{"gene":"RAB35","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2006,"finding":"Rab35 localizes to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway; inhibition of Rab35 leads to cytokinesis failure, and Rab35 is required for intercellular bridge localization of PIP2 and the septin SEPT2 during cytokinesis.","method":"Dominant-negative overexpression, RNAi knockdown, fluorescence microscopy, endocytic marker accumulation assay in dividing cells","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (DN mutant, RNAi, live imaging), replicated in subsequent studies across multiple labs","pmids":["16950109"],"is_preprint":false},{"year":2008,"finding":"In C. elegans, the DENN-domain protein RME-4 functions as a GEF activator of RAB-35 (Rab35 ortholog); RME-4 binds RAB-35 in its GDP-loaded conformation, localizes to clathrin-coated pits, and recruits RAB-35 to endosomes for receptor recycling. Genetic epistasis places RAB-35 downstream of clathrin, upstream of RAB-7, and synergistically with recycling regulators RAB-11 and RME-1.","method":"C. elegans genetic screen, yeast two-hybrid binding assay, epistasis analysis, GFP localization, RNAi","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in vivo, physical interaction assay, multiple orthogonal methods, replicated mechanistically by subsequent mammalian work","pmids":["18354496"],"is_preprint":false},{"year":2008,"finding":"EPI64C (TBC1D10C) is a Rab35-specific GAP (demonstrated by in vitro GAP activity assay); both EPI64C overexpression and Rab35 dominant-negative impair transferrin recycling in T cells and inhibit immunological synapse formation. Rab35 is recruited to the immunological synapse and regulates TCR transport there.","method":"In vitro GAP activity assay, dominant-negative transfection, immunofluorescence, T cell conjugate assay, mass spectrometry identification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro GAP activity established biochemically, functional cell assays, single lab but multiple orthogonal methods","pmids":["18450757"],"is_preprint":false},{"year":2009,"finding":"Rab35 regulates actin filament assembly during Drosophila bristle development and filopodia formation; the actin-bundling protein fascin directly associates with GTP-bound (active) Rab35 as an effector. Targeting Rab35 to the outer mitochondrial membrane is sufficient to trigger actin recruitment.","method":"Drosophila genetics, pulldown of fascin with active Rab35, mitochondrial targeting assay, cell culture filopodia assay","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical interaction (pulldown), ectopic targeting reconstitution, genetic model organism validation, replicated by subsequent studies","pmids":["19729655"],"is_preprint":false},{"year":2009,"finding":"Rab35 colocalizes with actin filaments and with Cdc42; active Rab35 stimulates neurite outgrowth in PC12 and N1E-115 cells via a Cdc42-dependent pathway, and siRNA knockdown of Rab35 abolishes neurite outgrowth.","method":"siRNA knockdown, dominant-active/dominant-negative transfection, Cdc42 GEF activity assay in vivo and in vitro, fluorescence colocalization","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo and in vitro Cdc42 activation assay, siRNA knockdown with defined phenotype, single lab","pmids":["19289122"],"is_preprint":false},{"year":2010,"finding":"The DENN domain of connecdenn/DENND1A functions as a guanine nucleotide exchange factor (GEF) for Rab35, activating it to regulate endosomal membrane trafficking; loss of Rab35 activity enlarges early endosomes, inhibits MHC class I recycling, and prevents EHD1 recruitment to endosomal tubules.","method":"In vitro GEF activity assay, dominant-negative/siRNA knockdown, endosomal morphology assay, receptor recycling assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — enzymatic GEF activity established in vitro, multiple functional readouts, replicated and extended in subsequent studies","pmids":["20159556"],"is_preprint":false},{"year":2010,"finding":"All three connecdenn family members (DENND1A, DENND1B, DENND1C) function as GEFs for Rab35 through their DENN domains; they interact with clathrin and AP-2, linking Rab35 activation to the clathrin machinery. Connecdenn 2 knockdown enlarges early endosomes similarly to Rab35 loss.","method":"In vitro GEF activity assay, Co-IP, siRNA knockdown, yeast two-hybrid, endosomal morphology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — enzymatic activity established in vitro for all three family members, multiple methods, independent confirmation of Rab35 GEF function","pmids":["20154091"],"is_preprint":false},{"year":2010,"finding":"Rab35 mediates transport of Cdc42 and Rac1 to the plasma membrane during phagocytosis in Drosophila immune cells; recruitment of these Rho GTPases to filopodium and lamellipodium formation sites is Rab35-dependent and occurs via microtubule tracks.","method":"Drosophila genetic screen, fluorescence microscopy, RNAi knockdown, microtubule disruption assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic screen in Drosophila, live imaging, multiple readouts, single lab","pmids":["20065041"],"is_preprint":false},{"year":2011,"finding":"Active GTP-bound Rab35 directly interacts with the PI(4,5)P2 5-phosphatase OCRL, which is an effector of Rab35; Rab35 controls OCRL localization at the intercellular bridge during cytokinesis. Depletion of either Rab35 or OCRL causes abnormal PI(4,5)P2 and F-actin accumulation at the intercellular bridge, inhibiting abscission.","method":"Pulldown/Co-IP of GTP-Rab35 with OCRL, siRNA knockdown, live-cell imaging, F-actin/PIP2 staining, patient-derived cell lines","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct protein-protein interaction reconstituted, multiple orthogonal methods, disease-relevant patient cells, replicated across labs","pmids":["21706022"],"is_preprint":false},{"year":2011,"finding":"MICAL-L1 is a Rab35 effector on tubular recycling endosomes; overexpression of active Rab35 impairs MICAL-L1 recruitment to tubular recycling endosomes, while Rab35 depletion promotes enhanced MICAL-L1 localization there. Arf6 also forms a complex with MICAL-L1, and Rab35 acts as an upstream regulator of both MICAL-L1 and Arf6 to control Rab8a function.","method":"Co-IP, siRNA knockdown, overexpression, fluorescence colocalization","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction, knockdown/OE phenotype, multiple methods, single lab","pmids":["21951725"],"is_preprint":false},{"year":2011,"finding":"Rab35 regulates phagosome formation during FcγR-mediated phagocytosis in macrophages by recruiting ACAP2 (an ARF6 GAP) to the phagocytic cup in a GTP-Rab35-dependent manner; GTP-Rab35-dependent ACAP2 recruitment controls actin-mediated pseudopod extension.","method":"RNAi knockdown, dominant-negative/GTP-locked mutant expression, live-cell imaging, co-localization","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging with GTP-locked mutant, RNAi, single lab, mechanistic pathway placement","pmids":["22045739"],"is_preprint":false},{"year":2011,"finding":"Connecdenn 3 (DENND1C) directly binds actin via a unique C-terminal motif and couples Rab35 activation to the actin cytoskeleton, producing membrane extensions. Connecdenn 3 colocalizes with fascin and actin filaments, identifying it as the relevant GEF for Rab35's actin-bundling activity.","method":"Direct actin-binding assay, Rab35 GEF assay, Co-IP, fluorescence colocalization, cell morphology assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding established biochemically, GEF activity confirmed, multiple methods, single lab","pmids":["22072793"],"is_preprint":false},{"year":2011,"finding":"Genome-wide RUN domain screen identified RUSC2 as a GTP-Rab35-specific binding protein; the Rab35-binding domain (RBD35, residues 982-1199 of RUSC2) specifically traps active Rab35 but not other Rabs, and RBD35 overexpression in PC12 cells inhibits NGF-dependent neurite outgrowth.","method":"Yeast two-hybrid genome-wide screen, in vitro pulldown, cell overexpression assay","journal":"Cell structure and function","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro specificity confirmed, cell-based functional consequence, single lab","pmids":["21737958"],"is_preprint":false},{"year":2012,"finding":"ARF6 GTPase negatively regulates Rab35 activation through EPI64B, a Rab35 GAP that is an ARF6 effector; constitutively active ARF6 reduces Rab35 loading into the endocytic pathway at clathrin-coated pits, establishing a hierarchical ARF6→EPI64B→Rab35 cascade controlling endocytic recycling and cytokinesis.","method":"Dominant mutant expression, epistasis analysis, fluorescence microscopy, GFP-ARF6 and Rab35 colocalization","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis established by multiple mutant combinations, mechanistic pathway defined, replicated in same lab and others","pmids":["22226746"],"is_preprint":false},{"year":2012,"finding":"Rab35 functions as an effector of Rab35 → centaurin-β2 (ACAP2) → ARF6-GAP cascade during neurite outgrowth: Rab35 accumulates at Arf6-positive endosomes upon NGF stimulation and recruits centaurin-β2 (ACAP2), whose ARF6-GAP activity is required for neurite outgrowth of PC12 cells.","method":"siRNA knockdown and rescue experiments, fluorescence colocalization, dominant mutant expression, NGF stimulation assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockdown-rescue with specific mutants, defined pathway placement, multiple orthogonal readouts","pmids":["22344257"],"is_preprint":false},{"year":2012,"finding":"Rab35 maintains cadherins at the cell surface to promote cell-cell adhesion and suppresses Arf6 activity, thereby downregulating Arf6-dependent recycling of β1-integrin and EGF receptors to inhibit cell migration; this coordinates inverse intracellular sorting of cadherin and integrin.","method":"siRNA knockdown, fluorescence microscopy, receptor trafficking assays, cell migration/adhesion assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with multiple cargo readouts, defined pathway placement, single lab","pmids":["23264734"],"is_preprint":false},{"year":2012,"finding":"In Drosophila tracheal terminal cells, Rab35 and its apical membrane-localized GAP Whacked polarize seamless tube growth along the proximodistal axis; constitutive Rab35 activation (or Whacked loss) causes tube overgrowth at tips, while dominant-negative Rab35 (or Whacked overexpression) causes ectopic tubes. Dynein motor complex is also required for seamless tube growth.","method":"Drosophila genetics, loss-of-function and gain-of-function mutants, live imaging","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple alleles, gain- and loss-of-function phenotypes, defined in vivo developmental context","pmids":["22407366"],"is_preprint":false},{"year":2012,"finding":"Rab35 regulates cadherin-mediated adherens junction formation: Rab35 accumulates at cell-cell contacts in a cadherin-dependent manner; its knockdown impairs N- and M-cadherin recruitment and their association with p120 catenin, and Rab35 function is required for PIP5KIγ accumulation and PI(4,5)P2 production at cell-cell contacts. Rab35 knockdown also blocks myoblast fusion.","method":"siRNA knockdown, dominant-negative expression, Co-IP, fluorescence microscopy, biochemical fractionation","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple mechanistic readouts, Co-IP, single lab","pmids":["23197472"],"is_preprint":false},{"year":2012,"finding":"TBC1D13 is a Rab35-specific GAP (demonstrated in comprehensive in vitro screen); TBC1D13 displays in vivo GAP activity toward Rab35 and inhibits insulin-stimulated GLUT4 translocation in adipocytes. Constitutively active Rab35 (but not Rab10) reverses this block, placing Rab35 in the insulin-stimulated GLUT4 pathway.","method":"In vitro GAP activity screen, in vivo GAP activity assay, constitutively active rescue, GLUT4 translocation assay","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical GAP activity with comprehensive specificity screen, in vivo rescue, single lab but multiple methods","pmids":["22762500"],"is_preprint":false},{"year":2014,"finding":"Rab35 promotes NGF-induced neurite outgrowth by recruiting MICAL-L1 to Arf6-positive recycling endosomes, which in turn recruits Rab8, Rab13, and Rab36; Rab35 acts as a master Rab upstream of MICAL-L1-dependent scaffolding of downstream Rabs. Knockdown-rescue experiments confirmed non-redundant roles of each downstream Rab.","method":"siRNA knockdown and rescue experiments, fluorescence colocalization, NGF stimulation assay","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via knockdown-rescue, multiple cargo readouts, single lab","pmids":["25086062"],"is_preprint":false},{"year":2014,"finding":"Rab35 and its effector ACAP2 (ARF6 