{"gene":"MYO5B","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2008,"finding":"Loss-of-function mutations in MYO5B (encoding myosin Vb motor protein) cause microvillus inclusion disease, characterized by lack of apical microvilli on enterocytes, formation of intracellular microvillus-containing vacuoles, and mislocalization of transferrin receptor, establishing MYO5B as required for apical and basolateral protein trafficking in epithelial cells.","method":"Homozygosity mapping, identification of nonsense/missense mutations, immunofluorescence microscopy in patient enterocytes","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetics, immunofluorescence, electron microscopy) replicated across subsequent studies","pmids":["18724368"],"is_preprint":false},{"year":2010,"finding":"siRNA knockdown of MYO5B in polarized CaCo-2 cells recapitulates MVID cellular phenotype: loss of surface microvilli, increased microvillus inclusion formation, and subapical enrichment of PAS-positive endomembrane compartments, confirming MYO5B loss-of-function as the disease mechanism.","method":"siRNA knockdown, fluorescence microscopy, Western blotting, electron microscopy in polarized CaCo-2 cells","journal":"Human mutation","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (siRNA, fluorescence microscopy, EM, WB) in a defined cell model, independently confirming genetic findings","pmids":["20186687"],"is_preprint":false},{"year":2011,"finding":"MYO5B functions as an effector for Rab8a and Rab11a GTPases, with distinct binding interfaces: mutations Q1300L and Y1307C abolish Rab8a association, while Y1714E and Q1748R uncouple Rab11a association. Rab11a-MYO5B interaction is required for transferrin recycling in non-polarized cells, while both Rab8a and Rab11a associations are required for apical membrane trafficking and de novo lumen formation in polarized epithelial cysts.","method":"Site-directed mutagenesis of MYO5B tail domain, expression of mutant tails in HeLa and MDCK cells, transferrin recycling assay, polarized cyst culture, immunofluorescence","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis with functional validation across multiple cell systems, multiple orthogonal readouts","pmids":["21282656"],"is_preprint":false},{"year":2013,"finding":"Myo5B works in association with a Rab11a-Rab8a module to promote regulated exocytosis in bladder umbrella cells, facilitating transit of discoidal/fusiform vesicles through a subapical cortical actin cytoskeleton before membrane fusion. Rab11a acts upstream of Rab8a in this pathway.","method":"Expression of dominant-negative/constitutively active Rab constructs, live cell imaging, stretch-induced exocytosis assay in bladder umbrella cells","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional epistasis established in a specific cell type with multiple Rab mutant constructs, single lab","pmids":["23389633"],"is_preprint":false},{"year":2014,"finding":"A tripartite complex of Rab11a, Rab11-FIP2, and MYO5B regulates recycling endosome trafficking. Point mutations S229P or G233E in Rab11-FIP2 disrupt interaction with MYO5B, and perturbation of this interaction increases vesicle speed and track length in live cells, consistent with impaired MYO5B tethering to the cytoskeleton.","method":"Random mutagenesis, yeast two-hybrid assay, co-expression in HeLa/MDCK cells, live cell imaging of Rab11a vesicle movement, Rab11-FIP2 knockdown","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with live imaging and knockdown in multiple cell lines, multiple orthogonal methods in single lab","pmids":["24372966"],"is_preprint":false},{"year":2014,"finding":"MYO5B deficiency in hepatocytes causes abnormal cytoplasmic distribution of RAB11A and BSEP (bile salt export pump), implicating the MYO5B/RAB11A apical recycling endosome pathway in targeting BSEP to the canalicular membrane in hepatocytes and thereby in bile homeostasis.","method":"Immunohistochemistry of liver biopsies from MVID patients, electron microscopy of bile canaliculi","journal":"Hepatology (Baltimore, Md.)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — immunohistochemistry in patient tissue, replicated across multiple patients but no in vitro functional manipulation","pmids":["24375397"],"is_preprint":false},{"year":2015,"finding":"Myo5B participates in a selective apical exocytosis cascade in polarized epithelial cells: apical delivery of NHE3, CFTR, and GLUT5 requires sequential interaction of Rab11-Myo5B-Slp4a-Munc18-2-Vamp7 with Syntaxin 3. Brush border enzymes DPPIV and sucrase-isomaltase traffic to the apical membrane independently of this pathway.","method":"CRISPR genome editing to introduce patient MYO5B mutation in human epithelial cell line, immunofluorescence, co-immunoprecipitation, cargo-specific apical trafficking assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — genome editing combined with Co-IP and cargo-specific functional assays, multiple orthogonal methods establishing pathway order","pmids":["26553929"],"is_preprint":false},{"year":2016,"finding":"Germline and intestine-specific MYO5B knockout mice develop diarrhea with loss of apical transporters (NHE3, brush border enzymes) and microvillus inclusions predominantly in the duodenum but not ileum, revealing a neonatal duodenum-specific apical trafficking pathway dependent on MYO5B. Inclusions require a neonatal-specific pathway as adult tamoxifen-induced KO produces diarrhea without inclusions.","method":"Germline KO, VillinCre conditional KO, tamoxifen-inducible VillinCreERT2 KO mice; electron microscopy, immunofluorescence, phenotypic analysis at different developmental stages","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"High","confidence_rationale":"Tier 2 / Strong — three independent mouse models with tissue-specific and inducible deletions, multiple orthogonal readouts, replicated across developmental stages","pmids":["27019864"],"is_preprint":false},{"year":2018,"finding":"Loss of MYO5B causes selective mislocalization of apical transporters: NHE3, SGLT1, AQP7, and DRA are lost from the apical membrane (causing malabsorption), while CFTR remains at the apical membrane and shows increased activity (driving chloride secretion). This differential trafficking establishes that CFTR trafficking is largely MYO5B-independent.","method":"MYO5B KO mice and tamoxifen-inducible intestine-specific KO, immunostaining, immunoelectron microscopy, enteroids, Ussing chamber electrophysiology, patient duodenal biopsies","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple mouse models, functional electrophysiology (Ussing chambers), patient tissue validation, multiple orthogonal methods","pmids":["30144427"],"is_preprint":false},{"year":2019,"finding":"Loss of MYO5B causes giant late endosome formation via a chloride channel-sensitive mechanism (involving redistribution of chloride channels from cell periphery to late endosomes), which physically hinders mitotic spindle orientation. MYO5B loss also delays cytokinesis independently of endosome size. Rab7 availability is a limiting factor for giant late endosome formation; increasing Rab7 corrects spindle misorientation