{"gene":"SSNA1","run_date":"2026-04-28T20:42:08","timeline":{"discoveries":[{"year":1998,"finding":"NA14 (SSNA1) is a nuclear autoantigen of 14 kDa containing an acidic N-terminal domain (aa 6-80) with heptad repeats characteristic of dimeric coiled-coil alpha-helices, and an alkaline C-terminal domain (aa 81-119). It localizes to numerous punctate structures scattered throughout the nucleus of HeLa and 3T3 cells.","method":"cDNA library screening with autoimmune serum, immunoprecipitation, recombinant protein expression, confocal immunofluorescence microscopy of transiently transfected cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (IP, IF, sequence analysis) in original discovery paper","pmids":["9430706"],"is_preprint":false},{"year":2003,"finding":"The Chlamydomonas orthologue DIP13 and human NA14 localize to microtubule structures including basal bodies, flagellar axonemes, and cytoplasmic microtubules; anti-DIP13 antibody cross-reacts with human NA14 at centrosomes and basal bodies of human sperm. Antisense knockdown of DIP13 in Chlamydomonas produces multinucleate, multiflagellate cells, implicating DIP13/NA14 in proper cell division.","method":"Antibody generation, immunofluorescence microscopy, antisense knockdown in Chlamydomonas","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — direct localization experiments with functional loss-of-function phenotype, cross-species validation","pmids":["12640030"],"is_preprint":false},{"year":2004,"finding":"Spastin physically interacts with the centrosomal protein NA14 (SSNA1), co-fractionates with the centrosomal marker gamma-tubulin, and is enriched at the spindle pole, central spindle, midbody, distal axon, and branching points. Deletion of the spastin region required for NA14 binding disrupts spastin's interaction with microtubules, suggesting NA14 targets spastin activity to the centrosome.","method":"Co-immunoprecipitation, subcellular fractionation, immunofluorescence microscopy in dividing cells and immortalized motoneurons, domain deletion analysis","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — reciprocal interaction confirmed by Co-IP and fractionation, functional domain deletion linking NA14 binding to microtubule interaction","pmids":["15269182"],"is_preprint":false},{"year":2008,"finding":"NA14 binds to the C-terminal region of the orphan receptor TPRA40/GPR175, identified by yeast two-hybrid and confirmed by GST pull-down and co-immunoprecipitation. NA14 mediates functional plasma membrane transport of TPRA40; an N-terminal deletion mutant of NA14 that cannot bind microtubules but retains TPRA40 binding inhibits TPRA40 translocation to the plasma membrane, impairing TPRA40's regulation of cell division in mouse embryos.","method":"Yeast two-hybrid screening, GST pull-down, co-immunoprecipitation, live-cell imaging, mouse embryo microinjection","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods (Y2H, GST-PD, Co-IP, functional embryo assay) but single lab","pmids":["18459117"],"is_preprint":false},{"year":2011,"finding":"Human NA14 forms a parallel coiled-coil structure spanning residues 14-104 that mediates self-association; N- and C-termini lack preferred structure. Leu83 and Leu93 are proposed to mediate interactions among NA14, spastin, and microtubules. NA14 tends to oligomerize and form fibrils in solution.","method":"Circular dichroism, NMR spectroscopy, site-directed mutagenesis, urea/thermal denaturation","journal":"Protein engineering, design & selection : PEDS","confidence":"Medium","confidence_rationale":"Tier 1 — NMR-based structural characterization with mutagenesis, but single lab","pmids":["22008182"],"is_preprint":false},{"year":2012,"finding":"The Trypanosoma brucei SSNA1 orthologue TbDIP13 self-assembles into fibril-like structures both in vitro and in vivo, partially co-localizes with acetylated alpha-tubulin, and deletion has little effect on parasite growth—suggesting possible functional redundancy. Comparative proteomics identified potential interacting partners.","method":"Electron microscopy of fibril structures, immunofluorescence co-localization with acetylated alpha-tubulin, gene deletion, comparative proteomics","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 — ortholog in a divergent parasite; single lab, limited mechanistic follow-up","pmids":["22363749"],"is_preprint":false},{"year":2014,"finding":"Endogenous NA14 (SSNA1) localizes specifically to centrioles in HeLa cells and rat cortical neurons. Stable NA14 knockdown dramatically impairs cytokinesis. Overexpression of NA14 in neurons significantly increases axon outgrowth, branching, and neuronal differentiation. NA14 is proposed to act as an adaptor regulating spastin localization to centrosomes.","method":"Stable shRNA knockdown in HeLa cells, immunofluorescence of endogenous proteins, neuronal overexpression in rat cortical primary cultures, time-lapse imaging of cell division","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — clean KD with defined cytokinesis phenotype, overexpression with neuronal phenotype, endogenous localization; multiple cell systems","pmids":["25390646"],"is_preprint":false},{"year":2016,"finding":"The SSNA1/DIP13 homologue in Toxoplasma gondii localizes to the conoid of the apical complex in mature and dividing cells, and to the basal complex during cell division. The protein self-associates into higher-order structures both in vitro and in vivo, and overexpression impairs parasite division.","method":"Immunofluorescence with parasite-specific markers, in vitro self-assembly assays, overexpression phenotypic analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization