vclb (vinculin b) is one of two zebrafish co-orthologs of vertebrate vinculin (VCL), retained from the teleost-specific genome duplication; its paralog is vcla. It encodes a cytoplasmic actin-binding adaptor of focal adhesions and adherens junctions that binds talin and alpha-catenin through its head domain and F-actin and paxillin through its tail. The protein conserves the binding residues of mammalian vinculin but carries phenylalanine at the position of the regulatory tyrosine Y822; GFP-tagged Vclb still localizes to focal adhesions and, via the alpha-catenin vinculin-binding site, to adherens junctions. vclb is the predominant vinculin copy in the embryonic heart, including epicardium and endocardium, and is the copy most prominently expressed in endothelial cells, where it marks focal adhesions and cell-cell junctions. vclb mutants develop epicardial hyperplasia, disorganized coronary vessels with elevated FAK and ERK phosphorylation, and die as juveniles. Combined loss with vcla causes leakage of small molecules from embryonic vessels. vclb mRNA is upregulated when vcla mutant transcripts undergo nonsense-mediated decay (transcriptional adaptation).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0002102 podosome | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Podosome localization mapped from the UniProt subcellular location vocabulary (ARBA rule). Reason: Vinculin is a podosome component in mammalian cells, and the ARBA-derived location is a family-level transfer. There is no zebrafish evidence for podosomes containing this copy, and podosomes are not where the zebrafish mutant phenotypes lie; kept as a plausible non-core location. |
| GO:0003779 actin binding | IEA GO_REF:0000002 | ACCEPT | Summary: Actin binding from the InterPro vinculin family signature. Reason: Vinculin binds F-actin through its tail domain, and the tail helices that mediate F-actin and paxillin binding are almost identical between the zebrafish copies and higher vertebrates. Correct, though less specific than the co-annotated actin filament binding (GO:0051015). Supporting Evidence: PMID:28767718 Especially Helices 3β5 which mediate paxillin and F-actin binding (Fig 1E) [52,53] are almost completely identical. |
| GO:0005198 structural molecule activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Structural molecule activity from an InterPro vinculin conserved-site mapping. Reason: Vinculin is a mechanosensitive linker that couples talin and alpha-catenin to F-actin; it is not a structural constituent in the sense of a filament or matrix protein. The informative activities (actin filament binding, alpha-catenin binding) are already annotated, so this vague parent adds nothing and implies a role the protein does not have. Supporting Evidence: PMID:28767718 Vinculin binds the adherens junction component Ξ±-catenin, which interacts with cadherin-bound Ξ²-catenin through its N-terminal head domain, and links the cadherin complex to F-actin using its C-terminal tail. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Cytoplasmic protein (IBA). Reason: Vinculin is a cytoplasmic, membrane-associated cytoskeletal protein. Both TGD copies descend from the PANTHER PTN005285701 vinculin node, so the IBA is expected on vclb and vcla alike; nothing in the vclb sequence or phenotype argues for loss of the function. Supporting Evidence: PMID:28767718 In Ξ±-catenin rescued cells, both vinculin A and vinculin B localize to integrin-based Focal Adhesion structures, as well as to the punctate Focal Adherens Junctions as evidenced by colocalization with Ξ±-catenin |
| GO:0005856 cytoskeleton | IBA GO_REF:0000033 | ACCEPT | Summary: Cytoskeleton (IBA). Reason: Vinculin is an actin-cytoskeleton-associated protein at adhesion sites. Both TGD copies descend from the PANTHER PTN005285701 vinculin node, so the IBA is expected on vclb and vcla alike; nothing in the vclb sequence or phenotype argues for loss of the function. Supporting Evidence: PMID:28767718 Vinculin binds the adherens junction component Ξ±-catenin, which interacts with cadherin-bound Ξ²-catenin through its N-terminal head domain, and links the cadherin complex to F-actin using its C-terminal tail. |