and cell delamination.","method":"MYO5B knockdown/KO in epithelial cells, live fluorescence microscopy, Rab7 overexpression rescue, chloride channel inhibitor treatment, quantitative spindle orientation analysis","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with rescue experiments, multiple orthogonal methods (live imaging, pharmacological inhibition, Rab7 rescue), single lab","pmids":["31682603"],"is_preprint":false},{"year":2020,"finding":"MYO5B motor domain-deficient mutants (but not full knockout) inhibit formation of specialized apical recycling endosomes and cause mislocalization of canalicular proteins (including BSEP) in hepatocytes by a dominant mechanism dependent on interaction with active Rab11a at the trans-Golgi Network/recycling endosome interface. MYO5B knockout alone does not produce canalicular localization defects in vitro or in vivo. This reveals a rab11a-mediated gain-of-toxic-function rather than loss-of-function as the mechanism for PFIC6.","method":"MYO5B KO (CRISPR) and mutant expression in vitro and in vivo, Rab11a mutant co-expression, immunofluorescence, live imaging of recycling endosomes, mouse models","journal":"Hepatology (Baltimore, Md.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — KO vs. mutant expression comparison with Rab11a epistasis, multiple orthogonal methods (KO, mutagenesis, in vivo), mechanistically rigorous","pmids":["31750554"],"is_preprint":false},{"year":2013,"finding":"MYO5B silencing by siRNA in gastric cancer cells promotes proliferation, invasion, and migration, and inhibits HGF-stimulated c-MET degradation, resulting in sustained c-MET levels and signaling. MYO5B promoter is epigenetically silenced in gastric cancer by aberrant DNA methylation and histone modification.","method":"siRNA knockdown, invasion/migration assays, Western blotting for c-MET, methylation-specific PCR, bisulfite sequencing, ChIP assay, 5-aza-dC/TSA treatment","journal":"Digestive diseases and sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with functional assays and mechanistic follow-up (c-MET signaling), epigenetic characterization by multiple methods, single lab","pmids":["23456500"],"is_preprint":false},{"year":2022,"finding":"UNC45A (a myosin co-chaperone) is required for myosin Vb protein expression; UNC45A depletion reduces MYO5B protein levels and disrupts RAB11A-positive recycling endosome positioning and microvilli development in intestinal and hepatic cells. Reintroduction of UNC45A or MYO5B rescues these defects.","method":"CRISPR-Cas9 KO of UNC45A, site-directed mutagenesis of UNC45A patient variant, Western blotting, confocal fluorescence and scanning electron microscopy, rescue experiments","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with rescue, multiple cell types, multiple orthogonal methods, single lab","pmids":["35421597"],"is_preprint":false},{"year":2022,"finding":"MYO5B directly binds GTP-bound Rab11b (but not GDP-bound Rab11b), and Rab11b-GTP activates the actin-activated ATPase activity of Myo5B. Myo5B and Rab11b co-localize on FN1-containing vesicles and move together in live cell imaging; siRNA knockdown of Myo5B or Rab11b reduces fibronectin secretion from pleural mesothelial cells without changing FN1 expression.","method":"Co-immunoprecipitation, in vitro ATPase activity assay, siRNA knockdown, live cell imaging, ELISA for FN1 secretion","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro ATPase assay with GTP/GDP-Rab11b, Co-IP, and live imaging, multiple orthogonal methods but single lab","pmids":["35563212"],"is_preprint":false},{"year":2021,"finding":"MYO5B loss in intestinal epithelial cells causes imbalance in Wnt/Notch signaling: Wnt ligand transcripts are significantly downregulated while Notch signaling molecules are unchanged, leading to reduced tuft cell populations and increased Paneth cells. Notch inhibition with dibenzazepine (DBZ) restores secretory cell populations in MYO5B-deficient intestine.","method":"Tamoxifen-inducible MYO5B KO mice, digital image analysis, RNA sequencing, organoid culture, pharmacological Notch inhibition with DBZ, LPA treatment","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO with RNA-seq, pharmacological rescue, and organoid validation, single lab","pmids":["34197342"],"is_preprint":false},{"year":2025,"finding":"MYO5B specifically governs MUC17 mucin levels at the brush border of enterocytes; MYO5B loss reduces MUC17 at the brush border without affecting overall MUC17 protein levels, distinguishing MYO5B's role from MYO1B which regulates total MUC17 protein levels.","method":"siRNA knockdown of MYO5B and MYO1B in enterocytes, confocal fluorescence microscopy, immunoblotting, brush border fractionation","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with multiple readouts distinguishing two motor proteins, single lab, peer-reviewed","pmids":["39661054"],"is_preprint":false},{"year":2025,"finding":"Expression of the MYO5B-p.(Arg824Cys) missense variant (but not liver-specific Myo5b knockout) in mice causes cholestasis with elevated serum liver enzymes and altered BSEP localization, establishing a toxic gain-of-function mechanism for MYO5B-associated PFIC rather than loss-of-function.","method":"CRISPR/Cas9 liver-specific Myo5b cKO mouse, adenoviral delivery of MYO5B missense variant to mice, serum biochemistry, liver histology, immunofluorescence for BSEP","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO vs. variant expression comparison with biochemical and histological readouts, single lab","pmids":["40127562"],"is_preprint":false},{"year":2026,"finding":"MYO5B deficiency impairs endosome-to-mitochondrion iron transfer: MYO5B-positive endosomes carrying transferrin show close physical associations with mitochondria, and MYO5B KO reduces mitochondrial iron content while causing endosomal iron accumulation, leading to mitochondrial dysfunction including fragmentation, reduced membrane potential, defective aerobic respiration, and increased oxidative stress. Introduction of full-length MYO5B restores mitochondrial membrane potential, while MVID-causing variants do not.","method":"CRISPR-Cas9 MYO5B KO in Caco2 cells, site-directed mutagenesis, fluorescence-based mitochondrial membrane potential and iron indicators, high-resolution respirometry, quantitative 3D fluorescence microscopy, carbonylated protein analysis from isolated mitochondria, rescue with full-length vs. mutant MYO5B","journal":"Gastroenterology report","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with mutagenesis rescue and multiple functional readouts, single lab, novel pathway claim","pmids":["41908891"],"is_preprint":false}],"current_model":"MYO5B encodes myosin Vb, an actin-based motor protein that functions as an effector for Rab8a and Rab11a GTPases to drive apical membrane recycling and exocytosis in polarized epithelial cells; it operates within a multiprotein cascade (Rab11a/Rab11-FIP2/Myo5B/Slp4a/Munc18-2/Vamp7/Stx3) to selectively deliver transporters (NHE3, SGLT1, DRA, AQP7) to the apical membrane while CFTR trafficking is largely MYO5B-independent, it traffics Rab11b-GTP-loaded vesicles via an ATPase activity stimulated by active Rab11b, and in hepatocytes motor-domain mutations cause a rab11a-dependent gain-of-toxic-function that mislocalizes BSEP from bile canaliculi; loss of MYO5B also disrupts late endosome size control (hindering mitotic spindle orientation), impairs endosome-to-mitochondrion iron transfer causing mitochondrial dysfunction, disrupts Wnt/Notch-dependent cell lineage differentiation, and regulates MUC17 mucin levels at the brush border."