experiments with functional overexpression phenotype, single lab","pmids":["27324377"],"is_preprint":false},{"year":2021,"finding":"Human SSNA1 directly modulates all parameters of microtubule dynamic instability in vitro: it slows growth, shrinkage, and catastrophe rates, and promotes rescue. SSNA1 forms stretches along growing microtubule ends, binds cooperatively to the lattice, becomes enriched at microtubule damage sites (both naturally occurring and induced by spastin severing), and protects microtubules against spastin-mediated severing.","method":"In vitro reconstitution with purified proteins, TIRF microscopy, dynamic instability analysis, spastin severing assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — rigorous in vitro reconstitution with quantitative single-molecule TIRF microscopy, multiple mechanistic parameters measured","pmids":["34970964"],"is_preprint":false},{"year":2024,"finding":"The cryo-EM structure of C. elegans SSNA-1 at 4.55 Å resolution reveals that SSNA1 forms an anti-parallel coiled-coil, with self-assembly driven by 16 C-terminal residue overhangs that dock on adjacent coiled-coils to form a triple-stranded helical junction. The microtubule-binding region maps to within this triple-stranded junction, indicating that self-assembly creates hubs for effective microtubule interaction. Deletion of SSNA-1 in C. elegans reduces embryonic viability and causes multipolar spindles; impairing self-assembly has a comparable effect on embryonic viability to knockout.","method":"Cryo-EM structure determination, genetic knockout and self-assembly mutants in C. elegans, embryonic viability assays, spindle imaging","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure at near-atomic resolution combined with functional genetic analysis and mutagenesis of self-assembly","pmids":["40804232"],"is_preprint":false},{"year":2025,"finding":"SAS-1 (C2CD3 homologue) is essential for SSNA-1 localization to centrioles during oogenesis and to the transition zone during ciliogenesis in C. elegans. In a heterologous human cell assay, SAS-1 recruits SSNA-1 to microtubules. Molecular epistasis with null alleles establishes that SSNA-1 is positioned downstream of SAS-1 in the centriole integrity pathway, and SSNA-1 localizes adjacent to the SAS-1 C-terminus within centriole architecture.","method":"Null allele generation, U-Ex-STED super-resolution microscopy, molecular epistasis experiments, heterologous human cell recruitment assay","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with null alleles, super-resolution localization, cross-species functional validation; multiple orthogonal methods","pmids":["41124206"],"is_preprint":false},{"year":2025,"finding":"SSNA1 increases microtubule rigidity and resistance to breakage under force (kinesin-driven gliding and microfluidic flow assays). SSNA1 localization to damage sites prevents incorporation of new tubulin dimers, thereby inhibiting microtubule lattice self-repair. SSNA1 does not recognize damage sites that have been repaired by tubulin incorporation.","method":"In vitro reconstitution, TIRF microscopy, kinesin gliding assays, microfluidic force application, tubulin incorporation assays with purified proteins","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — rigorous in vitro reconstitution with multiple force assays, but preprint not yet peer-reviewed","pmids":["41648615"],"is_preprint":true},{"year":2025,"finding":"Using KO-validated antibodies and super-resolution/expansion microscopy, SSNA1 is shown to localize to the distal lumen of centrioles in a ring-like 9-fold symmetric configuration, separate from centriolar microtubules, and not in the nucleus, midbody, or ciliary axoneme as previously reported. SSNA1 KO impairs ciliogenesis by failing to facilitate CP110 removal. A C2CD3-SSNA1-LRRCC1 hierarchical targeting axis was identified in the distal lumen. Tag-free SSNA1 and microtubule co-pelleting assays indicate SSNA1 does not bind microtubules in vitro under these conditions.","method":"KO-validated antibody, super-resolution microscopy with expansion microscopy (ExM), SSNA1 KO and oligomerization-deficient mutants, CP110 removal assays, microtubule co-pelleting, interactor screening","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods in single lab preprint; some findings (microtubule binding) contradict prior peer-reviewed reconstitution data, warranting caution","pmids":["bio_10.1101_2025.04.28.648957"],"is_preprint":true},{"year":2025,"finding":"C2CD3 organizes the distal centriolar lumen and physically interacts with MNR to recruit the DISCO complex; its depletion destabilizes a luminal ring network composed of C2CD3/SFI1/centrin-2/CEP135/NA14 (SSNA1), placing SSNA1 as a component of the distal luminal ring scaffold downstream of C2CD3.","method":"Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, in situ cryo-electron tomography, protein interaction assays, depletion experiments","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1-2 — cryo-ET and U-ExM with functional depletion, but preprint; SSNA1 finding is secondary to C2CD3 analysis","pmids":["bio_10.1101_2025.06.17.660204"],"is_preprint":true}],"current_model":"SSNA1/NA14 is a small coiled-coil protein that forms anti-parallel coiled-coil fibrils through C-terminal triple-stranded junctions, which create microtubule-interaction hubs; it stabilizes dynamic microtubules by slowing catastrophe and promoting rescue, acts as a damage sensor by preferentially binding to damaged lattice sites where it mechanically reinforces microtubules and blocks spastin-mediated severing and tubulin-mediated self-repair, localizes specifically to centrioles (distal lumen) and centrosomes in a pathway downstream of SAS-1/C2CD3, and is required for proper cytokinesis, cilia assembly (via CP110 removal), and axon outgrowth/branching—likely by acting as an adaptor that spatially and temporally regulates spastin's microtubule-severing activity at centrosomes."