{"gene":"SHROOM2","run_date":"2026-06-10T07:46:32","timeline":{"discoveries":[{"year":2007,"finding":"SHROOM2 directly interacts with the C-terminal MyTH4-FERM domain of myosin VIIa via yeast two-hybrid and binds F-actin; a SHROOM2 fragment containing the F-actin interaction region protects actin filaments from cytochalasin-D-induced disruption in MDCK cells. SHROOM2 also directly interacts with ZO-1 (TJP1) via its serine- and proline-rich region in vitro, and the two proteins co-immunoprecipitate from brain and cochlear extracts and co-localize at tight junctions.","method":"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation from tissue extracts, transfection/localization in MDCK and LE cells, cytochalasin-D protection assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP from tissue, direct in vitro binding, multiple orthogonal methods (Y2H, Co-IP, functional actin-protection assay) in single study","pmids":["17666436"],"is_preprint":false},{"year":2006,"finding":"SHROOM2 (APXL) is necessary and sufficient to govern localization of pigment granules (melanosomes) at the apical surface of retinal pigment epithelium cells in Xenopus. Loss of Shroom2 causes failure of melanosome maturation and apical association; ectopic expression in naive epithelial cells drives apical pigment accumulation in a manner requiring Rab27a GTPase. Additionally, Shroom2 induces apical accumulation of gamma-tubulin at apical cell surfaces.","method":"Morpholino knockdown in Xenopus embryos, ectopic expression, epistasis with Rab27a dominant-negative/knockdown, immunostaining","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with defined phenotype, epistasis with Rab27a establishing pathway position, gain-of-function rescue, multiple orthogonal methods","pmids":["16987870"],"is_preprint":false},{"year":2006,"finding":"SHROOM2 (Apxl) localizes to cortical actin in fibroblasts (unlike Shroom which associates with stress fibers). In epithelial cells, Apxl does not induce apical constriction as Shroom does, but acquires this capacity when artificially targeted to the apical junctional complex. SHROOM2 regulates cytoskeletal organization and architecture of endothelial cells.","method":"Transfection and fluorescence microscopy in fibroblasts and epithelial cells, chimeric protein targeting experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — localization by transfection/imaging with functional chimera experiments; single lab, multiple cell-type contexts","pmids":["16684770"],"is_preprint":false},{"year":2011,"finding":"SHROOM2 directly interacts with Rho kinase (ROCK) and facilitates the formation of a contractile network within endothelial cells. Depletion of Shrm2 results in decreased stress fiber organization, reduced collagen contraction, loss of ROCK and activated myosin II from cell-cell adhesion sites, and elevated branching/sprouting angiogenic behavior. This phenotype is recapitulated in HUVECs and embryonic stem cell-derived vasculogenesis assays.","method":"siRNA knockdown in HUVECs and ES cell-derived endothelial cells, collagen contraction assay, immunofluorescence for ROCK/pMLC, tube/sprouting angiogenesis assay, direct interaction assay with ROCK","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct interaction demonstrated, clean KD with defined molecular (ROCK/myosin II) and cellular (contractility, sprouting) phenotype; replicated in two endothelial cell systems","pmids":["21248203"],"is_preprint":false},{"year":2009,"finding":"In Xenopus, ectopic expression of Shroom2 increases epithelial cell height and loss of Shroom2 function results in failure of cell elongation in the neural epithelium, demonstrating Shroom2 is required for epithelial cell morphology in the neural tube.","method":"In situ hybridization for expression, morpholino knockdown (loss-of-function), ectopic expression in Xenopus embryos, cell height measurements","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific morphological phenotype plus gain-of-function in Xenopus, single lab","pmids":["19384856"],"is_preprint":false},{"year":2015,"finding":"SHROOM2 is identified as a candidate phosphorylation substrate for Rho-associated kinase (ROCK2) by the KISS method. SHROOM2 assembles into a ternary complex with Rho-kinase and Scrib in a phosphorylation-dependent manner, and this complex plays a crucial role in regulation of subcellular contractility.","method":"Kinase-interacting substrate screening (KISS), co-immunoprecipitation of ternary complex, phosphorylation-dependent interaction assays","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel substrate screen with biochemical validation of ternary complex; single lab but multiple methods (KISS + Co-IP)","pmids":["26101221"],"is_preprint":false},{"year":2019,"finding":"SHROOM2 participates in RhoA-ROCK-induced stress fiber formation and focal adhesion in nasopharyngeal carcinoma cells. Depletion of SHROOM2 also drives epithelial-to-mesenchymal transition (EMT) and enhances cell migration/invasion through a ROCK-independent mechanism; ROCK inhibitor Y-27632 did not cause EMT, and combined ROCK inhibition plus SHROOM2 depletion produced greater migration/invasion than either alone, indicating a synergistic rather than epistatic relationship.","method":"siRNA knockdown in NPC cells, ROCK inhibitor (Y-27632) treatment, wound-healing/invasion assays, immunofluorescence for EMT markers and stress fibers, clinical sample analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined cellular phenotype plus pharmacological pathway dissection; single lab, multiple orthogonal assays","pmids":["30683844"],"is_preprint":false},{"year":2018,"finding":"Rare deleterious missense variants in SHROOM2 (p.A1331S; p.R1557H) found in neural tube defect patients disrupt SHROOM2 binding to ROCK1 in vitro, as confirmed by mammalian two-hybrid assay and western blot.","method":"Targeted next-generation sequencing, Sanger sequencing confirmation, mammalian two-hybrid assay, western blot","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction assay (mammalian two-hybrid) with mutant vs. wild-type SHROOM2; single lab, two orthogonal methods","pmids":["29423651"],"is_preprint":false},{"year":2016,"finding":"ZO-1's U5-GuK domain binds shroom2 (among other partners), and the ZO-1/occludin interaction (via the OCEL domain) but not the shroom2 interaction is required for single lumen formation in 3D epithelial culture; deletion of shroom2 alone did not lead to multi-lumen cysts, distinguishing it from occludin's role.","method":"ZO-1 domain-deletion mutants in 3D MDCK cyst culture, siRNA knockdown of binding partners including shroom2, immunofluorescence for lumen number and spindle orientation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-deletion plus partner-specific KD with defined phenotypic readout; shroom2's non-essential role in this context is a mechanistically informative negative result, but interaction is confirmed","pmids":["27802160"],"is_preprint":false},{"year":2010,"finding":"During cochlear outer hair cell apical circumference remodeling in mice, SHROOM2 and F-actin transiently accumulate in the developing lateral lobes of the apical junctional complex, concurrent with polarized redistribution of myosin VIIa and myosin II, implicating SHROOM2 in actomyosin cytoskeleton-mediated apical shape remodeling.","method":"Immunostaining and geometric analysis of AJC in mouse cochlea (wild-type and hair bundle morphogenesis mutants), comparison across mutant backgrounds","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment with functional context, multiple mutant backgrounds tested; no functional manipulation of SHROOM2 itself in this study","pmids":["20332152"],"is_preprint":false},{"year":2023,"finding":"SCRIB serves as a molecular scaffold for SHROOM2/4 and ROCK1 during epithelial differentiation; SCRIB contains an evolutionarily conserved SHROOM-binding site in its C-terminus required for SCRIB function in controlling apical cell shape and apical contractility. SCRIB KO gut-like epithelia are flatter with reduced apical surface area and defective myosin light chain polarization.","method":"SCRIB KO in organ-on-chip gut epithelium model, site-directed mutagenesis of SHROOM-binding site in SCRIB, co-immunoprecipitation, immunofluorescence for myosin light chain, evolutionary conservation analysis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with defined phenotype, mutagenesis of interaction domain, Co-IP; single lab, multiple methods","pmids":["37930352"],"is_preprint":false},{"year":2004,"finding":"ApxL (SHROOM2) protein is expressed in LLC-PK1 renal epithelial cells and localizes to both the apical membrane and cytoplasmic compartments; vasopressin stimulation modifies ApxL cellular distribution, consistent with increased membrane-associated ApxL, suggesting a role in regulated membrane trafficking in the proximal nephron.","method":"RT-PCR, immunocytochemistry, vasopressin stimulation experiment in LLC-PK1 cells","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, immunocytochemistry localization with pharmacological stimulation; functional link to channel activity speculative and not directly tested for SHROOM2","pmids":["14982925"],"is_preprint":false},{"year":2025,"finding":"Claudin-11 (an HLD22-associated protein) directly interacts with SHROOM2 via its C-terminal PDZ ligand sequence binding to SHROOM2's PDZ domain. Knockdown of SHROOM2 in FBD-102b oligodendroglial cells reduces morphological differentiation and marker protein expression; this is linked to altered phosphorylation of Akt kinase. An HLD22-associated claudin-11 mutation abolishes its interaction with the SHROOM2 PDZ domain.","method":"Co-immunoprecipitation, siRNA knockdown, transfection of PDZ domain/ligand sequences, western blot for differentiation markers and Akt phosphorylation, morphological analysis","journal":"BBA advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction mapped to PDZ domain, KD phenotype, disease-mutant interaction loss; single lab, multiple orthogonal methods","pmids":["40230506"],"is_preprint":false}],"current_model":"SHROOM2 is an actin- and PDZ domain-containing scaffolding protein that localizes to cortical actin and tight/apical junctions in epithelial and endothelial cells, where it directly binds F-actin, myosin VIIa (via its MyTH4-FERM domain), ZO-1 (via its SP-rich region), and ROCK1/2; through recruitment of ROCK and myosin II to cell–cell adhesions, SHROOM2 regulates actomyosin contractility to control epithelial cell elongation, apical constriction, endothelial sprouting/angiogenesis, and melanosome apical positioning (the last requiring Rab27a), while also functioning as a scaffold component of SCRIB–SHROOM2–ROCK1 complexes and interacting with claudin-11 to influence oligodendroglial differentiation via Akt signaling."