GAP) negatively regulate oligodendrocyte differentiation and myelination by inactivating Arf6; during differentiation, Rab35 and ACAP2 activities decrease while Arf6 activity increases. Knockdown of Rab35 or ACAP2 promotes differentiation, while Arf6 knockdown inhibits it.","method":"siRNA knockdown, GTPase activity assays, oligodendrocyte-neuron cocultures, myelination assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via sequential knockdowns, GTPase activity measured, single lab","pmids":["24600047"],"is_preprint":false},{"year":2015,"finding":"Rab35 GTPase acts as a precise spatial and temporal switch for OCRL recruitment on newborn clathrin-coated vesicles immediately after scission from the plasma membrane; Rab35 loading on CCVs follows DENND1A recruitment and EPI64B disappearance. Depletion of Rab35 or OCRL retains internalized receptors (CI-MPR) in peripheral clathrin-positive endosomes with abnormal PI(4,5)P2 and actin.","method":"Live-cell imaging, siRNA knockdown, fluorescence microscopy with timed events, receptor trafficking assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — real-time imaging establishes temporal sequence, multiple cargo readouts, builds on replicated Rab35-OCRL interaction","pmids":["26725203"],"is_preprint":false},{"year":2015,"finding":"Two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are critical for binding ACAP2/centaurin-β2; two asparagine residues (Asn-610 and Asn-691) of centaurin-β2 are key for specific Rab35 recognition. Neither a centaurin-β2-binding-deficient Rab35(T76S/T81A) mutant nor a Rab35-binding-deficient centaurin-β2(N610A/N691A) mutant can support neurite outgrowth.","method":"Deletion and site-directed mutagenesis, pulldown interaction assay, knockdown-rescue functional assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis defines specific interacting residues, functional rescue confirms specificity, single lab","pmids":["25694427"],"is_preprint":false},{"year":2015,"finding":"Rab35 delivered by clathrin-mediated endocytosis is required for sorting of clathrin-independent endocytosis (CIE) cargo proteins (CD44, CD98, CD147) into Arf6-associated tubules; Rab35 (or its effector ACAPs) inhibits Arf6-GTP to prevent lysosomal degradation of CIE cargo. Rab35 siRNA knockdown elevates Arf6-GTP levels and mimics the phenotype of CME inhibition.","method":"CME inhibition, siRNA knockdown, constitutively active Rab35 expression, GTPase activity assay, flow cytometry","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via rescue, GTPase activity measured, defined pathway placement, single lab","pmids":["25988331"],"is_preprint":false},{"year":2015,"finding":"Recycling of the KCa2.3 channel depends on Rab35; dominant-negative Rab35 or EPI64C (Rab35 GAP) overexpression traps KCa2.3 in an intracellular compartment. Co-IP confirmed association between KCa2.3 and Rab35. EPI64C's effect was dependent on its GAP activity.","method":"Dominant-negative expression, Co-IP, surface biotinylation, EPI64C knockdown, fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction confirmed, surface expression quantified, multiple methods, single lab","pmids":["20360009"],"is_preprint":false},{"year":2016,"finding":"Rab35 plays a crucial role in polarity initiation and apical lumen positioning during the first cell division of MDCK cyst development; Rab35 physically couples cytokinesis with apico-basal polarity initiation by tethering vesicles containing aPKC, Cdc42, Crumbs3, and Podocalyxin through a direct interaction between Rab35 and the cytoplasmic tail of Podocalyxin. Rab35 inactivation leads to complete polarity inversion.","method":"3D MDCK cyst culture, dominant-negative/constitutively active mutants, pulldown/Co-IP for Rab35-Podocalyxin interaction, fluorescence microscopy","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical interaction established, 3D culture system, multiple cargo readouts, dramatic and specific phenotype","pmids":["27040773"],"is_preprint":false},{"year":2016,"finding":"Neuronal activity drives Rab35 activation; activated Rab35 binds the ESCRT-0 protein Hrs (a novel Rab35 effector), recruiting the downstream ESCRT machinery to synaptic vesicle pools to initiate activity-dependent SV protein degradation.","method":"Rab35 activation assay in neurons, Co-IP of Rab35-Hrs, siRNA knockdown, immunofluorescence, degradation assay","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction, activity-dependent assay, defined pathway placement, single lab","pmids":["27535913"],"is_preprint":false},{"year":2016,"finding":"PRPK (p53-related protein kinase) negatively regulates axon elongation by promoting Rab35 protein degradation via the ubiquitin-proteasome pathway; MAP1B interacts with PRPK to prevent this degradation and thereby allows Rab35-mediated axonal elongation. Rab35 also regulates Cdc42 activity in neurons.","method":"MAP1B Co-IP with PRPK, proteasome inhibitor rescue, Rab35 overexpression rescue in MAP1B KO neurons, epistasis","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, proteasomal pathway confirmed by inhibitor rescue, epistasis in KO neurons, single lab","pmids":["27383602"],"is_preprint":false},{"year":2016,"finding":"Rab35 controls cilium length and membrane composition; GFP-Rab35 localizes to cilia, and Rab35 loss (siRNA, morpholinos, knockout) reduces cilium length in mammalian cells and zebrafish. Rab35 controls ciliary levels of Smoothened, Arl13b, and INPP5E, and interacts with Arl13b. GEF (DENND1B) and GAP (TBC1D10A) for Rab35 also localize to cilia and regulate ciliary length.","method":"siRNA knockdown, morpholino in zebrafish, KO cells, GFP localization, immunofluorescence, Co-IP","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple loss-of-function approaches (siRNA, morpholino, KO) in multiple organisms, direct localization, Co-IP interaction","pmids":["31432619"],"is_preprint":false},{"year":2017,"finding":"Active GTP-bound Rab35 accumulates on bacteria-containing endosomes and directly binds and recruits the autophagy receptor NDP52 to internalized bacteria; Rab35 also promotes NDP52 interaction with ubiquitin. Rab35 regulates NDP52 recruitment to damaged mitochondria and autophagosomes for mitophagy and autophagosome maturation; TBC1D10A (GAP for Rab35) inhibits and TBK1 stimulates this process.","method":"Active Rab35 pulldown with NDP52, Co-IP, Rab35 knockdown, GFP-Rab35 localization, mitophagy assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding of Rab35-GTP with NDP52 established biochemically, multiple forms of autophagy examined, TBK1 pathway mechanistically linked","pmids":["28848034"],"is_preprint":false},{"year":2017,"finding":"Rab35 is dynamically planar polarized in Drosophila epithelial intercalation; Rab35 compartments are enriched at contractile interfaces and act as a ratchet ensuring unidirectional interface shortening. Actomyosin disruption causes Rab35 compartments to fail to terminate, while loss of Rab35 allows contraction but prevents ratcheting. Mesoderm invagination also fails when Rab35 is compromised.","method":"Live imaging in Drosophila embryo, genetic loss-of-function, Myosin II disruption, quantitative interface analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — live in vivo imaging, multiple genetic conditions, defined functional readout of ratchet mechanism","pmids":["28883443"],"is_preprint":false},{"year":2017,"finding":"Rab35 regulates evoked exocytosis of Weibel-Palade bodies (WPB) in endothelial cells; TBC1D10A is a Rab35 GAP in these cells, and Rab35(Q67A) (GAP-insensitive mutant) rescues TBC1D10A-mediated inhibition. ACAP2 is a Rab35 effector in this context (Co-IP/pulldown), acting as a negative regulator of WPB exocytosis upstream of Arf6.","method":"Genome-wide RabGAP screen, pulldown/Co-IP of Rab35-ACAP2, Rab35 siRNA knockdown, dominant-negative Rab35, WPB exocytosis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen combined with specific rescue, Co-IP interaction confirmed, multiple mechanistic layers, single lab but comprehensive","pmids":["28566286"],"is_preprint":false},{"year":2018,"finding":"LRRK2 kinase phosphorylates RAB35, and this phosphorylation mediates LRRK2's role in α-synuclein propagation; the PD-linked G2019S LRRK2 mutation (increased kinase activity) enhances propagation. Constitutive activation of RAB35 overrides reduced α-synuclein propagation in lrk-1 mutant C. elegans. LRRK2 kinase inhibitor reduced α-synuclein aggregation via enhanced lysosomal degradation.","method":"Cell culture, C. elegans genetics, rodent models; LRRK2 kinase inhibitor treatment, constitutively active RAB35 rescue, phosphorylation assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple model organisms, kinase inhibitor, constitutive activation rescue establishes epistasis, mechanistic pathway defined","pmids":["30150626"],"is_preprint":false},{"year":2018,"finding":"Rab35 maintains cadherins at cell surface and suppresses Arf6 to coordinate cell adhesion vs. migration; conversely, Rab35 knockdown promotes EMT-like behavior. RAB35 expression is suppressed in cancers characterized by Arf6 hyperactivity.","method":"siRNA knockdown, receptor trafficking assay, Arf6 activity assay, cancer cell migration assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with multiple cargo and phenotypic readouts, GTPase activity measured, single lab","pmids":["23264734"],"is_preprint":false},{"year":2018,"finding":"RAB35-p85/PI3K axis controls oscillatory apical protrusions (CDRs) required for chemotactic migration; RAB35 is necessary and sufficient for CDR waves and directly regulates p85/PI3K activity polarity to guide directional chemotaxis and chemoinvasion.","method":"RNAi screen, live imaging, PI3K activity assay, chemotaxis assay, RAB35 overexpression","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi screen combined with direct PI3K activity measurement, chemotaxis functional readout, single lab","pmids":["29662076"],"is_preprint":false},{"year":2018,"finding":"In C. elegans, RAB-35 binds CNT-1 (an ARF-6 GAP) and removes ARF-6 from phagosome membranes, facilitating PI(4,5)P2 removal and phagolysosome maturation to eliminate cells dying by linker cell-type death (LCD). Epistasis analysis showed this pathway is distinct from apoptotic engulfment genes.","method":"C. elegans genetics, in vivo imaging, epistasis analysis, Co-IP of RAB-35 and CNT-1","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis, direct protein interaction confirmed, real-time imaging, multiple alleles","pmids":["30220571"],"is_preprint":false},{"year":2018,"finding":"In C. elegans, RAB-35 defines a third apoptotic cell clearance pathway acting in parallel to CED-1 and CED-5 pathways; RAB-35 promotes the switch of phagosomal PI(4,5)P2 to PI(3)P and recruitment of RAB-5 to phagosomal surfaces. Candidate GAP TBC-10 and GEFs FLCN-1 and RME-4 regulate RAB-35 activity in this context.","method":"C. elegans genetic screen, epistasis analysis, phosphoinositide imaging, cell corpse clearance assay, GFP localization","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic screen, multiple epistasis combinations, phosphoinositide switching directly measured, in vivo","pmids":["30138370"],"is_preprint":false},{"year":2019,"finding":"IL-17A induces recruitment of Rab35 and its GEF DENND1C to the IL-17R/Act1 complex in airway smooth muscle cells; activated Rab35 is required for PKCα activation and fascin phosphorylation at Ser39, allowing F-actin interaction with myosin to form stress fibers and enhance smooth muscle contraction.","method":"Co-IP of Rab35 with IL-17R/Act1, Rab35 knockdown, PKCα activity assay, fascin phosphorylation assay, contraction assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction, Rab35 knockdown with functional phenotype, pathway defined, single lab","pmids":["30683702"],"is_preprint":false},{"year":2020,"finding":"Rab35 promotes formation of tunneling nanotubes (TNTs) and TNT-mediated vesicle transfer in neuronal cells; Rab35-GTP, ACAP2, ARF6-GDP, and EHD1 act in a cascade to promote TNT formation. MICAL-L1, required for Rab35's effect on neurite outgrowth, has no effect on TNTs, indicating distinct downstream pathways.","method":"Overexpression, dominant-negative expression, quantitative TNT assay, vesicle transfer assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain/loss-of-function for pathway components, defined cascade, single lab","pmids":["33033331"],"is_preprint":false},{"year":2020,"finding":"Rab35 controls myelin growth by forming a complex with myotubularin-related phosphatases MTMR13 and MTMR2, downregulating PI 3-phosphate levels and thereby suppressing mTORC1 activation. Targeted disruption of Rab35 leads to elevated PI 3-phosphates, hyperactive mTORC1 signaling, and focal hypermyelination in vivo; pharmacological