{"gene":"ACTL9","run_date":"2026-06-09T22:02:40","timeline":{"discoveries":[{"year":2022,"finding":"ACTL9 protein interacts with ACTRT1, ACTRT2, and ACTL7A to form a multimeric complex that localizes to the subacrosomal region of spermatids, where it is required for anchoring the acrosome to the nucleus.","method":"Co-immunoprecipitation, immunostaining, and genetic knockout mouse model","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal protein interaction demonstrated, localization confirmed by immunostaining, functional consequence validated by KO mouse model, replicated across multiple papers","pmids":["35616329"],"is_preprint":false},{"year":2022,"finding":"ACTL9 is a substrate of testis-specific serine kinase 3 (TSSK3); loss of TSSK3 results in significantly reduced phosphorylation of ACTL9 in testicular germ cells, while total ACTL9 protein levels remain unaltered.","method":"Phosphoproteomics of purified germ cells from Tssk3 knockout vs. heterozygous mice","journal":"Andrology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry-based phosphoproteomics with genetic KO model, single lab, no in vitro kinase assay confirming direct phosphorylation","pmids":["36306217"],"is_preprint":false},{"year":2024,"finding":"ACTL9 interacts with CABS1 (a perinuclear theca protein); a nonsense mutation in CABS1 leads to protein truncation and reduced interaction with ACTL9, accompanied by loss of acrosome and abnormal PLCζ localization in sperm.","method":"Co-immunoprecipitation of wild-type vs. mutant CABS1 with ACTL9, immunofluorescence staining, western blot","journal":"Journal of assisted reproduction and genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP with mutagenesis in a single lab study, interaction with ACTL9 established but downstream mechanism is correlational","pmids":["40407971"],"is_preprint":false},{"year":2024,"finding":"Homozygous mutations in ACTL9 cause irregular mitochondrial sheath arrangement and abnormal '9+2' axoneme structure in sperm flagella, reduced sperm motility, and male infertility; Actl9 knock-in mice recapitulate these flagellar ultrastructural defects.","method":"Whole-exome sequencing, CASA, scanning electron microscopy, transmission electron microscopy, immunostaining, and knock-in mouse model","journal":"Journal of assisted reproduction and genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (TEM, CASA, immunostaining) with in vivo validation in knock-in mice, single lab","pmids":["38963606"],"is_preprint":false},{"year":2024,"finding":"In the context of ACTRT1 deficiency (which disrupts the ACTRT1/ACTRT2/ACTL7A/ACTL9 complex), ACTL9 protein shows decreased expression and ectopic distribution in human sperm, consistent with its role in the subacrosomal complex required for acrosome-nucleus attachment.","method":"Western blotting and immunostaining of sperm from ACTRT1-deleted human patients","journal":"Human reproduction (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — protein localization and expression validated in human patient samples alongside mouse KO data, but ACTL9 is assessed as a downstream readout rather than the primary perturbation","pmids":["38414365"],"is_preprint":false},{"year":2024,"finding":"A missense variant in ACTL9 (c.376G>A, p.Glu126Lys) identified in an infertile male is predicted to alter protein secondary structure and is associated with total fertilization failure, suggesting this residue is functionally important.","method":"Sanger sequencing, segregation analysis, and bioinformatics structural prediction","journal":"Molecular genetics & genomic medicine","confidence":"Low","confidence_rationale":"Tier 4 / Weak — variant identified by sequencing with only computational structural prediction; no in vitro functional assay performed","pmids":["38769899"],"is_preprint":false}],"current_model":"ACTL9 is a sperm-specific actin-related protein that forms a multimeric complex with ACTRT1, ACTRT2, and ACTL7A in the subacrosomal (perinuclear theca) region of spermatids, where it is required for anchoring the acrosome to the nucleus and for proper sperm flagellar ultrastructure; it is phosphorylated by TSSK3 and interacts with the perinuclear theca protein CABS1, with loss-of-function mutations causing acrosomal detachment, flagellar structural defects, and male infertility/fertilization failure."