},"narrative":{"mechanistic_narrative":"SHROOM2 is an actin-associated scaffolding protein that organizes the actomyosin cytoskeleton at cortical and apical junctional sites to control epithelial and endothelial cell shape and contractility [PMID:17666436, PMID:21248203]. It binds F-actin directly and protects actin filaments from depolymerization, and through distinct domains assembles a network of cytoskeletal and junctional partners: the MyTH4-FERM domain of myosin VIIa, the tight-junction protein ZO-1 via its serine/proline-rich region, and Rho-associated kinase (ROCK), which it both binds and serves as a phosphorylation substrate for [PMID:17666436, PMID:26101221]. By recruiting ROCK and activated myosin II to cell-cell adhesion sites, SHROOM2 drives contractile network formation; its depletion in endothelial cells disorganizes stress fibers, reduces collagen contraction, and enhances angiogenic sprouting [PMID:21248203]. SHROOM2 operates as part of a ternary SCRIB-SHROOM2-ROCK1 scaffold, with SCRIB providing a conserved SHROOM-binding site required for apical cell shape control and myosin light chain polarization during epithelial differentiation [PMID:26101221, PMID:37930352]. In development, SHROOM2 governs apical positioning of melanosomes in retinal pigment epithelium in a Rab27a-dependent manner [PMID:16987870] and is required for epithelial cell elongation in the neural tube [PMID:19384856]. Through its PDZ domain SHROOM2 binds the C-terminal PDZ ligand of claudin-11, and its loss in oligodendroglial cells impairs differentiation in association with altered Akt phosphorylation [PMID:40230506]. Rare deleterious SHROOM2 missense variants found in neural tube defect patients disrupt its binding to ROCK1 [PMID:29423651], and an HLD22-associated claudin-11 mutation abolishes its interaction with the SHROOM2 PDZ domain [PMID:40230506].","teleology":[{"year":2004,"claim":"Establishing where SHROOM2 acts within an epithelial cell, this work placed the protein at the apical membrane and showed its distribution responds to hormonal stimulation, hinting at a role in regulated membrane trafficking.","evidence":"RT-PCR and immunocytochemistry with vasopressin stimulation in LLC-PK1 renal epithelial cells","pmids":["14982925"],"confidence":"Low","gaps":["Localization by immunocytochemistry only, not independently confirmed","No functional manipulation of SHROOM2","Link to membrane trafficking is correlative"]},{"year":2006,"claim":"Two studies defined SHROOM2's baseline cytoskeletal behavior and a developmental output: it localizes to cortical actin (distinct from the stress-fiber-associated Shroom) and is necessary and sufficient for apical melanosome positioning in retinal pigment epithelium.","evidence":"Transfection/imaging and chimeric targeting in fibroblasts/epithelia; morpholino knockdown, ectopic expression, and Rab27a epistasis in Xenopus","pmids":["16684770","16987870"],"confidence":"Medium","gaps":["Direct binding partners mediating cortical actin localization not yet mapped","Mechanism linking SHROOM2 to Rab27a-dependent melanosome maturation undefined"]},{"year":2007,"claim":"This work built the molecular interaction map underlying SHROOM2 scaffolding by demonstrating direct binding to myosin VIIa, F-actin, and ZO-1, anchoring the protein to both the actin cytoskeleton and tight junctions.","evidence":"Yeast two-hybrid, in vitro binding, reciprocal Co-IP from brain/cochlear tissue, actin-protection assay in MDCK cells","pmids":["17666436"],"confidence":"High","gaps":["Functional consequence of the myosin VIIa interaction not tested in vivo","Domain on SHROOM2 binding myosin VIIa not finely mapped"]},{"year":2009,"claim":"Connecting SHROOM2 to tissue morphogenesis, this study showed it is both required and sufficient for epithelial cell elongation in the neural tube.","evidence":"Morpholino knockdown, ectopic expression, and cell-height measurement in Xenopus neural epithelium","pmids":["19384856"],"confidence":"Medium","gaps":["Molecular effectors downstream of SHROOM2 in cell elongation not identified","Single-organism/single-lab evidence"]},{"year":2010,"claim":"This work tied SHROOM2 to dynamic apical shape remodeling by showing transient co-accumulation with F-actin, myosin VIIa, and myosin II at the developing apical junctional complex of cochlear hair cells.","evidence":"Immunostaining and geometric analysis of the apical junctional complex across mouse cochlear mutant backgrounds","pmids":["20332152"],"confidence":"Medium","gaps":["No functional manipulation of SHROOM2 in this system","Causal role in apical remodeling inferred from localization only"]},{"year":2011,"claim":"This study mechanistically linked SHROOM2 to contractility by showing it directly binds ROCK and is required to recruit ROCK and active myosin II to cell-cell adhesions, controlling endothelial contractile network formation and angiogenic sprouting.","evidence":"siRNA in HUVECs and ES-cell-derived endothelium, collagen contraction, sprouting assays, ROCK/pMLC immunofluorescence, direct interaction assay","pmids":["21248203"],"confidence":"High","gaps":["Whether ROCK recruitment depends on the same domains binding actin/ZO-1 not resolved","In vivo angiogenesis role not directly tested"]},{"year":2015,"claim":"This work refined the SHROOM2-ROCK relationship by identifying SHROOM2 as a ROCK2 phosphorylation substrate and showing it assembles a phosphorylation-dependent ternary complex with Rho-kinase and Scrib that controls subcellular contractility.","evidence":"Kinase-interacting substrate screening (KISS) and Co-IP