},"narrative":{"teleology":[{"year":1998,"claim":"Identification of SSNA1 (NA14) as a novel 14 kDa coiled-coil nuclear autoantigen established the gene's existence and its predicted capacity for self-association through heptad repeats.","evidence":"cDNA library screening with autoimmune serum, immunoprecipitation, confocal immunofluorescence in HeLa and 3T3 cells","pmids":["9430706"],"confidence":"High","gaps":["Nuclear punctate localization later contested by KO-validated antibody studies","No functional role established","Binding partners unknown"]},{"year":2003,"claim":"Cross-species localization to basal bodies, centrosomes, and flagella, combined with antisense knockdown producing multinucleate cells, established that SSNA1/DIP13 is a microtubule-associated protein required for proper cell division.","evidence":"Immunofluorescence and antisense knockdown in Chlamydomonas, cross-reactivity with human NA14 at centrosomes and sperm basal bodies","pmids":["12640030"],"confidence":"High","gaps":["Mechanism of cell division failure unknown","Direct microtubule binding not demonstrated biochemically","Mammalian loss-of-function not yet performed"]},{"year":2004,"claim":"Discovery that SSNA1 physically interacts with the microtubule-severing enzyme spastin and that their binding domain overlaps with spastin's microtubule-interaction region revealed SSNA1 as a spatial regulator of spastin activity at centrosomes.","evidence":"Co-immunoprecipitation, subcellular fractionation, immunofluorescence, domain deletion analysis in dividing cells and motoneurons","pmids":["15269182"],"confidence":"High","gaps":["Whether SSNA1 activates or inhibits spastin severing not determined","In vitro reconstitution of SSNA1-spastin interaction absent","Relevance to hereditary spastic paraplegia not tested"]},{"year":2011,"claim":"NMR and biophysical characterization defined SSNA1's parallel coiled-coil architecture and identified Leu83/Leu93 as critical residues for spastin and microtubule interactions, providing the first structural framework for understanding SSNA1 oligomerization and partner binding.","evidence":"Circular dichroism, NMR spectroscopy, site-directed mutagenesis, urea/thermal denaturation of purified human NA14","pmids":["22008182"],"confidence":"Medium","gaps":["No high-resolution atomic structure obtained","Functional validation of Leu83/Leu93 mutations in cells not performed","Parallel coiled-coil assignment later revised to anti-parallel by cryo-EM"]},{"year":2014,"claim":"Endogenous localization to centrioles and functional studies demonstrating that SSNA1 knockdown impairs cytokinesis while overexpression promotes axon branching established SSNA1 as a centrosomal adaptor with dual roles in cell division and neuronal morphogenesis.","evidence":"Stable shRNA knockdown in HeLa, immunofluorescence of endogenous protein, neuronal overexpression in rat cortical cultures, time-lapse imaging","pmids":["25390646"],"confidence":"High","gaps":["Mechanism linking centriolar SSNA1 to cytokinesis not defined","Axon branching phenotype based on overexpression only","Relationship to spastin in neurons not directly tested"]},{"year":2021,"claim":"In vitro reconstitution revealed that SSNA1 directly modulates all parameters of microtubule dynamic instability, preferentially accumulates at lattice damage sites, and protects microtubules from spastin severing, establishing SSNA1 as a bona fide microtubule stabilizer and damage sensor.","evidence":"Purified protein reconstitution with TIRF microscopy, quantitative dynamic instability analysis, spastin severing assays","pmids":["34970964"],"confidence":"High","gaps":["Physiological relevance of damage sensing in cells not demonstrated","Whether damage-site binding is the primary in vivo function unclear","Structural basis for lattice recognition unknown"]},{"year":2024,"claim":"Cryo-EM structure at 4.55 Å resolution revealed that SSNA1 forms anti-parallel coiled-coils with C-terminal triple-stranded junctions that serve as microtubule-binding hubs, and genetic analysis showed that self-assembly is essential for embryonic viability and spindle bipolarity.","evidence":"Cryo-EM of C. elegans SSNA-1 fibrils, genetic knockout and self-assembly mutants, embryonic viability and spindle imaging in C. elegans","pmids":["40804232"],"confidence":"High","gaps":["Human SSNA1 structure not yet determined","How triple-stranded junctions engage the microtubule lattice at atomic resolution unknown","In vivo fibril formation in mammalian cells not confirmed"]},{"year":2025,"claim":"Genetic epistasis and super-resolution microscopy established that SSNA1 localizes downstream of SAS-1/C2CD3 at centrioles and the transition zone, placing SSNA1 within a hierarchical centriole integrity and ciliogenesis pathway.","evidence":"Null allele epistasis, U-Ex-STED super-resolution, heterologous human cell recruitment assay in C. elegans","pmids":["41124206"],"confidence":"High","gaps":["Direct physical interaction between SSNA1 and C2CD3 not biochemically demonstrated","Role of SSNA1 at the transition zone not mechanistically defined","Whether the C2CD3-SSNA1 axis is conserved in mammals requires further testing"]},{"year":2025,"claim":"SSNA1 was shown to increase microtubule rigidity, block lattice self-repair by preventing tubulin incorporation at damage sites, and