inhibition of PI(3,5)P2 synthesis or mTORC1 rescues this.","method":"Co-IP of Rab35-MTMR13/MTMR2, conditional KO mice, phosphoinositide measurements, mTORC1 activity assay, pharmacological rescue","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical complex formation, in vivo KO with specific lipid and signaling readouts, pharmacological rescue, multiple methods","pmids":["32503983"],"is_preprint":false},{"year":2020,"finding":"Rab35's C-terminal polybasic cluster is required for plasma membrane targeting; exchanging the C-terminal region of Rab35 with Rab10 shifts localization, and reducing basic amino acids in this region shifts Rab35 to Golgi membranes.","method":"Rab35-Rab10 chimera construction, confocal microscopy of RAW264 cells expressing EGFP-fused chimeras","journal":"Acta histochemica et cytochemica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-swap mutagenesis with direct localization readout, single lab","pmids":["32873993"],"is_preprint":false},{"year":2021,"finding":"BAG3 forms a complex with HSP70 and TBC1D10B (EPI64B, a Rab35 GAP); this complex attenuates TBC1D10B's ability to inactivate RAB35, supporting RAB35 activation and HRS recruitment for ESCRT-mediated endosomal tau clearance. Loss of BAG3 leads to impaired tau clearance.","method":"Mass spectrometry of BAG3 interactors, Co-IP, live-cell imaging, immunohistochemistry in AD brains, P301S tau mouse model","journal":"Biological psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction confirmed, disease model validation, multiple methods, single lab","pmids":["35000752"],"is_preprint":false},{"year":2021,"finding":"Rab35 negatively regulates Aβ production by sorting APP and BACE1 out of the endosomal network; Rab35 coordinates distinct trafficking steps for BACE1 (via effector OCRL) and APP (via effector ACAP2). Rab35 overexpression prevents amyloidogenic trafficking induced by high glucocorticoid levels.","method":"Rab35 overexpression/knockdown, APP/BACE1 trafficking assay, Aβ production measurement, effector-specific rescue","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined effector-specific mechanisms for two cargos, GC-stress model, single lab","pmids":["34876559"],"is_preprint":false},{"year":2023,"finding":"Prior to migrasome formation, PIP5K1A generates PI(4,5)P2 at migrasome formation sites; accumulated PI(4,5)P2 recruits Rab35 via interaction with the C-terminal polybasic cluster of Rab35. Active Rab35 then promotes migrasome formation by recruiting and concentrating integrin α5, mediated by a direct Rab35-integrin α5 interaction.","method":"PIP5K1A recruitment assay, PI(4,5)P2 measurement, Rab35 recruitment/localization, Co-IP of Rab35-integrin α5, migrasome quantification","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1 / Strong — sequential biochemical interactions established, direct Co-IP, functional migrasome readout, defined signaling cascade","pmids":["37142675"],"is_preprint":false},{"year":2023,"finding":"DENND2B functions as a Rab35 GEF that recruits and activates Rab35 at the intercellular cytokinetic bridge (ICB); DENND2B also interacts with active Rab35 as an effector. DENND2B knockdown delays abscission, causes F-actin accumulation at the ICB, impairs ESCRT-III recruitment, and triggers Aurora B kinase activation (NoCut checkpoint).","method":"GEF activity assay, Co-IP, siRNA knockdown, live-cell imaging, ESCRT-III localization assay, Aurora B activity assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical GEF activity established, Co-IP, multiple downstream mechanistic readouts, single lab but comprehensive","pmids":["37454296"],"is_preprint":false},{"year":2023,"finding":"The Shigella effector IcsB (a lysine Nε-fatty acylase) post-translationally acylates Rab35 in its polybasic region, non-canonically entrapping Rab35 at the bacterial-containing vacuole (BCV). Rab35 and IcsB are dispensable for initial BCV breakage but required for unwrapping damaged BCV remnants from Shigella.","method":"STED super-resolution imaging, IcsB fatty acylase activity assay, post-translational modification assay, genetic complementation, Shigella invasion assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — enzymatic post-translational modification established biochemically, super-resolution localization, functional invasion phenotype","pmids":["38568808"],"is_preprint":false},{"year":2023,"finding":"DENND1A and folliculin (FLCN) are distinct Rab35 GEFs required for Podocalyxin (PODXL) trafficking under different conditions: in 3D cysts, only DENND1A KO causes polarity inversion similar to Rab35 KO; in 2D culture, FLCN knockdown causes PODXL accumulation in actin-rich structures as in Rab35 knockdown. This defines two functional cascades: FLCN-Rab35-OCRL (2D) and DENND1A-Rab35-ACAP2 (3D).","method":"CRISPR KO, siRNA knockdown, rescue with GEF-activity domain, fluorescence microscopy, MDCK cell culture","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO combined with GEF-activity domain rescue, two distinct culture conditions, defined effector cascades","pmids":["31992598"],"is_preprint":false},{"year":2023,"finding":"USP32 (a deubiquitylating enzyme) binds Rab35 and protects it from proteasomal degradation by reducing Lys48-ubiquitination of Rab35, thereby maintaining elevated Rab35 levels in imatinib-resistant GISTs and promoting exosome secretion and drug resistance transmission.","method":"Co-IP of USP32-Rab35, ubiquitination assay (K48-specific), proteasome inhibitor rescue, siRNA knockdown, exosome secretion assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, specific ubiquitin linkage identified, rescue experiment, single lab","pmids":["36725886"],"is_preprint":false},{"year":2024,"finding":"A homozygous missense variant in the GTPase fold of RAB35 (R27H) found in a patient with neurodevelopmental disorder causes enhanced interaction with DENND1A and decreased interaction with effector MICAL1, indicating an inactive conformation. This variant activates Arf6 (normally under negative Rab35 control), delays cytokinesis, and alters primary cilia length, number, and Arl13b composition.","method":"Exome sequencing, Co-IP interaction assays for DENND1A and MICAL1, Arf6 activity assay, cytokinesis assay, cilia analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — human disease variant with multiple biochemical readouts, functional cellular consequences, defined molecular mechanism","pmids":["38432637"],"is_preprint":false},{"year":2024,"finding":"RAB35 is essential for early embryogenesis in mice (homozygous KO arrests at E7.5); conditional brain-specific KO causes severe hippocampal lamination defects due to impaired pyramidal neuron distribution. Loss of RAB35 affects levels of other RABs and cell adhesion molecules including contactin-2.","method":"Conditional KO mice, LacZ reporter, histology, immunofluorescence, quantitative proteomics","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KO with specific developmental phenotype, quantitative proteomics, multiple complementary approaches","pmids":["37085665"],"is_preprint":false},{"year":2024,"finding":"Congenital Rab35 KO in mice causes embryonic lethality; conditional loss in kidney/ureter causes hydronephrosis associated with disrupted actin, altered Arf6 polarity, reduced adherens junctions, loss of tight junctions, defective EGFR localization, and shortened primary cilia.","method":"Conditional KO mice, histology, immunofluorescence, western blotting","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional KO with multiple molecular and cellular readouts, replicated by preprint version","pmids":["38530365"],"is_preprint":false},{"year":2024,"finding":"MICAL1 is recruited by active Rab35 to HIV-1 budding sites, where it locally depolymerizes cortical actin to promote viral budding and ESCRT scission machinery recruitment; depletion of Rab35 impairs viral release and mimics MICAL1 depletion.","method":"siRNA depletion, super-resolution microscopy, live imaging, viral release assay, ESCRT recruitment assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct functional link between Rab35 and MICAL1 at budding sites, single preprint, not yet peer-reviewed","pmids":["bio_10.1101_2024.10.07.616958"],"is_preprint":true}],"current_model":"RAB35 is a small GTPase that cycles between GTP-bound (active) and GDP-bound (inactive) states controlled by multiple GEFs (DENND1A/B/C, DENND2B, folliculin, RME-4) and GAPs (EPI64B/TBC1D10B, EPI64C/TBC1D10C, TBC1D13, TBC1D10A, Whacked); active RAB35 localizes to the plasma membrane and early/recycling endosomes via its C-terminal polybasic cluster, where it recruits effectors including OCRL (to hydrolyze PI(4,5)P2 on newborn endosomes), ACAP2/centaurin-β2 (an Arf6 GAP linking RAB35 to actin remodeling via Arf6 inactivation), fascin (for actin bundling), MICAL-L1, Hrs, NDP52, MICAL1, and MTMR phosphatases, thereby coordinating fast endocytic recycling, cytokinetic abscission (including actin depolymerization at the intercellular bridge and ESCRT-III recruitment), apico-basal polarity establishment, ciliogenesis, neurite outgrowth, phagocytosis, autophagy, synaptic vesicle protein turnover, myelin growth control through mTORC1 suppression, migrasome biogenesis, and α-synuclein propagation downstream of LRRK2-mediated phosphorylation."},"narrative":{"mechanistic_narrative":"RAB35 is a small GTPase that operates as a master regulator of fast endocytic recycling and plasma-membrane-proximal membrane dynamics, coordinating cytokinesis, cell polarity, ciliogenesis, actin remodeling, and selective autophagy [PMID:16950109, PMID:27040773, PMID:31432619]. Its activity is set by a GTPase switch controlled by multiple DENN-domain GEFs (DENND1A/B/C, DENND2B) and folliculin that activate RAB35 at clathrin-coated structures, recycling endosomes, and the cytokinetic bridge [PMID:20159556, PMID:20154091, PMID:37454296, PMID:31992598], opposed by a panel of TBC-domain GAPs (EPI64B/TBC1D10B, EPI64C/TBC1D10C, TBC1D13, TBC1D10A) [PMID:18450757, PMID:22226746, PMID:22762500, PMID:28566286]; plasma-membrane and migrasome targeting depends on its C-terminal polybasic cluster, which reads PI(4,5)P2 [PMID:32873993, PMID:37142675]. A central output of active RAB35 is suppression of Arf6, achieved by recruiting the Arf6-GAP ACAP2/centaurin-β2, which links RAB35 to actin remodeling, neurite outgrowth, cadherin-based adhesion, and inhibition of migration [PMID:22344257, PMID:23264734, PMID:25694427]. In parallel RAB35 recruits the inositol 5-phosphatase OCRL to newborn endocytic vesicles and the cytokinetic intercellular bridge to hydrolyze PI(4,5)P2, controlling F-actin disassembly and abscission, while DENND2B-driven RAB35 activation enables ESCRT-III recruitment for bridge resolution [PMID:21706022, PMID:26725203, PMID:37454296]. RAB35 effectors also include the actin-bundler fascin [PMID:19729655], MICAL-L1 scaffolding of downstream Rabs [PMID:21951725, PMID:25086062], the ESCRT-0 component Hrs and the autophagy receptor NDP52, the latter directing antibacterial autophagy and mitophagy [PMID:27535913, PMID:28848034], and the myotubularin phosphatases MTMR2/MTMR13, through which RAB35 limits PI(3)P levels and mTORC1 to control myelin growth [PMID:32503983]. RAB35 establishes apico-basal polarity by tethering Podocalyxin-containing vesicles during the first division of epithelial cysts [PMID:27040773], and its activity is regulated by LRRK2 phosphorylation, linking it to α-synuclein propagation [PMID:30150626]. Loss of RAB35 is embryonic-lethal in mice and causes hippocampal lamination and renal (hydronephrosis) defects [PMID:37085665, PMID:38530365], and a homozygous R27H GTPase-fold variant that locks RAB35 in an inactive conformation underlies a human neurodevelopmental disorder [PMID:38432637].","teleology":[{"year":2006,"claim":"Established RAB35 as the GTPase governing a fast endocytic recycling route and revealed an unexpected requirement in cytokinesis, defining its core cell-biological remit.","evidence":"Dominant-negative and RNAi perturbation with live imaging and endocytic-marker assays in dividing cells","pmids":["16950109"],"confidence":"High","gaps":["GEFs/GAPs setting RAB35 activity unidentified at this stage","molecular effectors mediating recycling and abscission not yet known"]},{"year":2008,"claim":"Identified the first GEF (RME-4) and a specific GAP (EPI64C/TBC1D10C), placing RAB35 within the clathrin pathway and showing the GTPase cycle controls receptor recycling and immune-cell function.","evidence":"C. elegans genetic epistasis and Y2H binding; in vitro GAP activity assay and T cell conjugate/immunological synapse assays","pmids":["18354496","18450757"],"confidence":"High","gaps":["mammalian GEF identity not yet established","downstream effectors of recycling