},"narrative":{"mechanistic_narrative":"MYO5B encodes myosin Vb, an actin-based motor that drives apical membrane recycling and exocytosis in polarized epithelial cells, where its loss-of-function mutations cause microvillus inclusion disease through failure of apical and basolateral protein trafficking [PMID:18724368, PMID:20186687]. MYO5B operates as an effector for the Rab8a and Rab11a GTPases through distinct tail-domain binding interfaces, with the Rab11a interaction supporting transferrin recycling and both interactions required for apical trafficking and de novo lumen formation [PMID:21282656]; it is tethered to the recycling pathway within a Rab11a–Rab11-FIP2–MYO5B complex whose disruption deregulates vesicle motility [PMID:24372966]. Within this framework MYO5B directs a selective apical exocytosis cascade in which Rab11–MYO5B–Slp4a–Munc18-2–Vamp7 act sequentially with Syntaxin 3 to deliver specific cargo, including NHE3, SGLT1, DRA, AQP7, and GLUT5, to the apical membrane, while CFTR and certain brush border enzymes traffic independently of MYO5B [PMID:26553929, PMID:30144427]. The motor binds GTP-loaded Rab11b, which stimulates its actin-activated ATPase activity to move cargo vesicles [PMID:35563212], and its function requires the myosin co-chaperone UNC45A for stable protein expression [PMID:35421597]. Beyond canonical trafficking, MYO5B controls late endosome size in a manner that governs mitotic spindle orientation [PMID:31682603], mediates endosome-to-mitochondrion iron transfer required for mitochondrial function [PMID:41908891], and shapes intestinal cell lineage through Wnt/Notch signaling balance [PMID:34197342]. In hepatocytes, MYO5B motor-domain missense variants—but not knockout—drive a Rab11a-dependent toxic gain-of-function that mislocalizes the bile salt export pump BSEP, establishing this dominant mechanism as the basis of MYO5B-associated progressive familial intrahepatic cholestasis (PFIC6) [PMID:31750554, PMID:40127562].","teleology":[{"year":2008,"claim":"Established that MYO5B is genetically required for epithelial protein trafficking, answering what gene underlies microvillus inclusion disease and what cellular process it serves.","evidence":"Homozygosity mapping and mutation identification with immunofluorescence in patient enterocytes","pmids":["18724368"],"confidence":"High","gaps":["Did not define MYO5B molecular partners","Did not distinguish loss- vs gain-of-function in non-intestinal tissues"]},{"year":2010,"claim":"Confirmed MYO5B loss-of-function as the direct disease mechanism by recapitulating the MVID cellular phenotype in a defined cell model.","evidence":"siRNA knockdown with fluorescence/electron microscopy in polarized CaCo-2 cells","pmids":["20186687"],"confidence":"High","gaps":["Did not identify the trafficking machinery MYO5B engages"]},{"year":2011,"claim":"Defined MYO5B as a dual Rab8a/Rab11a effector and mapped separable binding interfaces, explaining how one motor serves both recycling and apical morphogenesis.","evidence":"Tail-domain mutagenesis with transferrin recycling and polarized cyst assays in HeLa and MDCK cells","pmids":["21282656"],"confidence":"High","gaps":["Did not order Rab8a/Rab11a in the pathway","Did not identify downstream fusion machinery"]},{"year":2013,"claim":"Placed Rab11a upstream of Rab8a in a MYO5B-associated exocytosis module facilitating vesicle transit through cortical actin before fusion.","evidence":"Dominant-negative/constitutively active Rab constructs and stretch-induced exocytosis imaging in bladder umbrella cells","pmids":["23389633"],"confidence":"Medium","gaps":["Single cell type and single lab","Did not resolve fusion-machinery components"]},{"year":2013,"claim":"Linked MYO5B to cancer biology, showing its silencing sustains c-MET signaling and drives invasive behavior in gastric cancer cells.","evidence":"siRNA knockdown, invasion/migration assays, c-MET Western blotting, and epigenetic profiling (MSP, bisulfite sequencing, ChIP) in gastric cancer cells","pmids":["23456500"],"confidence":"Medium","gaps":["Mechanism linking MYO5B trafficking to c-MET degradation not resolved","Single lab"]},{"year":2014,"claim":"Resolved a tripartite Rab11a–Rab11-FIP2–MYO5B tethering complex whose disruption deregulates recycling-endosome vesicle motility.","evidence":"Random mutagenesis, yeast two-hybrid, co-expression and live imaging in HeLa/MDCK cells","pmids":["24372966"],"confidence":"High","gaps":["Did not link FIP2 mutations to disease","In vivo relevance not tested"]},{"year":2014,"claim":"Extended the MYO5B/Rab11a apical recycling pathway to hepatocytes by linking it to canalicular BSEP targeting and bile homeostasis.","evidence":"Immunohistochemistry of MVID patient liver biopsies and electron microscopy of bile canaliculi","pmids":["24375397"],"confidence":"Medium","gaps":["No in vitro functional manipulation","Could not distinguish loss- from gain-of-function"]},{"year":2015,"claim":"Defined the ordered selective apical exocytosis cascade (Rab11–MYO5B–Slp4a–Munc18-2–Vamp7–Stx3) and showed cargo selectivity for NHE3/CFTR/GLUT5 versus pathway-independent brush border enzymes.","evidence":"CRISPR knock-in of patient MYO5B mutation, Co-IP, and cargo-specific apical trafficking assays in human epithelial cells","pmids":["26553929"],"confidence":"High","gaps":["Cargo recognition specificity not mechanistically explained"]},{"year":2016,"claim":"Revealed developmental and regional specificity of MYO5B trafficking, showing duodenum- and neonatal-specific dependence for microvillus inclusion formation.","evidence":"Germline, conditional, and inducible MYO5B knockout mice with EM and immunofluorescence across developmental stages","pmids":["27019864"],"confidence":"High","gaps":["Molecular basis of the neonatal-specific pathway unknown","Why ileum is spared not resolved"]},{"year":2018,"claim":"Established differential cargo trafficking, defining CFTR as largely MYO5B-independent and explaining the secretory-versus-malabsorptive phenotype of MVID.","evidence":"Multiple knockout mouse models, immunoelectron microscopy, enteroids, Ussing chamber electrophysiology, and patient biopsies","pmids":["30144427"],"confidence":"High","gaps":["Why CFTR escapes MYO5B dependence is unexplained"]},{"year":2019,"claim":"Connected MYO5B to endosome size control and mitotic fidelity, showing giant late endosomes physically impair spindle orientation in a Rab7/chloride-channel-sensitive manner.","evidence":"MYO5B knockdown/KO with live imaging, chloride channel inhibition, and Rab7 overexpression rescue in epithelial cells","pmids":["31682603"],"confidence":"Medium","gaps":["Mechanism of chloride channel redistribution unclear","Single