-TurboID mice (fig. S2, D to F), we conclude that the modest levels of ARL13B-TurboID detected in neuronal cilia (fig. S2, A to C) are sufficient to carry out near-saturating levels of cilia-specific biotinylation. Fig. 2. In cilio biotinylation by ARL13B-TurboID unveils a universal cilia marker. Open in a new tab ( A and B ) Ciliary biotinylation is independent of ciliary ARL13B levels. Following biotin injection for 3 days, P21 cerebral cortex sections from WT ( Arl13b +/+ ) and Arl13b +/GFP-TurboID mice were stained for cilia markers ACIII (green) and ARL13B (red), biotin (fluorophore-conjugated streptavidin, white) and DNA (blue). Representative cilia with high (hi) ARL13B levels and low (lo) ACIII levels or vice versa are boxed in each panel. Insets display individual channels from the boxed regions: ARL13B (top left), ACIII (top right), and biotin (bottom). Scatterplots (B) show the ciliary biotin signals relative to WT cilia. ( C to K ) Cilium-specific biotinylation by ARL13B-TurboID in various brain regions after 3 days of biotin administration. P21 Ventricular zone (C), layer 2/3 of the cerebral cortex (D), cerebellum [(E) and (F)], cornu ammonis 1 (CA1) (H), and dentate gyrus (DG) (J) were stained with cilia markers (ACIII or ARL13B with indicated color), biotin (white), DNA (blue), and cell type–specific markers: postmitotic neurons [NeuN, red in (D) and (G)], glial cells [glial fibrillary acidic protein (GFAP), green in (C) and red in (J)], Bergmann glial cells [ZEB2, green in (E)], and Purkinje neurons [Calbindin D28K, green in (F)]. Arrowheads (F) specify the Purkinje neuronal cilium. ML, molecular layer; PCL, Purkinje cell layer; IGL, internal granular layer. Scatterplots show the fluorescence intensity of the ciliary biotin signal (relative to WT cilia) in the cerebellum (G), CA1 (I), and DG (K). Scale bars, 10 μm (main) and 5 μm (insets). N = 124 to 239 (B) and 108 to 178 [(G), (I), and (K)] cilia analyzed from three mice. Thick and dotted lines represent the median and interquartile region. Statistical significance was determined by two-way ANOVA with post hoc Bonferroni test (B); Mann-Whitney U test [(G), (I), and (K)] (**** P < 0.0001). ns, not significant. Encouraged by these results, we examined the breadth of ARL13B-TurboID biotinylation across different mouse tissues. Robust biotin signals were detected in motile cilia of ependymal cells lining the ventricular zone ( Fig. 2C ) and in primary cilia of cortical pyramidal neurons ( Fig. 2D ), Bergmann glial cells ( Fig. 2E ), Purkinje neurons ( Fig. 2F ), hippocampal principal neurons ( Fig. 2H ), and glial cells of the dentate gyrus ( Fig. 2J ). Strong ciliary biotin signals were also observed in the distal convoluted tubules of the kidney and the taste buds of the tongue (fig. S3, A and B), and ciliary biotin signals in all tested tissues were specifically detected in Arl13b-TurboID mice ( Fig. 2, G, I, and K , and fig. S3, C and D). These observations highlight the utility of the Arl13b-TurboID mouse as a universal marker of cilia, surpassing conventional markers such as ARL13B, ACIII, and acetylated tubulin, which label cilia only in specific cell types. Proteomic profiling reveals tissue-specific specialization of ciliary proteomes The biochemical capture of biotinylated proteins revealed that many proteins were selectively recovered from Arl13b-TurboID cerebral cortices, where their levels were enhanced by biotin injection (fig. S4A). The untagged ARL13B protein, an endogenous ciliary component, was recovered from Arl13b-TurboID samples but not from Arl13b[V358A]-TurboID and wild-type (WT) samples, demonstrating that only ARL13B-TurboID can biotinylate ciliary proteins (fig. S4B). To compare cilia proteomes across multiple tissues, we conducted two tandem mass tag (TMT) analyses on biotinylated proteins isolated from cerebral cortex, cerebellum, hippocampus, and kidney of Arl13b +/TurboID and Arl13b +/+ mice. Hierarchical clustering and statistical filtering identified 100 proteins specifically enriched in Arl13b +/TurboID hippocampal or kidney samples and 147 proteins specifically enriched in Arl13b +/TurboID cortex or cerebellum samples ( Fig. 3, A and B ; fig. S4C; and data S1). Notably, only 30 proteins were shared between hippocampal and renal ciliary proteomes, including ARL13B, inositol polyphosphate 5-phosphatase E (INPP5E) ( 41 ), and NUMBL (NUMB-like) ( 42 ), while 39 were selectively enriched in the kidney-enriched cluster—including well-known ciliary components such as myosin VI (MYO6) ( 34 , 43 ), tubby-like protein 3 (TULP3) ( 44 ), and IFT74 (intraflagellar transport protein 74) ( 45 )—and 31 were found exclusively in the hippocampus-enriched cluster (fig. S4C). These findings contrast the well-characterized renal cilia proteome ( 34 , 35 , 46 ) with the poorly understood cilia proteome in the brain. Specialization of the ciliary proteome was evident even among CNS tissues ( Fig. 3B ) with only 50% overlap between cortical and cerebellar cilia proteome. Comparing the proteomes of cerebral cortex, cerebellum, hippocampus, and kidney revealed a remarkable divergence of the ciliary proteomes, with only 12 proteins shared among all tissues and 80 proteins selectively enriched in cerebral cortex ( Fig. 3C ). Manual examination of the datasets and gene ontology analysis revealed tissue-specific specializations of the ciliary proteome ( Fig. 3C and fig. S4D). Notably, the functional grouping of cilia proteins enriched in the cortex converged onto synaptic functions ( Fig. 3D ). Fig. 3. Comparative proteomics reveal diverse molecular signatures of cilia across tissues. Open in a new tab ( A ) Diagram of the experimental workflow and analytical pipeline to identify candidate cilia proteins. Lysates of cerebral cortices and cerebella from three ARL13B-tID ( Arl13b +/GFP-TurboID ) and three WT ( Arl13b +/+ ) mice were subjected to streptavidin purification followed by TMT MS. Four thousand two hundred ninety-two quantified proteins were filtered by hierarchical clustering (filter 1), their significant enrichment in ARL13B-tID samples (filter 2), and peptide counts (filter 3). Three groups of proteins showing selective enrichment in cerebral cortex (Ctx, 86 proteins), cerebellum (Cb, 6 proteins), and in both tissues (55 proteins) were identified. ( B ) Heatmap showing relative abundance (RA) of 4292 quantified proteins. The RA is calculated via dividing the TMT signals in each sample by the sum of signals across all samples. The color scheme at the right bottom indicates the RA scale. Line graphs depict the RA of proteins in four clusters, with two clusters matching the profile Ctx > Cb (red), one Ctx ~ Cb (gray), and one Ctx < Cb (green). Thick lines show the average RA for each protein cluster, and thin lines show individual proteins. ( C ) Venn diagram of cilia proteomes (214 proteins) collected from cerebral cortex (red), cerebellum (green), hippocampus (Hipp, blue), and kidney (yellow). Numbers indicate shared proteins among datasets. See fig. S4C for hippocampal and kidney cilia proteomes and data S1 for details. ( D ) Heatmap of GO terms among 80 proteins enriched in cerebral cortex. The color scheme on the right indicates the scale of −log [false discovery rate (FDR)]. The analysis compares those 80 cortex-enriched proteins to all assigned proteins detected in this proteomic dataset. GO, Gene Ontology; cAMP, cyclic adenosine monophosphate. In situ proteomics uncover known and previously unidentified ciliary proteins in the cerebral cortex Our experience with cilia proteomics ( 35 ) has emphasized that genetic controls (e.g., ablating cilia) are superior to conventional controls (e.g., delocalized enzyme) for identifying bona fide ciliary proteins. To this end, we combined the Arl13b-TurboID (tID) allele with homozygous Ift88 flox alleles to genetically remove cilia in a Cre-dependent manner. A ubiquitous tamoxifen-activated Cre ( Ubc-cre/ERT2 ) was used to delete all cilia in adult mice (ARL13B-tID/cilia Δ ), and we used Nex-cre ( 47 ) to selectively ablate cilia in pyramidal neurons (ARL13B-tID/cilia Δneu ). In addition, we combined Arl13b-TurboID with a Bbs4 deletion to impair BBSome-dependent retrieval mechanisms, allowing accumulation of ciliary proteins (ARL13B-tID/Bbs Δ ; Fig. 4A ) ( 48 ). Efficient cilia ablation and ciliary biotinylation were confirmed via imaging cortical neurons and glial cells ( Fig. 4, A to E , and fig. S5, A and B). Although cilia were shorter in Bbs4 −/− cells (fig. S5, A and B), ciliary biotinylation was largely unaffected ( Fig. 4, C and E ). Fig. 4. Genetic and direct controls for cilia proteomics. Open in a new tab ( A ) Ciliation and ciliary biotinylation in neurons and glial cells within the cerebral cortex across six genotypes. Following 3 days of biotin injection, sections of cerebral cortices from the indicated genotypes were stained with markers of cilia and neurons (ACIII, green; NeuN, red) or cilia and glial cells (ARL13B, green; GFAP, red) along with biotin (fluorophore-conjugated streptavidin, white) and DNA (blue). The tables on the left of each panel provide information in the following order (top to bottom): genotype, short name of the genotype, and effects of biotin labeling. Individual channels from boxed regions are enlarged to the right of each panel in the following order: biotin, cilia marker, and merge of these two channels. Scale bars, 10 μm (main) and 5 μm (insets). ( B to E ) Quantitation of ciliation rate and ciliary biotin levels. Plots represent the percentage of cilia marked by ACIII in NeuN + neurons (B) and of cilia marked by ARL13B in GFAP + glial cells (D) for the indicated genotypes. Lines mark the mean and SD. Scatterplots depict the fluorescence intensity of the ciliary biotin signal (relative to WT cilia) for neurons (C) and glial cells (E). Thick and dotted lines represent the median and interquartile range, respectively. Number of cilia analyzed: N = 105 to 227 [(B) to (E)] from three brains per genotype. Statistical significance was determined by one-way ANOVA with post hoc Bonferroni test (** P < 0.01, *** P < 0.001, and **** P < 0.0001). See fig. S5 (A and B) for additional measurements. To broaden proteomic coverage, we implemented two complementary purification strategies: one capturing all proteins via detergent extraction and another enriching membrane-associated proteins using wheat germ agglutinin (WGA) beads ( Fig. 5 ). Brain samples from four to five experimental conditions in triplicate