of the ternary complex","pmids":["26101221"],"confidence":"Medium","gaps":["Specific SHROOM2 phosphosites and their functional effect not defined","Stoichiometry and assembly order of the ternary complex unknown"]},{"year":2016,"claim":"By dissecting ZO-1 domain interactions in lumen formation, this study confirmed the ZO-1/SHROOM2 interaction but showed SHROOM2, unlike occludin, is dispensable for single-lumen formation, delimiting the contexts where the interaction is functionally required.","evidence":"ZO-1 domain-deletion mutants and partner-specific siRNA in 3D MDCK cyst culture","pmids":["27802160"],"confidence":"Medium","gaps":["Functional role of the ZO-1/SHROOM2 interaction in other epithelial contexts not addressed","Negative result does not exclude redundancy"]},{"year":2018,"claim":"This work linked SHROOM2 to human disease by showing neural-tube-defect patient missense variants disrupt SHROOM2 binding to ROCK1, connecting the ROCK-scaffolding function to a pathological phenotype.","evidence":"Targeted sequencing of patients plus mammalian two-hybrid and western blot of mutant versus wild-type SHROOM2","pmids":["29423651"],"confidence":"Medium","gaps":["Causality of variants for NTD not established beyond interaction disruption","Cellular/developmental consequence of binding loss not shown"]},{"year":2019,"claim":"This study distinguished SHROOM2's roles in cancer, showing it contributes to RhoA-ROCK stress-fiber formation yet restrains EMT and invasion through a ROCK-independent mechanism, indicating SHROOM2 functions beyond simple ROCK epistasis.","evidence":"siRNA, Y-27632 ROCK inhibition, migration/invasion and EMT-marker assays in nasopharyngeal carcinoma cells","pmids":["30683844"],"confidence":"Medium","gaps":["The ROCK-independent effector restraining EMT not identified","In vivo tumor relevance not established"]},{"year":2023,"claim":"This work established SCRIB as the scaffold organizing SHROOM2/4 and ROCK1, mapping a conserved SHROOM-binding site in SCRIB required for apical cell shape and myosin light chain polarization during epithelial differentiation.","evidence":"SCRIB KO and SHROOM-binding-site mutagenesis in organ-on-chip gut epithelium with Co-IP and myosin light chain immunofluorescence","pmids":["37930352"],"confidence":"Medium","gaps":["Relative contributions of SHROOM2 versus SHROOM4 not separated","How SCRIB scaffolding integrates with the ZO-1/myosin VIIa interactions unknown"]},{"year":2025,"claim":"This study extended SHROOM2 scaffolding into glial biology, showing its PDZ domain binds the claudin-11 PDZ ligand and that SHROOM2 loss impairs oligodendroglial differentiation via altered Akt phosphorylation, with a disease mutation abolishing the interaction.","evidence":"Co-IP, PDZ domain/ligand transfection, siRNA, and western blot for differentiation markers and Akt phosphorylation in FBD-102b cells","pmids":["40230506"],"confidence":"Medium","gaps":["Mechanism connecting SHROOM2/claudin-11 to Akt signaling undefined","In vivo relevance to HLD22 not tested"]},{"year":null,"claim":"How SHROOM2's multiple interactions (actin, myosin VIIa, ZO-1, ROCK, SCRIB, claudin-11) are coordinated in space and time, and which are required for each tissue-specific output, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model of SHROOM2 domain usage","Tissue-specific partner requirements not systematically dissected","Phosphorylation regulation of complex assembly incompletely defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,5,10,12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,5]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,2,3]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,11]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,5,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,4,10]}],"complexes":["SCRIB-SHROOM2-ROCK1 ternary complex"],"partners":["MYO7A","TJP1","ROCK1","ROCK2","SCRIB","CLDN11"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13796","full_name":"Protein Shroom2","aliases":["Apical-like protein","Protein APXL"],"length_aa":1616,"mass_kda":176.4,"function":"May be involved in endothelial cell morphology changes during cell spreading. In the retinal pigment epithelium, may regulate the biogenesis of melanosomes and promote their association with the apical cell surface by inducing gamma-tubulin redistribution (By similarity)","subcellular_location":"Apical cell membrane; Cell junction, tight junction; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q13796/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SHROOM2","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/SHROOM2","total_profiled":1310},"omim":[{"mim_id":"611179","title":"SHROOM FAMILY MEMBER 1; SHROOM1","url":"https://www.omim.org/entry/611179"},{"mim_id":"604570","title":"SHROOM FAMILY MEMBER 3; SHROOM3","url":"https://www.omim.org/entry/604570"},{"mim_id":"300808","title":"G PROTEIN-COUPLED RECEPTOR 143; GPR143","url":"https://www.omim.org/entry/300808"},{"mim_id":"300500","title":"ALBINISM, OCULAR, TYPE I; OA1","url":"https://www.omim.org/entry/300500"},{"mim_id":"300103","title":"SHROOM FAMILY MEMBER 2; SHROOM2","url":"https://www.omim.org/entry/300103"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cell Junctions","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"retina","ntpm":19.3}],"url":"https://www.proteinatlas.org/search/SHROOM2"},"hgnc":{"alias_symbol":[],"prev_symbol":["APXL"]},"alphafold":{"accession":"Q13796","domains":[{"cath_id":"2.30.42.10","chopping":"25-108","consensus_level":"high","plddt":89.3458,"start":25,"end":108},{"cath_id":"-","chopping":"1312-1364_1434-1608","consensus_level":"high","plddt":89.6227,"start":1312,"end":1608}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13796","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13796-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13796-F1-predicted_aligned_error_v6.png","plddt_mean":48.