lab"]},{"year":2020,"claim":"Reframed MYO5B-associated cholestasis as a Rab11a-dependent toxic gain-of-function of motor-domain mutants rather than simple loss-of-function.","evidence":"CRISPR KO versus mutant expression with Rab11a epistasis, live imaging, and mouse models","pmids":["31750554"],"confidence":"High","gaps":["Molecular nature of the toxic species not defined","Why hepatocytes tolerate full KO but not mutant unclear"]},{"year":2021,"claim":"Showed MYO5B loss perturbs intestinal cell lineage by downregulating Wnt ligands and shifting secretory cell populations, a defect reversible by Notch inhibition.","evidence":"Inducible MYO5B KO mice, RNA-seq, organoids, and pharmacological Notch inhibition (DBZ)","pmids":["34197342"],"confidence":"Medium","gaps":["Link between trafficking defect and Wnt transcription not established","Single lab"]},{"year":2022,"claim":"Identified UNC45A as a co-chaperone required for MYO5B protein stability and recycling-endosome positioning, placing MYO5B downstream of chaperone control.","evidence":"CRISPR KO of UNC45A with rescue, microscopy in intestinal and hepatic cells","pmids":["35421597"],"confidence":"Medium","gaps":["Direct physical interaction not biochemically resolved","Single lab"]},{"year":2022,"claim":"Demonstrated GTP-bound Rab11b directly binds and activates MYO5B ATPase activity to drive fibronectin-vesicle secretion, extending Rab effector control to Rab11b.","evidence":"Co-IP, in vitro actin-activated ATPase assay with GTP/GDP-Rab11b, siRNA, live imaging, and FN1 ELISA in pleural mesothelial cells","pmids":["35563212"],"confidence":"Medium","gaps":["Single lab","Physiological role of Rab11b regulation in epithelia not tested"]},{"year":2025,"claim":"Distinguished MYO5B from MYO1B in mucin handling, showing MYO5B specifically governs MUC17 levels at the brush border without altering total protein.","evidence":"siRNA knockdown of MYO5B and MYO1B with confocal microscopy, immunoblotting, and brush border fractionation in enterocytes","pmids":["39661054"],"confidence":"Medium","gaps":["Mechanism of brush-border MUC17 retention unresolved","Single lab"]},{"year":2025,"claim":"Confirmed in vivo that a MYO5B missense variant, but not liver-specific knockout, causes cholestasis with BSEP mislocalization, cementing the toxic gain-of-function model for PFIC.","evidence":"Liver-specific Myo5b cKO and adenoviral missense-variant expression in mice with serum biochemistry and BSEP immunofluorescence","pmids":["40127562"],"confidence":"Medium","gaps":["Molecular identity of the toxic interaction not defined","Single lab"]},{"year":2026,"claim":"Revealed a non-canonical role for MYO5B in endosome-to-mitochondrion iron transfer, linking its loss to mitochondrial dysfunction and oxidative stress.","evidence":"CRISPR KO in Caco2 cells with mutagenesis rescue, mitochondrial iron/membrane potential indicators, respirometry, and 3D microscopy","pmids":["41908891"],"confidence":"Medium","gaps":["Mechanism of endosome-mitochondrion contact and iron handoff unresolved","Single lab and novel pathway claim"]},{"year":null,"claim":"How MYO5B achieves cargo-selective recognition—delivering specific apical transporters while excluding CFTR and certain enzymes—and the molecular basis of its toxic gain-of-function in cholestasis remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of MYO5B cargo selectivity","Toxic species in PFIC not molecularly defined","Mechanism coupling trafficking to Wnt and iron-transfer phenotypes unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[13]},{"term_id":"GO:0003774","term_label":"cytoskeletal motor activity","supporting_discovery_ids":[0,3,4]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[3,4,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,13]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[4,9,17]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,8]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[3,13]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3,4]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,6,8]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[2,6]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[8]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[14]}],"complexes":["Rab11a-Rab11-FIP2-MYO5B complex","Rab11-MYO5B-Slp4a-Munc18-2-Vamp7-Syntaxin 3 exocytic cascade"],"partners":["RAB11A","RAB8A","RAB11FIP2","RAB11B","UNC45A","STX3","STXBP2","VAMP7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9ULV0","full_name":"Unconventional myosin-Vb","aliases":[],"length_aa":1848,"mass_kda":213.7,"function":"May be involved in vesicular trafficking via its association with the CART complex. The CART complex is necessary for efficient transferrin receptor recycling but not for EGFR degradation. Required in a complex with RAB11A and RAB11FIP2 for the transport of NPC1L1 to the plasma membrane. Together with RAB11A participates in CFTR trafficking to the plasma membrane and TF (transferrin) recycling in nonpolarized cells. Together with RAB11A and RAB8A participates in epithelial cell polarization. Together with RAB25 regulates transcytosis. Required for proper localization of bile salt export pump ABCB11 at the apical/canalicular plasma membrane of hepatocytes (PubMed:34816459)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9ULV0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MYO5B","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":[{"gene":"CALM3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MYO5B","total_profiled":1310},"omim":[{"mim_id":"619868","title":"CHOLESTASIS, PROGRESSIVE FAMILIAL INTRAHEPATIC, 10; PFIC10","url":"https://www.omim.org/entry/619868"},{"mim_id":"619446","title":"RETINAL DYSTROPHY AND MICROVILLUS INCLUSION DISEASE; RDMVID","url":"https://www.omim.org/entry/619446"},{"mim_id":"619445","title":"DIARRHEA 12, WITH MICROVILLUS ATROPHY; DIAR12","url":"https://www.omim.org/entry/619445"},{"mim_id":"619377","title":"OSTEOOTOHEPATOENTERIC SYNDROME; OOHE","url":"https://www.omim.org/entry/619377"},{"mim_id":"613101","title":"HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS, FAMILIAL, 5, WITH OR WITHOUT MICROVILLUS INCLUSION DISEASE; FHL5","url":"https://www.omim.org/entry/613101"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":58.3}],"url":"https://www.proteinatlas.org/search/MYO5B"},"hgnc":{"alias_symbol":["KIAA1119"],"prev_symbol":[]},"alphafold":{"accession":"Q9ULV0","domains":[{"cath_id":"2.30.30,2.30.30","chopping":"2-64","consensus_level":"medium","plddt":83.1721,"start":2,"end":64},{"cath_id":"-","chopping":"700-750","consensus_level":"medium","plddt":84.298,"start":700,"end":750},{"cath_id":"-","chopping":"1473-1613_1825-1848","consensus_level":"medium","plddt":87.8621,"start":1473,"end":1848}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULV0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULV0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULV0-F1-predicted_aligned_error_v6.png","plddt_mean":75.