unresolved"]},{"year":2009,"claim":"Connected RAB35 to actin by identifying fascin as a direct GTP-dependent effector and a Cdc42-dependent role in neurite outgrowth, broadening RAB35 from trafficking into cytoskeletal control.","evidence":"Drosophila genetics, active-RAB35 pulldown of fascin, mitochondrial ectopic-targeting reconstitution; siRNA and Cdc42 activation assays in neuronal cell lines","pmids":["19729655","19289122"],"confidence":"High","gaps":["how RAB35 links to Cdc42 activation mechanistically unclear","relationship between fascin bundling and recycling roles undefined"]},{"year":2010,"claim":"Defined the connecdenn/DENND1 family as mammalian RAB35 GEFs coupled to clathrin/AP-2, explaining how RAB35 activation is spatially tied to endocytosis and recycling.","evidence":"In vitro GEF assays for DENND1A/B/C, Co-IP/Y2H with clathrin and AP-2, siRNA endosomal-morphology and receptor recycling readouts","pmids":["20159556","20154091"],"confidence":"High","gaps":["division of labor among the three GEF paralogs not resolved","structural basis of GEF specificity unknown"]},{"year":2011,"claim":"Resolved the abscission mechanism by showing active RAB35 recruits the PI(4,5)P2 5-phosphatase OCRL to the intercellular bridge to clear PI(4,5)P2 and F-actin, and catalogued additional effectors (MICAL-L1, RUSC2, ACAP2 in phagocytosis).","evidence":"GTP-RAB35 pulldown/Co-IP with OCRL, patient cells, Y2H RUN-domain screen, and RNAi/live imaging in macrophages","pmids":["21706022","21951725","21737958","22045739","22072793"],"confidence":"High","gaps":["temporal ordering of OCRL recruitment not yet defined","how multiple effectors are coordinated on one GTPase unclear"]},{"year":2012,"claim":"Built the RAB35–Arf6 regulatory axis: active RAB35 recruits ACAP2 to inactivate Arf6 driving neurite outgrowth, adhesion, and migration control, while Arf6→EPI64B feedback and additional GAPs (TBC1D13) place RAB35 in defined hierarchies and the GLUT4 pathway.","evidence":"Knockdown-rescue with specific mutants, epistasis, in vitro GAP screen, and cargo/GLUT4 trafficking assays","pmids":["22344257","22226746","23264734","22762500","22407366","23197472"],"confidence":"High","gaps":["precise residues mediating effector selection not yet mapped","how cadherin vs integrin sorting is partitioned mechanistically unresolved"]},{"year":2014,"claim":"Positioned RAB35 as a master Rab upstream of a MICAL-L1 scaffold that recruits Rab8/Rab13/Rab36, and showed RAB35–ACAP2 negatively regulates oligodendrocyte differentiation and myelination.","evidence":"siRNA knockdown-rescue with non-redundant downstream Rab tests; GTPase activity assays in oligodendrocyte cocultures","pmids":["25086062","24600047"],"confidence":"Medium","gaps":["direct vs indirect recruitment of downstream Rabs not distinguished","in vivo relevance of myelination role not yet shown at this stage"]},{"year":2015,"claim":"Established real-time temporal logic of the GTPase cycle on nascent vesicles and mapped specific switch-II residues governing effector selection, while extending cargo scope to CIE proteins and ion channels.","evidence":"Live-imaging of timed DENND1A/EPI64B/RAB35/OCRL events, site-directed mutagenesis of T76/T81, and trafficking/flow-cytometry assays","pmids":["26725203","25694427","25988331","20360009"],"confidence":"High","gaps":["structural mechanism of differential effector binding incomplete","generality of timed cascade across cargo types untested"]},{"year":2016,"claim":"Linked RAB35 to apico-basal polarity initiation via direct Podocalyxin tethering, to synaptic vesicle protein degradation via Hrs/ESCRT, and to ciliogenesis, and identified PRPK-mediated RAB35 turnover regulated by MAP1B.","evidence":"3D MDCK cyst culture with RAB35-Podocalyxin pulldown; Co-IP of RAB35-Hrs and activity assays in neurons; GFP localization and Co-IP in cilia; proteasome-inhibitor rescue","pmids":["27040773","27535913","31432619","27383602"],"confidence":"High","gaps":["how cytokinesis is coupled to polarity initiation mechanistically incompletely defined","ciliary effector hierarchy not fully resolved"]},{"year":2017,"claim":"Extended RAB35 effector logic to selective autophagy (direct NDP52 recruitment for xenophagy and mitophagy), regulated WPB exocytosis, and epithelial morphogenesis as a planar-polarized ratchet.","evidence":"Active-RAB35 pulldown of NDP52 and TBK1 linkage; genome-wide RabGAP screen with rescue for WPB; Drosophila live imaging of intercalation","pmids":["28848034","28566286","28883443"],"confidence":"High","gaps":["upstream signal triggering RAB35 activation in autophagy partly undefined","structural basis of NDP52 vs other effector binding unknown"]},{"year":2018,"claim":"Connected RAB35 to neurodegeneration via LRRK2 phosphorylation driving α-synuclein propagation, and defined roles in chemotactic migration (PI3K/p85), TNT formation, and conserved phagocytic clearance pathways.","evidence":"Multi-organism LRRK2 inhibitor and constitutively active RAB35 rescue; RNAi/PI3K assays; C. elegans genetics and Co-IP of RAB-35 with CNT-1","pmids":["30150626","29662076","30220571","30138370","23264734"],"confidence":"High","gaps":["functional consequence of RAB35 phosphorylation on its GTPase cycle not fully resolved","how PI3K activity is directly regulated by RAB35 unclear"]},{"year":2020,"claim":"Defined an in vivo myelin-growth-control mechanism whereby RAB35 complexes with MTMR2/MTMR13 to limit PI(3)P and mTORC1, and dissected RAB35's polybasic membrane-targeting determinant.","evidence":"Co-IP, conditional KO mice with phosphoinositide and mTORC1 readouts and pharmacological rescue; RAB35-Rab10 chimera localization","pmids":["32503983","32873993","33033331"],"confidence":"High","gaps":["tissue specificity of MTMR complex vs other effectors unresolved","how membrane targeting integrates with effector choice unclear"]},{"year":2021,"claim":"Implicated RAB35 in proteostasis and Alzheimer-relevant trafficking, showing BAG3/HSP70 stabilizes active RAB35 for ESCRT-mediated tau clearance and that RAB35 sorts APP/BACE1 to limit Aβ via distinct OCRL and ACAP2 cascades.","evidence":"Mass spectrometry, Co-IP, AD brain/tau-mouse models; effector-specific rescue with Aβ measurements","pmids":["35000752","34876559"],"confidence":"Medium","gaps":["causal contribution to human disease not established from cell models alone","how single RAB35 directs two cargos via different effectors mechanistically unresolved"]},{"year":2023,"claim":"Identified new regulatory inputs and outputs: PIP5K1A/PI(4,5)P2-driven RAB35 recruitment with direct integrin-α5 capture for migrasome biogenesis, DENND2B as the cytokinetic-bridge GEF, USP32 deubiquitination stabilizing RAB35, a Shigella acyltransferase that traps RAB35, and dual GEF cascades (FLCN vs DENND1A) for Podocalyxin.","evidence":"Sequential interaction/Co-IP assays, migrasome and abscission readouts, K48-ubiquitination assays, STED imaging of IcsB acylation, and CRISPR KO with GEF-domain rescue","pmids":["37142675","37454296","36725886","38568808","31992598"],"confidence":"High","gaps":["how distinct GEFs select context-specific effector cascades unresolved","stoichiometry of RAB35 with integrin-α5 at migrasomes unknown"]},{"year":2024,"claim":"Cemented physiological and pathological importance through a human inactivating R27H variant causing neurodevelopmental disease, in vivo KO mice revealing embryonic lethality and brain/kidney developmental defects, and a host-pathway role in HIV-1 budding via MICAL1.","evidence":"Exome sequencing with Co-IP/Arf6/cilia readouts; conditional KO mice with histology and proteomics; siRNA/super-resolution viral-release assays (preprint for HIV-1)","pmids":["38432637","37085665","38530365"],"confidence":"High","gaps":["genotype-phenotype spectrum of human RAB35 variants undefined","tissue-specific effector dependencies underlying KO phenotypes not fully dissected"]},{"year":null,"claim":"How a single GTPase selects among its many GEFs, GAPs, and competing effectors to execute context-specific outputs—and the structural and post-translational logic governing this selectivity—remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["no unifying structural model of effector discrimination","post-translational modification (phosphorylation, ubiquitination, acylation) integration into the GTPase cycle incompletely mapped","tissue-level effector hierarchies underlying in vivo phenotypes undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[0,2,18,48]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,14,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[13,14,15,23]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,40,43]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,5,9,21]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[28,48]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[40]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,5,21]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,8,44]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[29,39]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[13,14,34]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[16,25,49]}],"complexes":["RAB35-MTMR2/MTMR13 phosphatase complex","BAG3-HSP70-TBC1D10B complex"],"partners":["OCRL","ACAP2","DENND1A","MICAL-L1","NDP52","ARL13B","PODOCALYXIN","DENND2B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q15286","full_name":"Ras-related protein Rab-35","aliases":["GTP-binding protein RAY","Ras-related protein Rab-1C"],"length_aa":201,"mass_kda":23.0,"function":"The small GTPases Rab are key regulators of intracellular membrane trafficking, from the formation of transport vesicles to their fusion with membranes. Rabs cycle between an inactive GDP-bound form and an active GTP-bound form that is able to recruit to membranes different sets of downstream effectors directly responsible for vesicle formation, movement, tethering and fusion (PubMed:30905672). RAB35 is involved in the process of endocytosis and is an essential rate-limiting regulator of the fast recycling pathway back to the plasma membrane (PubMed:21951725). During cytokinesis, required for the postfurrowing terminal steps, namely for intercellular bridge stability and abscission, possibly by controlling phosphatidylinositol 4,5-bis phosphate (PIP2) and SEPT2 localization at the intercellular bridge (PubMed:16950109). May indirectly regulate neurite outgrowth. Together with TBC1D13 may be involved in regulation of insulin-induced glucose transporter SLC2A4/GLUT4 translocation to the plasma membrane in adipocytes (By similarity)","subcellular_location":"Cell membrane; Membrane, clathrin-coated pit; Cytoplasmic vesicle, clathrin-coated vesicle; Endosome; Melanosome","url":"https://www.uniprot.org/uniprotkb/Q15286/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAB35","classification":"Not Classified","n_dependent_lines":122,"n_total_lines":1208,"dependency_fraction":0.10099337748344371},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000111737","cell_line_id":"CID000433","localizations":[{"compartment":"membrane","grade":3},{"compartment":"vesicles","grade":3},{"compartment":"cell_contact","grade":2},{"compartment":"cytoplasmic","grade":2}],"interactors":[{"gene":"GDI1","stoichiometry":10.0},{"gene":"GDI2","stoichiometry":10.0},{"gene":"CHM","stoichiometry":0.2},{"gene":"TOMM40","stoichiometry":0.2},{"gene":"CHML","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000433","total_profiled":1310},"omim":[{"mim_id":"619563","title":"MICAL-LIKE PROTEIN 1; MICALL1","url":"https://www.omim.org/entry/619563"},{"mim_id":"616218","title":"TBC1 DOMAIN FAMILY, MEMBER 13; TBC1D13","url":"https://www.omim.org/entry/616218"},{"mim_id":"614222","title":"WARBURG MICRO SYNDROME 3; WARBM3","url":"https://www.omim.org/entry/614222"},{"mim_id":"613634","title":"DENN/MADD DOMAIN-CONTAINING PROTEIN 1C; DENND1C","url":"https://www.omim.org/entry/613634"},{"mim_id":"613633","title":"DENN/MADD DOMAIN-CONTAINING PROTEIN 1A; DENND1A","url":"https://www.omim.org/entry/613633"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RAB35"},"hgnc":{"alias_symbol":["H-ray"],"prev_symbol":[]},"alphafold":{"accession":"Q15286","domains":[{"cath_id":"3.40.50.300","chopping":"6-194","consensus_level":"high","plddt":91.2972,"start":6,"end":194}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15286","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q15286-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q15286-F1-predicted_aligned_error_v6.png","plddt_mean":89.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RAB35","jax_strain_url":"https://www.jax.org/strain/search?query=RAB35"},"sequence":{"accession":"Q15286","fasta_url":"https://rest.uniprot.org/uniprotkb/Q15286.