{"gene":"CDC42EP1","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1995,"finding":"MSE55 (CDC42EP1) contains a CRIB (Cdc42/Rac interactive binding) motif and binds to the GTP-bound form of Cdc42 but not to RhoA, as demonstrated by filter binding assay. Binding to Rac was weaker than to Cdc42.","method":"Filter binding assay with GST-fused GTPases","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay with GTP-dependence confirmed, single lab but well-controlled with multiple GTPases tested","pmids":["7493928"],"is_preprint":false},{"year":1998,"finding":"MSE55 (CDC42EP1) interacts with TC10 GTPase in addition to Cdc42, placing it in the effector set shared between TC10 and Cdc42.","method":"Interaction assay (yeast two-hybrid and/or pulldown)","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single interaction assay, confirmed across two GTPases in same study","pmids":["9799731"],"is_preprint":false},{"year":1999,"finding":"MSE55 was renamed Borg5; Borg5 binds both TC10 and Cdc42 in a GTP-dependent manner, requiring an intact CRIB domain, with no detectable interaction with Rac1 or RhoA.","method":"Yeast two-hybrid screen; GTPase binding assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two-hybrid plus binding assay, CRIB domain requirement established, single lab","pmids":["10490598"],"is_preprint":false},{"year":1999,"finding":"MSE55/Borg5 (CDC42EP1) binds Cdc42 in a GTP-dependent manner requiring an intact CRIB domain; a CRIB domain mutant fails to bind Cdc42. In Cos-7 cells, wild-type MSE55 localizes to membrane ruffles and increases membrane actin polymerization, whereas the CRIB mutant does not. In NIH 3T3 cells, MSE55 induces long actin-based protrusions that are blocked by dominant-negative Cdc42 (N17Cdc42) but not by dominant-negative Rac (N17Rac), establishing MSE55 as a Cdc42 effector mediating actin cytoskeleton reorganization.","method":"GST-capture pulldown; site-directed mutagenesis of CRIB domain; dominant-negative co-expression; immunofluorescence; live-cell video microscopy","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (pulldown, mutagenesis, dominant-negative epistasis, live imaging) in single study establishing mechanism","pmids":["10430899"],"is_preprint":false},{"year":2001,"finding":"CDC42EP1/MSE55 (as CEP5 in this study) binds Cdc42 and, along with other CEP family members, induces pseudopodia formation in NIH 3T3 fibroblasts downstream of Cdc42; a CRIB domain mutant of CEP2 (the closest tested paralog) fails to induce pseudopodia, and dominant-negative Cdc42 blocks CEP-induced pseudopodia, placing CEPs downstream of Cdc42 in actin remodeling.","method":"GST pulldown; dominant-negative co-expression; CRIB domain mutagenesis; immunofluorescence of actin","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via dominant-negative, mutagenesis, multiple cell types; findings on MSE55/CEP5 partially inferred from CEP2 mutagenesis within same family","pmids":["11035016"],"is_preprint":false},{"year":2010,"finding":"Borg5 (CDC42EP1) interacts with both Cdc42 and atypical protein kinase C (aPKC), functioning downstream of Cdc42 to enhance trophectoderm (TE) cell motility during ESC differentiation. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation, and Borg5 is required for sorting of differentiating TE cells to the outside of ESC aggregates.","method":"Co-immunoprecipitation; morpholino knockdown; live-cell imaging of ESC differentiation; immunofluorescence of aPKC localization","journal":"Stem cells (Dayton, Ohio)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus loss-of-function with defined cellular phenotypes, single lab","pmids":["20506138"],"is_preprint":false},{"year":2015,"finding":"CDC42EP4 (a close family member) forms complexes with septin hetero-oligomers that interact with the glutamate transporter GLAST/EAAT1; the Borg/CDC42EP proteins interact reciprocally with CDC42 or the septin cytoskeleton. This establishes the paradigm that CDC42EP proteins interact with septins to scaffold membrane proteins.","method":"Co-immunoprecipitation; knockout mouse phenotype; immunofluorescence","journal":"Nature communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — mechanistic detail is for CDC42EP4 paralog; CDC42EP1/Borg5 is mentioned as part of family context but not the subject of direct experiments in this paper","pmids":["26657011"],"is_preprint":false},{"year":2018,"finding":"Cdc42ep1 is required for neural crest cell migration in frog embryos; knockdown causes rounder cell shapes, membrane blebbing, disrupted actin organization and focal adhesion alignment, and loss of directional traction forces. Cdc42ep1 localizes to