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SHROOM2","jax_strain_url":"https://www.jax.org/strain/search?query=SHROOM2"},"sequence":{"accession":"Q13796","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13796.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13796/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13796"}},"corpus_meta":[{"pmid":"8180623","id":"PMC_8180623","title":"Regulation 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SHROOM2 also directly interacts with ZO-1 (TJP1) via its serine- and proline-rich region in vitro, and the two proteins co-immunoprecipitate from brain and cochlear extracts and co-localize at tight junctions.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation from tissue extracts, transfection/localization in MDCK and LE cells, cytochalasin-D protection assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP from tissue, direct in vitro binding, multiple orthogonal methods (Y2H, Co-IP, functional actin-protection assay) in single study\",\n      \"pmids\": [\"17666436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SHROOM2 (APXL) is necessary and sufficient to govern localization of pigment granules (melanosomes) at the apical surface of retinal pigment epithelium cells in Xenopus. Loss of Shroom2 causes failure of melanosome maturation and apical association; ectopic expression in naive epithelial cells drives apical pigment accumulation in a manner requiring Rab27a GTPase. Additionally, Shroom2 induces apical accumulation of gamma-tubulin at apical cell surfaces.\",\n      \"method\": \"Morpholino knockdown in Xenopus embryos, ectopic expression, epistasis with Rab27a dominant-negative/knockdown, immunostaining\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with defined phenotype, epistasis with Rab27a establishing pathway position, gain-of-function rescue, multiple orthogonal methods\",\n      \"pmids\": [\"16987870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SHROOM2 (Apxl) localizes to cortical actin in fibroblasts (unlike Shroom which associates with stress fibers). In epithelial cells, Apxl does not induce apical constriction as Shroom does, but acquires this capacity when artificially targeted to the apical junctional complex. SHROOM2 regulates cytoskeletal organization and architecture of endothelial cells.\",\n      \"method\": \"Transfection and fluorescence microscopy in fibroblasts and epithelial cells, chimeric protein targeting experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — localization by transfection/imaging with functional chimera experiments; single lab, multiple cell-type contexts\",\n      \"pmids\": [\"16684770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHROOM2 directly interacts with Rho kinase (ROCK) and facilitates the formation of a contractile network within endothelial cells. Depletion of Shrm2 results in decreased stress fiber organization, reduced collagen contraction, loss of ROCK and activated myosin II from cell-cell adhesion sites, and elevated branching/sprouting angiogenic behavior. This phenotype is recapitulated in HUVECs and embryonic stem cell-derived vasculogenesis assays.\",\n      \"method\": \"siRNA knockdown in HUVECs and ES cell-derived endothelial cells, collagen contraction assay, immunofluorescence for ROCK/pMLC, tube/sprouting angiogenesis assay, direct interaction assay with ROCK\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct interaction demonstrated, clean KD with defined molecular (ROCK/myosin II) and cellular (contractility, sprouting) phenotype; replicated in two endothelial cell systems\",\n      \"pmids\": [\"21248203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In Xenopus, ectopic expression of Shroom2 increases epithelial cell height and loss of Shroom2 function results in failure of cell elongation in the neural epithelium, demonstrating Shroom2 is required for epithelial cell morphology in the neural tube.\",\n      \"method\": \"In situ hybridization for expression, morpholino knockdown (loss-of-function), ectopic expression in Xenopus embryos, cell height measurements\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific morphological phenotype plus gain-of-function in Xenopus, single lab\",\n      \"pmids\": [\"19384856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SHROOM2 is identified as a candidate phosphorylation substrate for Rho-associated kinase (ROCK2) by the KISS method. SHROOM2 assembles into a ternary complex with Rho-kinase and Scrib in a phosphorylation-dependent manner, and this complex plays a crucial role in regulation of subcellular contractility.