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q15286/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15286"}},"corpus_meta":[{"pmid":"16950109","id":"PMC_16950109","title":"Rab35 regulates an endocytic recycling pathway essential for the terminal steps of cytokinesis.","date":"2006","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/16950109","citation_count":311,"is_preprint":false},{"pmid":"21706022","id":"PMC_21706022","title":"Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis.","date":"2011","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/21706022","citation_count":241,"is_preprint":false},{"pmid":"30943982","id":"PMC_30943982","title":"Long non-coding RNA HOTAIR promotes exosome secretion by regulating RAB35 and SNAP23 in hepatocellular carcinoma.","date":"2019","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30943982","citation_count":230,"is_preprint":false},{"pmid":"20159556","id":"PMC_20159556","title":"The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes.","date":"2010","source":"Molecular 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\"finding\": \"Rab35 localizes to the plasma membrane and endocytic compartments and controls a fast endocytic recycling pathway; inhibition of Rab35 leads to cytokinesis failure, and Rab35 is required for intercellular bridge localization of PIP2 and the septin SEPT2 during cytokinesis.\",\n      \"method\": \"Dominant-negative overexpression, RNAi knockdown, fluorescence microscopy, endocytic marker accumulation assay in dividing cells\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (DN mutant, RNAi, live imaging), replicated in subsequent studies across multiple labs\",\n      \"pmids\": [\"16950109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In C. elegans, the DENN-domain protein RME-4 functions as a GEF activator of RAB-35 (Rab35 ortholog); RME-4 binds RAB-35 in its GDP-loaded conformation, localizes to clathrin-coated pits, and recruits RAB-35 to endosomes for receptor recycling. Genetic epistasis places RAB-35 downstream of clathrin, upstream of RAB-7, and synergistically with recycling regulators RAB-11 and RME-1.\",\n      \"method\": \"C. elegans genetic screen, yeast two-hybrid binding assay, epistasis analysis, GFP localization, RNAi\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in vivo, physical interaction assay, multiple orthogonal methods, replicated mechanistically by subsequent mammalian work\",\n      \"pmids\": [\"18354496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"EPI64C (TBC1D10C) is a Rab35-specific GAP (demonstrated by in vitro GAP activity assay); both EPI64C overexpression and Rab35 dominant-negative impair transferrin recycling in T cells and inhibit immunological synapse formation. Rab35 is recruited to the immunological synapse and regulates TCR transport there.\",\n      \"method\": \"In vitro GAP activity assay, dominant-negative transfection, immunofluorescence, T cell conjugate assay, mass spectrometry identification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro GAP activity established biochemically, functional cell assays, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"18450757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Rab35 regulates actin filament assembly during Drosophila bristle development and filopodia formation; the actin-bundling protein fascin directly associates with GTP-bound (active) Rab35 as an effector. Targeting Rab35 to the outer mitochondrial membrane is sufficient to trigger actin recruitment.\",\n      \"method\": \"Drosophila genetics, pulldown of fascin with active Rab35, mitochondrial targeting assay, cell culture filopodia assay\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical interaction (pulldown), ectopic targeting reconstitution, genetic model organism validation, replicated by subsequent studies\",\n      \"pmids\": [\"19729655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Rab35 colocalizes with actin filaments and with Cdc42; active Rab35 stimulates neurite outgrowth in PC12 and N1E-115 cells via a Cdc42-dependent pathway, and siRNA knockdown of Rab35 abolishes neurite outgrowth.\",\n      \"method\": \"siRNA knockdown, dominant-active/dominant-negative transfection, Cdc42 GEF activity assay in vivo and in vitro, fluorescence colocalization\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo and in vitro Cdc42 activation assay, siRNA knockdown with defined phenotype, single lab\",\n      \"pmids\": [\"19289122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The DENN domain of connecdenn/DENND1A functions as a guanine nucleotide exchange factor (GEF) for Rab35, activating it to regulate endosomal membrane trafficking; loss of Rab35 activity enlarges early endosomes, inhibits MHC class I recycling, and prevents EHD1 recruitment to endosomal tubules.\",\n      \"method\": \"In vitro GEF activity assay, dominant-negative/siRNA knockdown, endosomal morphology assay, receptor recycling assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enzymatic GEF activity established in vitro, multiple functional readouts, replicated and extended in subsequent studies\",\n      \"pmids\": [\"20159556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"All three connecdenn family members (DENND1A, DENND1B, DENND1C) function as GEFs for Rab35 through their DENN domains; they interact with clathrin and AP-2, linking Rab35 activation to the clathrin machinery. Connecdenn 2 knockdown enlarges early endosomes similarly to Rab35 loss.\",\n      \"method\": \"In vitro GEF activity assay, Co-IP, siRNA knockdown, yeast two-hybrid, endosomal morphology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enzymatic activity established in vitro for all three family members, multiple methods, independent confirmation of Rab35 GEF function\",\n      \"pmids\": [\"20154091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Rab35 mediates transport of Cdc42 and Rac1 to the plasma membrane during phagocytosis in Drosophila immune cells; recruitment of these Rho GTPases to filopodium and lamellipodium formation sites is Rab35-dependent and occurs via microtubule tracks.\",\n      \"method\": \"Drosophila genetic screen, fluorescence microscopy, RNAi knockdown, microtubule disruption assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen in Drosophila, live imaging, multiple readouts, single lab\",\n      \"pmids\": [\"20065041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Active GTP-bound Rab35 directly interacts with the PI(4,5)P2 5-phosphatase OCRL, which is an effector of Rab35; Rab35 controls OCRL localization at the intercellular bridge during cytokinesis. Depletion of either Rab35 or OCRL causes abnormal PI(4,5)P2 and F-actin accumulation at the intercellular bridge, inhibiting abscission.\",\n      \"method\": \"Pulldown/Co-IP of GTP-Rab35 with OCRL, siRNA knockdown, live-cell imaging, F-actin/PIP2 staining, patient-derived cell lines\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct protein-protein interaction reconstituted, multiple orthogonal methods, disease-relevant patient cells, replicated across labs\",\n      \"pmids\": [\"21706022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MICAL-L1 is a Rab35 effector on tubular recycling endosomes; overexpression of active Rab35 impairs MICAL-L1 recruitment to tubular recycling endosomes, while Rab35 depletion promotes enhanced MICAL-L1 localization there. Arf6 also forms a complex with MICAL-L1, and Rab35 acts as an upstream regulator of both MICAL-L1 and Arf6 to control Rab8a function.\",\n      \"method\": \"Co-IP, siRNA knockdown, overexpression, fluorescence colocalization\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction, knockdown/OE phenotype, multiple methods, single lab\",\n      \"pmids\": [\"21951725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Rab35 regulates phagosome formation during FcγR-mediated phagocytosis in macrophages by recruiting ACAP2 (an ARF6 GAP) to the phagocytic cup in a GTP-Rab35-dependent manner; GTP-Rab35-dependent ACAP2 recruitment controls actin-mediated pseudopod extension.\",\n      \"method\": \"RNAi knockdown, dominant-negative/GTP-locked mutant expression, live-cell imaging, co-localization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging with GTP-locked mutant, RNAi, single lab, mechanistic pathway placement\",\n      \"pmids\": [\"22045739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Connecdenn 3 (DENND1C) directly binds actin via a unique C-terminal motif and couples Rab35 activation to the actin cytoskeleton, producing membrane extensions. Connecdenn 3 colocalizes with fascin and actin filaments, identifying it as the relevant GEF for Rab35's actin-bundling activity.\",\n      \"method\": \"Direct actin-binding assay, Rab35 GEF assay, Co-IP, fluorescence colocalization, cell morphology assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding established biochemically, GEF activity confirmed, multiple methods, single lab\",\n      \"pmids\": [\"22072793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Genome-wide RUN domain screen identified RUSC2 as a GTP-Rab35-specific binding protein; the Rab35-binding domain (RBD35, residues 982-1199 of RUSC2) specifically traps active Rab35 but not other Rabs, and RBD35 overexpression in PC12 cells inhibits NGF-dependent neurite outgrowth.\",\n      \"method\": \"Yeast two-hybrid genome-wide screen, in vitro pulldown, cell overexpression assay\",\n      \"journal\": \"Cell structure and function\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro specificity confirmed, cell-based functional consequence, single lab\",\n      \"pmids\": [\"21737958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ARF6 GTPase negatively regulates Rab35 activation through EPI64B, a Rab35 GAP that is an ARF6 effector; constitutively active ARF6 reduces Rab35 loading into the endocytic pathway at clathrin-coated pits, establishing a hierarchical ARF6→EPI64B→Rab35 cascade controlling endocytic recycling and cytokinesis.\",\n      \"method\": \"Dominant mutant expression, epistasis analysis, fluorescence microscopy, GFP-ARF6 and Rab35 colocalization\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis established by multiple mutant combinations, mechanistic pathway defined, replicated in same lab and others\",\n      \"pmids\": [\"22226746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Rab35 functions as an effector of Rab35 → centaurin-β2 (ACAP2) → ARF6-GAP cascade during neurite outgrowth: Rab35 accumulates at Arf6-positive endosomes upon NGF stimulation and recruits centaurin-β2 (ACAP2), whose ARF6-GAP activity is required for neurite outgrowth of PC12 cells.\",\n      \"method\": \"siRNA knockdown and rescue experiments, fluorescence colocalization, dominant mutant expression, NGF stimulation assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockdown-rescue with specific mutants, defined pathway placement, multiple orthogonal readouts\",\n      \"pmids\": [\"22344257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Rab35 maintains cadherins at the cell surface to promote cell-cell adhesion and suppresses Arf6 activity, thereby downregulating Arf6-dependent recycling of β1-integrin and EGF receptors to inhibit cell migration; this coordinates inverse intracellular sorting of cadherin and integrin.\",\n      \"method\": \"siRNA knockdown, fluorescence microscopy, receptor trafficking assays, cell migration/adhesion assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with multiple cargo readouts, defined pathway placement, single lab\",\n      \"pmids\": [\"23264734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In Drosophila tracheal terminal cells, Rab35 and its apical membrane-localized GAP Whacked polarize seamless tube growth along the proximodistal axis; constitutive Rab35 activation (or Whacked loss) causes tube overgrowth at tips, while dominant-negative Rab35 (or Whacked overexpression) causes ectopic tubes. Dynein motor complex is also required for seamless tube growth.\",\n      \"method\": \"Drosophila genetics, loss-of-function and gain-of-function mutants, live imaging\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple alleles, gain- and loss-of-function phenotypes, defined in vivo developmental context\",\n      \"pmids\": [\"22407366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Rab35 regulates cadherin-mediated adherens junction formation: Rab35 accumulates at cell-cell contacts in a cadherin-dependent manner; its knockdown impairs N- and M-cadherin recruitment and their association with p120 catenin, and Rab35 function is required for PIP5KIγ accumulation and PI(4,5)P2 production at cell-cell contacts. Rab35 knockdown also blocks myoblast fusion.