| GO:0005856 cytoskeleton | IEA GO_REF:0000044 | ACCEPT | Summary: Cytoskeleton from UniProt subcellular location mapping. Reason: Consistent with the IBA row and the known actin association of vinculin. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Plasma membrane (IBA); vinculin sits on the cytoplasmic face of adhesion sites. Reason: Vinculin is a peripheral protein at the cytoplasmic face of integrin and cadherin adhesions. Both TGD copies descend from the PANTHER PTN005285701 vinculin node, so the IBA is expected on vclb and vcla alike; nothing in the vclb sequence or phenotype argues for loss of the function. Supporting Evidence: PMID:28767718 In Ξ±-catenin rescued cells, both vinculin A and vinculin B localize to integrin-based Focal Adhesion structures, as well as to the punctate Focal Adherens Junctions as evidenced by colocalization with Ξ±-catenin |
| GO:0005911 cell-cell junction | IEA GO_REF:0000117 | ACCEPT | Summary: Cell-cell junction (ARBA). Reason: Correct but general; the more specific adherens junction term is supported experimentally for both copies in the same cell assay. Supporting Evidence: PMID:28767718 In Ξ±-catenin rescued cells, both vinculin A and vinculin B localize to integrin-based Focal Adhesion structures, as well as to the punctate Focal Adherens Junctions as evidenced by colocalization with Ξ±-catenin |
| GO:0005912 adherens junction | IBA GO_REF:0000033 | ACCEPT | Summary: Adherens junction (IBA). Reason: Both zebrafish copies are among the descendant evidences (ZFIN:ZDB-GENE-050506-61 = vcla, ZFIN:ZDB-GENE-131017-1 = vclb) used for this node, which is expected: each has its own IDA from PMID:28767718. Recruitment to adherens junctions depends on the alpha-catenin vinculin-binding site for both copies. Both TGD copies descend from the PANTHER PTN005285701 vinculin node, so the IBA is expected on vclb and vcla alike; nothing in the vclb sequence or phenotype argues for loss of the function. Supporting Evidence: PMID:28767718 In Ξ±-catenin rescued cells, both vinculin A and vinculin B localize to integrin-based Focal Adhesion structures, as well as to the punctate Focal Adherens Junctions as evidenced by colocalization with Ξ±-catenin PMID:28767718 Here it is apparent that both vinculin A and vinculin B are now excluded from Focal Adherens Junctions and Linear Adherens Junctions (arrows), but are still present in Focal Adhesions (arrows). |
| GO:0005912 adherens junction | IDA PMID:28767718 Zygotic vinculin is not essential for embryonic development ... | ACCEPT | Summary: GFP-tagged zebrafish vclb localizes to focal and linear adherens junctions in MDCK cells, dependent on the alpha-catenin vinculin-binding site. Reason: Direct localization assay of the zebrafish protein in epithelial cells; junctional recruitment is lost when alpha-catenin lacks its vinculin-binding site, showing the canonical recruitment mechanism. Both copies behave the same in this assay. Supporting Evidence: PMID:28767718 In Ξ±-catenin rescued cells, both vinculin A and vinculin B localize to integrin-based Focal Adhesion structures, as well as to the punctate Focal Adherens Junctions as evidenced by colocalization with Ξ±-catenin PMID:28767718 Here it is apparent that both vinculin A and vinculin B are now excluded from Focal Adherens Junctions and Linear Adherens Junctions (arrows), but are still present in Focal Adhesions (arrows). |
| GO:0005912 adherens junction | IEA GO_REF:0000044 | ACCEPT | Summary: Adherens junction from UniProt subcellular location mapping. Reason: Consistent with the IDA and IBA rows for the same term. |