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MYO5B","jax_strain_url":"https://www.jax.org/strain/search?query=MYO5B"},"sequence":{"accession":"Q9ULV0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9ULV0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9ULV0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULV0"}},"corpus_meta":[{"pmid":"18724368","id":"PMC_18724368","title":"MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity.","date":"2008","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18724368","citation_count":292,"is_preprint":false},{"pmid":"21282656","id":"PMC_21282656","title":"Rab GTPase-Myo5B complexes control membrane recycling and epithelial polarization.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21282656","citation_count":170,"is_preprint":false},{"pmid":"27532546","id":"PMC_27532546","title":"MYO5B mutations cause cholestasis with normal serum gamma-glutamyl transferase activity in children without microvillous inclusion disease.","date":"2016","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/27532546","citation_count":111,"is_preprint":false},{"pmid":"20186687","id":"PMC_20186687","title":"Loss-of-function of MYO5B is the main cause of microvillus inclusion disease: 15 novel mutations and a CaCo-2 RNAi cell model.","date":"2010","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/20186687","citation_count":104,"is_preprint":false},{"pmid":"24375397","id":"PMC_24375397","title":"MYO5B and bile salt export pump contribute to cholestatic liver disorder in microvillous inclusion disease.","date":"2014","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/24375397","citation_count":89,"is_preprint":false},{"pmid":"26553929","id":"PMC_26553929","title":"Cargo-selective apical exocytosis in epithelial cells is conducted by Myo5B, Slp4a, Vamp7, and Syntaxin 3.","date":"2015","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/26553929","citation_count":83,"is_preprint":false},{"pmid":"19006234","id":"PMC_19006234","title":"Navajo microvillous inclusion disease is due to a mutation in MYO5B.","date":"2008","source":"American journal of medical genetics. 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cells.\",\n      \"method\": \"Homozygosity mapping, identification of nonsense/missense mutations, immunofluorescence microscopy in patient enterocytes\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetics, immunofluorescence, electron microscopy) replicated across subsequent studies\",\n      \"pmids\": [\"18724368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"siRNA knockdown of MYO5B in polarized CaCo-2 cells recapitulates MVID cellular phenotype: loss of surface microvilli, increased microvillus inclusion formation, and subapical enrichment of PAS-positive endomembrane compartments, confirming MYO5B loss-of-function as the disease mechanism.\",\n      \"method\": \"siRNA knockdown, fluorescence microscopy, Western blotting, electron microscopy in polarized CaCo-2 cells\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (siRNA, fluorescence microscopy, EM, WB) in a defined cell model, independently confirming genetic findings\",\n      \"pmids\": [\"20186687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MYO5B functions as an effector for Rab8a and Rab11a GTPases, with distinct binding interfaces: mutations Q1300L and Y1307C abolish Rab8a association, while Y1714E and Q1748R uncouple Rab11a association. Rab11a-MYO5B interaction is required for transferrin recycling in non-polarized cells, while both Rab8a and Rab11a associations are required for apical membrane trafficking and de novo lumen formation in polarized epithelial cysts.\",\n      \"method\": \"Site-directed mutagenesis of MYO5B tail domain, expression of mutant tails in HeLa and MDCK cells, transferrin recycling assay, polarized cyst culture, immunofluorescence\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis with functional validation across multiple cell systems, multiple orthogonal readouts\",\n      \"pmids\": [\"21282656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Myo5B works in association with a Rab11a-Rab8a module to promote regulated exocytosis in bladder umbrella cells, facilitating transit of discoidal/fusiform vesicles through a subapical cortical actin cytoskeleton before membrane fusion. Rab11a acts upstream of Rab8a in this pathway.\",\n      \"method\": \"Expression of dominant-negative/constitutively active Rab constructs, live cell imaging, stretch-induced exocytosis assay in bladder umbrella cells\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional epistasis established in a specific cell type with multiple Rab mutant constructs, single lab\",\n      \"pmids\": [\"23389633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A tripartite complex of Rab11a, Rab11-FIP2, and MYO5B regulates recycling endosome trafficking. Point mutations S229P or G233E in Rab11-FIP2 disrupt interaction with MYO5B, and perturbation of this interaction increases vesicle speed and track length in live cells, consistent with impaired MYO5B tethering to the cytoskeleton.\",\n      \"method\": \"Random mutagenesis, yeast two-hybrid assay, co-expression in HeLa/MDCK cells, live cell imaging of Rab11a vesicle movement, Rab11-FIP2 knockdown\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with live imaging and knockdown in multiple cell lines, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"24372966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MYO5B deficiency in hepatocytes causes abnormal cytoplasmic distribution of RAB11A and BSEP (bile salt export pump), implicating the MYO5B/RAB11A apical recycling endosome pathway in targeting BSEP to the canalicular membrane in hepatocytes and thereby in bile homeostasis.\",\n      \"method\": \"Immunohistochemistry of liver biopsies from MVID patients, electron microscopy of bile canaliculi\",\n      \"journal\": \"Hepatology (Baltimore, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — immunohistochemistry in patient tissue, replicated across multiple patients but no in vitro functional manipulation\",\n      \"pmids\": [\"24375397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Myo5B participates in a selective apical exocytosis cascade in polarized epithelial cells: apical delivery of NHE3, CFTR, and GLUT5 requires sequential interaction of Rab11-Myo5B-Slp4a-Munc18-2-Vamp7 with Syntaxin 3. Brush border enzymes DPPIV and sucrase-isomaltase traffic to the apical membrane independently of this pathway.\",\n      \"method\": \"CRISPR genome editing to introduce patient MYO5B mutation in human epithelial cell line, immunofluorescence, co-immunoprecipitation, cargo-specific apical trafficking assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genome editing combined with Co-IP and cargo-specific functional assays, multiple orthogonal methods establishing pathway order\",\n      \"pmids\": [\"26553929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Germline and intestine-specific MYO5B knockout mice develop diarrhea with loss of apical transporters (NHE3, brush border enzymes) and microvillus inclusions predominantly in the duodenum but not ileum, revealing a neonatal duodenum-specific apical trafficking pathway dependent on MYO5B. Inclusions require a neonatal-specific pathway as adult tamoxifen-induced KO produces diarrhea without inclusions.