two subcellular compartments: membrane protrusions (together with Cdc42) and perinuclear patches (where Cdc42 is absent). Cdc42 directly interacts with Cdc42ep1 through the CRIB domain, and changes in Cdc42 levels shift the distribution of Cdc42ep1 between these locations, controlling membrane protrusion formation and migration directionality.","method":"Morpholino-mediated knockdown; co-immunoprecipitation; live-cell imaging; immunofluorescence of actin and focal adhesions; traction force microscopy","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with multiple orthogonal phenotypic readouts, direct interaction confirmed, subcellular localization linked to functional consequences, single lab with rigorous methods","pmids":["29040749"],"is_preprint":false},{"year":2023,"finding":"In neural crest cells, Cdc42ep1 colocalizes with septin filaments at the cell center and interacts with them in a reciprocal manner: septin filaments recruit Cdc42ep1 to the cell center, and Cdc42ep1 supports the formation of septin filaments. Loss of septin filaments impairs stability and contractility of actin stress fibers and alters their orientation; these septin activities depend on Cdc42ep1.","method":"Morpholino-mediated knockdown of septins and Cdc42ep1; co-localization by fluorescence imaging; rescue experiments; live-cell imaging of actin dynamics","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function epistasis between Cdc42ep1 and septins with defined actin phenotype, single lab, multiple imaging methods","pmids":["36923257"],"is_preprint":false},{"year":2023,"finding":"CDC42EP1 associates with SEPTIN-7 and Villin in human enterocytes. Upon Salmonella Typhimurium infection, CDC42EP1 is rapidly redistributed and aggregates around invading bacteria in a manner dependent on host CDC42 and bacterial activation of CDC42. CDC42EP1 is not required for initial bacterial entry but associates with Salmonella-containing vacuoles during long-term infection, contributing to intracellular bacterial growth/replication.","method":"Proteomic analysis of CDC42 interactome; co-localization by immunofluorescence; siRNA knockdown; bacterial infection assays","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic identification of binding partners confirmed by localization and functional knockdown, single lab","pmids":["37877586"],"is_preprint":false},{"year":2024,"finding":"In epithelial MDCK cells, Borg5/CDC42EP1 limits actomyosin contractility, cell-cell adhesion tension, and motility. Borg5 depletion inhibits lateral F-actin cortex development and stimulates radial stress fibers and microtubule-dependent leading-edge lamellae. Borg5 limits colocalization of septin proteins with microtubules. Borg5 physically interacts with the rod domain of myosin IIA (MYH9 heavy chain), and this interaction is reduced in the presence of septins, suggesting Borg5 counteracts septin-associated myosin activation to restrict contractility.","method":"siRNA depletion; co-immunoprecipitation; immunofluorescence; live-cell imaging; traction force/adhesion tension measurements","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple phenotypic readouts plus Co-IP identifying myosin IIA interaction, single lab, two orthogonal methods","pmids":["39503295"],"is_preprint":false}],"current_model":"CDC42EP1/MSE55/Borg5 is a CRIB-domain-containing effector that binds activated (GTP-bound) Cdc42 and TC10 but not Rac1 or RhoA, and acts downstream of Cdc42 to reorganize the actin cytoskeleton; it also interacts reciprocally with septin filaments (and the myosin IIA rod domain) to regulate actin stress fiber stability, contractility, and directed cell migration in contexts including neural crest cells, trophectoderm differentiation, and epithelial morphogenesis."},"narrative":{"mechanistic_narrative":"CDC42EP1 (MSE55/Borg5) is a CRIB-domain effector that links the Rho-family GTPases Cdc42 and TC10 to the actin and septin cytoskeletons, thereby controlling cell shape, contractility, and directed migration [PMID:10430899, PMID:29040749]. It binds the GTP-bound forms of Cdc42 and TC10—but not Rac1 or RhoA—through an intact CRIB domain, and a CRIB mutant abolishes both Cdc42 binding and downstream activity [PMID:7493928, PMID:10490598, PMID:10430899]. Acting downstream of Cdc42, CDC42EP1 localizes to membrane ruffles and protrusions where it drives actin polymerization and pseudopodia/protrusion formation in a manner blocked by dominant-negative Cdc42 [PMID:10430899, PMID:11035016]. CDC42EP1 also partitions to perinuclear and cell-center compartments where it reciprocally interacts with septin filaments: septins recruit CDC42EP1 to the cell