selectively recognize unrepaired damage, refining the model of SSNA1 as a mechanical reinforcer that competes with lattice turnover.","evidence":"In vitro reconstitution with kinesin gliding assays, microfluidic force application, tubulin incorporation assays (preprint)","pmids":["41648615"],"confidence":"Medium","gaps":["Preprint; not yet peer-reviewed","Physiological consequence of blocked self-repair in cells untested","Whether rigidity increase requires fibril formation or monomeric binding not resolved"]},{"year":2025,"claim":"KO-validated antibody and expansion microscopy localized SSNA1 to a 9-fold symmetric ring in the distal centriolar lumen and identified a C2CD3-SSNA1-LRRCC1 targeting hierarchy required for CP110 removal and ciliogenesis, while challenging previous reports of nuclear and midbody localization.","evidence":"SSNA1 KO cells, KO-validated antibody, super-resolution/expansion microscopy, co-pelleting assays, CP110 removal analysis (preprint)","pmids":["bio_10.1101_2025.04.28.648957"],"confidence":"Medium","gaps":["Preprint; not yet peer-reviewed","Contradiction with in vitro microtubule binding data from peer-reviewed studies remains unresolved","Role of SSNA1 oligomerization versus monomeric form in centriolar lumen function not fully dissected"]},{"year":null,"claim":"Key unresolved questions include: what is the atomic-resolution structure of mammalian SSNA1 on microtubules, how does SSNA1 coordinate spastin severing versus damage protection in vivo, and what is the precise mechanism by which centriolar SSNA1 facilitates CP110 removal during ciliogenesis.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of SSNA1 bound to microtubules at atomic resolution","In vivo function of damage sensing not established","Molecular mechanism of CP110 removal by SSNA1 unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,2,8,9]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[8,9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,6]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1,2,6,9,10]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,8]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,6,9]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[10,9]}],"complexes":["distal centriolar lumen ring (C2CD3/SFI1/centrin-2/CEP135/SSNA1)"],"partners":["SPAST","C2CD3","LRRCC1","CP110","GPR175"],"other_free_text":[]},"mechanistic_narrative":"SSNA1 (NA14) is a small coiled-coil protein that self-assembles into fibrillar structures and functions as a microtubule-associated damage sensor, stabilizer, and centriolar scaffold component essential for cytokinesis, cilia assembly, and neuronal morphogenesis. SSNA1 forms anti-parallel coiled-coil fibrils through C-terminal triple-stranded junctions that create microtubule-interaction hubs; it cooperatively binds the microtubule lattice, preferentially accumulates at damage sites, slows catastrophe, promotes rescue, and protects microtubules from spastin-mediated severing [PMID:34970964, PMID:40804232]. SSNA1 localizes to the distal lumen of centrioles downstream of C2CD3/SAS-1 in a hierarchical targeting pathway, where it participates in a luminal ring scaffold and is required for CP110 removal during ciliogenesis [PMID:41124206, PMID:25390646]. Through its physical interaction with the microtubule-severing ATPase spastin, SSNA1 acts as a spatial adaptor at centrosomes and along neuronal microtubules, and its loss causes multipolar spindles, cytokinesis failure, and impaired axon branching [PMID:15269182, PMID:25390646, PMID:40804232]."},"prefetch_data":{"uniprot":{"accession":"O43805","full_name":"Microtubule nucleation factor SSNA1","aliases":["Nuclear autoantigen of 14 kDa","Sjoegren syndrome nuclear autoantigen 1"],"length_aa":119,"mass_kda":13.6,"function":"Microtubule-binding protein which stabilizes dynamic microtubules by slowing growth and shrinkage at both plus and minus ends and serves as a sensor of microtubule damage, protecting microtubules from the microtubule-severing enzyme SPAST (PubMed:34970964). Induces microtubule branching which is mediated by the formation of long SSNA1 fibrils which guide microtubule protofilaments to split apart from the mother microtubule and form daughter microtubules (By similarity). Plays a role in axon outgrowth and branching (PubMed:25390646). Required for cell division (PubMed:25390646)","subcellular_location":"Nucleus; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriole; Midbody; Cytoplasm, cytoskeleton, flagellum basal body; Cytoplasm, cytoskeleton, flagellum axoneme; Cell projection, axon","url":"https://www.uniprot.org/uniprotkb/O43805/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SSNA1","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SSNA1","total_profiled":1310},"omim":[{"mim_id":"616720","title":"MYASTHENIC SYNDROME, CONGENITAL, 19; CMS19","url":"https://www.omim.org/entry/616720"},{"mim_id":"610882","title":"SS NUCLEAR AUTOANTIGEN 1; SSNA1","url":"https://www.omim.org/entry/610882"},{"mim_id":"604277","title":"SPASTIN; SPAST","url":"https://www.omim.org/entry/604277"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Basal body","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SSNA1"},"hgnc":{"alias_symbol":["NA14","N14"],"prev_symbol":[]},"alphafold":{"accession":"O43805","domains":[{"cath_id":"1.20.5","chopping":"1-73","consensus_level":"medium","plddt":97.0499,"start":1,"end":73}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43805","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43805-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43805-F1-predicted_aligned_error_v6.png","plddt_mean":93.