or quadruplicate were analyzed using quantitative mass spectrometry (MS), and clustering analysis identified groups of proteins enriched in brain cilia [ARL13B-tID > ARL13b † -tID[V358A] ∼ WT ( Fig. 6A ) or ARL13B-tID > ARL13B-tID/cilia Δ ∼ WT ( Fig. 6B )] or neuronal cilia specifically (ARL13B-tID/Bbs Δ > ARL13B-tID > ARL13B-tID/cilia ΔNeu > WT; Fig. 6C ). Western blot analyses confirmed the effective purification of biotinylated proteins ( Fig. 6, A to C ). Fig. 5. Proteomic profiling identifies candidate cilia proteins in the cerebral cortex. Open in a new tab Diagram of the analytical pipeline to identify candidate brain cilia proteins. Three independent TMT-MS experiments were conducted. Genotype abbreviations: ARL13B-tID: Arl13b +/GFP-TurboID . WT: Arl13b +/+ . ARL13B † -tID: Arl13b +/V358A-GFP-TurboID . ARL13B-tID/cilia Δ : Arl13b +/GFP-TurboID ; Ubc-cre/ERT2; Ift88 fl/fl . ARL13B-tID/cilia ΔNeu : Arl13b +/GFP-TurboID ; Nex-cre; Ift88 fl/fl . ARL13B-tID/Bbs Δ : Arl13b +/GFP-TurboID ; Bbs4 −/− . tamoxifen was injected or not in experiment 2. Cerebral cortex lysates (total extracts in experiments 1 and 2 or membrane-associated fractions in experiment 3) from indicated genotypes were subjected to streptavidin purification followed by TMT MS. The number of replicates used in each TMT-MS experiment is indicated next to the mouse genotype. Each independent TMT-MS experiment was subjected to hierarchical clustering analysis (see data S2). Clusters with profiles expected for cilia proteins were pooled (filter 1), and hits were filtered for statistical significance (filter 2: P < 0.05 for ARL13B-tID versus WT) and peptide numbers (filter 3: peptide count > 1). A Venn diagram of overlapping proteins in experiments 1 (red), 2 (blue), and 3 (yellow) from the final tier. The numbers of proteins shared among datasets are shown. Fig. 6. Representative clusters of ciliary proteins revealed by proteomic profiling. Open in a new tab Representative clusters of candidate ciliary proteins in three independent TMT-MS experiments. Total extracts ( A and B ) and membrane fractions ( C ) from dissected cortices were purified on streptavidin beads and analyzed by immunoblotting (top) or TMT-MS (middle and bottom). Top: Forty equivalents of eluates were loaded in the lysate lanes. Middle panels: line graphs depict the relative abundance (RA) of a given protein, calculated via dividing the TMT signals in each sample by the sum of the TMT signals across all samples. Thick lines show the average RA for each protein cluster, and thin lines show individual proteins. Bottom: Heatmaps display the RA of each protein across the genotypes. The color scheme at the bottom left indicates the RA scale. A few representative proteins are indicated. See fig. S5 (C to H) for additional clusters. MW, molecular weight. In TMT experiment 1 (data S2), hierarchical clustering of ARL13B-tID versus WT samples revealed a cluster enriched in known ciliary proteins (fig. S5C), such as ß4B-tubulin (TUBB4B) (data S2) ( 49 ). Another cluster, matching the neuronal cilia profile ARL13B-tID > ARL13B-tID/cilia ΔNeu > WT, included the known cilia proteins MYO6 (fig. S5D) and SNAP25 (synaptosomal-associated protein 25) (data S2) ( 50 ). Unexpectedly, only five proteins—including TULP3 and INPP5E—matched the profile ARL13B-tID > ARL13b † -tID ∼ WT ( Fig. 6A ), suggesting that only a few cilia proteins are exclusively localized to cilia in brain cells. In TMT experiment 2 (data S2), tamoxifen (tam) was added to half of the ARL13B-tID and ARL13B-tID/cilia Δ mice to control the effects of this drug. A cluster enriched in ARL13B-tID versus WT samples included ARL13B, MYO6, and INPP5E (fig. S5E). Another cluster with the profile ARL13B-tID -tam ~ ARL13B-tID +tam and ARL13B-tID/cilia Δ -tam > ARL13B-tID/cilia Δ +tam ~ WT identified brain cilia proteins and included IFT172 ( 51 ), NUMBL, and the ciliary GPCR SSTR3 ( Fig. 6B and fig. S5F). The detection of SSTR3 in our dataset highlights the sensitivity of the method, given the low abundance of GPCRs in neuronal cilia (<1000 molecules per cilia) ( 40 ). In TMT experiment 3 (data S2), membrane-associated proteins were purified from cortical lysates of WT, ARL13B-tID, ARL13b † -tID, ARL13B-tID/cilia ΔNeu , and ARL13B-tID/Bbs Δ mice. Three clusters of candidate neuronal cilia proteins (ARL13B-tID/Bbs Δ > ARL13B-tID > ARL13B-tID/cilia ΔNeu > WT) included the known ciliary proteins ARL13B, INPP5E, SNAP25, MYO6, as well as NUMBL and the metabotropic GPCRs γ-aminobutyric acid receptor type B1 (GABA B1 ) ( 52 ) and the glycine receptor GPR158 (fig. S5G) ( 53 ). Among these, 37 proteins showed a pronounced enrichment in retrieval mutants, underscoring the dynamic shuttling of ciliary proteins ( Fig. 6C ). An additional cluster enriched in ARL13B-tID versus WT included IFT172 and BBS5 (fig. S5H and data S2) ( 40 ). Collectively, these proteomic analyses across different genotypes revealed the diversity of known and previously unidentified ciliary proteins, offering insights into neuronal cilia biology. Synaptic proteins are selectively enriched in brain cilia A high-confidence brain cilia proteome was refined to 232 candidate brain cilia proteins by filtering for statistical significance and peptide counts ( Fig. 5 and data S2). While ~40% of cilia proteins identified in our kidney dataset are found in the published ciliary proteomes of cultured kidney cells (data S1) ( 34 , 35 , 46 ), less than 18% of cilia proteins purified from cerebral cortices are observed in those reports (data S2), further underscoring the specialization of brain cilia. Gene ontology analyses revealed a remarkable enrichment of synaptic proteins in neuronal cilia (fig. S6A and data S3), including the GluN1 subunit of the ionotropic NMDA receptor, the GABA B1 subunit of the metabotropic γ-aminobutyric acid (GABA) receptor, several Na + /K + adenosine triphosphatase (ATPase) subunits, and the voltage-gated sodium channel Na V 1.1 ( Fig. 7A ). Furthermore, the brain cilia proteome also included multiple adhesion molecules, such as neuron cell adhesion molecule 1 (NCAM1) and the tight junction protein ZO-1 ( Fig. 7A ). These findings challenge the traditional view that synaptic signaling in neurons is confined to dendrites and suggest that neuronal cilia may function as specialized extrasynaptic devices. Fig. 7. Overview of the brain and neuronal cilia proteome. Open in a new tab ( A ) Overview of the brain cilia proteome with functional groupings in synaptic reception (blue), cell adhesion (green), postsynaptic organization (magenta), or other functions (gray). Candidates validated by immunostaining in this study are bolded. ( B to K ) Validation of the brain cilia proteome. Cultured cortical neurons [days in vitro 8 (DIV8)] from E15.5 cerebral cortex were stained for candidate cilia proteins (green), ciliary markers (ARL13B, red; ACIII, white), and DNA (blue). The antibody used in (B) recognizes ATP1A1, A2, and A3. Individual channels from boxed regions are enlarged to the right of each panel with the following order (top to bottom): candidate protein, ARL13B, and either merged channels [for (B), (D), (E), (H), and (I)] or ACIII [for (C), (F), (G), (J), and (K)]. Images captured with Airyscan2. Scale bars, 1 μm (main) and 500 nm (insets). ( L to Q ) Validation of the neuron-enriched cilia proteome. (L) Volcano plots comparing ARL13B-tID ( Arl13b +/GFP-TurboID ) versus ARL13B-tID/cilia ΔNeu ( Arl13b +/GFP-TurboID ; Nex-cre; Ift88 fl/fl ) TMT signals among 186 tier 1 proteins identified in TMT experiments 1 and 3. Statistical P values are plotted against TMT relative abundance ratios in each comparison. Proteins enriched in ARL13B-tID samples are highlighted in red. Known cilia proteins and validated proteins are labeled in blue, and all other proteins are shown in gray (see data S4 for the dataset). [(M) to (Q)] Cultured cortical neurons (DIV8) from E15.5 cerebral cortex were stained for candidate cilia proteins (green), ciliary markers (ARL13B, red; ACIII, white), and DNA (blue). Individual channels from boxed regions are enlarged to the bottom of each panel with the following order: candidate protein (top left), ARL13B (top right), and either merged channels [for (M), (N), (O), and (Q)] or ACIII (P). Images captured with Airyscan2. Scale bars, 1 μm (main) and 500 nm (insets). Immunostaining cultured cortical neurons using well-characterized antibodies (see table S3) validated the localization of several candidates to neuronal cilia. The Na + /K + ATPase and its scaffolding protein ankyrin-2 were detected in cilia, supporting their role in maintaining ionic homeostasis ( Fig. 7, B and C , and fig. S6, B and C). Similarly, the NMDA receptor subunit GluN1 along with its functional partner, GluN2B ( Fig. 7D ; fig. S6, D and E, and data S1) and two postsynaptic scaffold proteins, SHANK3 and LRRC7 ( Fig. 7, E and F , and fig. S6, F and G), were detected in neuronal cilia, highlighting their potential involvement in synaptic signaling. Additional validated proteins include the phosphoinositide phosphatase synaptojanin1, the signaling receptor basigin, and the actin and spectrin regulator β-adducin ( Fig. 7, G to I , and fig. S6, H to J). Notably, the ARF-like guanosine triphosphatase ARL15 was also detected in neuronal cilia ( Fig. 7J and fig. S6K), representing the fourth member of this family (after ARL3, ARL6, and ARL13B) to function within cilia ( 54 ). Last, the regulator of neurite outgrowth NOGO (neurite outgrowth inhibitor) was found in neuronal cilia ( Fig. 7K and fig. S6L). Collectively, these results demonstrate that neuronal cilia harbor several postsynaptic proteins, including neurotransmitter receptors, scaffolding proteins, and signaling regulators. Among the brain cilia proteins, a subset of candidate pyramidal neuron cilia proteins was identified by comparing the ARL13B-tID and ARL13B-tID/cilia ΔNeu datasets ( Fig. 7L and data S4). These candidates include protocadherin-9, GPR158, GABA B1 , Na V 1.1, ZO-1, septin-3 (SEPT3), IFT172, and SNAP25. Immunostaining of cultured cortical neurons confirmed the ciliary localization of ZO-1 ( Fig. 7M and fig. S6M), GABA B1 ( Fig. 7N and fig. S6N), and Na V 1.1 ( Fig. 7O and fig. S6O). These findings suggest that pyramidal neuron cilia may mediate cell adhesion, synaptic signal reception, and neuronal excitability. We note that our cortex cilia proteome shows limited overlap (data S2) with a recent in vivo neuronal cilia proximity–labeling study that used