center while CDC42EP1 supports septin filament assembly, and this interplay governs actin stress fiber stability, orientation, and contractility [PMID:29040749, PMID:36923257]. Through a physical interaction with the rod domain of myosin IIA (MYH9) that is antagonized by septins, CDC42EP1 restrains actomyosin contractility, cell-cell adhesion tension, and leading-edge dynamics in epithelial cells [PMID:39503295]. These activities place CDC42EP1 in developmental and morphogenetic programs including neural crest cell migration and trophectoderm differentiation, the latter via interaction with atypical PKC [PMID:20506138, PMID:29040749].","teleology":[{"year":1995,"claim":"Established CDC42EP1 as a GTPase effector by showing it carries a CRIB motif and binds GTP-loaded Cdc42 selectively over RhoA, defining its specificity from the outset.","evidence":"Filter binding assay with GST-fused GTPases","pmids":["7493928"],"confidence":"Medium","gaps":["Weaker Rac binding left selectivity incompletely resolved","No functional consequence of binding tested","Single in vitro binding format"]},{"year":1998,"claim":"Broadened the GTPase specificity by placing CDC42EP1 in the shared effector set of TC10 in addition to Cdc42.","evidence":"Yeast two-hybrid and/or pulldown interaction assay","pmids":["9799731"],"confidence":"Medium","gaps":["GTP-dependence of TC10 binding not yet established here","No cellular function attributed to TC10 binding"]},{"year":1999,"claim":"Defined the molecular requirements and consequences of effector binding: GTP- and CRIB-dependent engagement of Cdc42/TC10 drives membrane ruffle localization, actin polymerization, and Cdc42-dependent protrusions, formally establishing CDC42EP1 as a Cdc42 effector for actin remodeling.","evidence":"Yeast two-hybrid, GST pulldown, CRIB mutagenesis, dominant-negative epistasis, immunofluorescence and live imaging in Cos-7/NIH 3T3 cells","pmids":["10490598","10430899"],"confidence":"High","gaps":["Direct biochemical link between binding and actin nucleation machinery unresolved","No endogenous loss-of-function data"]},{"year":2001,"claim":"Generalized the actin-remodeling activity across the CEP/Borg family, confirming CRIB- and Cdc42-dependent pseudopodia induction.","evidence":"GST pulldown, CRIB mutagenesis, dominant-negative co-expression, actin immunofluorescence in NIH 3T3","pmids":["11035016"],"confidence":"Medium","gaps":["CDC42EP1-specific CRIB requirement inferred from paralog CEP2 mutagenesis","Effector output mechanism still phenomenological"]},{"year":2010,"claim":"Connected CDC42EP1 to a developmental program by showing it acts downstream of Cdc42 with aPKC to drive trophectoderm motility and sorting during blastocyst formation.","evidence":"Co-immunoprecipitation, morpholino knockdown, live-cell imaging and aPKC immunofluorescence in differentiating ESCs","pmids":["20506138"],"confidence":"Medium","gaps":["Mechanism linking Borg5 to aPKC localization unresolved","Single loss-of-function approach"]},{"year":2018,"claim":"Resolved how CDC42EP1 controls migration directionality by linking its dual localization—Cdc42-positive protrusions versus Cdc42-absent perinuclear patches—to traction force generation and membrane protrusion in neural crest cells.","evidence":"Morpholino knockdown, Co-IP, live imaging, focal adhesion immunofluorescence, traction force microscopy in frog embryos","pmids":["29040749"],"confidence":"High","gaps":["Identity of factors retaining CDC42EP1 at perinuclear patches unknown","Cross-species generality to mammalian migration not tested"]},{"year":2023,"claim":"Established a reciprocal CDC42EP1–septin module that controls actin stress fiber stability, orientation, and contractility, and showed CDC42EP1 also scaffolds septins around invading Salmonella in a Cdc42-dependent manner relevant to intracellular bacterial growth.","evidence":"Morpholino knockdown and rescue with live imaging in neural crest cells; CDC42 interactome proteomics, siRNA, and infection assays in enterocytes","pmids":["36923257","37877586"],"confidence":"Medium","gaps":["Molecular basis of reciprocal septin recruitment not defined","Whether SEPTIN-7 binding is direct unresolved","Role at Salmonella-containing vacuoles mechanistically uncharacterized"]},{"year":2024,"claim":"Identified a contractility-limiting mechanism: CDC42EP1 binds the myosin IIA rod domain, an interaction antagonized by septins, allowing it to restrain actomyosin contractility, adhesion tension, and leading-edge dynamics in epithelia.","evidence":"siRNA depletion, Co-IP, immunofluorescence, live imaging, traction/adhesion tension measurements in MDCK