Integrin beta 2 (CAOG_05058) is one of four integrin beta subunits of the amoeboid filasterean Capsaspora owczarzaki, a close unicellular relative of animals, and the most highly expressed of them. It is a 1056-residue single-pass type I membrane protein. It has a signal peptide, a vWA-like beta-I domain with conserved MIDAS, ADMIDAS and LIMBS cation-binding sites, a PSI domain, a cysteine-rich EGF-like stalk that is longer than in animal integrins, a transmembrane helix, and a short cytoplasmic tail with the alpha-subunit-interacting region and NPxY-type motifs that bind talin in animals. Capsaspora also encodes integrin alpha subunits and the main intracellular adhesome proteins (talin, vinculin, paxillin, the ILK-PINCH-parvin complex, FAK and Src), so the protein presumably forms alpha/beta heterodimers, although this has not been tested. In adherent cells, integrin beta 2 is found on the cell body and in distinct patches along the actin-dependent filopodia that attach the cells to surfaces, partly overlapping with vinculin. Mammalian fibronectin increases adhesion and the amount of integrin beta 2 in filopodia. An antibody against its ligand-binding beta-I domain strongly reduces adhesion to fibronectin-coated surfaces, so the protein mediates cell-substrate adhesion. Capsaspora has no fibronectin ortholog, and its natural ligand is unknown; candidates include secreted Capsaspora proteins with fibronectin-type domains or RGD sequences. Whether integrin beta 2 also signals or takes part in cell-cell adhesion during aggregation has not been examined.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005178 integrin binding | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: TreeGrafter transfer from PTN002560695, the vertebrate integrin beta-like protein 1 (ITGBL1) node in PTHR10082. The term is inherited there from the integrin-beta family root, so it is a family-wide claim. For a beta subunit, the "integrin" it binds is its alpha partner. Capsaspora has four integrin alpha subunits, and the alpha-interacting tail motif is conserved in beta 2 [PMID:20479219], but heterodimer formation has not been tested. Reason: The claim is plausible from conserved motifs and from the presence of alpha partners, and it is not specific to multicellular animals, since it concerns heterodimer assembly rather than any animal ligand. The authors who described the repertoire state explicitly that heterodimerisation is assumed, not shown. Kept as non-core, consistent with the human ITGB1 review, where the same IBA is kept as a non-core subunit-association term best captured by integrin complex. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node SUPPORTS SOURCE BUT NOT TARGET Graft node is the vertebrate ITGBL1 clade (all leaves vertebrate ITGBL1), not an integrin beta orthology group containing Capsaspora; the PTHR10082 tree is rooted at Eumetazoa (PTN000801545) and has no unicellular sequences. The term itself is a family-root inheritance that fits a canonical integrin beta, so the outcome is acceptable despite the wrong graft. Supporting Evidence: PMID:20479219 Other key motifs in metazoan integrin β proteins are the cytoplasmic integrin α-interacting motif and the NPXY motif PMID:20479219 Thus, we can assume that they too work as heterodimers and that they interact and function similarly to metazoan homologs. Functional analysis will be needed to test this hypothesis. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Cell membrane; single-pass type I). The protein has a signal peptide and one transmembrane helix, and immunostaining places it on the cell body and in patches along filopodia [DOI:10.1101/2020.02.27.967653]. Reason: Signal peptide plus a single transmembrane helix and filopodial staining are consistent with plasma membrane localization. Blocking adhesion by an antibody added to live cells also implies an exposed ectodomain. Supporting Evidence: file:CAPO3/coITGB2/coITGB2-uniprot.txt Single-pass type I membrane protein DOI:10.1101/2020.02.27.967653 We observed that both antibodies stain the cell body and multiple distinct patches along filopodia |
| GO:0005925 focal adhesion | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: TreeGrafter transfer from the vertebrate ITGBL1 node PTN002560695. In Capsaspora, integrin beta 2 and vinculin form distinct, partly overlapping patches along filopodia, which the authors suggest may be anchorage sites of adhesion [DOI:10.1101/2020.02.27.967653]. These patches have not been shown to be focal adhesions, which GO defines as cell-substrate junctions that anchor the cell to the extracellular matrix and terminate actin filaments. Reason: Focal adhesions are a specific structure of animal cells, mature integrin adhesions at the ends of stress fibres. In Capsaspora the integrin and vinculin patches lie within filopodia, and the authors themselves describe this as an unexpected role for integrin adhesion within filopodia. They characterise the patches only as possible anchorage sites. The localization that is shown is captured by the NEW filopodium annotation. A focal-adhesion (or cell-substrate junction) assignment would need evidence of a junctional plaque, for example talin and paxillin co-recruitment and actin-filament termination. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG LINEAGE OR TAXON MISMATCH Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node SUPPORTS SOURCE BUT NOT TARGET Vertebrate ITGBL1 node in a Eumetazoa-rooted tree; focal adhesion is inherited from animal integrin betas (PTN000801545). Capsaspora sits outside that clade and its integrin adhesions are filopodial patches not characterised as focal adhesions. Supporting Evidence: DOI:10.1101/2020.02.27.967653 Taken together, these results suggest that the integrin and vinculin patches may represent anchorage sites of adhesion. DOI:10.1101/2020.02.27.967653 which is an unexpected role for integrin adhesion within filopodia |
| GO:0007160 cell-matrix adhesion | IEA GO_REF:0000118 | MODIFY | Summary: TreeGrafter transfer from the vertebrate ITGBL1 node. Capsaspora integrin beta 2 does mediate substrate adhesion: an antibody to its beta-I domain inhibits adhesion to fibronectin-coated surfaces [PMID:32857975]. The surfaces are plastic coated with mammalian fibronectin or with material the cells secrete; Capsaspora has no fibronectin ortholog, and no Capsaspora extracellular matrix ligand has been identified [DOI:10.1101/2020.02.27.967653]. Reason: The process is supported, but "matrix" implies an extracellular matrix that has not been defined for Capsaspora. The experiments measure attachment to a coated substrate, which is exactly what cell-substrate adhesion (GO:0031589), the parent of cell-matrix adhesion, describes. The integrin is the adhesion receptor itself, so it does the work of the process. The antibody-blocking result (DOI:10.1101/2020.02.27.967653, PMID:32857975) is direct perturbation evidence for the replacement term. Revisit cell-matrix adhesion if the endogenous secreted ligand is identified. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node SUPPORTS SOURCE BUT NOT TARGET Wrong graft (vertebrate ITGBL1 clade); the inherited family-root term presupposes an animal-type extracellular matrix that is not established in Capsaspora. The parent term is supported directly. Proposed replacements: cell-substrate adhesion Supporting Evidence: PMID:32857975 Finally, using a specific antibody for integrin β2, we inhibited cell adhesion to a fibronectin-coated surface. DOI:10.1101/2020.02.27.967653 Taken together, these results show that the major integrin β subunit in Capsaspora makes a substantial contribution to substrate adhesion. DOI:10.1101/2020.02.27.967653 This suggests that a protein secreted by Capsaspora sticks to the plastic substrate and provides a ligand for adhesion |
| GO:0007229 integrin-mediated signaling pathway | IEA GO_REF:0000118 | UNDECIDED | Summary: TreeGrafter transfer from the vertebrate ITGBL1 node. Capsaspora encodes the intracellular signalling partners of animal integrins (FAK, Src, the ILK-PINCH-parvin complex) [PMID:20479219], and fibronectin increases filopodium number and integrin recruitment to filopodia [DOI:10.1101/2020.02.27.967653]. No signalling downstream of integrin beta 2 has been measured. Reason: The pathway is plausible, since the signalling components are present and ligand-dependent changes in filopodia are observed. But those changes could equally reflect adhesion mechanics, and no signalling output has been tested. The 2010 authors also caution that scaffold proteins may have had other ancestral roles. Left undecided pending, for example, FAK phosphorylation assays after ligand engagement. Propagation Review Root cause: UNRESOLVED Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node UNRESOLVED Wrong graft (vertebrate ITGBL1); the family-root signalling term may or may not apply to a Capsaspora integrin, which has the FAK/Src/IPP partners but no signalling data. Supporting Evidence: PMID:20479219 bona fide FAK are only present in Metazoa and C. owczarzaki DOI:10.1101/2020.02.27.967653 These results suggest that Fibronectin promotes cell adhesion by increasing the number and length of filopodia as well as by promoting recruitment of integrin and vinculin to the filopodia. |
| GO:0008305 integrin complex | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from the vertebrate ITGBL1 node. Capsaspora encodes four integrin alpha subunits with conserved FG-GAP repeats and cytoplasmic KXGFFXR-type motifs, and beta 2 has the alpha-interacting cytoplasmic motif [PMID:20479219]. Reason: Membership of an alpha/beta heterodimer is the defining property of an integrin beta subunit. Every element needed for it is conserved, so the inference is sound even though the Capsaspora heterodimer has not been isolated. Note that the graft node itself (vertebrate ITGBL1, a TM-less protein) is a poor source for this term; the conclusion rests on the Capsaspora sequence features. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node SUPPORTS SOURCE BUT NOT TARGET Wrong graft (vertebrate ITGBL1 clade); the term is a family-root inheritance that fits the Capsaspora protein on its own sequence features. Supporting Evidence: PMID:20479219 Both motifs are well conserved in C. owczarzaki integrin β1–β3 and Amastigomonas sp. integrin β PMID:20479219 we found four integrin β and four integrin α genes in C. owczarzaki |