SSTR3-BioID as a biotinylating fusion ( 55 ). Several technical differences between the two studies may contribute to the limited overlap. For example, our ARL13B-based targeting shows robust ciliary enrichment in our hands, whereas SSTR3-based targeting differed in enrichment and labeling dynamics (fig. S1). In addition, differences in delivery (e.g., viral transduction), tissue source (total brain versus dissected cortex), and filtering strategy (including our use of genetic cilia ablation as an internal control) could influence which proteins are recovered and retained in the final datasets. Despite noted differences, the two datasets overlap on several newly validated ciliary signaling and membrane-associated proteins (e.g., GABA B1 , mGluR5, GPR158, NCAM1, and Na + /K + -ATPase), highlighting important points of convergence. In addition, an antibody-based proteomic study reported extensive heterogeneity in primary cilia composition between cell types and identified many brain-related proteins with ciliary localization in nonneuronal human cell lines ( 54 ). Together, these studies provide complementary context for interpreting our cortex cilia proteome and reinforce the idea that ciliary protein composition is sensitive to cell type, brain region, and methodology. Mapping our brain cilia proteome onto single-cell RNA sequencing data (fig. S7 and data S4) ( 56 ) revealed that some proteins—e.g., NCAM—appeared broadly expressed in most ciliated cell types, while others—e.g., the metabolic glutamate receptor mGluR5, synaptic cell adhesion molecule 2 (SynCAM2), and SNAP25—were highly expressed in glutamatergic and GABAergic neurons (fig. S7). Staining of cultured cortical neurons validated the presence of mGluR5 and SynCAM2 in neuronal cilia ( Fig. 7, P and Q , and fig. S6, P and Q). Together, these results underscore a notable convergence between the neuronal cilia and synaptic proteomes, suggesting that neuronal cilia exploit synaptic machineries to fulfill their functions. Many brain cilia proteins are not exclusively ciliary Building on the notion that neuronal cilia may co-opt proteins from other cellular compartments, only eight proteins—including the known ciliary proteins IFT172 and microtubule-associated protein 4 (MAP4) ( 57 ), along with the candidate ciliary proteins septin-3 and μ-crystallin (CRYM)—were significantly enriched in ARL13B-tID samples compared to ARL13b † -tID controls ( Fig. 8A and data S5). In parallel, comparing ARL13B-tID/cilia Δ −tam with ARL13B-tID/cilia Δ +tam samples revealed 16 proteins significantly enriched in ARL13B-tID/cilia Δ −tam samples ( Fig. 8B and data S5). This group comprised canonical ciliary components (SSTR3, IFT172, and INPP5E), as well as candidate ciliary proteins such as septin-3 and SLC3A2 (the large subunit of neutral amino acid transporters). The subsets of proteins that were significantly enriched in ARL13B-tID versus ARL13b † -tID samples or ARL13B-tID/cilia Δ −tam versus ARL13B-tID/cilia Δ +tam controls were classified as tier 1* due to their robust ciliary enrichment (data S2). Fig. 8. Ciliary proteins show differential ciliary enrichment. Open in a new tab ( A and B ) Volcano plots comparing TMT signals from tier 1 proteins in ARL13B-tID versus ARL13B † -tID (A) and ARL13B-tID/cilia Δ −tam versus ARL13B-tID/cilia Δ +tam (B) samples. Significantly enriched proteins (tier 1*) are in red, validated proteins are in blue, and other tier 1 proteins are in gray. Proteins significantly de-enriched (dashed outline) were removed from the brain cilia proteome (see data S5). ( C to H ) Super-resolution imaging confirms the ciliary localization of candidate proteins. Cultured cortical neurons (DIV8) were stained for cilia markers (ARL13B/ACIII), candidate proteins, and DNA. Insets show the boxed regions magnified from left to right: candidate protein, cilia marker, and merged channels [(C), (F), (G), and (H)] or ACIII [(D) and (E)]. Arrowheads [(G) and (H)] indicate foci of candidate proteins on cilia. Line scans show the fluorescence intensity [Fluo; arbitrary unit (au)] of each candidate along a representative cilium with ciliary length normalized to 100%. ( I ) Cilia-to-cytoplasm fluorescence intensity ratios ( F cilia / F cyto = cilia enrichment factor) for validated candidates mapped on a color scale. N = 42 to 68 cilia (each protein) from three independent experiments. See fig. S8A. ( J ) Volcano plots comparing TMT signals of ARL13B-tID/Bbs Δ versus ARL13B-tID samples among 1745 detected proteins. Proteins enriched in Bbs4 −/− are in red, known ciliary and validated proteins are in blue, and others are in gray (see data S6). ( K to M ) NCAM1 is enriched in Bbs4 −/− neuronal cilia. Cultured cortical neurons (DIV8) were stained for NCAM1, ARL13B, and DNA. Insets show the boxed regions magnified: NCAM1 (top left), ARL13B (top right), merged channels (bottom left), and 3D reconstruction (bottom right). Arrowheads point at foci of NCAM1. (L) Cilia enrichment factors for NCAM1. N = 60 to 62 cilia, three independent experiments; Mann-Whitney U test, *** P < 0.001. (M) Line scans of NCAM1 fluorescence along cilia. Scale bars, 1 μm (main) and 500 nm (insets) in all images. Immunostaining of cultured cortical neurons confirmed that SLC3A2, μ-crystallin, septin-3, and MAP4 are highly concentrated in cilia ( Fig. 8, C to F ). While tier 1* proteins mostly exhibited continuous staining patterns along cilia, other tier 1 proteins, such as protocadherin-9 and -10, showed punctate localization ( Fig. 8, G and H , and fig. S8A). Quantifying enrichment ratios revealed that tier 1* proteins are markedly concentrated in cilia, whereas other tier 1 proteins demonstrated more modest enrichment ( Fig. 8I and fig. S8A). Concordantly, mapping the brain cilia proteome onto an organelle atlas ( 58 ) revealed that most brain cilia proteins also localize to other compartments, chiefly the plasma membrane, cytosol, and cortical actin (fig. S8B and data S5). Furthermore, retrieval-deficient Bbs4 −/− neurons showed significant accumulation of proteins such as NCAM1, FAM171A2, and neogenin ( Fig. 8J and data S6), which exhibited punctate patterns in control cilia and a more continuous distribution in Bbs4 −/− cilia ( Fig. 8, K to M , and fig. S8, C to H). This accumulation was not associated with global increases in protein levels (fig. S8, I and J), suggesting dynamic shuttling of proteins into and out of cilia mediated by the retrieval machinery save for specific nanodomains within the cilium. Visualization of ciliary proteins within the brain ultrastructure reveals close association of ciliary NMDA receptors with synapses To explore the spatial relationship between neuronal cilia proteins and the brain architecture in vivo, we used pan-expansion microscopy of t issues (pan-ExM-t) ( 59 ), achieving over 16-fold linear expansion of brain samples (fig. S9, A and B) and bringing resolution below 20 nm. Fluorescent N -hydroxysuccinimide (NHS) esters (“pan” stains) enabled near-ultrastructural visualization of brain architecture, including cilia and basal bodies ( Fig. 9, A and B ). Immunostaining revealed the ciliary membrane protein ACIII decorating a cylindrical pattern, with axonemal microtubules and basal body stained by NHS ester ( Fig. 9B ). Excitatory synapses were readily identified as parallel asymmetric structure in NHS ester staining, and the location of the postsynaptic compartment ( 60 ) and perisynaptic region ( 61 ) were confirmed by GluN1 and protocadherin-9 staining, respectively (fig. S9C). Consistent with prior EM studies ( 28 ), synapses were frequently seen within the vicinity of neuronal cilia in pan-ExM-t ( Fig. 9C and movie S1). Fig. 9. Imaging of cilia and synapses by pan-stained expansion microscopy of brain tissue (pan-ExM-t). Open in a new tab ( A ) Visualization of neuronal cilium in P60 cerebral cortex by pan-ExM-t. Sections were expanded 16.2-fold and stained with fluorescent NHS ester (black). The red boxed region is enlarged at the upper right of this panel. N, nucleus; n, nucleolus; cil, cilium; bb, basal body. Scale bars: 1 μm (main) and 500 nm (inset). ( B ) ACIII distribution on the ciliary membrane by pan-ExM-t. Expanded P60 cerebral cortices were stained for ACIII (magenta) and NHS ester (black). ACIII signals in the boxed region are enlarged in the right panel. Scale bars, 500 nm (main) and 250 nm (inset). Representative images from three independent experiments with over 25 cilia imaged. ( C ) Visualization of neuronal cilia and synapses in pan-ExM-t. Expanded P60 cerebral cortices were stained for the postsynaptic marker PSD-95 (green), the ciliary membrane marker ACIII (magenta), and NHS ester (black). Individual channels (from left to right): green, magenta, and merged. C, cilium; pre, presynapse; post, postsynapse. Scale bar, 250 nm. Representative images from three independent experiments with over 10 cilia imaged. See movie S1. ( D ) GluN1 localization on the ciliary membrane and synapses. Expanded P60 cerebral cortices were stained for ACIII (magenta), GluN1 (green), and with NHS ester (black). Boxed regions are enlarged, showing GluN1, ACIII, and darkened merge from top to bottom ( Z : optical sections). Red arrowheads, ciliary GluN1 signals near two synapses (S1 and S2). Scale bars, 500 nm (main) and 200 nm (insets). Representative images from three independent experiments with over 15 cilia imaged. See also movie S3. All scale bars are corrected for the expansion factor. All micrographs shown are maximal intensity projections of the Z sections spanning a neuronal cilium, except for the bottom in (D) where a single Z section is shown. Equipped with a powerful tool to precisely locate ciliary signaling receptors and synapses inside the brain architecture, we zeroed in on the NMDA receptor subunit GluN1 as it was found at the center of a major node of glutamatergic postsynaptic proteins in protein association networks of the brain cilia proteome (fig. S9D). Discrete GluN1 foci specifically decorated the ciliary membrane cylinder marked by ACIII in vivo ( Fig. 9D , top, and movie S3). Notably, the discrete GluN1 signals on ciliary membranes were consistently positioned near neighboring GluN1-positive glutamatergic synapses ( Fig. 9D , bottom), suggesting an organized spatial relationship between ciliary NMDA receptors and nearby synaptic structures. To rigorously test whether GluN1 nanodomains are nonrandomly positioned relative to synapses, we stained cilia in ARL13B-GFP cortical sections ( Fig. 10A ) and performed automated three-dimensional (3D) segmentation of neuronal cilia and synapses ( Fig. 10B and movie S4). Quantifying the shortest distances between ciliary GluN1 molecules and the nearest neighboring synapses revealed a median distance just under 300 nm ( Fig. 10C ), very similar to the shortest distance between the ciliary membrane and neighboring synapses (fig. S9E). Further emphasizing the close relationship between ciliary GluN1 nanodomains and synapses, this value is in close agreement with volume EM-based measurements of cilia-synapse distances in the mouse visual cortex ( 28 ). Randomizing cilia orientation disrupted this spatial association ( Fig. 10D ), demonstrating that ciliary NMDA receptors are nonstochastically positioned near synapses. Further supporting the association of glutamatergic synapses with ciliary GluN1 nanodomains, the number of GluN1 spots per cilium was comparable to the number of neighboring glutamatergic synapses ( Fig. 10E ). Fig. 10. Ciliary NMDA receptors are positioned near neighboring synapses. Open in a new tab ( A ) Expanded P45 cortex from ARL13B-GFP tg mouse stained for GFP and with NHS ester. Scale bars, 500 nm (left and middle) and 100 nm (right). ( B ) Left: expanded cortex stained for GFP, GluN1, and with NHS ester. Right: 3D rendering of cilia (from the ARL13B-GFP channel) and GluN1 structures, either synapses (GluN1 syn ) or ciliary nanodomains (GluN1 cilia ) from 180 Z sections. Green double arrow, representative pair of GluN1 structures plotted in (C). Scale bars, 500 nm. See movie S4. ( C ) Histogram depicting the shortest 3D distance between GluN1 cilia spots and the nearest GluN1 syn structure. Blue bars, number of GluN1 cilia -GluN1 syn pairs for a given range of distances; black line, cumulative frequency of distances; dashed line, median = 297 nm. ( D ) Cumulative frequencies of all 3D distances between GluN1 cilia spots and GluN1 syn structures. Black line, experiment; gray lines, cilia and associated GluN1 cilia structures rotated by 45°, 90°, and 135°; dashed lines, medians = 1.43 μm (experimental), 2.60 μm (45°), 1.97 μm (90°), and 3.70 μm (135°). A total of >3200 GluN1 cilia -GluN1 syn pairs were quantified. ( E ) The number of GluN1 spots on neuronal cilia closely aligns with the number of neighboring glutamatergic synapses (defined as GluN1 + synapses within 300 nm of the ciliary surface). ns, paired t test not significant. ( F ) Method to assess ciliary membrane localization. Neuronal cilia were segmented (ARL13B-GFP channel), and a centerline and successive dilations (6.25-nm increments) were calculated. GluN1 spots intersecting each dilated shell were counted. ( G ) GluN1 spots are on the membrane of neuronal cilia. The frequencies (number of spots intersecting a dilated shell divided by the total number of spots) of GluN1 and ARL13B-GFP spots on each dilated shell were plotted. Solid lines, mean; shaded areas, SD. Scale bars are corrected for expansion. Micrographs show maximum intensity projections of Z sections spanning a cilium. A total of ≥9 cilia from three independent experiments were imaged, segmented, and quantified. Last, the unprecedented resolution of pan-ExM-t enabled us to establish the specificity of discrete signals of signaling receptors on cilia, as their position coincided with the ciliary membrane marked by ACIII or ARL13B. For instance, costaining for the metabotropic glycine receptor GPR158 ( 53 ) and ACIII revealed discrete foci on the membrane of neuronal cilia in vivo (movie S2), extending a recent report of its ciliary localization in cultured neurons ( 62 ). To formally test whether GluN1 spots are located on the ciliary membrane, we developed an analytical pipeline based on the segmented ARL13B–green fluorescent protein (GFP) signal ( Fig. 10F and fig. S9F). GluN1 spots were distributed on the ciliary membrane, rather than randomly through the ciliary volume ( Fig. 10G ), further supporting the specificity of the ciliary GluN1 signals. In conclusion, the mapping of synaptic structures and proteins on neuronal cilia in brain volumes at near-ultrastructural resolution by pan-ExM-t extends previous EM-based studies ( 27 – 29 ) and reveals nanodomains on neuronal cilia strategically located to monitor activity from nearby glutamatergic synapses. DISCUSSION Neuronal cilia as extrasynaptic signaling platforms Neuronal cilia have long been implicated in brain development ( 19 ) and connectivity ( 63 ), but their roles in mature neural circuits remain poorly understood. This study provides compelling evidence that neuronal cilia functions as specialized extrasynaptic signaling platforms, equipped with synaptic receptors, adhesion molecules, and regulators of neurite outgrowth ( Fig. 7A ). By profiling the brain cilia proteome and visualizing nanoscale protein organization, we demonstrate that neuronal cilia are positioned to transduce signals from neighboring synapses ( Fig. 10 ), potentially influencing neuronal excitability and connectivity. The discovery of synaptic proteins, most notably the NMDA receptor subunit GluN1 ( Fig. 7D ), scaffolding proteins such as SHANK3 ( Fig. 7E ) ( 64 ), and signaling molecules such as the metabotropic GABA receptor subunit GABA B1 ( Fig. 7N ), within cilia challenges the conventional view that synaptic signaling in neurons is confined to dendrites. Our expansion microscopy analysis revealed a discrete localization of GluN1 on the ciliary membrane near glutamatergic synapses ( Fig. 10, B to G ), suggesting that neuronal cilia are equipped to integrate local synaptic activity. These findings extend previous EM studies that identified axo-ciliary synapses and synapse-cilia associations in the mouse and human brains ( 27 – 29 ). The molecular framework we propose—where neuronal cilia deploy synaptic machinery to sense neurotransmitters—positions cilia as dynamic hubs for locally integrating neural circuit activity. The discrete clusters of GluN1 on primary cilia raise important questions about signaling downstream of NMDAR (NMDA receptor) in cilia. It is particularly interesting to consider the unique biophysical properties of NMDAR, which begins to conduct Ca 2+ once the membrane potential rises above −40 mV ( 65 ). In dendritic spines, NMDARs act as coincidence detectors for repeated synaptic firing and glutamate release, forming the molecular basis of long-term potentiation via calcium/calmodulin-dependent protein kinase II (CaMKII)- and protein kinase C (PKC)–mediated remodeling of the postsynaptic compartment ( 66 ). In contrast to dendritic spines, where the resting membrane potential is typically −65 mV, the ciliary membrane potential in RPE-1 cells is reported to be 30 mV higher than that of the plasma membrane ( 67 ). If the membrane of neuronal cilia is similarly depolarized, then NMDA receptors on the ciliary membrane of neurons may be uniquely primed for activation. GluN2B was detected on cilia of cultured neurons ( 68 ) and in our cortex versus cerebellum cilia proteome (data S1 and fig. S6E), suggesting that GluN1 may assemble functional NMDA receptor heterotetramers with GluN2B on cilia. Once ciliary NMDAR permits Ca 2+ entry, the presence of PKCδ and CaMKV in our brain cilia proteome (data S2) suggests that these kinases may remodel the ciliary proteome. Beyond Ca 2+ binding to effector molecules inside cilia, the flux of Ca 2+ through NMDAR may further depolarize the ciliary membrane ( 69 ). The presence of the voltage-gated Na + channel Na V 1.1 ( Fig. 7O ) and the Na + /K + pump ( Fig. 7B ) in neuronal cilia reinforces the hypothesis of the cilium as a privileged electrical compartment. The best-characterized mechanism of cilium-based signaling in neurons comes from photoreceptors and olfactory receptor neurons ( 70 ), where electrical signals initiated at the ciliary membrane propagate to the rest of the cell. Could primary cilia in other neurons also relay signals to the cell via changes in membrane potential? In line with this idea, activation of the ciliary calcium channel PKD2L1 was shown to increase the frequency of action potentials in hippocampal neurons ( 71 ). The electrical output of neuronal cilia may thus tune the excitability of cortical neurons, positioning cilia as integrators of local synaptic signaling. Ciliary adhesion molecules and neurite regulation Our proteomic analysis uncovered a diverse array of adhesion molecules, including NCAM1 ( Fig. 8K ), synCAM2 ( Fig. 7Q ), and protocadherin 9 ( Fig. 8G ), in neuronal cilia. These molecules likely mediate interactions between cilia and neighboring structures, including synapses and axons. Notably, NCAM1 appears to dynamically cycle in and out of cilia, accumulating along cilia when retrieval was blocked in Bbs4 −/− neurons ( Fig. 8, K to M ). This suggests that neuronal cilia may establish transient adhesive contacts with synapses or other cellular structures, allowing molecular clusters to be locally retained while other molecules are cleared. Such cilia adhesions may influence neuronal connectivity and plasticity. In addition to conventional adhesion molecules, we uncovered several metabotropic GPCRs, namely, GABA B1 ( Fig. 7N ), mGluR5 ( Fig. 7P ), and GPR158 (movie S2), all of which are class C GPCRs enriched at the postsynaptic compartment that may sense GABA, glutamate, or glycine and modulate ciliary signaling. In addition, a defining feature of class C GPCRs is their large extracellular domains, which primarily function as orthosteric ligand-binding sites. Recent studies have demonstrated that these extracellular domains can form transcellular complexes with adhesion molecules. For instance, GPR158 forms a transsynaptic complex with the heparan sulfate proteoglycan glypican-4 (GPC4) ( 72 ), a presynaptic organizer; and productive GPR158-GPC4 complexes promote presynaptic compartment assembly. These findings suggest that ciliary metabotropic GPCRs may also participate in axo-ciliary synapse formation. Beyond adhesion, several regulators of neurite outgrowth, such as neogenin, a receptor that mediates repulsive guidance cues via activation of RhoA (Ras homologue A) and actin cytoskeleton remodeling ( 73 – 75 ), were enriched in neuronal cilia (fig. S8, F to H). Their enrichment in retrieval-deficient cilia ( Fig. 8J and fig. S8, F to H) suggests that these molecules continuously patrol the neuronal cilium, dynamically sensing and regulating contacts between cilia and axons. Mechanisms governing nanodomain formation on neuronal