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SSNA1","jax_strain_url":"https://www.jax.org/strain/search?query=SSNA1"},"sequence":{"accession":"O43805","fasta_url":"https://rest.uniprot.org/uniprotkb/O43805.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43805/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43805"}},"corpus_meta":[{"pmid":"11518718","id":"PMC_11518718","title":"Murine notch homologs (N1-4) undergo 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Replication of the backcross test and molecular characterization of the N14 locus.","date":"1998","source":"Genetica","url":"https://pubmed.ncbi.nlm.nih.gov/9949700","citation_count":13,"is_preprint":false},{"pmid":"36940584","id":"PMC_36940584","title":"Production of acetone, butanol, and ethanol by electro-fermentation with Clostridium saccharoperbutylacetonicum N1-4.","date":"2023","source":"Bioelectrochemistry (Amsterdam, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/36940584","citation_count":12,"is_preprint":false},{"pmid":"22008182","id":"PMC_22008182","title":"Characterization of the structure and self-recognition of the human centrosomal protein NA14: implications for stability and function.","date":"2011","source":"Protein engineering, design & selection : PEDS","url":"https://pubmed.ncbi.nlm.nih.gov/22008182","citation_count":11,"is_preprint":false},{"pmid":"32103460","id":"PMC_32103460","title":"Investigation of secondary metabolism in the industrial butanol 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It localizes to numerous punctate structures scattered throughout the nucleus of HeLa and 3T3 cells.\",\n      \"method\": \"cDNA library screening with autoimmune serum, immunoprecipitation, recombinant protein expression, confocal immunofluorescence microscopy of transiently transfected cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (IP, IF, sequence analysis) in original discovery paper\",\n      \"pmids\": [\"9430706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The Chlamydomonas orthologue DIP13 and human NA14 localize to microtubule structures including basal bodies, flagellar axonemes, and cytoplasmic microtubules; anti-DIP13 antibody cross-reacts with human NA14 at centrosomes and basal bodies of human sperm. Antisense knockdown of DIP13 in Chlamydomonas produces multinucleate, multiflagellate cells, implicating DIP13/NA14 in proper cell division.\",\n      \"method\": \"Antibody generation, immunofluorescence microscopy, antisense knockdown in Chlamydomonas\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiments with functional loss-of-function phenotype, cross-species validation\",\n      \"pmids\": [\"12640030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Spastin physically interacts with the centrosomal protein NA14 (SSNA1), co-fractionates with the centrosomal marker gamma-tubulin, and is enriched at the spindle pole, central spindle, midbody, distal axon, and branching points. Deletion of the spastin region required for NA14 binding disrupts spastin's interaction with microtubules, suggesting NA14 targets spastin activity to the centrosome.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, immunofluorescence microscopy in dividing cells and immortalized motoneurons, domain deletion analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction confirmed by Co-IP and fractionation, functional domain deletion linking NA14 binding to microtubule interaction\",\n      \"pmids\": [\"15269182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"NA14 binds to the C-terminal region of the orphan receptor TPRA40/GPR175, identified by yeast two-hybrid and confirmed by GST pull-down and co-immunoprecipitation. NA14 mediates functional plasma membrane transport of TPRA40; an N-terminal deletion mutant of NA14 that cannot bind microtubules but retains TPRA40 binding inhibits TPRA40 translocation to the plasma membrane, impairing TPRA40's regulation of cell division in mouse embryos.\",\n      \"method\": \"Yeast two-hybrid screening, GST pull-down, co-immunoprecipitation, live-cell imaging, mouse embryo microinjection\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods (Y2H, GST-PD, Co-IP, functional embryo assay) but single lab\",\n      \"pmids\": [\"18459117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Human NA14 forms a parallel coiled-coil structure spanning residues 14-104 that mediates self-association; N- and C-termini lack preferred structure. Leu83 and Leu93 are proposed to mediate interactions among NA14, spastin, and microtubules. NA14 tends to oligomerize and form fibrils in solution.\",\n      \"method\": \"Circular dichroism, NMR spectroscopy, site-directed mutagenesis, urea/thermal denaturation\",\n      \"journal\": \"Protein engineering, design & selection : PEDS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — NMR-based structural characterization with mutagenesis, but single lab\",\n      \"pmids\": [\"22008182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The Trypanosoma brucei SSNA1 orthologue TbDIP13 self-assembles into fibril-like structures both in vitro and in vivo, partially co-localizes with acetylated alpha-tubulin, and deletion has little effect on parasite growth—suggesting possible functional redundancy. Comparative proteomics identified potential interacting partners.