\",\n      \"method\": \"Germline KO, VillinCre conditional KO, tamoxifen-inducible VillinCreERT2 KO mice; electron microscopy, immunofluorescence, phenotypic analysis at different developmental stages\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three independent mouse models with tissue-specific and inducible deletions, multiple orthogonal readouts, replicated across developmental stages\",\n      \"pmids\": [\"27019864\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Loss of MYO5B causes selective mislocalization of apical transporters: NHE3, SGLT1, AQP7, and DRA are lost from the apical membrane (causing malabsorption), while CFTR remains at the apical membrane and shows increased activity (driving chloride secretion). This differential trafficking establishes that CFTR trafficking is largely MYO5B-independent.\",\n      \"method\": \"MYO5B KO mice and tamoxifen-inducible intestine-specific KO, immunostaining, immunoelectron microscopy, enteroids, Ussing chamber electrophysiology, patient duodenal biopsies\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple mouse models, functional electrophysiology (Ussing chambers), patient tissue validation, multiple orthogonal methods\",\n      \"pmids\": [\"30144427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Loss of MYO5B causes giant late endosome formation via a chloride channel-sensitive mechanism (involving redistribution of chloride channels from cell periphery to late endosomes), which physically hinders mitotic spindle orientation. MYO5B loss also delays cytokinesis independently of endosome size. Rab7 availability is a limiting factor for giant late endosome formation; increasing Rab7 corrects spindle misorientation and cell delamination.\",\n      \"method\": \"MYO5B knockdown/KO in epithelial cells, live fluorescence microscopy, Rab7 overexpression rescue, chloride channel inhibitor treatment, quantitative spindle orientation analysis\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with rescue experiments, multiple orthogonal methods (live imaging, pharmacological inhibition, Rab7 rescue), single lab\",\n      \"pmids\": [\"31682603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MYO5B motor domain-deficient mutants (but not full knockout) inhibit formation of specialized apical recycling endosomes and cause mislocalization of canalicular proteins (including BSEP) in hepatocytes by a dominant mechanism dependent on interaction with active Rab11a at the trans-Golgi Network/recycling endosome interface. MYO5B knockout alone does not produce canalicular localization defects in vitro or in vivo. This reveals a rab11a-mediated gain-of-toxic-function rather than loss-of-function as the mechanism for PFIC6.\",\n      \"method\": \"MYO5B KO (CRISPR) and mutant expression in vitro and in vivo, Rab11a mutant co-expression, immunofluorescence, live imaging of recycling endosomes, mouse models\",\n      \"journal\": \"Hepatology (Baltimore, Md.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — KO vs. mutant expression comparison with Rab11a epistasis, multiple orthogonal methods (KO, mutagenesis, in vivo), mechanistically rigorous\",\n      \"pmids\": [\"31750554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MYO5B silencing by siRNA in gastric cancer cells promotes proliferation, invasion, and migration, and inhibits HGF-stimulated c-MET degradation, resulting in sustained c-MET levels and signaling. MYO5B promoter is epigenetically silenced in gastric cancer by aberrant DNA methylation and histone modification.\",\n      \"method\": \"siRNA knockdown, invasion/migration assays, Western blotting for c-MET, methylation-specific PCR, bisulfite sequencing, ChIP assay, 5-aza-dC/TSA treatment\",\n      \"journal\": \"Digestive diseases and sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with functional assays and mechanistic follow-up (c-MET signaling), epigenetic characterization by multiple methods, single lab\",\n      \"pmids\": [\"23456500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UNC45A (a myosin co-chaperone) is required for myosin Vb protein expression; UNC45A depletion reduces MYO5B protein levels and disrupts RAB11A-positive recycling endosome positioning and microvilli development in intestinal and hepatic cells. Reintroduction of UNC45A or MYO5B rescues these defects.\",\n      \"method\": \"CRISPR-Cas9 KO of UNC45A, site-directed mutagenesis of UNC45A patient variant, Western blotting, confocal fluorescence and scanning electron microscopy, rescue experiments\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with rescue, multiple cell types, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"35421597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MYO5B directly binds GTP-bound Rab11b (but not GDP-bound Rab11b), and Rab11b-GTP activates the actin-activated ATPase activity of Myo5B. Myo5B and Rab11b co-localize on FN1-containing vesicles and move together in live cell imaging; siRNA knockdown of Myo5B or Rab11b reduces fibronectin secretion from pleural mesothelial cells without changing FN1 expression.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ATPase activity assay, siRNA knockdown, live cell imaging, ELISA for FN1 secretion\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ATPase assay with GTP/GDP-Rab11b, Co-IP, and live imaging, multiple orthogonal methods but single lab\",\n      \"pmids\": [\"35563212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MYO5B loss in intestinal epithelial cells causes imbalance in Wnt/Notch signaling: Wnt ligand transcripts are significantly downregulated while Notch signaling molecules are unchanged, leading to reduced tuft cell populations and increased Paneth cells. Notch inhibition with dibenzazepine (DBZ) restores secretory cell populations in MYO5B-deficient intestine.\",\n      \"method\": \"Tamoxifen-inducible MYO5B KO mice, digital image analysis, RNA sequencing, organoid culture, pharmacological Notch inhibition with DBZ, LPA treatment\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO with RNA-seq, pharmacological rescue, and organoid validation, single lab\",\n      \"pmids\": [\"34197342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MYO5B specifically governs MUC17 mucin levels at the brush border of enterocytes; MYO5B loss reduces MUC17 at the brush border without affecting overall MUC17 protein levels, distinguishing MYO5B's role from MYO1B which regulates total MUC17 protein levels.\",\n      \"method\": \"siRNA knockdown of MYO5B and MYO1B in enterocytes, confocal fluorescence microscopy, immunoblotting, brush border fractionation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with multiple readouts distinguishing two motor proteins, single lab, peer-reviewed\",\n      \"pmids\": [\"39661054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Expression of the MYO5B-p.(Arg824Cys) missense variant (but not liver-specific Myo5b knockout) in mice causes cholestasis with elevated serum liver enzymes and altered BSEP localization, establishing a toxic gain-of-function mechanism for MYO5B-associated PFIC rather than loss-of-function.