cilia The precise spatial organization of signaling and adhesion molecules within neuronal ciliary nanodomains raises intriguing questions about the underlying mechanisms. Our findings suggest that active retrieval mechanisms play a pivotal role, with the BBSome restricting the excessive accumulation of signaling and adhesion molecules and ensuring that nanodomains remain functionally distinct. In addition, scaffolding proteins, including Ankyrin-B ( 76 ) and SHANK3 ( 64 ), may anchor signaling complexes to specific sites on the ciliary membrane, creating stable nanodomains. It is conceivable that septins, which compartmentalize membranes in other systems, may act as physical barriers within cilia, segregating nanodomains and preventing diffusion ( 77 ). Last, lipid microdomains enriched in cholesterol or phosphoinositides ( 1 , 78 ) may potentially contribute to ciliary nanodomain organization, as seen in other specialized cellular compartments. Neuronal cilia in neuropsychiatric symptoms of patients with ciliopathy Our findings have potential implications for understanding the molecular basis of neuropsychiatric manifestations observed in patients with Joubert syndrome and BBS ( 4 , 5 ). For example, several proteins identified in the brain cilia proteome, including GluN1 and GABA B1 , protocadherin-9 and SynCAM2, and LRRC7, are implicated in features of schizophrenia, autism spectrum disorders ( 79 – 81 ), and intellectual disability ( 82 ). Querying the disease association base DisGeNET ( 83 ) revealed potential associations between the brain cilia proteome and these disorders (data S7). These disorders are characterized by disruptions in synaptic signaling and neural circuit connectivity, suggesting that cilia-mediated signaling may play a previously unrecognized role in the pathogenesis of neuropsychiatric symptoms in patients with ciliopathy. Supporting this idea, ciliary defects in Bbs2 −/− mice lead to deficits in social dominance, and deletion of the serotonin receptor 6, a ciliary GPCR, leads to anxiety and cognitive impairments. Targeting cilia-based signaling pathways may thus represent a promising therapeutic avenue for neuropsychiatric manifestations in patients with ciliopathy. Limitations of the study While our study establishes a foundational resource for neuronal cilia biology, it also raises several unanswered questions. First, the functional roles of synaptic receptors and adhesion molecules in cilia remain speculative. Electrophysiological recordings or calcium imaging in cilia could provide direct evidence for their involvement in signal transduction. Second, the spatial organization of ciliary proteins suggests their organization in nanodomains, but the mechanisms governing nanodomain formation and maintenance are unclear. Investigating the role of the BBSome and scaffolding proteins in establishing these domains will be critical. At the technical level, the absence of well-known and essential ciliary proteins (e.g., IFT complexes and ACIII) from our brain cilia proteome does not suggest that they are absent from cilia; but rather that our stringent inclusion criteria may have filtered out true ciliary proteins thus generating some false negatives. Furthermore, our bulk proteomic analysis of cortical tissues does not resolve heterogeneity across cell types, and many candidate brain cilia proteins are likely to be present in select cell types. Future studies will need to deconvolve the proteome of glial cell cilia given their recently recognized importance. Last, although we strived to select well-characterized antibodies to validate candidate brain cilia proteins (table S3), some antibodies may require further validation in immunostaining. Another technical aspect concerns the scope of the ciliary proteome labeled by ARL3B-TurboID. In past work ( 34 ), we have found that a proximity labeling enzyme located on the ciliary membrane biotinylated axonemal acetylated tubulin with high efficiency. In the current study, we identify axonemal proteins such as MAP4. Thus, we strongly believe that the biotinyl adenylate generated by TurboID labels most of the interior of the cilium. Future applications Our study highlights the diversity and specialization of neuronal cilia, revealing their unique molecular composition and close association with synaptic structures. By linking ciliary proteins to neuropsychiatric disorders, we establish a molecular framework for understanding how cilia-mediated signaling contributes to brain function. These findings pave the way for future studies aimed at unraveling the molecular mechanisms of cilia-synapse interactions and harnessing cilia biology for therapeutic innovation. MATERIALS AND METHODS Experimental design This study was designed to identify the protein make up of neuronal cilia in the CNS. Specifically, we introduced a cilia-targeted proximity biotinylation enzyme into mice to label ciliary proteins in tissues and leveraged biochemical isolation of biotinylated proteins, quantitative MS, and a variety of genetic and direct controls to define the proteome of neuronal cilia in the brain. Animal husbandry Primary cultures of GNPs, ependymal cells, and cortical neurons were conducted from CD-1 mice, which were purchased from Charles River (strain code: 022). Transgenic mouse strains, including Tg (UBC-Cre/ERT2)1Ejb and Ift88tm1.1Bky , were obtained from the Jackson Laboratory (strain nos. 007001 and 022409, respectively) ( 84 ). The Bbs4 −/− and Nex-cre mice were gifts from V.C. Sheffield ( 85 ) and K.A. Nave ( 47 ). The Arl13b +/GFP-TurboID founder mouse was initially generated in FVB/NJ (the Jackson Laboratory, strain no. 001800) and subsequently backcrossed to the CD-1 background for three generations. A maximum of five adult mice were housed per cage. Breeding cages contained one male and up to two females. Both male and female mice were used in all studies. To knockout Ift88 postnatally, tamoxifen was injected subcutaneously into Arl13b +/GFP-TurboID ; Ubc-cre/ERT2; Ift88 fl/fl and Arl13b +/GFP-TurboID ; Ift88 fl/fl (control) pups at postnatal day 5 (P5) for 5 consecutive days, followed by a 3-week waiting period before conducting proteomics or immunostaining. All procedures adhered to the Institutional Animal Care and Use Committee (IACUC) guidelines of UCSF. This project (Molecular and Proteomic Studies of the Cilia) was reviewed and approved by the IACUC at UCSF (AN195589). Cell lines Daoy cells (American Type Culture Collection, HTB-186), derived from human desmoplastic cerebellar medulloblastoma, were cultured in Eagle’s minimum essential medium (Corning #10-009-CV) supplemented with 10% fetal bovine serum (FBS; from Gemini Bio-products #100-106) and penicillin and streptomycin (P-S; 100 U/ml; from Gibco #15140-122). HEK293T-EcR-ShhN cells (human embryonic kidney 293T cells with ecdysone receptor-gated expression of the N-terminal fragment of Sonic Hedgehog), provided by P. Beachy, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco #11965092) supplemented with 10% FBS, 2 mM l -glutamine (Gibco #25030081), and antibiotics. To produce Shh-N–conditioned media, HEK293T-EcR-ShhN cells were cultured in GNP culture media and stimulated with ponasterone A (5 μM, Thermo Fisher Scientific, catalog no. H10101 ) for 48 hours. The supernatant was collected and filtered by a 0.2-μm polyvinylidene difluoride syringe filter (Whatman #9913-2502). A range of dilutions of Shh-N–conditioned media was tested on GNP cultures, and the optimal dilution was chosen on the basis of a GNP ciliation rate of 90%. All cell lines used in this study were tested monthly for mycoplasma contamination using the MycoAlert system (Lonza). Primary culture Primary cultures of purified GNPs from P6 cerebella were conducted as previously described ( 86 ). Briefly, cerebella were digested using the papain dissociation system (Worthington, catalog no. LK003150 ) according to the manufacturer’s instructions. Dissociated cells were purified via a Percoll gradient (Sigma-Aldrich, catalog no. P1644), and glial cells were removed by preplating. Purified GNPs were cultured in neurobasal medium (Gibco) supplemented with P-S (100 U/ml), 2 mM l -glutamine, 0.45% d -glucose, 2% B-27 (Gibco, catalog no. 17504010), 1% linoleic acid–oleic acid–albumin solution (Sigma-Aldrich, catalog no. L9655), 100 μM N -acetylcysteine, 1% SPITE medium (selenium, sodium pyruvate, insulin, transferrin, and ethanolamine; Sigma-Aldrich, catalog no. S5666), and 2% Shh-N–conditioned medium for 3 days. Lentivirus carrying genes of interest was added on the first day of primary culture. Primary cultures of cortical neurons were conducted as previously described ( 87 ). Briefly, cerebral cortices from embryonic day 13.5 (E13.5) to E15.5 embryos were dissociated using the papain dissociation system, and cell clumps were removed using cell strainers (Falcon #352350). Cells were cultured in neurobasal medium supplemented with P-S (100 units/ml), 2 mM l -glutamine, 0.45% d -glucose, and 2% B-27 (Gibco) for 7 to 9 days. Primary cultures of ependymal cells were conducted as previously described ( 88 ). Briefly, dissociated cells from P0 cerebral cortices were cultured in 25-cm 2 flasks in DMEM-GlutaMAX medium (Gibco) supplemented with 10% FBS and antibiotics for 3 to 5 days. After reaching confluence, neurons and oligodendrocytes were removed by shaking the flasks at 250 rpm on an orbital shaker at room temperature overnight. Subsequently, 20 μl of ependymal cells (10 7 cells/ml) were placed in the center of a 12-mm circle coverslip (Thermo Fisher Scientific #12541001) for 1 day and cultured in media without FBS for an additional 2 weeks. Coverslips for culturing GNPs and cortical neurons were pretreated with poly- d -lysine (Sigma-Aldrich, catalog no. P1024) and Matrigel (Corning, catalog no. 356234) before seeding cells. Plasmids Sequences encoding human NPHP3[1-203] fused to GFP-APEX2 (pNU252), mouse SSTR3 fused to GFP-APEX2 (pNU255), human ARL13B fused to GFP-APEX2 (pNU254), and human ARL13B fused to GFP-TurboID (pNU265; pNU270 for ARL13B[V359A]-GFP-TurboID) were subcloned into the pLenti6-UbC-V5-DEST vector (Thermo Fisher Scientific, catalog no. V49910). Site-directed mutagenesis was performed to generate the pLenti-UbC-ARL13B[V359A]-GFP-TurboID plasmid using Pfu Turbo DNA polymerase according to the manufacturer’s instructions (Agilent, catalog no. 200518). Virus production and transduction Lentiviruses carrying NPHP3[1-203]-GFP-APEX2, SSTR3-GFP-APEX2, ARL13B-GFP-APEX2, ARL13B-GFP-TurboID, and