\",\n      \"method\": \"siRNA knockdown, dominant-negative expression, Co-IP, fluorescence microscopy, biochemical fractionation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple mechanistic readouts, Co-IP, single lab\",\n      \"pmids\": [\"23197472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TBC1D13 is a Rab35-specific GAP (demonstrated in comprehensive in vitro screen); TBC1D13 displays in vivo GAP activity toward Rab35 and inhibits insulin-stimulated GLUT4 translocation in adipocytes. Constitutively active Rab35 (but not Rab10) reverses this block, placing Rab35 in the insulin-stimulated GLUT4 pathway.\",\n      \"method\": \"In vitro GAP activity screen, in vivo GAP activity assay, constitutively active rescue, GLUT4 translocation assay\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical GAP activity with comprehensive specificity screen, in vivo rescue, single lab but multiple methods\",\n      \"pmids\": [\"22762500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Rab35 promotes NGF-induced neurite outgrowth by recruiting MICAL-L1 to Arf6-positive recycling endosomes, which in turn recruits Rab8, Rab13, and Rab36; Rab35 acts as a master Rab upstream of MICAL-L1-dependent scaffolding of downstream Rabs. Knockdown-rescue experiments confirmed non-redundant roles of each downstream Rab.\",\n      \"method\": \"siRNA knockdown and rescue experiments, fluorescence colocalization, NGF stimulation assay\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via knockdown-rescue, multiple cargo readouts, single lab\",\n      \"pmids\": [\"25086062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Rab35 and its effector ACAP2 (ARF6 GAP) negatively regulate oligodendrocyte differentiation and myelination by inactivating Arf6; during differentiation, Rab35 and ACAP2 activities decrease while Arf6 activity increases. Knockdown of Rab35 or ACAP2 promotes differentiation, while Arf6 knockdown inhibits it.\",\n      \"method\": \"siRNA knockdown, GTPase activity assays, oligodendrocyte-neuron cocultures, myelination assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via sequential knockdowns, GTPase activity measured, single lab\",\n      \"pmids\": [\"24600047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Rab35 GTPase acts as a precise spatial and temporal switch for OCRL recruitment on newborn clathrin-coated vesicles immediately after scission from the plasma membrane; Rab35 loading on CCVs follows DENND1A recruitment and EPI64B disappearance. Depletion of Rab35 or OCRL retains internalized receptors (CI-MPR) in peripheral clathrin-positive endosomes with abnormal PI(4,5)P2 and actin.\",\n      \"method\": \"Live-cell imaging, siRNA knockdown, fluorescence microscopy with timed events, receptor trafficking assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — real-time imaging establishes temporal sequence, multiple cargo readouts, builds on replicated Rab35-OCRL interaction\",\n      \"pmids\": [\"26725203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are critical for binding ACAP2/centaurin-β2; two asparagine residues (Asn-610 and Asn-691) of centaurin-β2 are key for specific Rab35 recognition. Neither a centaurin-β2-binding-deficient Rab35(T76S/T81A) mutant nor a Rab35-binding-deficient centaurin-β2(N610A/N691A) mutant can support neurite outgrowth.\",\n      \"method\": \"Deletion and site-directed mutagenesis, pulldown interaction assay, knockdown-rescue functional assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis defines specific interacting residues, functional rescue confirms specificity, single lab\",\n      \"pmids\": [\"25694427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Rab35 delivered by clathrin-mediated endocytosis is required for sorting of clathrin-independent endocytosis (CIE) cargo proteins (CD44, CD98, CD147) into Arf6-associated tubules; Rab35 (or its effector ACAPs) inhibits Arf6-GTP to prevent lysosomal degradation of CIE cargo. Rab35 siRNA knockdown elevates Arf6-GTP levels and mimics the phenotype of CME inhibition.\",\n      \"method\": \"CME inhibition, siRNA knockdown, constitutively active Rab35 expression, GTPase activity assay, flow cytometry\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via rescue, GTPase activity measured, defined pathway placement, single lab\",\n      \"pmids\": [\"25988331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Recycling of the KCa2.3 channel depends on Rab35; dominant-negative Rab35 or EPI64C (Rab35 GAP) overexpression traps KCa2.3 in an intracellular compartment. Co-IP confirmed association between KCa2.3 and Rab35. EPI64C's effect was dependent on its GAP activity.\",\n      \"method\": \"Dominant-negative expression, Co-IP, surface biotinylation, EPI64C knockdown, fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction confirmed, surface expression quantified, multiple methods, single lab\",\n      \"pmids\": [\"20360009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rab35 plays a crucial role in polarity initiation and apical lumen positioning during the first cell division of MDCK cyst development; Rab35 physically couples cytokinesis with apico-basal polarity initiation by tethering vesicles containing aPKC, Cdc42, Crumbs3, and Podocalyxin through a direct interaction between Rab35 and the cytoplasmic tail of Podocalyxin. Rab35 inactivation leads to complete polarity inversion.\",\n      \"method\": \"3D MDCK cyst culture, dominant-negative/constitutively active mutants, pulldown/Co-IP for Rab35-Podocalyxin interaction, fluorescence microscopy\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical interaction established, 3D culture system, multiple cargo readouts, dramatic and specific phenotype\",\n      \"pmids\": [\"27040773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Neuronal activity drives Rab35 activation; activated Rab35 binds the ESCRT-0 protein Hrs (a novel Rab35 effector), recruiting the downstream ESCRT machinery to synaptic vesicle pools to initiate activity-dependent SV protein degradation.\",\n      \"method\": \"Rab35 activation assay in neurons, Co-IP of Rab35-Hrs, siRNA knockdown, immunofluorescence, degradation assay\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction, activity-dependent assay, defined pathway placement, single lab\",\n      \"pmids\": [\"27535913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PRPK (p53-related protein kinase) negatively regulates axon elongation by promoting Rab35 protein degradation via the ubiquitin-proteasome pathway; MAP1B interacts with PRPK to prevent this degradation and thereby allows Rab35-mediated axonal elongation. Rab35 also regulates Cdc42 activity in neurons.\",\n      \"method\": \"MAP1B Co-IP with PRPK, proteasome inhibitor rescue, Rab35 overexpression rescue in MAP1B KO neurons, epistasis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, proteasomal pathway confirmed by inhibitor rescue, epistasis in KO neurons, single lab\",\n      \"pmids\": [\"27383602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rab35 controls cilium length and membrane composition; GFP-Rab35 localizes to cilia, and Rab35 loss (siRNA, morpholinos, knockout) reduces cilium length in mammalian cells and zebrafish. Rab35 controls ciliary levels of Smoothened, Arl13b, and INPP5E, and interacts with Arl13b. GEF (DENND1B) and GAP (TBC1D10A) for Rab35 also localize to cilia and regulate ciliary length.\",\n      \"method\": \"siRNA knockdown, morpholino in zebrafish, KO cells, GFP localization, immunofluorescence, Co-IP\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple loss-of-function approaches (siRNA, morpholino, KO) in multiple organisms, direct localization, Co-IP interaction\",\n      \"pmids\": [\"31432619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Active GTP-bound Rab35 accumulates on bacteria-containing endosomes and directly binds and recruits the autophagy receptor NDP52 to internalized bacteria; Rab35 also promotes NDP52 interaction with ubiquitin. Rab35 regulates NDP52 recruitment to damaged mitochondria and autophagosomes for mitophagy and autophagosome maturation; TBC1D10A (GAP for Rab35) inhibits and TBK1 stimulates this process.\",\n      \"method\": \"Active Rab35 pulldown with NDP52, Co-IP, Rab35 knockdown, GFP-Rab35 localization, mitophagy assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding of Rab35-GTP with NDP52 established biochemically, multiple forms of autophagy examined, TBK1 pathway mechanistically linked\",\n      \"pmids\": [\"28848034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Rab35 is dynamically planar polarized in Drosophila epithelial intercalation; Rab35 compartments are enriched at contractile interfaces and act as a ratchet ensuring unidirectional interface shortening. Actomyosin disruption causes Rab35 compartments to fail to terminate, while loss of Rab35 allows contraction but prevents ratcheting. Mesoderm invagination also fails when Rab35 is compromised.\",\n      \"method\": \"Live imaging in Drosophila embryo, genetic loss-of-function, Myosin II disruption, quantitative interface analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live in vivo imaging, multiple genetic conditions, defined functional readout of ratchet mechanism\",\n      \"pmids\": [\"28883443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Rab35 regulates evoked exocytosis of Weibel-Palade bodies (WPB) in endothelial cells; TBC1D10A is a Rab35 GAP in these cells, and Rab35(Q67A) (GAP-insensitive mutant) rescues TBC1D10A-mediated inhibition. ACAP2 is a Rab35 effector in this context (Co-IP/pulldown), acting as a negative regulator of WPB exocytosis upstream of Arf6.\",\n      \"method\": \"Genome-wide RabGAP screen, pulldown/Co-IP of Rab35-ACAP2, Rab35 siRNA knockdown, dominant-negative Rab35, WPB exocytosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen combined with specific rescue, Co-IP interaction confirmed, multiple mechanistic layers, single lab but comprehensive\",\n      \"pmids\": [\"28566286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LRRK2 kinase phosphorylates RAB35, and this phosphorylation mediates LRRK2's role in α-synuclein propagation; the PD-linked G2019S LRRK2 mutation (increased kinase activity) enhances propagation. Constitutive activation of RAB35 overrides reduced α-synuclein propagation in lrk-1 mutant C. elegans. LRRK2 kinase inhibitor reduced α-synuclein aggregation via enhanced lysosomal degradation.\",\n      \"method\": \"Cell culture, C. elegans genetics, rodent models; LRRK2 kinase inhibitor treatment, constitutively active RAB35 rescue, phosphorylation assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple model organisms, kinase inhibitor, constitutive activation rescue establishes epistasis, mechanistic pathway defined\",\n      \"pmids\": [\"30150626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Rab35 maintains cadherins at cell surface and suppresses Arf6 to coordinate cell adhesion vs. migration; conversely, Rab35 knockdown promotes EMT-like behavior. RAB35 expression is suppressed in cancers characterized by Arf6 hyperactivity.\",\n      \"method\": \"siRNA knockdown, receptor trafficking assay, Arf6 activity assay, cancer cell migration assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with multiple cargo and phenotypic readouts, GTPase activity measured, single lab\",\n      \"pmids\": [\"23264734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RAB35-p85/PI3K axis controls oscillatory apical protrusions (CDRs) required for chemotactic migration; RAB35 is necessary and sufficient for CDR waves and directly regulates p85/PI3K activity polarity to guide directional chemotaxis and chemoinvasion.\",\n      \"method\": \"RNAi screen, live imaging, PI3K activity assay, chemotaxis assay, RAB35 overexpression\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi screen combined with direct PI3K activity measurement, chemotaxis functional readout, single lab\",\n      \"pmids\": [\"29662076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In C. elegans, RAB-35 binds CNT-1 (an ARF-6 GAP) and removes ARF-6 from phagosome membranes, facilitating PI(4,5)P2 removal and phagolysosome maturation to eliminate cells dying by linker cell-type death (LCD). Epistasis analysis showed this pathway is distinct from apoptotic engulfment genes.\",\n      \"method\": \"C. elegans genetics, in vivo imaging, epistasis analysis, Co-IP of RAB-35 and CNT-1\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis, direct protein interaction confirmed, real-time imaging, multiple alleles\",\n      \"pmids\": [\"30220571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In C. elegans, RAB-35 defines a third apoptotic cell clearance pathway acting in parallel to CED-1 and CED-5 pathways; RAB-35 promotes the switch of phagosomal PI(4,5)P2 to PI(3)P and recruitment of RAB-5 to phagosomal surfaces. Candidate GAP TBC-10 and GEFs FLCN-1 and RME-4 regulate RAB-35 activity in this context.