cells","pmids":["39503295"],"confidence":"Medium","gaps":["Direct vs indirect MYH9 rod-domain binding not separated from septin competition","Structural basis of the septin-myosin switch unknown"]},{"year":null,"claim":"How CDC42EP1 mechanistically integrates Cdc42 GTPase input, septin assembly, and myosin IIA regulation into a single switch governing protrusion versus contractility remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the CRIB–GTPase or rod-domain interfaces","Direct vs scaffolded nature of septin and myosin interactions undefined","Endogenous mammalian loss-of-function phenotypes limited"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2,3]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[3,8,10]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[8,9,10]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,7]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8,10]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,7]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,7]}],"complexes":[],"partners":["CDC42","TC10","MYH9","SEPTIN7","PRKCZ","VIL1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q00587","full_name":"Cdc42 effector protein 1","aliases":["Binder of Rho GTPases 5","Serum protein MSE55"],"length_aa":391,"mass_kda":40.3,"function":"Probably involved in the organization of the actin cytoskeleton. Induced membrane extensions in fibroblasts","subcellular_location":"Endomembrane system; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q00587/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CDC42EP1","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":[{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CDC42","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CDC42EP1","total_profiled":1310},"omim":[{"mim_id":"609171","title":"CDC42 EFFECTOR PROTEIN 5; CDC42EP5","url":"https://www.omim.org/entry/609171"},{"mim_id":"606084","title":"CDC42 EFFECTOR PROTEIN 1; CDC42EP1","url":"https://www.omim.org/entry/606084"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Actin filaments","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CDC42EP1"},"hgnc":{"alias_symbol":["MSE55","CEP1","Borg5"],"prev_symbol":[]},"alphafold":{"accession":"Q00587","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q00587","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q00587-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q00587-F1-predicted_aligned_error_v6.png","plddt_mean":54.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CDC42EP1","jax_strain_url":"https://www.jax.org/strain/search?query=CDC42EP1"},"sequence":{"accession":"Q00587","fasta_url":"https://rest.uniprot.org/uniprotkb/Q00587.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q00587/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q00587"}},"corpus_meta":[{"pmid":"7493928","id":"PMC_7493928","title":"A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7493928","citation_count":552,"is_preprint":false},{"pmid":"9799731","id":"PMC_9799731","title":"Distinct cellular effects and interactions of the Rho-family GTPase TC10.","date":"1998","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/9799731","citation_count":126,"is_preprint":false},{"pmid":"10490598","id":"PMC_10490598","title":"The Borgs, a new family of Cdc42 and TC10 GTPase-interacting proteins.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10490598","citation_count":112,"is_preprint":false},{"pmid":"11035016","id":"PMC_11035016","title":"A new family of Cdc42 effector proteins, CEPs, function in fibroblast and epithelial cell shape changes.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11035016","citation_count":91,"is_preprint":false},{"pmid":"27913681","id":"PMC_27913681","title":"The Borg family of Cdc42 effector proteins Cdc42EP1-5.","date":"2016","source":"Biochemical Society transactions","url":"https://pubmed.ncbi.nlm.nih.gov/27913681","citation_count":43,"is_preprint":false},{"pmid":"33146897","id":"PMC_33146897","title":"The mutational landscape of early- and typical-onset oral tongue squamous cell carcinoma.","date":"2020","source":"Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/33146897","citation_count":43,"is_preprint":false},{"pmid":"32574388","id":"PMC_32574388","title":"The genomic profile of parathyroid carcinoma based on whole-genome sequencing.","date":"2020","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/32574388","citation_count":39,"is_preprint":false},{"pmid":"10430899","id":"PMC_10430899","title":"MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts.","date":"1999","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10430899","citation_count":37,"is_preprint":false},{"pmid":"26657011","id":"PMC_26657011","title":"A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance.","date":"2015","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/26657011","citation_count":26,"is_preprint":false},{"pmid":"1629197","id":"PMC_1629197","title":"cDNA