\",\n      \"method\": \"Kinase-interacting substrate screening (KISS), co-immunoprecipitation of ternary complex, phosphorylation-dependent interaction assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel substrate screen with biochemical validation of ternary complex; single lab but multiple methods (KISS + Co-IP)\",\n      \"pmids\": [\"26101221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SHROOM2 participates in RhoA-ROCK-induced stress fiber formation and focal adhesion in nasopharyngeal carcinoma cells. Depletion of SHROOM2 also drives epithelial-to-mesenchymal transition (EMT) and enhances cell migration/invasion through a ROCK-independent mechanism; ROCK inhibitor Y-27632 did not cause EMT, and combined ROCK inhibition plus SHROOM2 depletion produced greater migration/invasion than either alone, indicating a synergistic rather than epistatic relationship.\",\n      \"method\": \"siRNA knockdown in NPC cells, ROCK inhibitor (Y-27632) treatment, wound-healing/invasion assays, immunofluorescence for EMT markers and stress fibers, clinical sample analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined cellular phenotype plus pharmacological pathway dissection; single lab, multiple orthogonal assays\",\n      \"pmids\": [\"30683844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Rare deleterious missense variants in SHROOM2 (p.A1331S; p.R1557H) found in neural tube defect patients disrupt SHROOM2 binding to ROCK1 in vitro, as confirmed by mammalian two-hybrid assay and western blot.\",\n      \"method\": \"Targeted next-generation sequencing, Sanger sequencing confirmation, mammalian two-hybrid assay, western blot\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction assay (mammalian two-hybrid) with mutant vs. wild-type SHROOM2; single lab, two orthogonal methods\",\n      \"pmids\": [\"29423651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ZO-1's U5-GuK domain binds shroom2 (among other partners), and the ZO-1/occludin interaction (via the OCEL domain) but not the shroom2 interaction is required for single lumen formation in 3D epithelial culture; deletion of shroom2 alone did not lead to multi-lumen cysts, distinguishing it from occludin's role.\",\n      \"method\": \"ZO-1 domain-deletion mutants in 3D MDCK cyst culture, siRNA knockdown of binding partners including shroom2, immunofluorescence for lumen number and spindle orientation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-deletion plus partner-specific KD with defined phenotypic readout; shroom2's non-essential role in this context is a mechanistically informative negative result, but interaction is confirmed\",\n      \"pmids\": [\"27802160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"During cochlear outer hair cell apical circumference remodeling in mice, SHROOM2 and F-actin transiently accumulate in the developing lateral lobes of the apical junctional complex, concurrent with polarized redistribution of myosin VIIa and myosin II, implicating SHROOM2 in actomyosin cytoskeleton-mediated apical shape remodeling.\",\n      \"method\": \"Immunostaining and geometric analysis of AJC in mouse cochlea (wild-type and hair bundle morphogenesis mutants), comparison across mutant backgrounds\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment with functional context, multiple mutant backgrounds tested; no functional manipulation of SHROOM2 itself in this study\",\n      \"pmids\": [\"20332152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SCRIB serves as a molecular scaffold for SHROOM2/4 and ROCK1 during epithelial differentiation; SCRIB contains an evolutionarily conserved SHROOM-binding site in its C-terminus required for SCRIB function in controlling apical cell shape and apical contractility. SCRIB KO gut-like epithelia are flatter with reduced apical surface area and defective myosin light chain polarization.\",\n      \"method\": \"SCRIB KO in organ-on-chip gut epithelium model, site-directed mutagenesis of SHROOM-binding site in SCRIB, co-immunoprecipitation, immunofluorescence for myosin light chain, evolutionary conservation analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with defined phenotype, mutagenesis of interaction domain, Co-IP; single lab, multiple methods\",\n      \"pmids\": [\"37930352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ApxL (SHROOM2) protein is expressed in LLC-PK1 renal epithelial cells and localizes to both the apical membrane and cytoplasmic compartments; vasopressin stimulation modifies ApxL cellular distribution, consistent with increased membrane-associated ApxL, suggesting a role in regulated membrane trafficking in the proximal nephron.\",\n      \"method\": \"RT-PCR, immunocytochemistry, vasopressin stimulation experiment in LLC-PK1 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, immunocytochemistry localization with pharmacological stimulation; functional link to channel activity speculative and not directly tested for SHROOM2\",\n      \"pmids\": [\"14982925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Claudin-11 (an HLD22-associated protein) directly interacts with SHROOM2 via its C-terminal PDZ ligand sequence binding to SHROOM2's PDZ domain. Knockdown of SHROOM2 in FBD-102b oligodendroglial cells reduces morphological differentiation and marker protein expression; this is linked to altered phosphorylation of Akt kinase. An HLD22-associated claudin-11 mutation abolishes its interaction with the SHROOM2 PDZ domain.