\",\n      \"method\": \"C. elegans genetic screen, epistasis analysis, phosphoinositide imaging, cell corpse clearance assay, GFP localization\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic screen, multiple epistasis combinations, phosphoinositide switching directly measured, in vivo\",\n      \"pmids\": [\"30138370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"IL-17A induces recruitment of Rab35 and its GEF DENND1C to the IL-17R/Act1 complex in airway smooth muscle cells; activated Rab35 is required for PKCα activation and fascin phosphorylation at Ser39, allowing F-actin interaction with myosin to form stress fibers and enhance smooth muscle contraction.\",\n      \"method\": \"Co-IP of Rab35 with IL-17R/Act1, Rab35 knockdown, PKCα activity assay, fascin phosphorylation assay, contraction assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction, Rab35 knockdown with functional phenotype, pathway defined, single lab\",\n      \"pmids\": [\"30683702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Rab35 promotes formation of tunneling nanotubes (TNTs) and TNT-mediated vesicle transfer in neuronal cells; Rab35-GTP, ACAP2, ARF6-GDP, and EHD1 act in a cascade to promote TNT formation. MICAL-L1, required for Rab35's effect on neurite outgrowth, has no effect on TNTs, indicating distinct downstream pathways.\",\n      \"method\": \"Overexpression, dominant-negative expression, quantitative TNT assay, vesicle transfer assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain/loss-of-function for pathway components, defined cascade, single lab\",\n      \"pmids\": [\"33033331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Rab35 controls myelin growth by forming a complex with myotubularin-related phosphatases MTMR13 and MTMR2, downregulating PI 3-phosphate levels and thereby suppressing mTORC1 activation. Targeted disruption of Rab35 leads to elevated PI 3-phosphates, hyperactive mTORC1 signaling, and focal hypermyelination in vivo; pharmacological inhibition of PI(3,5)P2 synthesis or mTORC1 rescues this.\",\n      \"method\": \"Co-IP of Rab35-MTMR13/MTMR2, conditional KO mice, phosphoinositide measurements, mTORC1 activity assay, pharmacological rescue\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical complex formation, in vivo KO with specific lipid and signaling readouts, pharmacological rescue, multiple methods\",\n      \"pmids\": [\"32503983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Rab35's C-terminal polybasic cluster is required for plasma membrane targeting; exchanging the C-terminal region of Rab35 with Rab10 shifts localization, and reducing basic amino acids in this region shifts Rab35 to Golgi membranes.\",\n      \"method\": \"Rab35-Rab10 chimera construction, confocal microscopy of RAW264 cells expressing EGFP-fused chimeras\",\n      \"journal\": \"Acta histochemica et cytochemica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-swap mutagenesis with direct localization readout, single lab\",\n      \"pmids\": [\"32873993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BAG3 forms a complex with HSP70 and TBC1D10B (EPI64B, a Rab35 GAP); this complex attenuates TBC1D10B's ability to inactivate RAB35, supporting RAB35 activation and HRS recruitment for ESCRT-mediated endosomal tau clearance. Loss of BAG3 leads to impaired tau clearance.\",\n      \"method\": \"Mass spectrometry of BAG3 interactors, Co-IP, live-cell imaging, immunohistochemistry in AD brains, P301S tau mouse model\",\n      \"journal\": \"Biological psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction confirmed, disease model validation, multiple methods, single lab\",\n      \"pmids\": [\"35000752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rab35 negatively regulates Aβ production by sorting APP and BACE1 out of the endosomal network; Rab35 coordinates distinct trafficking steps for BACE1 (via effector OCRL) and APP (via effector ACAP2). Rab35 overexpression prevents amyloidogenic trafficking induced by high glucocorticoid levels.\",\n      \"method\": \"Rab35 overexpression/knockdown, APP/BACE1 trafficking assay, Aβ production measurement, effector-specific rescue\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined effector-specific mechanisms for two cargos, GC-stress model, single lab\",\n      \"pmids\": [\"34876559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Prior to migrasome formation, PIP5K1A generates PI(4,5)P2 at migrasome formation sites; accumulated PI(4,5)P2 recruits Rab35 via interaction with the C-terminal polybasic cluster of Rab35. Active Rab35 then promotes migrasome formation by recruiting and concentrating integrin α5, mediated by a direct Rab35-integrin α5 interaction.\",\n      \"method\": \"PIP5K1A recruitment assay, PI(4,5)P2 measurement, Rab35 recruitment/localization, Co-IP of Rab35-integrin α5, migrasome quantification\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — sequential biochemical interactions established, direct Co-IP, functional migrasome readout, defined signaling cascade\",\n      \"pmids\": [\"37142675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DENND2B functions as a Rab35 GEF that recruits and activates Rab35 at the intercellular cytokinetic bridge (ICB); DENND2B also interacts with active Rab35 as an effector. DENND2B knockdown delays abscission, causes F-actin accumulation at the ICB, impairs ESCRT-III recruitment, and triggers Aurora B kinase activation (NoCut checkpoint).\",\n      \"method\": \"GEF activity assay, Co-IP, siRNA knockdown, live-cell imaging, ESCRT-III localization assay, Aurora B activity assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical GEF activity established, Co-IP, multiple downstream mechanistic readouts, single lab but comprehensive\",\n      \"pmids\": [\"37454296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The Shigella effector IcsB (a lysine Nε-fatty acylase) post-translationally acylates Rab35 in its polybasic region, non-canonically entrapping Rab35 at the bacterial-containing vacuole (BCV). Rab35 and IcsB are dispensable for initial BCV breakage but required for unwrapping damaged BCV remnants from Shigella.\",\n      \"method\": \"STED super-resolution imaging, IcsB fatty acylase activity assay, post-translational modification assay, genetic complementation, Shigella invasion assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enzymatic post-translational modification established biochemically, super-resolution localization, functional invasion phenotype\",\n      \"pmids\": [\"38568808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"DENND1A and folliculin (FLCN) are distinct Rab35 GEFs required for Podocalyxin (PODXL) trafficking under different conditions: in 3D cysts, only DENND1A KO causes polarity inversion similar to Rab35 KO; in 2D culture, FLCN knockdown causes PODXL accumulation in actin-rich structures as in Rab35 knockdown. This defines two functional cascades: FLCN-Rab35-OCRL (2D) and DENND1A-Rab35-ACAP2 (3D).\",\n      \"method\": \"CRISPR KO, siRNA knockdown, rescue with GEF-activity domain, fluorescence microscopy, MDCK cell culture\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO combined with GEF-activity domain rescue, two distinct culture conditions, defined effector cascades\",\n      \"pmids\": [\"31992598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP32 (a deubiquitylating enzyme) binds Rab35 and protects it from proteasomal degradation by reducing Lys48-ubiquitination of Rab35, thereby maintaining elevated Rab35 levels in imatinib-resistant GISTs and promoting exosome secretion and drug resistance transmission.\",\n      \"method\": \"Co-IP of USP32-Rab35, ubiquitination assay (K48-specific), proteasome inhibitor rescue, siRNA knockdown, exosome secretion assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, specific ubiquitin linkage identified, rescue experiment, single lab\",\n      \"pmids\": [\"36725886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A homozygous missense variant in the GTPase fold of RAB35 (R27H) found in a patient with neurodevelopmental disorder causes enhanced interaction with DENND1A and decreased interaction with effector MICAL1, indicating an inactive conformation. This variant activates Arf6 (normally under negative Rab35 control), delays cytokinesis, and alters primary cilia length, number, and Arl13b composition.\",\n      \"method\": \"Exome sequencing, Co-IP interaction assays for DENND1A and MICAL1, Arf6 activity assay, cytokinesis assay, cilia analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human disease variant with multiple biochemical readouts, functional cellular consequences, defined molecular mechanism\",\n      \"pmids\": [\"38432637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RAB35 is essential for early embryogenesis in mice (homozygous KO arrests at E7.5); conditional brain-specific KO causes severe hippocampal lamination defects due to impaired pyramidal neuron distribution. Loss of RAB35 affects levels of other RABs and cell adhesion molecules including contactin-2.\",\n      \"method\": \"Conditional KO mice, LacZ reporter, histology, immunofluorescence, quantitative proteomics\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KO with specific developmental phenotype, quantitative proteomics, multiple complementary approaches\",\n      \"pmids\": [\"37085665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Congenital Rab35 KO in mice causes embryonic lethality; conditional loss in kidney/ureter causes hydronephrosis associated with disrupted actin, altered Arf6 polarity, reduced adherens junctions, loss of tight junctions, defective EGFR localization, and shortened primary cilia.\",\n      \"method\": \"Conditional KO mice, histology, immunofluorescence, western blotting\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional KO with multiple molecular and cellular readouts, replicated by preprint version\",\n      \"pmids\": [\"38530365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MICAL1 is recruited by active Rab35 to HIV-1 budding sites, where it locally depolymerizes cortical actin to promote viral budding and ESCRT scission machinery recruitment; depletion of Rab35 impairs viral release and mimics MICAL1 depletion.\",\n      \"method\": \"siRNA depletion, super-resolution microscopy, live imaging, viral release assay, ESCRT recruitment assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct functional link between Rab35 and MICAL1 at budding sites, single preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.10.07.616958\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RAB35 is a small GTPase that cycles between GTP-bound (active) and GDP-bound (inactive) states controlled by multiple GEFs (DENND1A/B/C, DENND2B, folliculin, RME-4) and GAPs (EPI64B/TBC1D10B, EPI64C/TBC1D10C, TBC1D13, TBC1D10A, Whacked); active RAB35 localizes to the plasma membrane and early/recycling endosomes via its C-terminal polybasic cluster, where it recruits effectors including OCRL (to hydrolyze PI(4,5)P2 on newborn endosomes), ACAP2/centaurin-β2 (an Arf6 GAP linking RAB35 to actin remodeling via Arf6 inactivation), fascin (for actin bundling), MICAL-L1, Hrs, NDP52, MICAL1, and MTMR phosphatases, thereby coordinating fast endocytic recycling, cytokinetic abscission (including actin depolymerization at the intercellular bridge and ESCRT-III recruitment), apico-basal polarity establishment, ciliogenesis, neurite outgrowth, phagocytosis, autophagy, synaptic vesicle protein turnover, myelin growth control through mTORC1 suppression, migrasome biogenesis, and α-synuclein propagation downstream of LRRK2-mediated phosphorylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAB35 is a small GTPase that operates as a master regulator of fast endocytic recycling and plasma-membrane-proximal membrane dynamics, coordinating cytokinesis, cell polarity, ciliogenesis, actin remodeling, and selective autophagy [#0, #25, #28]. Its activity is set by a GTPase switch controlled by multiple DENN-domain GEFs (DENND1A/B/C, DENND2B) and folliculin that activate RAB35 at clathrin-coated structures, recycling endosomes, and the cytokinetic bridge [#5, #6, #44, #46], opposed by a panel of TBC-domain GAPs (EPI64B/TBC1D10B, EPI64C/TBC1D10C, TBC1D13, TBC1D10A) [#2, #13, #18, #31]; plasma-membrane and migrasome targeting depends on its C-terminal polybasic cluster, which reads PI(4,5)P2 [#40, #43]. A central output of active RAB35 is suppression of Arf6, achieved by recruiting the Arf6-GAP ACAP2/centaurin-β2, which links RAB35 to actin remodeling, neurite outgrowth, cadherin-based adhesion, and inhibition of migration [#14, #15, #22]. In parallel RAB35 recruits the inositol 5-phosphatase OCRL to newborn endocytic vesicles and the cytokinetic intercellular bridge to hydrolyze PI(4,5)P2, controlling F-actin disassembly and abscission, while DENND2B-driven RAB35 activation enables ESCRT-III recruitment for bridge resolution [#8, #21, #44]. RAB35 effectors also include the actin-bundler fascin [#3], MICAL-L1 scaffolding of downstream Rabs [#9, #19], the ESCRT-0 component Hrs and the autophagy receptor NDP52, the latter directing antibacterial autophagy and mitophagy [#26, #29], and the myotubularin phosphatases MTMR2/MTMR13, through which RAB35 limits PI(3)P levels and mTORC1 to control myelin growth [#39]. RAB35 establishes apico-basal polarity by tethering Podocalyxin-containing vesicles during the first division of epithelial cysts [#25], and its activity is regulated by LRRK2 phosphorylation, linking it to α-synuclein propagation [#32]. Loss of RAB35 is embryonic-lethal in mice and causes hippocampal lamination and renal (hydronephrosis) defects [#49, #50], and a homozygous R27H GTPase-fold variant that locks RAB35 in an inactive conformation underlies a human neurodevelopmental disorder [#48].