\",\n      \"method\": \"CRISPR/Cas9 liver-specific Myo5b cKO mouse, adenoviral delivery of MYO5B missense variant to mice, serum biochemistry, liver histology, immunofluorescence for BSEP\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO vs. variant expression comparison with biochemical and histological readouts, single lab\",\n      \"pmids\": [\"40127562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MYO5B deficiency impairs endosome-to-mitochondrion iron transfer: MYO5B-positive endosomes carrying transferrin show close physical associations with mitochondria, and MYO5B KO reduces mitochondrial iron content while causing endosomal iron accumulation, leading to mitochondrial dysfunction including fragmentation, reduced membrane potential, defective aerobic respiration, and increased oxidative stress. Introduction of full-length MYO5B restores mitochondrial membrane potential, while MVID-causing variants do not.\",\n      \"method\": \"CRISPR-Cas9 MYO5B KO in Caco2 cells, site-directed mutagenesis, fluorescence-based mitochondrial membrane potential and iron indicators, high-resolution respirometry, quantitative 3D fluorescence microscopy, carbonylated protein analysis from isolated mitochondria, rescue with full-length vs. mutant MYO5B\",\n      \"journal\": \"Gastroenterology report\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with mutagenesis rescue and multiple functional readouts, single lab, novel pathway claim\",\n      \"pmids\": [\"41908891\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MYO5B encodes myosin Vb, an actin-based motor protein that functions as an effector for Rab8a and Rab11a GTPases to drive apical membrane recycling and exocytosis in polarized epithelial cells; it operates within a multiprotein cascade (Rab11a/Rab11-FIP2/Myo5B/Slp4a/Munc18-2/Vamp7/Stx3) to selectively deliver transporters (NHE3, SGLT1, DRA, AQP7) to the apical membrane while CFTR trafficking is largely MYO5B-independent, it traffics Rab11b-GTP-loaded vesicles via an ATPase activity stimulated by active Rab11b, and in hepatocytes motor-domain mutations cause a rab11a-dependent gain-of-toxic-function that mislocalizes BSEP from bile canaliculi; loss of MYO5B also disrupts late endosome size control (hindering mitotic spindle orientation), impairs endosome-to-mitochondrion iron transfer causing mitochondrial dysfunction, disrupts Wnt/Notch-dependent cell lineage differentiation, and regulates MUC17 mucin levels at the brush border.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MYO5B encodes myosin Vb, an actin-based motor that drives apical membrane recycling and exocytosis in polarized epithelial cells, where its loss-of-function mutations cause microvillus inclusion disease through failure of apical and basolateral protein trafficking [#0, #1]. MYO5B operates as an effector for the Rab8a and Rab11a GTPases through distinct tail-domain binding interfaces, with the Rab11a interaction supporting transferrin recycling and both interactions required for apical trafficking and de novo lumen formation [#2]; it is tethered to the recycling pathway within a Rab11a–Rab11-FIP2–MYO5B complex whose disruption deregulates vesicle motility [#4]. Within this framework MYO5B directs a selective apical exocytosis cascade in which Rab11–MYO5B–Slp4a–Munc18-2–Vamp7 act sequentially with Syntaxin 3 to deliver specific cargo, including NHE3, SGLT1, DRA, AQP7, and GLUT5, to the apical membrane, while CFTR and certain brush border enzymes traffic independently of MYO5B [#6, #8]. The motor binds GTP-loaded Rab11b, which stimulates its actin-activated ATPase activity to move cargo vesicles [#13], and its function requires the myosin co-chaperone UNC45A for stable protein expression [#12]. Beyond canonical trafficking, MYO5B controls late endosome size in a manner that governs mitotic spindle orientation [#9], mediates endosome-to-mitochondrion iron transfer required for mitochondrial function [#17], and shapes intestinal cell lineage through Wnt/Notch signaling balance [#14]. In hepatocytes, MYO5B motor-domain missense variants—but not knockout—drive a Rab11a-dependent toxic gain-of-function that mislocalizes the bile salt export pump BSEP, establishing this dominant mechanism as the basis of MYO5B-associated progressive familial intrahepatic cholestasis (PFIC6) [#10, #16].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established that MYO5B is genetically required for epithelial protein trafficking, answering what gene underlies microvillus inclusion disease and what cellular process it serves.\",\n      \"evidence\": \"Homozygosity mapping and mutation identification with immunofluorescence in patient enterocytes\",\n      \"pmids\": [\"18724368\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define MYO5B molecular partners\", \"Did not distinguish loss- vs gain-of-function in non-intestinal tissues\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Confirmed MYO5B loss-of-function as the direct disease mechanism by recapitulating the MVID cellular phenotype in a defined cell model.\",\n      \"evidence\": \"siRNA knockdown with fluorescence/electron microscopy in polarized CaCo-2 cells\",\n      \"pmids\": [\"20186687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the trafficking machinery MYO5B engages\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined MYO5B as a dual Rab8a/Rab11a effector and mapped separable binding interfaces, explaining how one motor serves both recycling and apical morphogenesis.\",\n      \"evidence\": \"Tail-domain mutagenesis with transferrin recycling and polarized cyst assays in HeLa and MDCK cells\",\n      \"pmids\": [\"21282656\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not order Rab8a/Rab11a in the pathway\", \"Did not identify downstream fusion machinery\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed Rab11a upstream of Rab8a in a MYO5B-associated exocytosis module facilitating vesicle transit through cortical actin before fusion.\",\n      \"evidence\": \"Dominant-negative/constitutively active Rab constructs and stretch-induced exocytosis imaging in bladder umbrella cells\",\n      \"pmids\": [\"23389633\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cell type and single lab\", \"Did not resolve fusion-machinery components\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Linked MYO5B to cancer biology, showing its silencing sustains c-MET signaling and drives invasive behavior in gastric cancer cells.\",\n      \"evidence\": \"siRNA knockdown, invasion/migration assays, c-MET Western blotting, and epigenetic profiling (MSP, bisulfite sequencing, ChIP) in gastric cancer cells\",\n      \"pmids\": [\"23456500\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking MYO5B trafficking to c-MET degradation not resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved a tripartite Rab11a–Rab11-FIP2–MYO5B tethering complex whose disruption deregulates recycling-endosome vesicle motility.