| GO:0009986 cell surface | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: TreeGrafter transfer from the vertebrate ITGBL1 node. An antibody to the extracellular beta-I domain added to cells before plating blocks adhesion, which implies the ectodomain is exposed at the cell surface [DOI:10.1101/2020.02.27.967653]. Reason: Correct for a type I transmembrane adhesion receptor and indirectly supported by the antibody-blocking experiment. It adds little beyond plasma membrane. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node SUPPORTS SOURCE BUT NOT TARGET Wrong graft (vertebrate ITGBL1 clade); the generic location fits the Capsaspora protein independently. Supporting Evidence: DOI:10.1101/2020.02.27.967653 Pre-incubation of this cells with this antibody prior to plating substantially reduced adhesion to fibronectin-treated surfaces |
| GO:0016020 membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt subcellular-location mapping (Membrane; single-pass type I). Correct but less informative than plasma membrane. Reason: True (one transmembrane helix) but redundant with the more specific plasma membrane annotation. Supporting Evidence: file:CAPO3/coITGB2/coITGB2-uniprot.txt Single-pass type I membrane protein |
| GO:0016477 cell migration | IEA GO_REF:0000118 | UNDECIDED | Summary: TreeGrafter transfer from the vertebrate ITGBL1 node. Capsaspora adherent cells attach by filopodia, but the cached papers report no measurement of cell migration or of any effect of integrin beta 2 inhibition on movement. The preprint notes that in animal cells filopodia are associated with dynamic exploration of the substrate, in contrast to the stable adhesive role seen in Capsaspora [DOI:10.1101/2020.02.27.967653]. Reason: Integrin traction is a plausible contributor to amoeboid crawling, but no data exist for Capsaspora. The process term should wait for a motility assay with the blocking antibody or a knockout. Propagation Review Root cause: UNRESOLVED Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node UNRESOLVED Wrong graft (vertebrate ITGBL1 clade); family-root migration term untested in Capsaspora. Supporting Evidence: DOI:10.1101/2020.02.27.967653 In animal cells filopodia are more generally associated with dynamic exploration of the substrate rather than forming stable adherent contacts |
| GO:0033627 cell adhesion mediated by integrin | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer from the vertebrate ITGBL1 node. Directly supported in Capsaspora: an antibody to the beta-I domain of integrin beta 2 inhibits cell adhesion to fibronectin-coated surfaces, whereas an antibody to the stalk does not [PMID:32857975; DOI:10.1101/2020.02.27.967653]. Reason: The term (attachment of a cell to another cell or to a substrate via an integrin) matches the experiment. The integrin is the adhesion receptor, so it does the work of the process. The domain-specific antibody result (ligand-binding domain blocks, stalk does not) argues against a non-specific steric effect. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node SUPPORTS SOURCE BUT NOT TARGET Wrong graft (vertebrate ITGBL1 clade); the family-root term is confirmed independently for the Capsaspora protein by antibody blocking. Supporting Evidence: PMID:32857975 Our results suggest that adhesion to the substrate in C. owczarzaki is mediated by integrins. DOI:10.1101/2020.02.27.967653 in contrast to the antibody against the cysteine-rich stalk, which did not impair adhesion |
| GO:0098609 cell-cell adhesion | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: TreeGrafter transfer from the vertebrate ITGBL1 node. Capsaspora forms multicellular aggregates, so it has cell-cell adhesion, but no experiment links integrin beta 2 to it. The functional data concern adhesion to substrate, and the preprint names the aggregative stage as a subject for future work [DOI:10.1101/2020.02.27.967653]. In coYki knockouts, where CAOG_05058 is up-regulated, cell-substrate adhesion increases without a change in cell-cell adhesion [PMID:35659869]. Reason: In animals, integrin-dependent cell-cell adhesion comes from binding to counter-receptors on other cells (for example ICAM and VCAM on leukocytes and endothelium). These counter-receptors are animal innovations, and none has been identified in Capsaspora. The term reaches this protein only through a family-root inheritance on a wrong (vertebrate ITGBL1) graft node. It should not be asserted until integrin involvement in aggregation is tested. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG CONTEXT OR TISSUE MISMATCH Sources checked: PANTHER:PTN002560695 · PTHR10082:SF3 integrin beta-like protein 1, Euteleostomi node SUPPORTS SOURCE BUT NOT TARGET Vertebrate ITGBL1 node in a Eumetazoa-rooted tree; integrin cell-cell adhesion in animals depends on animal-specific counter-receptors, and there is no Capsaspora evidence for it. Supporting Evidence: DOI:10.1101/2020.02.27.967653 Further investigation of integrin-mediated adhesion in Capsaspora, particularly during the aggregative stage, by examining its ligands and intracellular binding partners PMID:35659869 two integrin-β genes (CAOG_05058 and CAOG_01283) are upregulated in coYki -/- cells |
| GO:0030175 filopodium | IDA PMID:32857975 Integrin-Mediated Adhesion in the Unicellular Holozoan Capsa... | NEW | Summary: Immunostaining of adherent Capsaspora cells with antibodies against integrin beta 2 shows the protein in distinct patches along the actin-dependent filopodia, partly overlapping with vinculin. Its filopodial intensity increases on fibronectin [PMID:32857975; DOI:10.1101/2020.02.27.967653]. Reason: Direct localization evidence, stated in the cached abstract and detailed in the cached preprint; the antibodies were raised against CAOG_05058 regions. This is the most specific location shown for the protein. It replaces the unsupported focal-adhesion assignment. Supporting Evidence: PMID:32857975 We show that integrin β2 and its associated protein vinculin localize as distinct patches in the filopodia. DOI:10.1101/2020.02.27.967653 extracellular domain of the integrin β2 (CAOG_05058, aminoacid region 733-835) |
| GO:0098631 cell adhesion mediator activity | IMP PMID:32857975 Integrin-Mediated Adhesion in the Unicellular Holozoan Capsa... | NEW | Summary: An antibody against the ligand-binding beta-I domain of integrin beta 2, added to cells before plating, substantially reduces adhesion to fibronectin-coated surfaces; an antibody against the stalk does not [PMID:32857975; DOI:10.1101/2020.02.27.967653]. Reason: This is the molecular activity behind the accepted adhesion process terms: the cell-surface receptor binds an external substrate to attach the cell. Function-blocking antibody evidence is recorded as IMP. The term is deliberately generic because the substrate ligand is not known; it does not assert binding to fibronectin or any other animal extracellular matrix protein. Supporting Evidence: PMID:32857975 Finally, using a specific antibody for integrin β2, we inhibited cell adhesion to a fibronectin-coated surface. DOI:10.1101/2020.02.27.967653 Pre-incubation of this cells with this antibody prior to plating substantially reduced adhesion to fibronectin-treated surfaces |
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Download this section (compressed HTML)Q: What is the endogenous ligand of Capsaspora integrin beta 2? Candidates named by the authors are secreted Capsaspora proteins with fibronectin-type domains or RGD sequences; does adhesion depend on RGD, and on divalent cations at the conserved MIDAS site?
Q: Which of the four Capsaspora integrin alpha subunits pairs with beta 2, and does the pair form in vivo?
Q: Are the filopodial integrin-vinculin patches junctional plaques with talin and paxillin, comparable to animal nascent or focal adhesions, or a distinct filopodial adhesion structure?
Q: Does integrin beta 2 contribute to cell-cell adhesion or aggregate shape during the aggregative stage, where adhesome genes are up-regulated? Is its up-regulation in coYki knockouts the cause of their increased substrate adhesion?
Q: TreeGrafter grafts this and the other Capsaspora integrin betas onto the vertebrate ITGBL1 node (PTN002560695) of a Eumetazoa-rooted tree. Could PANTHER add a holozoan outgroup so that unicellular integrins graft at the family root instead?
Experiment: Knock out CAOG_05058 (genome editing now works in Capsaspora) and measure adhesion on uncoated and fibronectin-coated surfaces, filopodium number and length, and vinculin patch formation; rescue with wild-type and MIDAS-mutant transgenes.
Hypothesis: Integrin beta 2 is required for substrate adhesion and filopodial adhesion patches.
Type: genetic knockout / complementation
Experiment: Immunoprecipitate integrin beta 2 with the beta-I antibody from adherent cells and identify co-precipitating alpha subunits, talin and other adhesome proteins by mass spectrometry.
Hypothesis: Integrin beta 2 forms a heterodimer with a specific integrin alpha subunit.
Type: protein interaction / proteomics
Experiment: Plate cells on fibronectin with or without the beta-I blocking antibody and measure tyrosine phosphorylation of Capsaspora FAK and paxillin.
Hypothesis: Integrin beta 2 engagement triggers FAK/Src signalling.
Type: biochemical signalling assay
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