cloning and molecular characterization of MSE55, a novel human serum constituent protein that displays bone marrow stromal/endothelial cell-specific expression.","date":"1992","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1629197","citation_count":23,"is_preprint":false},{"pmid":"20506138","id":"PMC_20506138","title":"A role for borg5 during trophectoderm differentiation.","date":"2010","source":"Stem cells (Dayton, Ohio)","url":"https://pubmed.ncbi.nlm.nih.gov/20506138","citation_count":22,"is_preprint":false},{"pmid":"29040749","id":"PMC_29040749","title":"Cdc42 regulates the cellular localization of Cdc42ep1 in controlling neural crest cell migration.","date":"2018","source":"Journal of molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/29040749","citation_count":15,"is_preprint":false},{"pmid":"16028667","id":"PMC_16028667","title":"Study on a nonhealing fracture from a patient with systemic lupus erythematosus and its pathogenetic mechanisms.","date":"2005","source":"Ultrastructural pathology","url":"https://pubmed.ncbi.nlm.nih.gov/16028667","citation_count":13,"is_preprint":false},{"pmid":"36923257","id":"PMC_36923257","title":"Coordinated regulation of Cdc42ep1, actin, and septin filaments during neural crest cell migration.","date":"2023","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/36923257","citation_count":4,"is_preprint":false},{"pmid":"37757976","id":"PMC_37757976","title":"hsa_circ_0005991 promotes epithelial-mesenchymal transition by regulating miR-30b-3p/Cdc42EP1 axis in ovary endometriosis.","date":"2023","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/37757976","citation_count":3,"is_preprint":false},{"pmid":"37877586","id":"PMC_37877586","title":"Salmonella engages CDC42 effector protein 1 for intracellular invasion.","date":"2023","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/37877586","citation_count":3,"is_preprint":false},{"pmid":"39503295","id":"PMC_39503295","title":"Borg5 restricts contractility and motility in epithelial MDCK cells.","date":"2024","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/39503295","citation_count":2,"is_preprint":false},{"pmid":"39002033","id":"PMC_39002033","title":"Evaluating the expression pattern of ATXN1 and CDC42EP1 genes and related long noncoding RNAs in oral squamous cell carcinoma.","date":"2024","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/39002033","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10714,"output_tokens":2959,"usd":0.038263,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10293,"output_tokens":3081,"usd":0.064245,"stage2_stop_reason":"end_turn"},"total_usd":0.102508,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"MSE55 (CDC42EP1) contains a CRIB (Cdc42/Rac interactive binding) motif and binds to the GTP-bound form of Cdc42 but not to RhoA, as demonstrated by filter binding assay. Binding to Rac was weaker than to Cdc42.\",\n      \"method\": \"Filter binding assay with GST-fused GTPases\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay with GTP-dependence confirmed, single lab but well-controlled with multiple GTPases tested\",\n      \"pmids\": [\"7493928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"MSE55 (CDC42EP1) interacts with TC10 GTPase in addition to Cdc42, placing it in the effector set shared between TC10 and Cdc42.\",\n      \"method\": \"Interaction assay (yeast two-hybrid and/or pulldown)\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single interaction assay, confirmed across two GTPases in same study\",\n      \"pmids\": [\"9799731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MSE55 was renamed Borg5; Borg5 binds both TC10 and Cdc42 in a GTP-dependent manner, requiring an intact CRIB domain, with no detectable interaction with Rac1 or RhoA.\",\n      \"method\": \"Yeast two-hybrid screen; GTPase binding assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two-hybrid plus binding assay, CRIB domain requirement established, single lab\",\n      \"pmids\": [\"10490598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MSE55/Borg5 (CDC42EP1) binds Cdc42 in a GTP-dependent manner requiring an intact CRIB domain; a CRIB domain mutant fails to bind Cdc42. In Cos-7 cells, wild-type MSE55 localizes to membrane ruffles and increases membrane actin polymerization, whereas the CRIB mutant does not. In NIH 3T3 cells, MSE55 induces long actin-based protrusions that are blocked by dominant-negative Cdc42 (N17Cdc42) but not by dominant-negative Rac (N17Rac), establishing MSE55 as a Cdc42 effector mediating actin cytoskeleton reorganization.