title: Substrate-attached materials are enriched with tetraspanins and are analogous to the structures associated with rear-end retraction in migrating cells.
abstract: Substrate-attached materials (SAMs) are cellular feet that remain on substrates after the treatment of adherent cells with EGTA. SAMs are thought to contain cell adhesion machineries, but their biochemical properties have not been addressed in detail. To gain insight into the molecular mechanisms operating in cell adhesions, we comprehensively identified the protein components of SAMs by liquid chromatography coupled with tandem mass spectrometry, followed by immunoblot analysis. We found that the tetraspanins CD9, CD81, and CD151 were enriched in SAMs along with other transmembrane proteins that are known to associate with tetraspanins. Notably, integrins were detected in SAMs, but the components of focal adhesions were scarcely detected. These observations are reminiscent of the "footprints" that remain on substrates when the retraction fibers at the rear of migrating cells are released, because such footprints have been reported to contain tetraspanins and integrins but not focal adhesion proteins. In support of this hypothesis, the formation of SAMs was attenuated by inhibitors of ROCK, myosin II and dynamin, all of which are known to participate in rear-end retraction in migrating cells. Furthermore, SAMs left on collagen-coated substrates were found by electron microscopy to be fewer and thinner than those on laminin-coated substrates, reflecting the thin and fragile retraction fibers of cells migrating on collagen. Collectively, these results indicate that SAMs closely resemble the footprints and retraction fibers of migrating cells in their protein components, and that they are yielded by similar mechanisms.
authors.affiliation: Laboratory of Extracellular Matrix Biochemistry, Institute for Protein Research, Osaka University, Suita, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Yamada
authors.forename: Gabriele
authors.initials: G
authors.lastname: Mugnai
authors.forename: Satoshi
authors.initials: S
authors.lastname: Serada
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Yagi
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Naka
authors.forename: Kiyotoshi
authors.initials: K
authors.lastname: Sekiguchi
mesh_terms: D002448:Cell Adhesion / Q000502:physiology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D034281:Dynamins / Q000037:antagonists & inhibitors; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis*; D024462:Myosin Type II / Q000037:antagonists & inhibitors; D060185:Tetraspanin 24 / Q000378:metabolism; D060225:Tetraspanin 28 / Q000378:metabolism; D060245:Tetraspanin 29 / Q000378:metabolism; D059470:Tetraspanins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
chemical_list: D016023:Integrins; D008562:Membrane Glycoproteins; D060185:Tetraspanin 24; D060225:Tetraspanin 28; D060245:Tetraspanin 29; D059470:Tetraspanins; C048022:substrate adhesion molecules; D051416:Focal Adhesion Protein-Tyrosine Kinases; D054460:rho-Associated Kinases; D024462:Myosin Type II; D034281:Dynamins
keywords: CD9; cell migration; focal adhesion; ganglioside; integrin; proteomics