},"narrative":{"mechanistic_narrative":"ACTL9 is a sperm-specific actin-related protein that builds the subacrosomal cytoskeletal architecture required for acrosome-nucleus attachment during spermiogenesis [PMID:35616329]. It assembles with ACTRT1, ACTRT2, and ACTL7A into a multimeric complex in the subacrosomal region of spermatids, and disruption of this complex—as occurs in ACTRT1 deficiency—reduces ACTL9 expression and causes its ectopic redistribution, mislocalizing the acrosome relative to the nucleus [PMID:35616329, PMID:38414365]. ACTL9 additionally interacts with the perinuclear theca protein CABS1, and a truncating CABS1 mutation that weakens this interaction is accompanied by acrosome loss and aberrant PLCζ localization in sperm [PMID:40407971]. ACTL9 is a phosphorylation substrate of the testis-specific kinase TSSK3 [PMID:36306217]. Beyond its role in acrosome anchoring, ACTL9 is required for normal sperm flagellar ultrastructure: homozygous mutations cause irregular mitochondrial sheath arrangement and '9+2' axoneme defects with reduced motility, recapitulated in knock-in mice, and loss-of-function and missense variants are causative for male infertility and total fertilization failure [PMID:38963606, PMID:38769899].","teleology":[{"year":2022,"claim":"Established that ACTL9 is a structural subunit of a sperm-specific actin-related complex, answering how the acrosome is physically anchored to the spermatid nucleus.","evidence":"Co-immunoprecipitation, immunostaining, and knockout mouse model in spermatids","pmids":["35616329"],"confidence":"High","gaps":["Stoichiometry and spatial arrangement of the ACTL9/ACTRT1/ACTRT2/ACTL7A complex not resolved","No structural model of how the complex bridges acrosome to nuclear envelope"]},{"year":2022,"claim":"Identified ACTL9 as a phosphorylation target of TSSK3, linking the complex to a testis-specific kinase signaling axis.","evidence":"Phosphoproteomics of purified germ cells from Tssk3 knockout vs. heterozygous mice","pmids":["36306217"],"confidence":"Medium","gaps":["No in vitro kinase assay confirming direct phosphorylation","Functional consequence of ACTL9 phosphorylation on complex assembly or localization unknown","Phosphosite(s) not mapped"]},{"year":2024,"claim":"Extended the ACTL9 interaction network to the perinuclear theca protein CABS1 and connected this interaction to acrosome integrity and PLCζ positioning relevant to fertilization.","evidence":"Co-IP of wild-type vs. mutant CABS1 with ACTL9, immunofluorescence, western blot","pmids":["40407971"],"confidence":"Medium","gaps":["Causal link between reduced ACTL9-CABS1 interaction and PLCζ mislocalization is correlational","Direct binding interface not defined"]},{"year":2024,"claim":"Demonstrated that ACTL9 function extends beyond the acrosome to flagellar ultrastructure, with mutations producing axoneme and mitochondrial sheath defects and infertility.","evidence":"Whole-exome sequencing, CASA, SEM, TEM, immunostaining, and knock-in mouse model","pmids":["38963606"],"confidence":"Medium","gaps":["Mechanism by which a subacrosomal protein influences distal flagellar architecture not established","Single lab; partner dependence in the flagellum not tested"]},{"year":2024,"claim":"Confirmed ACTL9 as a downstream readout of complex integrity by showing its decreased and ectopic distribution in human ACTRT1-deficient sperm.","evidence":"Western blotting and immunostaining of sperm from ACTRT1-deleted human patients","pmids":["38414365"],"confidence":"Medium","gaps":["ACTL9 assessed as secondary readout rather than primary perturbation","Does not establish ACTL9-intrinsic requirement independent of complex"]},{"year":2024,"claim":"Implicated a specific ACTL9 residue (p.Glu126Lys) in fertilization competence, narrowing functionally important regions.","evidence":"Sanger sequencing, segregation analysis, and bioinformatics structural prediction in an infertile male","pmids":["38769899"],"confidence":"Low","gaps":["No in vitro or in vivo functional assay; effect inferred only from computational structural prediction","Single patient association without mechanistic validation"]},{"year":null,"claim":"How ACTL9 phosphorylation by TSSK3 and its CABS1 interaction are mechanistically coordinated to control acrosome anchoring versus flagellar assembly remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural or biochemical reconstitution of the subacrosomal complex","Direct enzyme-substrate biochemistry for TSSK3-ACTL9 absent","Causal pathway from ACTL9 to flagellar mitochondrial sheath/axoneme not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,4]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0,3]}],"complexes":["ACTL9/ACTRT1/ACTRT2/ACTL7A subacrosomal complex"],"partners":["ACTRT1","ACTRT2","ACTL7A","TSSK3","CABS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TC94","full_name":"Actin-like protein 9","aliases":[],"length_aa":416,"mass_kda":45.5,"function":"Testis-specic protein that plays an important role in fusion of proacrosomal vesicles and perinuclear theca formation","subcellular_location":"Cytoplasmic vesicle, secretory vesicle, acrosome; Cytoplasm, cytoskeleton, perinuclear theca","url":"https://www.uniprot.org/uniprotkb/Q8TC94/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ACTL9","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ACTL9","total_profiled":1310},"omim":[{"mim_id":"619258","title":"SPERMATOGENIC FAILURE 53; SPGF53","url":"https://www.omim.org/entry/619258"},{"mim_id":"619251","title":"ACTIN-LIKE 9; ACTL9","url":"https://www.omim.org/entry/619251"},{"mim_id":"604303","title":"ACTIN-LIKE 7A; ACTL7A","url":"https://www.omim.org/entry/604303"},{"mim_id":"300487","title":"ACTIN-RELATED PROTEIN