\",\n      \"evidence\": \"Random mutagenesis, yeast two-hybrid, co-expression and live imaging in HeLa/MDCK cells\",\n      \"pmids\": [\"24372966\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not link FIP2 mutations to disease\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended the MYO5B/Rab11a apical recycling pathway to hepatocytes by linking it to canalicular BSEP targeting and bile homeostasis.\",\n      \"evidence\": \"Immunohistochemistry of MVID patient liver biopsies and electron microscopy of bile canaliculi\",\n      \"pmids\": [\"24375397\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro functional manipulation\", \"Could not distinguish loss- from gain-of-function\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the ordered selective apical exocytosis cascade (Rab11–MYO5B–Slp4a–Munc18-2–Vamp7–Stx3) and showed cargo selectivity for NHE3/CFTR/GLUT5 versus pathway-independent brush border enzymes.\",\n      \"evidence\": \"CRISPR knock-in of patient MYO5B mutation, Co-IP, and cargo-specific apical trafficking assays in human epithelial cells\",\n      \"pmids\": [\"26553929\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cargo recognition specificity not mechanistically explained\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed developmental and regional specificity of MYO5B trafficking, showing duodenum- and neonatal-specific dependence for microvillus inclusion formation.\",\n      \"evidence\": \"Germline, conditional, and inducible MYO5B knockout mice with EM and immunofluorescence across developmental stages\",\n      \"pmids\": [\"27019864\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the neonatal-specific pathway unknown\", \"Why ileum is spared not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established differential cargo trafficking, defining CFTR as largely MYO5B-independent and explaining the secretory-versus-malabsorptive phenotype of MVID.\",\n      \"evidence\": \"Multiple knockout mouse models, immunoelectron microscopy, enteroids, Ussing chamber electrophysiology, and patient biopsies\",\n      \"pmids\": [\"30144427\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why CFTR escapes MYO5B dependence is unexplained\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected MYO5B to endosome size control and mitotic fidelity, showing giant late endosomes physically impair spindle orientation in a Rab7/chloride-channel-sensitive manner.\",\n      \"evidence\": \"MYO5B knockdown/KO with live imaging, chloride channel inhibition, and Rab7 overexpression rescue in epithelial cells\",\n      \"pmids\": [\"31682603\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of chloride channel redistribution unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Reframed MYO5B-associated cholestasis as a Rab11a-dependent toxic gain-of-function of motor-domain mutants rather than simple loss-of-function.\",\n      \"evidence\": \"CRISPR KO versus mutant expression with Rab11a epistasis, live imaging, and mouse models\",\n      \"pmids\": [\"31750554\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular nature of the toxic species not defined\", \"Why hepatocytes tolerate full KO but not mutant unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed MYO5B loss perturbs intestinal cell lineage by downregulating Wnt ligands and shifting secretory cell populations, a defect reversible by Notch inhibition.\",\n      \"evidence\": \"Inducible MYO5B KO mice, RNA-seq, organoids, and pharmacological Notch inhibition (DBZ)\",\n      \"pmids\": [\"34197342\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Link between trafficking defect and Wnt transcription not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified UNC45A as a co-chaperone required for MYO5B protein stability and recycling-endosome positioning, placing MYO5B downstream of chaperone control.\",\n      \"evidence\": \"CRISPR KO of UNC45A with rescue, microscopy in intestinal and hepatic cells\",\n      \"pmids\": [\"35421597\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical interaction not biochemically resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated GTP-bound Rab11b directly binds and activates MYO5B ATPase activity to drive fibronectin-vesicle secretion, extending Rab effector control to Rab11b.\",\n      \"evidence\": \"Co-IP, in vitro actin-activated ATPase assay with GTP/GDP-Rab11b, siRNA, live imaging, and FN1 ELISA in pleural mesothelial cells\",\n      \"pmids\": [\"35563212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Physiological role of Rab11b regulation in epithelia not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Distinguished MYO5B from MYO1B in mucin handling, showing MYO5B specifically governs MUC17 levels at the brush border without altering total protein.\",\n      \"evidence\": \"siRNA knockdown of MYO5B and MYO1B with confocal microscopy, immunoblotting, and brush border fractionation in enterocytes\",\n      \"pmids\": [\"39661054\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of brush-border MUC17 retention unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Confirmed in vivo that a MYO5B missense variant, but not liver-specific knockout, causes cholestasis with BSEP mislocalization, cementing the toxic gain-of-function model for PFIC.\",\n      \"evidence\": \"Liver-specific Myo5b cKO and adenoviral missense-variant expression in mice with serum biochemistry and BSEP immunofluorescence\",\n      \"pmids\": [\"40127562\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular identity of the toxic interaction not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed a non-canonical role for MYO5B in endosome-to-mitochondrion iron transfer, linking its loss to mitochondrial dysfunction and oxidative stress.\",\n      \"evidence\": \"CRISPR KO in Caco2 cells with mutagenesis rescue, mitochondrial iron/membrane potential indicators, respirometry, and 3D microscopy\",\n      \"pmids\": [\"41908891\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of endosome-mitochondrion contact and iron handoff unresolved\", \"Single lab and novel pathway claim\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MYO5B achieves cargo-selective recognition—delivering specific apical transporters while excluding CFTR and certain enzymes—and the molecular basis of its toxic gain-of-function in cholestasis remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of MYO5B cargo selectivity\", \"Toxic species in PFIC not molecularly defined\", \"Mechanism coupling trafficking to Wnt and iron-transfer phenotypes unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0003774\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [3, 4, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [4, 9, 17]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [3, 13]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 6, 8]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"complexes\": [\n      \"Rab11a-Rab11-FIP2-MYO5B complex\",\n      \"Rab11-MYO5B-Slp4a-Munc18-2-Vamp7-Syntaxin 3 exocytic cascade\"\n    ],\n    \"partners\": [\n      \"RAB11A\",\n      \"RAB8A\",\n      \"RAB11FIP2\",\n      \"RAB11B\",\n      \"UNC45A\",\n      \"STX3\",\n      \"STXBP2\",\n      \"VAMP7\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}