\",\n      \"method\": \"Electron microscopy of fibril structures, immunofluorescence co-localization with acetylated alpha-tubulin, gene deletion, comparative proteomics\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — ortholog in a divergent parasite; single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"22363749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Endogenous NA14 (SSNA1) localizes specifically to centrioles in HeLa cells and rat cortical neurons. Stable NA14 knockdown dramatically impairs cytokinesis. Overexpression of NA14 in neurons significantly increases axon outgrowth, branching, and neuronal differentiation. NA14 is proposed to act as an adaptor regulating spastin localization to centrosomes.\",\n      \"method\": \"Stable shRNA knockdown in HeLa cells, immunofluorescence of endogenous proteins, neuronal overexpression in rat cortical primary cultures, time-lapse imaging of cell division\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined cytokinesis phenotype, overexpression with neuronal phenotype, endogenous localization; multiple cell systems\",\n      \"pmids\": [\"25390646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The SSNA1/DIP13 homologue in Toxoplasma gondii localizes to the conoid of the apical complex in mature and dividing cells, and to the basal complex during cell division. The protein self-associates into higher-order structures both in vitro and in vivo, and overexpression impairs parasite division.\",\n      \"method\": \"Immunofluorescence with parasite-specific markers, in vitro self-assembly assays, overexpression phenotypic analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiments with functional overexpression phenotype, single lab\",\n      \"pmids\": [\"27324377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Human SSNA1 directly modulates all parameters of microtubule dynamic instability in vitro: it slows growth, shrinkage, and catastrophe rates, and promotes rescue. SSNA1 forms stretches along growing microtubule ends, binds cooperatively to the lattice, becomes enriched at microtubule damage sites (both naturally occurring and induced by spastin severing), and protects microtubules against spastin-mediated severing.\",\n      \"method\": \"In vitro reconstitution with purified proteins, TIRF microscopy, dynamic instability analysis, spastin severing assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — rigorous in vitro reconstitution with quantitative single-molecule TIRF microscopy, multiple mechanistic parameters measured\",\n      \"pmids\": [\"34970964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The cryo-EM structure of C. elegans SSNA-1 at 4.55 Å resolution reveals that SSNA1 forms an anti-parallel coiled-coil, with self-assembly driven by 16 C-terminal residue overhangs that dock on adjacent coiled-coils to form a triple-stranded helical junction. The microtubule-binding region maps to within this triple-stranded junction, indicating that self-assembly creates hubs for effective microtubule interaction. Deletion of SSNA-1 in C. elegans reduces embryonic viability and causes multipolar spindles; impairing self-assembly has a comparable effect on embryonic viability to knockout.\",\n      \"method\": \"Cryo-EM structure determination, genetic knockout and self-assembly mutants in C. elegans, embryonic viability assays, spindle imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure at near-atomic resolution combined with functional genetic analysis and mutagenesis of self-assembly\",\n      \"pmids\": [\"40804232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SAS-1 (C2CD3 homologue) is essential for SSNA-1 localization to centrioles during oogenesis and to the transition zone during ciliogenesis in C. elegans. In a heterologous human cell assay, SAS-1 recruits SSNA-1 to microtubules. Molecular epistasis with null alleles establishes that SSNA-1 is positioned downstream of SAS-1 in the centriole integrity pathway, and SSNA-1 localizes adjacent to the SAS-1 C-terminus within centriole architecture.\",\n      \"method\": \"Null allele generation, U-Ex-STED super-resolution microscopy, molecular epistasis experiments, heterologous human cell recruitment assay\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with null alleles, super-resolution localization, cross-species functional validation; multiple orthogonal methods\",\n      \"pmids\": [\"41124206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SSNA1 increases microtubule rigidity and resistance to breakage under force (kinesin-driven gliding and microfluidic flow assays). SSNA1 localization to damage sites prevents incorporation of new tubulin dimers, thereby inhibiting microtubule lattice self-repair. SSNA1 does not recognize damage sites that have been repaired by tubulin incorporation.\",\n      \"method\": \"In vitro reconstitution, TIRF microscopy, kinesin gliding assays, microfluidic force application, tubulin incorporation assays with purified proteins\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — rigorous in vitro reconstitution with multiple force assays, but preprint not yet peer-reviewed\",\n      \"pmids\": [\"41648615\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Using KO-validated antibodies and super-resolution/expansion microscopy, SSNA1 is shown to localize to the distal lumen of centrioles in a ring-like 9-fold symmetric configuration, separate from centriolar microtubules, and not in the nucleus, midbody, or ciliary axoneme as previously reported. SSNA1 KO impairs ciliogenesis by failing to facilitate CP110 removal. A C2CD3-SSNA1-LRRCC1 hierarchical targeting axis was identified in the distal lumen. Tag-free SSNA1 and microtubule co-pelleting assays indicate SSNA1 does not bind microtubules in vitro under these conditions.