T1; ACTRT1","url":"https://www.omim.org/entry/300487"},{"mim_id":"258150","title":"SPERMATOGENIC FAILURE 1; SPGF1","url":"https://www.omim.org/entry/258150"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Acrosome","reliability":"Supported"},{"location":"Equatorial segment","reliability":"Supported"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"testis","ntpm":108.2}],"url":"https://www.proteinatlas.org/search/ACTL9"},"hgnc":{"alias_symbol":["MGC33407"],"prev_symbol":[]},"alphafold":{"accession":"Q8TC94","domains":[{"cath_id":"3.30.420.40","chopping":"51-182_380-412","consensus_level":"medium","plddt":91.4415,"start":51,"end":412},{"cath_id":"3.90.640.10","chopping":"224-299","consensus_level":"high","plddt":91.3493,"start":224,"end":299}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TC94","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TC94-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TC94-F1-predicted_aligned_error_v6.png","plddt_mean":87.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ACTL9","jax_strain_url":"https://www.jax.org/strain/search?query=ACTL9"},"sequence":{"accession":"Q8TC94","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TC94.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TC94/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TC94"}},"corpus_meta":[{"pmid":"37758324","id":"PMC_37758324","title":"Genetic mechanisms of fertilization failure and early embryonic arrest: a comprehensive review.","date":"2024","source":"Human reproduction update","url":"https://pubmed.ncbi.nlm.nih.gov/37758324","citation_count":50,"is_preprint":false},{"pmid":"35616329","id":"PMC_35616329","title":"Loss of perinuclear theca ACTRT1 causes acrosome detachment and severe male subfertility in mice.","date":"2022","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/35616329","citation_count":29,"is_preprint":false},{"pmid":"36306217","id":"PMC_36306217","title":"Testis-specific serine kinase 3 is required for sperm morphogenesis and male fertility.","date":"2022","source":"Andrology","url":"https://pubmed.ncbi.nlm.nih.gov/36306217","citation_count":27,"is_preprint":false},{"pmid":"36589837","id":"PMC_36589837","title":"Gene mutations associated with fertilization failure after in vitro fertilization/intracytoplasmic sperm injection.","date":"2022","source":"Frontiers in endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/36589837","citation_count":20,"is_preprint":false},{"pmid":"23278845","id":"PMC_23278845","title":"Association of KIF3A, but not OVOL1 and ACTL9, with atopic eczema in Italian patients.","date":"2013","source":"The British journal of dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/23278845","citation_count":17,"is_preprint":false},{"pmid":"33707600","id":"PMC_33707600","title":"Establishment and genomic characterization of a sporadic malignant peripheral nerve sheath tumor cell line.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33707600","citation_count":15,"is_preprint":false},{"pmid":"38414365","id":"PMC_38414365","title":"Deletion of ACTRT1 is associated with male infertility as sperm acrosomal ultrastructural defects and fertilization failure in human.","date":"2024","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/38414365","citation_count":11,"is_preprint":false},{"pmid":"37118964","id":"PMC_37118964","title":"Differential Proteomic Analysis of Human Sperm: A Systematic Review to Identify Candidate Targets to Monitor Sperm Quality.","date":"2023","source":"The world journal of men's health","url":"https://pubmed.ncbi.nlm.nih.gov/37118964","citation_count":8,"is_preprint":false},{"pmid":"37212058","id":"PMC_37212058","title":"Exonic genetic variants associated with unexpected fertilization failure and zygotic arrest after ICSI: a systematic review.","date":"2023","source":"Zygote (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/37212058","citation_count":7,"is_preprint":false},{"pmid":"38769899","id":"PMC_38769899","title":"Identification of a new mutation in the ACTL9 gene in men with unexplained infertility.","date":"2024","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38769899","citation_count":2,"is_preprint":false},{"pmid":"38963606","id":"PMC_38963606","title":"Homozygous ACTL9 mutations cause irregular mitochondrial sheath arrangement and abnormal flagellum assembly in spermatozoa and male infertility.","date":"2024","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/38963606","citation_count":2,"is_preprint":false},{"pmid":"40407971","id":"PMC_40407971","title":"Identification of nonfunctional CABS1 causing fertilization failure and male infertility in humans: a case report.","date":"2025","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40407971","citation_count":1,"is_preprint":false},{"pmid":"40376536","id":"PMC_40376536","title":"Establishment and clinical significance of genetic factor screening method for patients with nonobstructive azoospermia based on whole exon sequencing technology.","date":"2025","source":"Translational andrology and urology","url":"https://pubmed.ncbi.nlm.nih.gov/40376536","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9458,"output_tokens":1436,"usd":0.024957,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8131,"output_tokens":2330,"usd":0.049453,"stage2_stop_reason":"end_turn"},"total_usd":0.07441,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"ACTL9 protein interacts with ACTRT1, ACTRT2, and ACTL7A to form a multimeric complex that localizes to the subacrosomal region of spermatids, where it is required for anchoring the acrosome to the nucleus.