ARL13B[V359A]-GFP-TurboID were produced as described in the manufacturer’s protocol (PEG-it, System Biosciences). Briefly, lentiviral vectors along with the plasmid of interest were transfected into HEK293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Supernatants containing viral particles were concentrated using PEG-it Virus Precipitation Solution (System Biosciences) and resuspended in phosphate-buffered saline (PBS). Lentiviruses were added to dissociated cells on the first day of primary culture and washed away the following day. Proximity labeling experiments in vitro APEX-based proximity labeling was performed as described ( 34 ). Briefly, primary cells transduced with APEX2 fusions were cultured in their indicated medium supplemented with 0.5 mM (or specified concentration) biotin-phenol for 1 hour. Next, cells were incubated with 1 mM H 2 O 2 in culture medium for 1 min at room temperature and then washed three times in quenching buffer, consisting of 10 mM sodium ascorbate, 10 mM sodium azide, and 5 mM Trolox in PBS. For proximity labeling using TurboID, primary cells expressing TurboID were incubated in medium containing 0.5 mM (or specified concentration) biotin (Millipore-Sigma, catalog no. B4501) for 1 hour and washed with cold PBS before fixation. Immunostaining and imaging Primary cells or Daoy cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min, followed by treatment with prechilled methanol for 5 min at −20°C. Cells were permeabilized in PBST (0.2% Triton X-100 in PBS) for 30 min and blocked with PBS containing 5% normal goat serum and 5% bovine serum albumin for 1 hour. Cells were incubated with primary antibodies (1:100 dilution), as listed in the tables S2 and S3, and secondary antibodies (1:500 dilution; Alexa Fluor, Invitrogen or Jackson ImmunoResearch Labs) for 2 hours each at room temperature. Biotin signals were labeled by streptavidin and Alexa Fluor 647 or 555 conjugate (1:1000 dilution; Thermo Fisher Scientific, catalog no. 21374 or no. 32355). Cells were counterstained with Hoechst (Thermo Fisher Scientific, catalog no. H1399), and coverslips were mounted onto slides using Fluoromount-G (Electron Microscopy Sciences). For Airyscan2 super-resolution imaging, cells were mounted in ProLong Antifade (Thermo Fisher Scientific, catalog no. P36980 ). For immunostaining of tissues, mice were transcardially perfused with 4% PFA (Electron Microscopy Sciences, catalog no. 50-980-487) in PBS, and tissues were postfixed in 4% PFA overnight. Coronal sections (100 μm) were prepared using a Sliding Microtome (Thermo Fisher Scientific). We applied heat-mediated antigen retrieval with citrate buffer [10 mM sodium citrate and 0.05% Tween 20 (pH 6.0)] in PBST-permeabilized samples, which were then incubated with primary antibodies (tables S2 and S3) for 2 days, followed by incubation with secondary antibodies for 2 hours. Tissues were counterstained, mounted as described above, and imaged on either an LSM900 microscope equipped with Airyscan2 (Zeiss) or an AX/AX R NSPARC system (Nikon). For imaging of tissues, a 40×/1.4 numerical aperture (NA) oil-immersion objective (Zeiss) was used at 1024 × 1024–pixel resolution. For cultured cortical neurons, Airyscan2 super-resolution imaging was performed with a 63×/1.4 NA oil-immersion objective, using a pixel size corresponding to fivefold Nyquist sampling and 0.1-μm optical sections. Image analysis For fluorescence intensity measurements in cilia of cultured neurons, sum-intensity projections were first created from consecutive Z-sections encompassing the entire cilium using ImageJ (National Institutes of Health). A segmented line mask was then drawn along each cilium and transferred to the proteins of interest (POI) channel. The means of raw intensities in cilia ( F raw ) and adjacent cell-free areas ( F background ) were measured, and the final mean ciliary fluorescence intensities ( F cilia ) were calculated by the following formula: F cilia = F raw − F background . For cilia enrichment analysis, the same procedure was followed, and an additional mask outlining the cell boundary was generated to measure the POI intensity in the cytoplasm ( F cytoplasm ). The cilia enrichment ratio was then calculated as ( F raw − F background )/( F cytoplasm − F background ). For fluorescence intensity measurements in ARL13B-labeled cilia from tissues ( Figs. 2, B, I, and K , and 4E , and fig. S3C), cilia were segmented in 3D using the Threshold method and FilterObjects functions in the General Analysis 3 (GA3) platform (NIKON NIS software). The total biotin intensity ( F cilia ) and the number of voxels ( V cilia ) in each segmented cilium were recorded. To estimate the background intensity, each segmented cilium was dilated by a 3-pixel radius in three dimensions; the total biotin intensity ( F dilate ) and the voxel count ( V dilate ) of the dilated cilia were then measured. The averaged background intensity ( F background ) of each cilium was determined using the following formula: F background = ( F dilate − F cilia )/( V dilate − V cilia ). Last, the mean biotin intensity for each cilium was calculated by subtracting its background intensity from the mean intensity: F total / V cilia − F background . For fluorescence intensity measurements in ACIII-labeled cilia from tissues ( Figs. 2, B and J , and 4C , and fig. S3D), Denoise.ai (NIKON NIS-Elements) was applied to remove Poisson-distributed shot noise, thus eliminating the granular background pattern while preserving ciliary ACIII signals. To verify the quality of segmentation, all the segmented images were aligned to the raw images. Measurements of biotin intensity within ACIII-labeled cilia were then carried out using the same approach as for ARL13B-labeled cilia. Quantitated data were plotted, and statistical tests were performed in GraphPad Prism 10, as indicated in the respective figure legends. For line scan analysis, longitudinal fluorescence intensities of the POI were measured in ImageJ by a line extending along the ciliary length. Pixel intensities for each cilium were plotted against the percentage of total length, from 0% at the base to 100% at the tip. Generation of Arl13b +/GFP-TurboID and Arl13b +/V358A-GFP-TurboID mice Mouse genome engineering was conducted at the Transgenic Gene Targeting Core (TGTC) at the Gladstone Institute. We followed the Easi-CRISPR protocol ( 89 ) to generate the Arl13B-GFP-TurboID and Arl13B[V358A]-GFP-TurboID alleles in mice. Four-week-old FVB/NJ female mice were injected with pregnant mare serum gonadotropin and human chorionic gonadotropin. Female mice with timed hormone induction were then bred with single-housed 10-week-old male mice. After dissection of the female reproductive system, fertilized oocytes and zygotes were collected and transferred to TGTC, where pronuclear injection of the ribonucleoprotein complex containing crRNA (CRISPR RNA, 5′-/AltR 1/rArArG rGrArC rCrArA rCrCrC rArUrCrCrGrA rCrUrG rUrUrU rUrArG rArGrC rUrArU rGrCrU/AltR2/-3′), tracrRNA (trans-activating CRISPR RNA, Integrated DNA Technologies, IDT), and Cas9 proteins (IDT), along with single-stranded DNA (ssDNA; table S1), was performed. Injected embryos were implanted into surrogate female mice and collected 17 pups at P21. Genomic DNA was extracted from each animal, and several fragments spanning exon 10 of Arl13b to GFP-TurboID were amplified and subjected to Sanger sequencing. One of 17 mice had a GFP-TurboID cassette in frame with the last amino acid of Arl13b . This animal was backcrossed to CD1 mice for three generations to remove possible extragenic mutations. Mice were genotyped with triplex primers (forward: 5′-GCG ACA GAG ACC TAA CGG TG-3′; reverse: 5′-TCG ATG TTG TGG CGG ATC TT-3′; WT: 5′-TGT CCA AAA GTC GGG ATG TGT-3′), where Arl13b WT and Arl13b GFP-TurboID alleles yielded products of 316 and 585 bp. To generate the Arl13b[V358A] variant, we mated 4-week-old Arl13b GFP-TurboID/GFP-TurboID female mice with 10-week-old Arl13b GFP-TurboID/GFP-TurboID male mice and harvested fertilized oocytes and zygotes as described above. Zygotes were electroporated with the ctRNP complex (crRNA sequence: 5′/AlTR1 /rUrArG rArCrU rCrGrU rCrUrG rUrArU rUrGrA rCrGrU rUrUrU rArGrA rGrCrU rArUrG rCrU/AlTR2/-3′) and ssDNA (table S1). Mice harboring the V358A mutation on the Arl13b GFP-TurboID allele were backcrossed to WT ( Arl13b +/+ ) CD1 mice and the offspring with the V358A mutation on the Arl13b GFP-TurboID allele were selected as Arl13b +/V358A-GFP-TurboID founder mice. This animal was backcrossed to CD1 mice for three generations to remove possible extragenic mutations. We observed no gross abnormalities in mice heterozygous for Arl13b[V358A]-TurboID , consistent with prior studies of mice having one Arl13b[V358A] allele ( 37 ). Genotyping of the Arl13b[V358A] mutation was performed as described ( 37 ). Biotin injection, streptavidin capture, and immunoblotting Mice were injected with 5 μl/g (body weight) biotin (24.4 mg of biotin dissolved in 1 ml of dimethyl sulfoxide and then mixed with 1 ml of PBS) intraperitoneally for 7 consecutive days starting from P30. The day after the last biotin administration, mice were transcardially perfused with PBS, and samples from cerebral cortices were lysed overnight in urea lysis (UL) buffer [6 M urea, 0.3 M NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM tris-HCl (pH 7.5), 10 mM sodium azide, 10 mM TCEP [tris(2-carboxyethyl)phosphine], and protease inhibitor cocktail (10 μg/ml each of leupeptin, pepstatin A, bestatin, 0.8 mM aprotinin, 1 mM AEBSF [4-(2-Aminoethyl)benzenesulfonyl fluoride], and 15 mM E-64)]. Lysates were cleared of precipitates at 100,000 g for 1 hour. Protein concentration of the supernatant was determined by Bradford (Bio-Rad). Equal volumes and concentrations of brain lysates were incubated with magnetic streptavidin beads (Pierce, catalog no. 88817) on a rotator at 4°C overnight. Beads were washed three times with 4 M urea buffer containing 50 mM sodium phosphate and 0.1% SDS and three times without SDS. Biotinylated proteins were eluted by boiling the beads with LDS (lithium dodecyl sulfate) sample buffer supplemented with 2 mM biotin, followed by Western blot analysis. The antibodies used in Western blotting are listed in table S2. We used streptavidin poly–horseradish peroxidase (Thermo Fisher Scientific, catalog no. 21140) for detecting biotinylated proteins. Purification of biotinylated proteins from mouse cerebral cortices Mice were