| GO:0005925 focal adhesion | IBA GO_REF:0000033 | ACCEPT | Summary: Focal adhesion (IBA). Reason: Both copies localize to integrin-based focal adhesions independently of alpha-catenin. Both TGD copies descend from the PANTHER PTN005285701 vinculin node, so the IBA is expected on vclb and vcla alike; nothing in the vclb sequence or phenotype argues for loss of the function. Supporting Evidence: PMID:28767718 In Ξ±-catenin rescued cells, both vinculin A and vinculin B localize to integrin-based Focal Adhesion structures, as well as to the punctate Focal Adherens Junctions as evidenced by colocalization with Ξ±-catenin PMID:28767718 Here it is apparent that both vinculin A and vinculin B are now excluded from Focal Adherens Junctions and Linear Adherens Junctions (arrows), but are still present in Focal Adhesions (arrows). |
| GO:0005925 focal adhesion | IEA GO_REF:0000120 | ACCEPT | Summary: Focal adhesion (combined IEA). Reason: Consistent with experimental localization of both copies to focal adhesions. Supporting Evidence: PMID:28767718 In Ξ±-catenin rescued cells, both vinculin A and vinculin B localize to integrin-based Focal Adhesion structures, as well as to the punctate Focal Adherens Junctions as evidenced by colocalization with Ξ±-catenin |
| GO:0007155 cell adhesion | IBA GO_REF:0000033 | ACCEPT | Summary: Cell adhesion (IBA). Reason: Vinculin reinforces integrin- and cadherin-based adhesions by linking them to actin; this is the core process of the protein. Both TGD copies descend from the PANTHER PTN005285701 vinculin node, so the IBA is expected on vclb and vcla alike; nothing in the vclb sequence or phenotype argues for loss of the function. Supporting Evidence: PMID:28767718 Vinculin binds the adherens junction component Ξ±-catenin, which interacts with cadherin-bound Ξ²-catenin through its N-terminal head domain, and links the cadherin complex to F-actin using its C-terminal tail. PMID:36260739 Together, the data show that vinculin tightens endothelial junctions in blood vessels. |
| GO:0007155 cell adhesion | IEA GO_REF:0000002 | ACCEPT | Summary: Cell adhesion from InterPro vinculin family signatures. Reason: Consistent with the IBA row and the conserved adhesion-linker function. |
| GO:0007507 heart development | IMP PMID:27578788 Vinculin b deficiency causes epicardial hyperplasia and coro... | ACCEPT | Summary: vclb gene-trap mutants have multiple cardiac defects, epicardial hyperplasia and coronary vessel disorganization, and die as juveniles. Reason: Direct loss-of-function evidence for this copy (abstract-only cache; defer to the ZFIN curator for details). Consistent with vclb being the predominant vinculin copy in the embryonic heart and the copy expressed in epicardium and endocardium. Heart development is broad; the more specific coronary vasculature term below captures the best-characterized defect. Supporting Evidence: PMID:27578788 We report a zebrafish gene-trap vinculin b (vclb) mutant that displays abnormal coronary vessel development among multiple cardiac defects. PMID:27578788 The mutant shows overproliferation of epicardium-derived cells and disorganization of coronary vessels, and they eventually die off at juvenile stages. PMID:30635353 A recent report showed that vclb is the predominant vcl gene expressed in the embryonic heart (Cheng et al., 2016). |
| GO:0008013 beta-catenin binding | IBA GO_REF:0000033 | UNDECIDED | Summary: Beta-catenin binding (IBA). Reason: The PAINT node is sound for vertebrate vinculin, but vclb carries F at the position of Y822, the tyrosine whose phosphorylation was reported to be needed for vinculin-beta-catenin binding, and the Y822F mutation of chicken vinculin abrogated the interaction. Vclb still reaches cell-cell junctions in MDCK cells via alpha-catenin, which does not test beta-catenin binding. This is a candidate target-specific divergence inside the clade, so the inheritance of this particular activity by vclb is unresolved until binding is tested directly. Propagation Review Root cause: UNRESOLVED Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN005285701 UNRESOLVED Vertebrate/metazoan vinculin node carrying the beta-catenin binding IBD. Sound for vinculin generally; vclb is inside the clade but carries a substitution at a residue linked to this specific interaction, and binding has not been tested for vclb. Supporting Evidence: PMID:28767718 The one notable difference between zebrafish vinculin A and B is the change of the otherwise conserved Y at position 822 to F in vinculin B. PMID:28767718 Curiously, the 822F residue in zebrafish vinculin B does not perturb its localization to cell-cell junctions. |