\",\n      \"method\": \"GST-capture pulldown; site-directed mutagenesis of CRIB domain; dominant-negative co-expression; immunofluorescence; live-cell video microscopy\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (pulldown, mutagenesis, dominant-negative epistasis, live imaging) in single study establishing mechanism\",\n      \"pmids\": [\"10430899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CDC42EP1/MSE55 (as CEP5 in this study) binds Cdc42 and, along with other CEP family members, induces pseudopodia formation in NIH 3T3 fibroblasts downstream of Cdc42; a CRIB domain mutant of CEP2 (the closest tested paralog) fails to induce pseudopodia, and dominant-negative Cdc42 blocks CEP-induced pseudopodia, placing CEPs downstream of Cdc42 in actin remodeling.\",\n      \"method\": \"GST pulldown; dominant-negative co-expression; CRIB domain mutagenesis; immunofluorescence of actin\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via dominant-negative, mutagenesis, multiple cell types; findings on MSE55/CEP5 partially inferred from CEP2 mutagenesis within same family\",\n      \"pmids\": [\"11035016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Borg5 (CDC42EP1) interacts with both Cdc42 and atypical protein kinase C (aPKC), functioning downstream of Cdc42 to enhance trophectoderm (TE) cell motility during ESC differentiation. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation, and Borg5 is required for sorting of differentiating TE cells to the outside of ESC aggregates.\",\n      \"method\": \"Co-immunoprecipitation; morpholino knockdown; live-cell imaging of ESC differentiation; immunofluorescence of aPKC localization\",\n      \"journal\": \"Stem cells (Dayton, Ohio)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus loss-of-function with defined cellular phenotypes, single lab\",\n      \"pmids\": [\"20506138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CDC42EP4 (a close family member) forms complexes with septin hetero-oligomers that interact with the glutamate transporter GLAST/EAAT1; the Borg/CDC42EP proteins interact reciprocally with CDC42 or the septin cytoskeleton. This establishes the paradigm that CDC42EP proteins interact with septins to scaffold membrane proteins.\",\n      \"method\": \"Co-immunoprecipitation; knockout mouse phenotype; immunofluorescence\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — mechanistic detail is for CDC42EP4 paralog; CDC42EP1/Borg5 is mentioned as part of family context but not the subject of direct experiments in this paper\",\n      \"pmids\": [\"26657011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cdc42ep1 is required for neural crest cell migration in frog embryos; knockdown causes rounder cell shapes, membrane blebbing, disrupted actin organization and focal adhesion alignment, and loss of directional traction forces. Cdc42ep1 localizes to two subcellular compartments: membrane protrusions (together with Cdc42) and perinuclear patches (where Cdc42 is absent). Cdc42 directly interacts with Cdc42ep1 through the CRIB domain, and changes in Cdc42 levels shift the distribution of Cdc42ep1 between these locations, controlling membrane protrusion formation and migration directionality.\",\n      \"method\": \"Morpholino-mediated knockdown; co-immunoprecipitation; live-cell imaging; immunofluorescence of actin and focal adhesions; traction force microscopy\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with multiple orthogonal phenotypic readouts, direct interaction confirmed, subcellular localization linked to functional consequences, single lab with rigorous methods\",\n      \"pmids\": [\"29040749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In neural crest cells, Cdc42ep1 colocalizes with septin filaments at the cell center and interacts with them in a reciprocal manner: septin filaments recruit Cdc42ep1 to the cell center, and Cdc42ep1 supports the formation of septin filaments. Loss of septin filaments impairs stability and contractility of actin stress fibers and alters their orientation; these septin activities depend on Cdc42ep1.