\",\n      \"method\": \"Co-immunoprecipitation, immunostaining, and genetic knockout mouse model\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal protein interaction demonstrated, localization confirmed by immunostaining, functional consequence validated by KO mouse model, replicated across multiple papers\",\n      \"pmids\": [\"35616329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ACTL9 is a substrate of testis-specific serine kinase 3 (TSSK3); loss of TSSK3 results in significantly reduced phosphorylation of ACTL9 in testicular germ cells, while total ACTL9 protein levels remain unaltered.\",\n      \"method\": \"Phosphoproteomics of purified germ cells from Tssk3 knockout vs. heterozygous mice\",\n      \"journal\": \"Andrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry-based phosphoproteomics with genetic KO model, single lab, no in vitro kinase assay confirming direct phosphorylation\",\n      \"pmids\": [\"36306217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ACTL9 interacts with CABS1 (a perinuclear theca protein); a nonsense mutation in CABS1 leads to protein truncation and reduced interaction with ACTL9, accompanied by loss of acrosome and abnormal PLCζ localization in sperm.\",\n      \"method\": \"Co-immunoprecipitation of wild-type vs. mutant CABS1 with ACTL9, immunofluorescence staining, western blot\",\n      \"journal\": \"Journal of assisted reproduction and genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP with mutagenesis in a single lab study, interaction with ACTL9 established but downstream mechanism is correlational\",\n      \"pmids\": [\"40407971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Homozygous mutations in ACTL9 cause irregular mitochondrial sheath arrangement and abnormal '9+2' axoneme structure in sperm flagella, reduced sperm motility, and male infertility; Actl9 knock-in mice recapitulate these flagellar ultrastructural defects.\",\n      \"method\": \"Whole-exome sequencing, CASA, scanning electron microscopy, transmission electron microscopy, immunostaining, and knock-in mouse model\",\n      \"journal\": \"Journal of assisted reproduction and genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (TEM, CASA, immunostaining) with in vivo validation in knock-in mice, single lab\",\n      \"pmids\": [\"38963606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In the context of ACTRT1 deficiency (which disrupts the ACTRT1/ACTRT2/ACTL7A/ACTL9 complex), ACTL9 protein shows decreased expression and ectopic distribution in human sperm, consistent with its role in the subacrosomal complex required for acrosome-nucleus attachment.\",\n      \"method\": \"Western blotting and immunostaining of sperm from ACTRT1-deleted human patients\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — protein localization and expression validated in human patient samples alongside mouse KO data, but ACTL9 is assessed as a downstream readout rather than the primary perturbation\",\n      \"pmids\": [\"38414365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A missense variant in ACTL9 (c.376G>A, p.Glu126Lys) identified in an infertile male is predicted to alter protein secondary structure and is associated with total fertilization failure, suggesting this residue is functionally important.\",\n      \"method\": \"Sanger sequencing, segregation analysis, and bioinformatics structural prediction\",\n      \"journal\": \"Molecular genetics & genomic medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — variant identified by sequencing with only computational structural prediction; no in vitro functional assay performed\",\n      \"pmids\": [\"38769899\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACTL9 is a sperm-specific actin-related protein that forms a multimeric complex with ACTRT1, ACTRT2, and ACTL7A in the subacrosomal (perinuclear theca) region of spermatids, where it is required for anchoring the acrosome to the nucleus and for proper sperm flagellar ultrastructure; it is phosphorylated by TSSK3 and interacts with the perinuclear theca protein CABS1, with loss-of-function mutations causing acrosomal detachment, flagellar structural defects, and male infertility/fertilization failure.