\",\n      \"method\": \"KO-validated antibody, super-resolution microscopy with expansion microscopy (ExM), SSNA1 KO and oligomerization-deficient mutants, CP110 removal assays, microtubule co-pelleting, interactor screening\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in single lab preprint; some findings (microtubule binding) contradict prior peer-reviewed reconstitution data, warranting caution\",\n      \"pmids\": [\"bio_10.1101_2025.04.28.648957\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"C2CD3 organizes the distal centriolar lumen and physically interacts with MNR to recruit the DISCO complex; its depletion destabilizes a luminal ring network composed of C2CD3/SFI1/centrin-2/CEP135/NA14 (SSNA1), placing SSNA1 as a component of the distal luminal ring scaffold downstream of C2CD3.\",\n      \"method\": \"Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, in situ cryo-electron tomography, protein interaction assays, depletion experiments\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — cryo-ET and U-ExM with functional depletion, but preprint; SSNA1 finding is secondary to C2CD3 analysis\",\n      \"pmids\": [\"bio_10.1101_2025.06.17.660204\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SSNA1/NA14 is a small coiled-coil protein that forms anti-parallel coiled-coil fibrils through C-terminal triple-stranded junctions, which create microtubule-interaction hubs; it stabilizes dynamic microtubules by slowing catastrophe and promoting rescue, acts as a damage sensor by preferentially binding to damaged lattice sites where it mechanically reinforces microtubules and blocks spastin-mediated severing and tubulin-mediated self-repair, localizes specifically to centrioles (distal lumen) and centrosomes in a pathway downstream of SAS-1/C2CD3, and is required for proper cytokinesis, cilia assembly (via CP110 removal), and axon outgrowth/branching—likely by acting as an adaptor that spatially and temporally regulates spastin's microtubule-severing activity at centrosomes.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SSNA1 (NA14) is a small coiled-coil protein that self-assembles into fibrillar structures and functions as a microtubule-associated damage sensor, stabilizer, and centriolar scaffold component essential for cytokinesis, cilia assembly, and neuronal morphogenesis. SSNA1 forms anti-parallel coiled-coil fibrils through C-terminal triple-stranded junctions that create microtubule-interaction hubs; it cooperatively binds the microtubule lattice, preferentially accumulates at damage sites, slows catastrophe, promotes rescue, and protects microtubules from spastin-mediated severing [PMID:34970964, PMID:40804232]. SSNA1 localizes to the distal lumen of centrioles downstream of C2CD3/SAS-1 in a hierarchical targeting pathway, where it participates in a luminal ring scaffold and is required for CP110 removal during ciliogenesis [PMID:41124206, PMID:25390646]. Through its physical interaction with the microtubule-severing ATPase spastin, SSNA1 acts as a spatial adaptor at centrosomes and along neuronal microtubules, and its loss causes multipolar spindles, cytokinesis failure, and impaired axon branching [PMID:15269182, PMID:25390646, PMID:40804232].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of SSNA1 (NA14) as a novel 14 kDa coiled-coil nuclear autoantigen established the gene's existence and its predicted capacity for self-association through heptad repeats.\",\n      \"evidence\": \"cDNA library screening with autoimmune serum, immunoprecipitation, confocal immunofluorescence in HeLa and 3T3 cells\",\n      \"pmids\": [\"9430706\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear punctate localization later contested by KO-validated antibody studies\", \"No functional role established\", \"Binding partners unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Cross-species localization to basal bodies, centrosomes, and flagella, combined with antisense knockdown producing multinucleate cells, established that SSNA1/DIP13 is a microtubule-associated protein required for proper cell division.\",\n      \"evidence\": \"Immunofluorescence and antisense knockdown in Chlamydomonas, cross-reactivity with human NA14 at centrosomes and sperm basal bodies\",\n      \"pmids\": [\"12640030\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of cell division failure unknown\", \"Direct microtubule binding not demonstrated biochemically\", \"Mammalian loss-of-function not yet performed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery that SSNA1 physically interacts with the microtubule-severing enzyme spastin and that their binding domain overlaps with spastin's microtubule-interaction region revealed SSNA1 as a spatial regulator of spastin activity at centrosomes.\",\n      \"evidence\": \"Co-immunoprecipitation, subcellular fractionation, immunofluorescence, domain deletion analysis in dividing cells and motoneurons\",\n      \"pmids\": [\"15269182\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SSNA1 activates or inhibits spastin severing not determined\", \"In vitro reconstitution of SSNA1-spastin interaction absent\", \"Relevance to hereditary spastic paraplegia not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"NMR and biophysical characterization defined SSNA1's parallel coiled-coil architecture and identified Leu83/Leu93 as critical residues for spastin and microtubule interactions, providing the first structural framework for understanding SSNA1 oligomerization and partner binding.