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established RAB35 as the GTPase governing a fast endocytic recycling route and revealed an unexpected requirement in cytokinesis, defining its core cell-biological remit.\",\n      \"evidence\": \"Dominant-negative and RNAi perturbation with live imaging and endocytic-marker assays in dividing cells\",\n      \"pmids\": [\"16950109\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"GEFs/GAPs setting RAB35 activity unidentified at this stage\", \"molecular effectors mediating recycling and abscission not yet known\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified the first GEF (RME-4) and a specific GAP (EPI64C/TBC1D10C), placing RAB35 within the clathrin pathway and showing the GTPase cycle controls receptor recycling and immune-cell function.\",\n      \"evidence\": \"C. elegans genetic epistasis and Y2H binding; in vitro GAP activity assay and T cell conjugate/immunological synapse assays\",\n      \"pmids\": [\"18354496\", \"18450757\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"mammalian GEF identity not yet established\", \"downstream effectors of recycling unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connected RAB35 to actin by identifying fascin as a direct GTP-dependent effector and a Cdc42-dependent role in neurite outgrowth, broadening RAB35 from trafficking into cytoskeletal control.\",\n      \"evidence\": \"Drosophila genetics, active-RAB35 pulldown of fascin, mitochondrial ectopic-targeting reconstitution; siRNA and Cdc42 activation assays in neuronal cell lines\",\n      \"pmids\": [\"19729655\", \"19289122\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"how RAB35 links to Cdc42 activation mechanistically unclear\", \"relationship between fascin bundling and recycling roles undefined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the connecdenn/DENND1 family as mammalian RAB35 GEFs coupled to clathrin/AP-2, explaining how RAB35 activation is spatially tied to endocytosis and recycling.\",\n      \"evidence\": \"In vitro GEF assays for DENND1A/B/C, Co-IP/Y2H with clathrin and AP-2, siRNA endosomal-morphology and receptor recycling readouts\",\n      \"pmids\": [\"20159556\", \"20154091\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"division of labor among the three GEF paralogs not resolved\", \"structural basis of GEF specificity unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the abscission mechanism by showing active RAB35 recruits the PI(4,5)P2 5-phosphatase OCRL to the intercellular bridge to clear PI(4,5)P2 and F-actin, and catalogued additional effectors (MICAL-L1, RUSC2, ACAP2 in phagocytosis).\",\n      \"evidence\": \"GTP-RAB35 pulldown/Co-IP with OCRL, patient cells, Y2H RUN-domain screen, and RNAi/live imaging in macrophages\",\n      \"pmids\": [\"21706022\", \"21951725\", \"21737958\", \"22045739\", \"22072793\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"temporal ordering of OCRL recruitment not yet defined\", \"how multiple effectors are coordinated on one GTPase unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Built the RAB35–Arf6 regulatory axis: active RAB35 recruits ACAP2 to inactivate Arf6 driving neurite outgrowth, adhesion, and migration control, while Arf6→EPI64B feedback and additional GAPs (TBC1D13) place RAB35 in defined hierarchies and the GLUT4 pathway.\",\n      \"evidence\": \"Knockdown-rescue with specific mutants, epistasis, in vitro GAP screen, and cargo/GLUT4 trafficking assays\",\n      \"pmids\": [\"22344257\", \"22226746\", \"23264734\", \"22762500\", \"22407366\", \"23197472\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"precise residues mediating effector selection not yet mapped\", \"how cadherin vs integrin sorting is partitioned mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Positioned RAB35 as a master Rab upstream of a MICAL-L1 scaffold that recruits Rab8/Rab13/Rab36, and showed RAB35–ACAP2 negatively regulates oligodendrocyte differentiation and myelination.\",\n      \"evidence\": \"siRNA knockdown-rescue with non-redundant downstream Rab tests; GTPase activity assays in oligodendrocyte cocultures\",\n      \"pmids\": [\"25086062\", \"24600047\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct vs indirect recruitment of downstream Rabs not distinguished\", \"in vivo relevance of myelination role not yet shown at this stage\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established real-time temporal logic of the GTPase cycle on nascent vesicles and mapped specific switch-II residues governing effector selection, while extending cargo scope to CIE proteins and ion channels.\",\n      \"evidence\": \"Live-imaging of timed DENND1A/EPI64B/RAB35/OCRL events, site-directed mutagenesis of T76/T81, and trafficking/flow-cytometry assays\",\n      \"pmids\": [\"26725203\", \"25694427\", \"25988331\", \"20360009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"structural mechanism of differential effector binding incomplete\", \"generality of timed cascade across cargo types untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked RAB35 to apico-basal polarity initiation via direct Podocalyxin tethering, to synaptic vesicle protein degradation via Hrs/ESCRT, and to ciliogenesis, and identified PRPK-mediated RAB35 turnover regulated by MAP1B.\",\n      \"evidence\": \"3D MDCK cyst culture with RAB35-Podocalyxin pulldown; Co-IP of RAB35-Hrs and activity assays in neurons; GFP localization and Co-IP in cilia; proteasome-inhibitor rescue\",\n      \"pmids\": [\"27040773\", \"27535913\", \"31432619\", \"27383602\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"how cytokinesis is coupled to polarity initiation mechanistically incompletely defined\", \"ciliary effector hierarchy not fully resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended RAB35 effector logic to selective autophagy (direct NDP52 recruitment for xenophagy and mitophagy), regulated WPB exocytosis, and epithelial morphogenesis as a planar-polarized ratchet.\",\n      \"evidence\": \"Active-RAB35 pulldown of NDP52 and TBK1 linkage; genome-wide RabGAP screen with rescue for WPB; Drosophila live imaging of intercalation\",\n      \"pmids\": [\"28848034\", \"28566286\", \"28883443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"upstream signal triggering RAB35 activation in autophagy partly undefined\", \"structural basis of NDP52 vs other effector binding unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected RAB35 to neurodegeneration via LRRK2 phosphorylation driving α-synuclein propagation, and defined roles in chemotactic migration (PI3K/p85), TNT formation, and conserved phagocytic clearance pathways.\",\n      \"evidence\": \"Multi-organism LRRK2 inhibitor and constitutively active RAB35 rescue; RNAi/PI3K assays; C. elegans genetics and Co-IP of RAB-35 with CNT-1\",\n      \"pmids\": [\"30150626\", \"29662076\", \"30220571\", \"30138370\", \"23264734\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"functional consequence of RAB35 phosphorylation on its GTPase cycle not fully resolved\", \"how PI3K activity is directly regulated by RAB35 unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined an in vivo myelin-growth-control mechanism whereby RAB35 complexes with MTMR2/MTMR13 to limit PI(3)P and mTORC1, and dissected RAB35's polybasic membrane-targeting determinant.\",\n      \"evidence\": \"Co-IP, conditional KO mice with phosphoinositide and mTORC1 readouts and pharmacological rescue; RAB35-Rab10 chimera localization\",\n      \"pmids\": [\"32503983\", \"32873993\", \"33033331\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"tissue specificity of MTMR complex vs other effectors unresolved\", \"how membrane targeting integrates with effector choice unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Implicated RAB35 in proteostasis and Alzheimer-relevant trafficking, showing BAG3/HSP70 stabilizes active RAB35 for ESCRT-mediated tau clearance and that RAB35 sorts APP/BACE1 to limit Aβ via distinct OCRL and ACAP2 cascades.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, AD brain/tau-mouse models; effector-specific rescue with Aβ measurements\",\n      \"pmids\": [\"35000752\", \"34876559\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"causal contribution to human disease not established from cell models alone\", \"how single RAB35 directs two cargos via different effectors mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified new regulatory inputs and outputs: PIP5K1A/PI(4,5)P2-driven RAB35 recruitment with direct integrin-α5 capture for migrasome biogenesis, DENND2B as the cytokinetic-bridge GEF, USP32 deubiquitination stabilizing RAB35, a Shigella acyltransferase that traps RAB35, and dual GEF cascades (FLCN vs DENND1A) for Podocalyxin.\",\n      \"evidence\": \"Sequential interaction/Co-IP assays, migrasome and abscission readouts, K48-ubiquitination assays, STED imaging of IcsB acylation, and CRISPR KO with GEF-domain rescue\",\n      \"pmids\": [\"37142675\", \"37454296\", \"36725886\", \"38568808\", \"31992598\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"how distinct GEFs select context-specific effector cascades unresolved\", \"stoichiometry of RAB35 with integrin-α5 at migrasomes unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cemented physiological and pathological importance through a human inactivating R27H variant causing neurodevelopmental disease, in vivo KO mice revealing embryonic lethality and brain/kidney developmental defects, and a host-pathway role in HIV-1 budding via MICAL1.\",\n      \"evidence\": \"Exome sequencing with Co-IP/Arf6/cilia readouts; conditional KO mice with histology and proteomics; siRNA/super-resolution viral-release assays (preprint for HIV-1)\",\n      \"pmids\": [\"38432637\", \"37085665\", \"38530365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"genotype-phenotype spectrum of human RAB35 variants undefined\", \"tissue-specific effector dependencies underlying KO phenotypes not fully dissected\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single GTPase selects among its many GEFs, GAPs, and competing effectors to execute context-specific outputs—and the structural and post-translational logic governing this selectivity—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"no unifying structural model of effector discrimination\", \"post-translational modification (phosphorylation, ubiquitination, acylation) integration into the GTPase cycle incompletely mapped\", \"tissue-level effector hierarchies underlying in vivo phenotypes undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [0, 2, 18, 48]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 14, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [13, 14, 15, 23]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 40, 43]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 5, 9, 21]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [28, 48]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [40]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 5, 21]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 8, 44]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [29, 39]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [13, 14, 34]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [16, 25, 49]}\n    ],\n    \"complexes\": [\"RAB35-MTMR2/MTMR13 phosphatase complex\", \"BAG3-HSP70-TBC1D10B complex\"],\n    \"partners\": [\"OCRL\", \"ACAP2\", \"DENND1A\", \"MICAL-L1\", \"NDP52\", \"Arl13b\", \"Podocalyxin\", \"DENND2B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}