| GO:0015629 actin cytoskeleton | IEA GO_REF:0000002 | ACCEPT | Summary: Actin cytoskeleton (InterPro). Reason: Vinculin binds F-actin at adhesion sites; correct location. Supporting Evidence: PMID:28767718 Vinculin binds the adherens junction component Ξ±-catenin, which interacts with cadherin-bound Ξ²-catenin through its N-terminal head domain, and links the cadherin complex to F-actin using its C-terminal tail. |
| GO:0016020 membrane | IEA GO_REF:0000117 | ACCEPT | Summary: Membrane (ARBA). Reason: Correct but uninformative; plasma membrane and adhesion-site terms are more specific. |
| GO:0030016 myofibril | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Myofibril (ARBA). Reason: In the zebrafish heart vinculin immunoreactivity is found at sarcomeric Z-disks and costameres (antibody not paralog-specific), consistent with a muscle location, but this is a tissue-specific attachment site, not the core location. Supporting Evidence: PMID:26954676 We show here for the first time that an orchestrated interplay of Paxillin, Focal Adhesion Kinase (FAK) and Vinculin localizing to sarcomeric Z-disks and costameres in cardiomyocytes is essential for the regulation of cardiac contractility. |
| GO:0042383 sarcolemma | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Sarcolemma (UniProt subcellular location mapping). Reason: Costameric vinculin lies at the sarcolemma of cardiomyocytes; the zebrafish antibody data do not distinguish the paralogs. Plausible, muscle-specific, non-core. Supporting Evidence: PMID:26954676 We show here for the first time that an orchestrated interplay of Paxillin, Focal Adhesion Kinase (FAK) and Vinculin localizing to sarcomeric Z-disks and costameres in cardiomyocytes is essential for the regulation of cardiac contractility. |
| GO:0044291 cell-cell contact zone | IBA GO_REF:0000033 | ACCEPT | Summary: Cell-cell contact zone (IBA). Reason: Vinculin is recruited to tensioned cadherin contacts; both copies reach cell-cell junctions in the same assay. Both TGD copies descend from the PANTHER PTN005285701 vinculin node, so the IBA is expected on vclb and vcla alike; nothing in the vclb sequence or phenotype argues for loss of the function. Supporting Evidence: PMID:28767718 In Ξ±-catenin rescued cells, both vinculin A and vinculin B localize to integrin-based Focal Adhesion structures, as well as to the punctate Focal Adherens Junctions as evidenced by colocalization with Ξ±-catenin |
| GO:0045294 alpha-catenin binding | IBA GO_REF:0000033 | ACCEPT | Summary: Alpha-catenin binding (IBA). Reason: Junctional recruitment of both zebrafish copies requires the vinculin-binding site of alpha-catenin, and the D1 head residue A50 that mediates talin/alpha-catenin binding is conserved. Both TGD copies descend from the PANTHER PTN005285701 vinculin node, so the IBA is expected on vclb and vcla alike; nothing in the vclb sequence or phenotype argues for loss of the function. Supporting Evidence: PMID:28767718 Here it is apparent that both vinculin A and vinculin B are now excluded from Focal Adherens Junctions and Linear Adherens Junctions (arrows), but are still present in Focal Adhesions (arrows). PMID:28767718 The key residue of which mutation perturbs all of these interactions, A50, is fully conserved [47,48]. |
| GO:0048471 perinuclear region of cytoplasm | IEA GO_REF:0000044 | MARK AS OVER ANNOTATED | Summary: Perinuclear region of cytoplasm (UniProt subcellular location mapping). Reason: No zebrafish or functional basis for a perinuclear vinculin pool; the protein acts at adhesion sites. The mapped keyword adds no information about function. |