\",\n      \"evidence\": \"Circular dichroism, NMR spectroscopy, site-directed mutagenesis, urea/thermal denaturation of purified human NA14\",\n      \"pmids\": [\"22008182\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution atomic structure obtained\", \"Functional validation of Leu83/Leu93 mutations in cells not performed\", \"Parallel coiled-coil assignment later revised to anti-parallel by cryo-EM\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Endogenous localization to centrioles and functional studies demonstrating that SSNA1 knockdown impairs cytokinesis while overexpression promotes axon branching established SSNA1 as a centrosomal adaptor with dual roles in cell division and neuronal morphogenesis.\",\n      \"evidence\": \"Stable shRNA knockdown in HeLa, immunofluorescence of endogenous protein, neuronal overexpression in rat cortical cultures, time-lapse imaging\",\n      \"pmids\": [\"25390646\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking centriolar SSNA1 to cytokinesis not defined\", \"Axon branching phenotype based on overexpression only\", \"Relationship to spastin in neurons not directly tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"In vitro reconstitution revealed that SSNA1 directly modulates all parameters of microtubule dynamic instability, preferentially accumulates at lattice damage sites, and protects microtubules from spastin severing, establishing SSNA1 as a bona fide microtubule stabilizer and damage sensor.\",\n      \"evidence\": \"Purified protein reconstitution with TIRF microscopy, quantitative dynamic instability analysis, spastin severing assays\",\n      \"pmids\": [\"34970964\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of damage sensing in cells not demonstrated\", \"Whether damage-site binding is the primary in vivo function unclear\", \"Structural basis for lattice recognition unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cryo-EM structure at 4.55 Å resolution revealed that SSNA1 forms anti-parallel coiled-coils with C-terminal triple-stranded junctions that serve as microtubule-binding hubs, and genetic analysis showed that self-assembly is essential for embryonic viability and spindle bipolarity.\",\n      \"evidence\": \"Cryo-EM of C. elegans SSNA-1 fibrils, genetic knockout and self-assembly mutants, embryonic viability and spindle imaging in C. elegans\",\n      \"pmids\": [\"40804232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human SSNA1 structure not yet determined\", \"How triple-stranded junctions engage the microtubule lattice at atomic resolution unknown\", \"In vivo fibril formation in mammalian cells not confirmed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Genetic epistasis and super-resolution microscopy established that SSNA1 localizes downstream of SAS-1/C2CD3 at centrioles and the transition zone, placing SSNA1 within a hierarchical centriole integrity and ciliogenesis pathway.\",\n      \"evidence\": \"Null allele epistasis, U-Ex-STED super-resolution, heterologous human cell recruitment assay in C. elegans\",\n      \"pmids\": [\"41124206\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical interaction between SSNA1 and C2CD3 not biochemically demonstrated\", \"Role of SSNA1 at the transition zone not mechanistically defined\", \"Whether the C2CD3-SSNA1 axis is conserved in mammals requires further testing\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"SSNA1 was shown to increase microtubule rigidity, block lattice self-repair by preventing tubulin incorporation at damage sites, and selectively recognize unrepaired damage, refining the model of SSNA1 as a mechanical reinforcer that competes with lattice turnover.\",\n      \"evidence\": \"In vitro reconstitution with kinesin gliding assays, microfluidic force application, tubulin incorporation assays (preprint)\",\n      \"pmids\": [\"41648615\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint; not yet peer-reviewed\", \"Physiological consequence of blocked self-repair in cells untested\", \"Whether rigidity increase requires fibril formation or monomeric binding not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"KO-validated antibody and expansion microscopy localized SSNA1 to a 9-fold symmetric ring in the distal centriolar lumen and identified a C2CD3-SSNA1-LRRCC1 targeting hierarchy required for CP110 removal and ciliogenesis, while challenging previous reports of nuclear and midbody localization.\",\n      \"evidence\": \"SSNA1 KO cells, KO-validated antibody, super-resolution/expansion microscopy, co-pelleting assays, CP110 removal analysis (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.04.28.648957\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint; not yet peer-reviewed\", \"Contradiction with in vitro microtubule binding data from peer-reviewed studies remains unresolved\", \"Role of SSNA1 oligomerization versus monomeric form in centriolar lumen function not fully dissected\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: what is the atomic-resolution structure of mammalian SSNA1 on microtubules, how does SSNA1 coordinate spastin severing versus damage protection in vivo, and what is the precise mechanism by which centriolar SSNA1 facilitates CP110 removal during ciliogenesis.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of SSNA1 bound to microtubules at atomic resolution\", \"In vivo function of damage sensing not established\", \"Molecular mechanism of CP110 removal by SSNA1 unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 2, 8, 9]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1, 2, 6, 9, 10]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 6, 9]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [10, 9]}\n    ],\n    \"complexes\": [\n      \"distal centriolar lumen ring (C2CD3/SFI1/centrin-2/CEP135/SSNA1)\"\n    ],\n    \"partners\": [\n      \"SPAST\",\n      \"C2CD3\",\n      \"LRRCC1\",\n      \"CP110\",\n      \"GPR175\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}