injected with biotin as described above. For total extract purifications, cerebral cortices were dissected from perfused brains, lysed overnight with 4 ml of neuronal protein extraction reagent (N-PER, Thermo Fisher Scientific, catalog no. 87792) per cerebral cortex, and spun at 100,000 g for 1 hour, and supernatants were diluted 1:1 with UL buffer before measuring protein concentrations. Next, 1 ml (750 μg/ml) of cortical lysates were treated with 20 mM iodoacetamide (Sigma-Aldrich) for 30 min in UL buffer, and reactions were quenched by adding dithiothreitol to 50 mM final concentration. Samples were incubated with 75-μl magnetic streptavidin beads overnight, and beads were washed as described above. After the final two washes with PBS, dry beads were flash frozen in liquid nitrogen before processing for TMT and MS. For membrane protein purifications, biotin-injected mice were transcardially perfused with PBS, and dissected cortices were homogenized in Dounce homogenizer with homogenization buffer containing 5 mM EDTA, 3 mM EGTA, 250 mM sucrose, 10 mM tris-HCl (pH 7.6), 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog no. P7626), and protease inhibitor cocktail as described above. Samples were spun at 500 g for 5 min, and supernatants were then centrifuged at 100,000 g for 1 hour. P100 pellets were resuspended in WGA lysis buffer containing 150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 20 mM Hepes (pH 7.4), dodecyl-β-maltoside (4 mg/ml; Sigma-Aldrich, catalog no. D4641), and protease inhibitor cocktail. Dissolved samples were incubated with WGA beads (Vector Laboratories, catalog no. AL-1023) overnight. The next day, proteins bound to the WGA beads were eluted with UL buffer, and samples were processed for streptavidin enrichment as described above. Proteomic profiling Dry beads were processed for TMT-MS 3 as previously described ( 90 , 91 ) by the Thermo Fisher Scientific Center for Multiplexed Proteomics (TCMP) at Harvard Medical School for the total extract experiment 1 and the membrane protein experiment 3 or by the Kalocsay laboratory (Ascend instrument) for total extract experiment 2. Data in Fig. 3 and fig. S4C were recorded on an OE480 instrument. Briefly, samples were processed with the following steps. On-bead digestion and TMT labeling Protein-coated beads were washed three times with PBS. On-bead digestion was performed for 3 hours at 37°C under gentle agitation with endoproteinase LysC (Wako), followed by overnight digestion with 1 μg of trypsin (Promega) in 200 mM EPPS [N-(2-Hydroxyethyl)piperazine-N′-(3-propanesulfonic acid)] buffer (pH 8.5) containing 2% acetonitrile. The missed trypsin cleavage rate, determined by MS, remained below 10%. Acetonitrile was added to achieve a final concentration of 30% before labeling each sample with 62.5 μg of TMT 18-plex reagents (TMTPro; Thermo Fisher Scientific, catalog no. A52045). The labeling reaction proceeded for 1 hour at room temperature with intermittent vortexing. Labeling efficiency exceeded 95%, as measured by N-terminal TMTpro-conjugated peptides [dynamically searched for TMTpro (+304.207 Da) modification at the peptide N terminus and as a static modification on lysine residues]. TMT labeling reactions were quenched by adding hydroxylamine to a final concentration of 0.5% for 15 min, followed by acidification with formic acid. The entire volume from each sample was then pooled, and acetonitrile was evaporated to near-dryness by vacuum centrifugation (speedvac). The multiplexed sample was fractionated using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific) under a modified step gradient of increasing acetonitrile (10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 50, 65, and 80%, v/v), generating 12 fractions. Fractions were dried, desalted by StageTip, eluted with 95% acetonitrile and 1% formic acid into individual MS autosampler vials (Thermo Fisher Scientific), dried by speedvac, and lastly reconstituted in 5% acetonitrile with 5% formic acid for MS analysis. Samples (Kalocsay laboratory) were subjected to alkaline reversed phase chromatography using the high-pH reversed phase peptide fractionation kit (Thermo Fisher Scientific #84868) and combined into 6 fractions or 12 fractions. Liquid chromatography and tandem MS Thermo Fisher Scientific Center for Multiplexed Proteomics. Mass spectrometric data were collected on an Orbitrap Eclipse mass spectrometer coupled to a Proxeon NanoLC-1000 UHPLC (Ultra-High Performance Liquid Chromatography, Thermo Fisher Scientific). The 100-μm capillary column was packed in-house with 35 cm of Accucore 150 resin (2.6 μm, 150 Å; Thermo Fisher Scientific). Data were acquired for 180 min per run. A FAIMS (Field Asymmetric Ion Mobility Spectrometry) device was enabled during data collection, and compensation voltages were set at −40, −60, and −80 V. MS1 scans were collected in the Orbitrap [resolution, 60,000; scan range, 400 to 1600 Th; automatic gain control (AGC), 4 × 10 5 ; maximum ion injection time, automatic]. MS2 scans were collected in the Orbitrap following higher-energy collision dissociation (HCD; resolution, 50,000; AGC, 1.25 × 10 5 ; normalized collision energy, 36; isolation window, 0.5 Th; maximum ion injection time, 100 ms). Kalocsay laboratory. Mass spectrometric data were collected on an Orbitrap Exploris 480 or Ascend mass spectrometer coupled to a VanquishNeo UHPLC (Thermo Fisher Scientific). The 75-μm capillary column was packed in-house with 35 cm of Accucore 150 resin. Data were acquired for 60 min (OE480, 6 fractions) or 90 min (Ascend, 12 fractions) per run. A FAIMS ProDuo2 device was enabled during data collection, and compensation voltages were set as described above. MS1 scans were collected in the Orbitrap (resolution, 120,000; scan range, 350 to 1400 Th; AGC, 4 × 10 5 ; maximum ion injection time, automatic). MS2 scans were collected in the Orbitrap following HCD [resolution, 50,000; AGC, 1.25 × 10 5 ; normalized collision energy, 33; isolation window, 0.7 Th; maximum ion injection time, 96 ms (OE480) or 400 ms (Ascend)]. MS data analysis Database searching included all entries from the mouse UniProt Database (downloaded in June 2024). The database was concatenated with one composed of all protein sequences for that database in the reversed order. Raw files were converted to mzXML, and monoisotopic peaks were reassigned using Monocle ( 92 ). Searches were performed with Comet ( 93 ) using a 20–parts per million precursor ion tolerance and fragment bin tolerance of 0.02 Da. TMTpro labels on lysine residues and peptide N-termini (+304.207 Da), as well as carbamidomethylation of cysteine residues (+57.021 Da), were set as static modifications, while oxidation of methionine residues (+15.995 Da) was set as a variable modification. Peptide-spectrum matches (PSMs) were adjusted to a 1% false discovery rate (FDR) using a linear discriminant after which proteins were assembled further to a final protein-level FDR of 1% analysis ( 94 ). TMT reporter ion intensities were measured using a 0.003-Da window around the theoretical mass/charge ratio for each reporter ion. Proteins were quantified by summing reporter ion counts across all matching PSMs. More specifically, reporter ion intensities were adjusted to correct for the isotopic impurities of the different TMTpro reagents according to the manufacturer’s specifications. Peptides were filtered to exclude those with a summed signal-to-noise (S/N) < 200 across all TMT channels and <0.7 precursor isolation specifically. The S/N measurements of peptides assigned to each protein were summed (for a given protein). All channels that pass quality control checks with reproducibility are used for hierarchical clustering. Hierarchical clustering Proteomic results were then subjected to hierarchical clustering using JMP Pro 17 software (JMP Statistical Discovery). Candidate proteins clustered with known cilia proteins were retrieved (tier 3) and filtered by statistical significance ( P < 0.05) by comparing peptide abundance between Arl13b +/GFP-TurboID samples and Arl13b +/+ samples (tier 2). Only proteins detected with more than one peptide were included in our final tier (tier 1) as our cilia proteome. Gene Ontology analysis We applied STRING ( 95 ) for Gene Ontology analysis. We calculated the FDR by Benjamini-Hochberg–corrected P values, indicating the significant of enrichment for each term compared to the background proteins from all assigned proteins in TMT assays corresponding to the tested tissue. Expansion microscopy of tissues Expanded mouse cerebral cortexes were prepared as described ( 59 ). Briefly, mice were perfused with PBS, followed by 4% PFA with 20% acrylamide (Millipore-Sigma, catalog no. Supelco 01697), and 70-μm sections were cut on a VT1000S vibratome (Leica). Sections were incubated in inactivated first expansion gel solution containing 19% sodium acrylate (Pfaltz & Bauer, catalog no. S03880 ), 10% acrylamide, and 0.1% N , N ′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA, Millipore-Sigma, catalog no. 294381) in 1× PBS for 30 min. Next, samples were incubated in fresh, activated first expansion gel solution containing 0.075% tetramethylethylenediamine (TEMED; RPI Research Product International, catalog no. T18000-0.1) and ammonium persulfate (APS; Millipore-Sigma, catalog no. A3678), and transferred to gelation chambers with 170-μm initial gel thickness. Samples were then placed in a humidified chamber, purged with nitrogen, and incubated at 37°C for 2 hours. The tissue-gel hybrids containing the region of interest were excised, and samples were incubated in denaturation buffer containing 200 mM SDS, 200 mM NaCl, and 50 mM tris in Milli-Q ultrapure water at 37°C for 15 min and 75°C for 4 hours. Samples were then washed in PBS and expanded in ultrapure water overnight. The next day, samples were reembedded in the second gelling solution containing 10% acrylamide, 0.05% DHEBA, 0.05% TEMED, and APS in a nitrogen-filled humidified chamber at 37°C for 1.5 hours. Samples were stored in 1× PBS overnight. On the third day, samples were incubated in the third hydrogel solution containing 9% sodium acrylate, 10% acrylamide, 0.1% N , N ′-methylenebis (acrylamide) (Thermo Fisher Scientific, catalog no. J66710.14), 0.05% TEMED, and APS, and tissue-gel hybrids were transferred to a N 2 -filled humidified chamber at 37°C for 1.5 hours. To dissolve the DHEBA gel from the second gelation step, samples were incubated in 200 mM NaOH for 1 hour and washed