| GO:0051015 actin filament binding | IEA GO_REF:0000002 | ACCEPT | Summary: Actin filament binding (InterPro). Reason: Core activity of the vinculin tail; the F-actin-binding tail helices are nearly identical to mammalian vinculin in both copies. Supporting Evidence: PMID:28767718 Especially Helices 3β5 which mediate paxillin and F-actin binding (Fig 1E) [52,53] are almost completely identical. |
| GO:0060977 coronary vasculature morphogenesis | IMP PMID:27578788 Vinculin b deficiency causes epicardial hyperplasia and coro... | ACCEPT | Summary: Vclb is required for organized coronary vessel formation; loss raises FAK/ERK signalling in epicardium and endocardium. Reason: The main phenotype of the vclb mutant and the one that distinguishes the copies: vcla is expressed in the myocardium but not the epicardium, so the coronary/epicardial role falls to vclb by expression. The mechanism given (paxillin released from the Vclb complex, FAK/ERK hyperphosphorylation) is the ancestral vinculin adhesion-site activity acting in a vclb-specific expression domain. Supporting Evidence: PMID:27578788 Mechanistically, Vclb deficiency results in the release of another cytoskeletal protein, paxillin, from the Vclb complex and the upregulation of ERK and FAK phosphorylation in epicardium and endocardium, causing disorganization of endothelial cells and pericytes during coronary vessel development. PMID:27578788 By contrast, cardiac muscle development is relatively normal, probably owing to redundancy with Vcla, a vinculin paralog that is expressed in the myocardium but not epicardium. |
| GO:0061028 establishment of endothelial barrier | IGI PMID:36260739 Vinculin strengthens the endothelial barrier during vascular... | NEW | Summary: Vinculin strengthens endothelial adherens junctions; vcla;vclb double and vcla-/-;vclb+/- embryos leak 10 kDa dextran from intersegmental vessels. Reason: Participation: vinculin is recruited to tensioned VE-cadherin junctions through alpha-catenin and reinforces their link to actin, so it contributes structure that barrier formation depends on (junctional vinculin, not a downstream signal). The in vivo evidence is genetic and involves both copies (vcla-/-;vclb+/- and vcla-/-;vclb-/- both leak), so the annotation is made to both copies as IGI rather than to one. vclb is the copy most prominently expressed in endothelium (PMID:36314606), so the relative contribution of each copy is not resolved. Supporting Evidence: PMID:36260739 Our analysis shows that vclaβ/β;vclb+/βand vcl full-KO embryos exhibited increased perivascular dextran levels PMID:36260739 we observed vascular leakage specifically for small molecules, 10 kDa, in vinculin knockout zebrafish, whereas the vasculature still acted as a barrier for larger molecules PMID:36260739 Together, the data show that vinculin tightens endothelial junctions in blood vessels. |
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Download this section (compressed HTML)Q: Does Vclb (F822) bind beta-catenin, and does the Y822F substitution change junction stabilization in a vinculin-null zebrafish background?
Q: Is the juvenile lethality of vclb mutants due to loss of an epicardial/endocardial expression domain that vcla lacks, i.e. would vcla expressed from the vclb locus rescue it?
Q: Can the vclb locus encode a metavinculin exon, or is metavinculin made only from vcla?
Experiment: Express vcla (and vclb as control) coding sequence in epicardium (tcf21 driver) of vclb mutants and score coronary vessel organization and FAK/ERK phosphorylation.
Hypothesis: vclb-specific coronary phenotypes reflect expression, not protein divergence.
Type: cross-rescue
Experiment: Co-immunoprecipitation of tagged Vcla, Vclb and Vclb F822Y with beta-catenin in zebrafish cells or embryos.
Hypothesis: F822 in Vclb reduces beta-catenin binding relative to Vcla.
Type: protein interaction assay
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