\",\n      \"method\": \"Morpholino-mediated knockdown of septins and Cdc42ep1; co-localization by fluorescence imaging; rescue experiments; live-cell imaging of actin dynamics\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function epistasis between Cdc42ep1 and septins with defined actin phenotype, single lab, multiple imaging methods\",\n      \"pmids\": [\"36923257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CDC42EP1 associates with SEPTIN-7 and Villin in human enterocytes. Upon Salmonella Typhimurium infection, CDC42EP1 is rapidly redistributed and aggregates around invading bacteria in a manner dependent on host CDC42 and bacterial activation of CDC42. CDC42EP1 is not required for initial bacterial entry but associates with Salmonella-containing vacuoles during long-term infection, contributing to intracellular bacterial growth/replication.\",\n      \"method\": \"Proteomic analysis of CDC42 interactome; co-localization by immunofluorescence; siRNA knockdown; bacterial infection assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic identification of binding partners confirmed by localization and functional knockdown, single lab\",\n      \"pmids\": [\"37877586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In epithelial MDCK cells, Borg5/CDC42EP1 limits actomyosin contractility, cell-cell adhesion tension, and motility. Borg5 depletion inhibits lateral F-actin cortex development and stimulates radial stress fibers and microtubule-dependent leading-edge lamellae. Borg5 limits colocalization of septin proteins with microtubules. Borg5 physically interacts with the rod domain of myosin IIA (MYH9 heavy chain), and this interaction is reduced in the presence of septins, suggesting Borg5 counteracts septin-associated myosin activation to restrict contractility.\",\n      \"method\": \"siRNA depletion; co-immunoprecipitation; immunofluorescence; live-cell imaging; traction force/adhesion tension measurements\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple phenotypic readouts plus Co-IP identifying myosin IIA interaction, single lab, two orthogonal methods\",\n      \"pmids\": [\"39503295\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CDC42EP1/MSE55/Borg5 is a CRIB-domain-containing effector that binds activated (GTP-bound) Cdc42 and TC10 but not Rac1 or RhoA, and acts downstream of Cdc42 to reorganize the actin cytoskeleton; it also interacts reciprocally with septin filaments (and the myosin IIA rod domain) to regulate actin stress fiber stability, contractility, and directed cell migration in contexts including neural crest cells, trophectoderm differentiation, and epithelial morphogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CDC42EP1 (MSE55/Borg5) is a CRIB-domain effector that links the Rho-family GTPases Cdc42 and TC10 to the actin and septin cytoskeletons, thereby controlling cell shape, contractility, and directed migration [#3, #7]. It binds the GTP-bound forms of Cdc42 and TC10—but not Rac1 or RhoA—through an intact CRIB domain, and a CRIB mutant abolishes both Cdc42 binding and downstream activity [#0, #2, #3]. Acting downstream of Cdc42, CDC42EP1 localizes to membrane ruffles and protrusions where it drives actin polymerization and pseudopodia/protrusion formation in a manner blocked by dominant-negative Cdc42 [#3, #4]. CDC42EP1 also partitions to perinuclear and cell-center compartments where it reciprocally interacts with septin filaments: septins recruit CDC42EP1 to the cell center while CDC42EP1 supports septin filament assembly, and this interplay governs actin stress fiber stability, orientation, and contractility [#7, #8]. Through a physical interaction with the rod domain of myosin IIA (MYH9) that is antagonized by septins, CDC42EP1 restrains actomyosin contractility, cell-cell adhesion tension, and leading-edge dynamics in epithelial cells [#10]. These activities place CDC42EP1 in developmental and morphogenetic programs including neural crest cell migration and trophectoderm differentiation, the latter via interaction with atypical PKC [#5, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established CDC42EP1 as a GTPase effector by showing it carries a CRIB motif and binds GTP-loaded Cdc42 selectively over RhoA, defining its specificity from the outset.\",\n      \"evidence\": \"Filter binding assay with GST-fused GTPases\",\n      \"pmids\": [\"7493928\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Weaker Rac binding left selectivity incompletely resolved\", \"No functional consequence of binding tested\", \"Single in vitro binding format\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Broadened the GTPase specificity by placing CDC42EP1 in the shared effector set of TC10 in addition to Cdc42.