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, transfection of PDZ domain/ligand sequences, western blot for differentiation markers and Akt phosphorylation, morphological analysis\",\n      \"journal\": \"BBA advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction mapped to PDZ domain, KD phenotype, disease-mutant interaction loss; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"40230506\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SHROOM2 is an actin- and PDZ domain-containing scaffolding protein that localizes to cortical actin and tight/apical junctions in epithelial and endothelial cells, where it directly binds F-actin, myosin VIIa (via its MyTH4-FERM domain), ZO-1 (via its SP-rich region), and ROCK1/2; through recruitment of ROCK and myosin II to cell–cell adhesions, SHROOM2 regulates actomyosin contractility to control epithelial cell elongation, apical constriction, endothelial sprouting/angiogenesis, and melanosome apical positioning (the last requiring Rab27a), while also functioning as a scaffold component of SCRIB–SHROOM2–ROCK1 complexes and interacting with claudin-11 to influence oligodendroglial differentiation via Akt signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SHROOM2 is an actin-associated scaffolding protein that organizes the actomyosin cytoskeleton at cortical and apical junctional sites to control epithelial and endothelial cell shape and contractility [#0, #3]. It binds F-actin directly and protects actin filaments from depolymerization, and through distinct domains assembles a network of cytoskeletal and junctional partners: the MyTH4-FERM domain of myosin VIIa, the tight-junction protein ZO-1 via its serine/proline-rich region, and Rho-associated kinase (ROCK), which it both binds and serves as a phosphorylation substrate for [#0, #5]. By recruiting ROCK and activated myosin II to cell-cell adhesion sites, SHROOM2 drives contractile network formation; its depletion in endothelial cells disorganizes stress fibers, reduces collagen contraction, and enhances angiogenic sprouting [#3]. SHROOM2 operates as part of a ternary SCRIB-SHROOM2-ROCK1 scaffold, with SCRIB providing a conserved SHROOM-binding site required for apical cell shape control and myosin light chain polarization during epithelial differentiation [#5, #10]. In development, SHROOM2 governs apical positioning of melanosomes in retinal pigment epithelium in a Rab27a-dependent manner [#1] and is required for epithelial cell elongation in the neural tube [#4]. Through its PDZ domain SHROOM2 binds the C-terminal PDZ ligand of claudin-11, and its loss in oligodendroglial cells impairs differentiation in association with altered Akt phosphorylation [#12]. Rare deleterious SHROOM2 missense variants found in neural tube defect patients disrupt its binding to ROCK1 [#7], and an HLD22-associated claudin-11 mutation abolishes its interaction with the SHROOM2 PDZ domain [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing where SHROOM2 acts within an epithelial cell, this work placed the protein at the apical membrane and showed its distribution responds to hormonal stimulation, hinting at a role in regulated membrane trafficking.\",\n      \"evidence\": \"RT-PCR and immunocytochemistry with vasopressin stimulation in LLC-PK1 renal epithelial cells\",\n      \"pmids\": [\"14982925\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Localization by immunocytochemistry only, not independently confirmed\", \"No functional manipulation of SHROOM2\", \"Link to membrane trafficking is correlative\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Two studies defined SHROOM2's baseline cytoskeletal behavior and a developmental output: it localizes to cortical actin (distinct from the stress-fiber-associated Shroom) and is necessary and sufficient for apical melanosome positioning in retinal pigment epithelium.\",\n      \"evidence\": \"Transfection/imaging and chimeric targeting in fibroblasts/epithelia; morpholino knockdown, ectopic expression, and Rab27a epistasis in Xenopus\",\n      \"pmids\": [\"16684770\", \"16987870\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding partners mediating cortical actin localization not yet mapped\", \"Mechanism linking SHROOM2 to Rab27a-dependent melanosome maturation undefined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"This work built the molecular interaction map underlying SHROOM2 scaffolding by demonstrating direct binding to myosin VIIa, F-actin, and ZO-1, anchoring the protein to both the actin cytoskeleton and tight junctions.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, reciprocal Co-IP from brain/cochlear tissue, actin-protection assay in MDCK cells\",\n      \"pmids\": [\"17666436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the myosin VIIa interaction not tested in vivo\", \"Domain on SHROOM2 binding myosin VIIa not finely mapped\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connecting SHROOM2 to tissue morphogenesis, this study showed it is both required and sufficient for epithelial cell elongation in the neural tube.\",\n      \"evidence\": \"Morpholino knockdown, ectopic expression, and cell-height measurement in Xenopus neural epithelium\",\n      \"pmids\": [\"19384856\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular effectors downstream of SHROOM2 in cell elongation not identified\", \"Single-organism/single-lab evidence\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"This work tied SHROOM2 to dynamic apical shape remodeling by showing transient co-accumulation with F-actin, myosin VIIa, and myosin II at the developing apical junctional complex of cochlear hair cells.