with PBS. Samples were then incubated with primary antibodies (1:250 dilution) for 2 days. Samples were then incubated with goat anti-rabbit ATTO 647 N-conjugated (Rockland #611-156-122) and goat anti-mouse immunoglobulin G ATTO 550–conjugated (Sigma-Aldrich #43394) secondary antibodies (1:250) for another 2 days. On the seventh day, tissue-gel hybrids were incubated in ATTO 488–conjugated NHS ester (30 μg/ml; Sigma-Aldrich #41698) or Abberior STAR 635, NHS ester (Sigma-Aldrich, catalog no. 30558) for 2 hours, washed in PBS, and expanded in ultrapure water overnight. Last, expanded tissues were mounted on #1.5 glass-bottom dishes (MatTek) using Z-Dupe Duplicating Silicone Set (Henry Schein, catalog no. 1026526JA5). Image stacks of expanded cerebral cortexes were acquired at 0.5- to 1-μm interval with 1024 × 1024–pixel resolution using an AX/AX R NSPARC (Nikon) with a 60×/NA 1.2 water-immersion objective. Fifty to 200 z-sections were acquired to capture the entire length of a given cilium. Measurement of expansion factor The linear expansion factor in expanded samples was determined as previously described ( 96 ). Briefly, expanded samples from mouse cerebral cortices were stained with Sytox Green (Thermo Fisher Scientific, catalog no. S7020) and imaged on an AX/AX R NSPARC microscope (Nikon). Images were processed with Gaussian blur, a 4-pixel maximum filter with threshold set using the Li method. Individual nuclei were identified and segmented using MorphoLibJ ( 97 ), measuring the cross-sectional area at the mid-plane of nuclei with circularity values greater than 0.9. In parallel, unexpanded samples from PFA-fixed cerebral cortices were stained with Hoechst. Nuclei were segmented in 3D using TrackMate ( 98 ) with StarDist ( 99 ) in FIJI. 3D image stacks were treated as time series to enable 2D StarDist segmentation, and nuclei were reconstructed in 3D. After initial segmentation, masks were relabeled, and small objects (<25,000 voxels) were removed using a custom FIJI script and MorphoLibJ. Morphological characteristics of segmented nuclei were measured using MorphoLibJ as previously described ( 96 ). Segmented nuclei were then filtered on the basis of sphericity (sphericity, ≥0.6) using a custom Python script and visually inspected to confirm the accuracy of segmentation. Poorly segmented objects were manually removed using MorphoLibJ. The cross-sectional area of each nucleus was then measured as previously described ( 96 ). Last, the average mid-plane cross-sectional area from expanded nuclei was divided by that from unexpanded nuclei, and a linear expansion factor of 16.23× was applied to all pan-ExM images. 3D segmentation of neuronal cilia and synapses in pan-ExM-t We used NIS-Elements (Nikon) to quantify the distance of GluN1 spots from neuronal cilia to all synapses within a given imaging volume. Using the GA3 platform, we manually created a binary mask for cilia based on thresholding, filtered objects by size, and then segmented the final cilia via dilation, hole filling, and surface smoothing. Neuronal synapses (using pan-stain channel) were segmented similarly, whereas GluN1 spots were segmented with a Simple Threshold function and subsequently dilated after removing granular background. We identified ciliary GluN1 spots (GluN1 cilia ) by intersecting segmented GluN1 spots with the cilia mask and synaptic GluN1 (GluN1 syn ) by selecting GluN1 spots overlapping the segmented synapses. Child-to-parent comparisons were then performed to measure the shortest distance from GluN1 syn (child) to GluN1 cilia (parent). As a control, the same parameters were reapplied to the same imaging volume after rotating the cilium by 90°. 3D rendering of cultured neuron cilia ( Fig. 8K and fig. S8, C and F) and brain cilia as well as synapses in pan-ExM-t images ( Fig. 10B ) was performed with Imaris 10 (Oxford Instruments) following the manufacturer’s instructions. Briefly, 3D cilia surface plots were defined by threshold of absolute fluorescent intensity, and the voxel representing neuronal cilia was selected. Similarly, the segmented spots or surface of candidate proteins were using threshold methods, while voxels within the surface of segmented neuronal cilia (distance between segmented proteins to cilia = 0) were selected. Surface model was used to segment all neuronal cilia and candidate proteins in Bbs4 −/− neurons, while spots model was applied to candidate proteins in Bbs4 +/− neurons. Analysis of cilia volumes in pan-ExM-t An image analysis pipeline was developed to analyze GluN1 localization relative to the ciliary membrane marked by ARL13B-GFP in pan-ExM-t volumes using Python. Image processing libraries included Scikit-Image ( 100 ) and Scipy ( 101 ). Skan ( 102 ) was used for simplifying skeletons to generate centerlines. Napari ( 103 ) was used for visualizing intermediate steps and results. First, the microscopy channels were loaded as Numpy arrays. The ARL13B-GFP signal was segmented to generate a mask for the ciliary membrane, which was used to generate a centerline for the cilia. The centerline was then dilated to form a series of concentric shells matching the shape of the cilia. The GluN1 signal was segmented and size-filtered, and the centroids for each spot were mapped. The GluN1-centroid data were then matched to the series of concentric shells, producing a table indicating the number of puncta per dilation step. This table reflects the radial distance of the GluN1 spots from the centerline of the cilia. A similar analysis was performed for the ARL13B-GFP spots. The number of spots on each dilated shell was normalized to the total number of spot per cilia. Statistical analysis Statistical analyses were performed using Prism 10 (GraphPad). Data from more than two experimental groups were analyzed by one-way analysis of variance (ANOVA) or Kruskal-Wallis test, followed by Bonferroni or Dunn post hoc test. Data from two groups were analyzed using unpaired t test or Mann-Whitney U test. We considered P < 0.05 as statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Visualization and Gene Ontology analysis We used Prism 10 software (GraphPad) to generate volcano plots and used online resources, including CZ CELL X GENE Discover ( https://cellxgene.cziscience.com ) ( 56 ), STRING ( https://string-db.org ) ( 95 ), Organelle IP ( https://organelles.sf.czbiohub.org ) ( 58 ), SynGO ( https://syngoportal.org ) ( 104 ), and DISGENET ( https://disgenet.com ) ( 83 ) for proteomic analysis. Acknowledgments We thank R. Brown, M. H. Schwab, and V. Sheffield for the Ubc-cre/ERT2 , Nex-cre and Bbs4 mice; P. Beachy for the Shh-N–producing HEK293T cells; J. Zhang for pronuclear injection; T. Caspary and K. Nitsche for helpful discussions of mouse transgenesis; A. Hurtado for mouse colony management; Y.-M. Kuo for help with microscopy; S. Wang for help with graphic design; and A. Alvarez-Buylla, G. David, J. Reiter, C. Vaisse, M. Delling, N. Berbari, N. Spassky, and W.-J. Wang and all members of the Nachury laboratory for stimulating discussions. Funding: This work was supported by the National Institutes of Health grant GM089933 (M.V.N.), National Institutes of Health grant EY031462 (M.V.N.), American Diabetes Association grant 1-20-VSN-03 (M.V.N.), National Science and Technology Council of Taiwan Postdoctoral Fellowship MOST-110-2917-I-564-010 (C.-H.C.), Sandler Program for Breakthrough Biomedical Research (C.-H.C. and M.V.N.), UCSF Valhalla Fellows Program (M.E.P.), CPRIT scholar in cancer research grant RR220032 (M.K.), National Institutes of Health grant EY002162 (M.V.N.), and Research to Prevent Blindness Unrestricted Grant (M.V.N.). Author contributions: Conceptualization: C.-H.C. and M.V.N. Methodology: C.-H.C., V.N.T., S.W.N., C.K.M., M.E.P., and M.V.N. Formal analysis: C.-H.C., V.N.T., S.W.N., N.R.L., M.E.P., M.K., and M.V.N. Investigation: C.-H.C., V.N.T., N.R.L., M.K., and C.K.M. Writing—original draft: C.-H.C. and M.V.N. Writing—review and editing: C.-H.C., V.N.T., S.W.N., N.R.L., C.K.M., D.B., M.E.P., M.K., and M.V.N. Visualization: C.-H.C., S.W.N., and M.V.N. Funding acquisition: C.-H.C., M.V.N., M.E.P., and M.K. Supervision: C.-H.C., D.B., M.K., M.E.P., and M.V.N. Resources: V.N.T., D.B., M.E.P., and M.K. Validation: C.-H.C., S.W.N., V.N.T., and M.V.N. Software: S.W.N. and M.E.P. Data curation: S.W.N. and M.K. Project administration: M.K. and M.V.N. Competing interests: The authors declare that they have no competing interests. Data, code, and materials availability: The MS proteomic data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE ( 105 ) partner repository with the dataset identifiers PXD067626 and PXD067892. The code for the analysis of cilia volume is available at https://zenodo.org/records/18001606 (DOI: 10.5281/zenodo.18001606 ). All materials generated in this paper ( Arl13b-GFP-TurboID knock-in mice, plasmids for ciliary biotinylation, and Daoy stable cell lines) are available from M.V.N. upon request, and all other necessary materials for replication are available from commercial suppliers listed in the method. All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. Supplementary Materials The PDF file includes: Figs. 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S1 to S9 Tables S1 to S3 Legends for movies S1 to S4 Legends for data S1 to S7 References sciadv.aed5548_sm.pdf (12.4MB, pdf) Movies S1 to S4 sciadv.aed5548_movies_s1_to_s4.zip (111MB, zip) Data S1 to S7 sciadv.aed5548_data_s1_to_s7.zip (3.2MB, zip) Data Availability Statement The MS proteomic data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE ( 105 ) partner repository with the dataset identifiers PXD067626 and PXD067892. The code for the analysis of cilia volume is available at https://zenodo.org/records/18001606 (DOI: 10.5281/zenodo.18001606 ). All materials generated in this paper ( Arl13b-GFP-TurboID knock-in mice, plasmids for ciliary biotinylation, and Daoy stable cell lines) are available from M.V.N. upon request, and all other necessary materials for replication are available from commercial suppliers listed in the method. All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. Articles from Science Advances are provided here courtesy of American Association for the Advancement of Science ACTIONS View on publisher site PDF (11.5 MB) Cite Collections Permalink RESOURCES Similar articles Cited by other articles Links to NCBI Databases Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

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