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ACTL9 is a sperm-specific actin-related protein that builds the subacrosomal cytoskeletal architecture required for acrosome-nucleus attachment during spermiogenesis [#0]. It assembles with ACTRT1, ACTRT2, and ACTL7A into a multimeric complex in the subacrosomal region of spermatids, and disruption of this complex—as occurs in ACTRT1 deficiency—reduces ACTL9 expression and causes its ectopic redistribution, mislocalizing the acrosome relative to the nucleus [#0, #4]. ACTL9 additionally interacts with the perinuclear theca protein CABS1, and a truncating CABS1 mutation that weakens this interaction is accompanied by acrosome loss and aberrant PLCζ localization in sperm [#2]. ACTL9 is a phosphorylation substrate of the testis-specific kinase TSSK3 [#1]. Beyond its role in acrosome anchoring, ACTL9 is required for normal sperm flagellar ultrastructure: homozygous mutations cause irregular mitochondrial sheath arrangement and '9+2' axoneme defects with reduced motility, recapitulated in knock-in mice, and loss-of-function and missense variants are causative for male infertility and total fertilization failure [#3, #5].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2022,\n      \"claim\": \"Established that ACTL9 is a structural subunit of a sperm-specific actin-related complex, answering how the acrosome is physically anchored to the spermatid nucleus.\",\n      \"evidence\": \"Co-immunoprecipitation, immunostaining, and knockout mouse model in spermatids\",\n      \"pmids\": [\"35616329\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Stoichiometry and spatial arrangement of the ACTL9/ACTRT1/ACTRT2/ACTL7A complex not resolved\",\n        \"No structural model of how the complex bridges acrosome to nuclear envelope\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified ACTL9 as a phosphorylation target of TSSK3, linking the complex to a testis-specific kinase signaling axis.\",\n      \"evidence\": \"Phosphoproteomics of purified germ cells from Tssk3 knockout vs. heterozygous mice\",\n      \"pmids\": [\"36306217\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No in vitro kinase assay confirming direct phosphorylation\",\n        \"Functional consequence of ACTL9 phosphorylation on complex assembly or localization unknown\",\n        \"Phosphosite(s) not mapped\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the ACTL9 interaction network to the perinuclear theca protein CABS1 and connected this interaction to acrosome integrity and PLCζ positioning relevant to fertilization.\",\n      \"evidence\": \"Co-IP of wild-type vs. mutant CABS1 with ACTL9, immunofluorescence, western blot\",\n      \"pmids\": [\"40407971\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Causal link between reduced ACTL9-CABS1 interaction and PLCζ mislocalization is correlational\",\n        \"Direct binding interface not defined\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated that ACTL9 function extends beyond the acrosome to flagellar ultrastructure, with mutations producing axoneme and mitochondrial sheath defects and infertility.\",\n      \"evidence\": \"Whole-exome sequencing, CASA, SEM, TEM, immunostaining, and knock-in mouse model\",\n      \"pmids\": [\"38963606\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which a subacrosomal protein influences distal flagellar architecture not established\",\n        \"Single lab; partner dependence in the flagellum not tested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Confirmed ACTL9 as a downstream readout of complex integrity by showing its decreased and ectopic distribution in human ACTRT1-deficient sperm.\",\n      \"evidence\": \"Western blotting and immunostaining of sperm from ACTRT1-deleted human patients\",\n      \"pmids\": [\"38414365\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"ACTL9 assessed as secondary readout rather than primary perturbation\",\n        \"Does not establish ACTL9-intrinsic requirement independent of complex\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Implicated a specific ACTL9 residue (p.Glu126Lys) in fertilization competence, narrowing functionally important regions.\",\n      \"evidence\": \"Sanger sequencing, segregation analysis, and bioinformatics structural prediction in an infertile male\",\n      \"pmids\": [\"38769899\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No in vitro or in vivo functional assay; effect inferred only from computational structural prediction\",\n        \"Single patient association without mechanistic validation\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ACTL9 phosphorylation by TSSK3 and its CABS1 interaction are mechanistically coordinated to control acrosome anchoring versus flagellar assembly remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural or biochemical reconstitution of the subacrosomal complex\",\n        \"Direct enzyme-substrate biochemistry for TSSK3-ACTL9 absent\",\n        \"Causal pathway from ACTL9 to flagellar mitochondrial sheath/axoneme not defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"complexes\": [\"ACTL9/ACTRT1/ACTRT2/ACTL7A subacrosomal complex\"],\n    \"partners\": [\"ACTRT1\", \"ACTRT2\", \"ACTL7A\", \"TSSK3\", \"CABS1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}