\",\n      \"evidence\": \"Immunostaining and geometric analysis of the apical junctional complex across mouse cochlear mutant backgrounds\",\n      \"pmids\": [\"20332152\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional manipulation of SHROOM2 in this system\", \"Causal role in apical remodeling inferred from localization only\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"This study mechanistically linked SHROOM2 to contractility by showing it directly binds ROCK and is required to recruit ROCK and active myosin II to cell-cell adhesions, controlling endothelial contractile network formation and angiogenic sprouting.\",\n      \"evidence\": \"siRNA in HUVECs and ES-cell-derived endothelium, collagen contraction, sprouting assays, ROCK/pMLC immunofluorescence, direct interaction assay\",\n      \"pmids\": [\"21248203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ROCK recruitment depends on the same domains binding actin/ZO-1 not resolved\", \"In vivo angiogenesis role not directly tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"This work refined the SHROOM2-ROCK relationship by identifying SHROOM2 as a ROCK2 phosphorylation substrate and showing it assembles a phosphorylation-dependent ternary complex with Rho-kinase and Scrib that controls subcellular contractility.\",\n      \"evidence\": \"Kinase-interacting substrate screening (KISS) and Co-IP of the ternary complex\",\n      \"pmids\": [\"26101221\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific SHROOM2 phosphosites and their functional effect not defined\", \"Stoichiometry and assembly order of the ternary complex unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"By dissecting ZO-1 domain interactions in lumen formation, this study confirmed the ZO-1/SHROOM2 interaction but showed SHROOM2, unlike occludin, is dispensable for single-lumen formation, delimiting the contexts where the interaction is functionally required.\",\n      \"evidence\": \"ZO-1 domain-deletion mutants and partner-specific siRNA in 3D MDCK cyst culture\",\n      \"pmids\": [\"27802160\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of the ZO-1/SHROOM2 interaction in other epithelial contexts not addressed\", \"Negative result does not exclude redundancy\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"This work linked SHROOM2 to human disease by showing neural-tube-defect patient missense variants disrupt SHROOM2 binding to ROCK1, connecting the ROCK-scaffolding function to a pathological phenotype.\",\n      \"evidence\": \"Targeted sequencing of patients plus mammalian two-hybrid and western blot of mutant versus wild-type SHROOM2\",\n      \"pmids\": [\"29423651\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causality of variants for NTD not established beyond interaction disruption\", \"Cellular/developmental consequence of binding loss not shown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"This study distinguished SHROOM2's roles in cancer, showing it contributes to RhoA-ROCK stress-fiber formation yet restrains EMT and invasion through a ROCK-independent mechanism, indicating SHROOM2 functions beyond simple ROCK epistasis.\",\n      \"evidence\": \"siRNA, Y-27632 ROCK inhibition, migration/invasion and EMT-marker assays in nasopharyngeal carcinoma cells\",\n      \"pmids\": [\"30683844\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The ROCK-independent effector restraining EMT not identified\", \"In vivo tumor relevance not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"This work established SCRIB as the scaffold organizing SHROOM2/4 and ROCK1, mapping a conserved SHROOM-binding site in SCRIB required for apical cell shape and myosin light chain polarization during epithelial differentiation.\",\n      \"evidence\": \"SCRIB KO and SHROOM-binding-site mutagenesis in organ-on-chip gut epithelium with Co-IP and myosin light chain immunofluorescence\",\n      \"pmids\": [\"37930352\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contributions of SHROOM2 versus SHROOM4 not separated\", \"How SCRIB scaffolding integrates with the ZO-1/myosin VIIa interactions unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"This study extended SHROOM2 scaffolding into glial biology, showing its PDZ domain binds the claudin-11 PDZ ligand and that SHROOM2 loss impairs oligodendroglial differentiation via altered Akt phosphorylation, with a disease mutation abolishing the interaction.\",\n      \"evidence\": \"Co-IP, PDZ domain/ligand transfection, siRNA, and western blot for differentiation markers and Akt phosphorylation in FBD-102b cells\",\n      \"pmids\": [\"40230506\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting SHROOM2/claudin-11 to Akt signaling undefined\", \"In vivo relevance to HLD22 not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SHROOM2's multiple interactions (actin, myosin VIIa, ZO-1, ROCK, SCRIB, claudin-11) are coordinated in space and time, and which are required for each tissue-specific output, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model of SHROOM2 domain usage\", \"Tissue-specific partner requirements not systematically dissected\", \"Phosphorylation regulation of complex assembly incompletely defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 5, 10, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 2, 3]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 4, 10]}\n    ],\n    \"complexes\": [\n      \"SCRIB-SHROOM2-ROCK1 ternary complex\"\n    ],\n    \"partners\": [\n      \"MYO7A\",\n      \"TJP1\",\n      \"ROCK1\",\n      \"ROCK2\",\n      \"SCRIB\",\n      \"CLDN11\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}