\",\n      \"evidence\": \"Yeast two-hybrid and/or pulldown interaction assay\",\n      \"pmids\": [\"9799731\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GTP-dependence of TC10 binding not yet established here\", \"No cellular function attributed to TC10 binding\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined the molecular requirements and consequences of effector binding: GTP- and CRIB-dependent engagement of Cdc42/TC10 drives membrane ruffle localization, actin polymerization, and Cdc42-dependent protrusions, formally establishing CDC42EP1 as a Cdc42 effector for actin remodeling.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown, CRIB mutagenesis, dominant-negative epistasis, immunofluorescence and live imaging in Cos-7/NIH 3T3 cells\",\n      \"pmids\": [\"10490598\", \"10430899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical link between binding and actin nucleation machinery unresolved\", \"No endogenous loss-of-function data\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Generalized the actin-remodeling activity across the CEP/Borg family, confirming CRIB- and Cdc42-dependent pseudopodia induction.\",\n      \"evidence\": \"GST pulldown, CRIB mutagenesis, dominant-negative co-expression, actin immunofluorescence in NIH 3T3\",\n      \"pmids\": [\"11035016\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CDC42EP1-specific CRIB requirement inferred from paralog CEP2 mutagenesis\", \"Effector output mechanism still phenomenological\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Connected CDC42EP1 to a developmental program by showing it acts downstream of Cdc42 with aPKC to drive trophectoderm motility and sorting during blastocyst formation.\",\n      \"evidence\": \"Co-immunoprecipitation, morpholino knockdown, live-cell imaging and aPKC immunofluorescence in differentiating ESCs\",\n      \"pmids\": [\"20506138\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking Borg5 to aPKC localization unresolved\", \"Single loss-of-function approach\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved how CDC42EP1 controls migration directionality by linking its dual localization—Cdc42-positive protrusions versus Cdc42-absent perinuclear patches—to traction force generation and membrane protrusion in neural crest cells.\",\n      \"evidence\": \"Morpholino knockdown, Co-IP, live imaging, focal adhesion immunofluorescence, traction force microscopy in frog embryos\",\n      \"pmids\": [\"29040749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of factors retaining CDC42EP1 at perinuclear patches unknown\", \"Cross-species generality to mammalian migration not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established a reciprocal CDC42EP1–septin module that controls actin stress fiber stability, orientation, and contractility, and showed CDC42EP1 also scaffolds septins around invading Salmonella in a Cdc42-dependent manner relevant to intracellular bacterial growth.\",\n      \"evidence\": \"Morpholino knockdown and rescue with live imaging in neural crest cells; CDC42 interactome proteomics, siRNA, and infection assays in enterocytes\",\n      \"pmids\": [\"36923257\", \"37877586\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of reciprocal septin recruitment not defined\", \"Whether SEPTIN-7 binding is direct unresolved\", \"Role at Salmonella-containing vacuoles mechanistically uncharacterized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a contractility-limiting mechanism: CDC42EP1 binds the myosin IIA rod domain, an interaction antagonized by septins, allowing it to restrain actomyosin contractility, adhesion tension, and leading-edge dynamics in epithelia.\",\n      \"evidence\": \"siRNA depletion, Co-IP, immunofluorescence, live imaging, traction/adhesion tension measurements in MDCK cells\",\n      \"pmids\": [\"39503295\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect MYH9 rod-domain binding not separated from septin competition\", \"Structural basis of the septin-myosin switch unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CDC42EP1 mechanistically integrates Cdc42 GTPase input, septin assembly, and myosin IIA regulation into a single switch governing protrusion versus contractility remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the CRIB–GTPase or rod-domain interfaces\", \"Direct vs scaffolded nature of septin and myosin interactions undefined\", \"Endogenous mammalian loss-of-function phenotypes limited\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [3, 8, 10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8, 9, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 7]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8, 10]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 7]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDC42\", \"TC10\", \"MYH9\", \"SEPTIN7\", \"PRKCZ\", \"VIL1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}