Rapid induction and isolation of focal adhesion complexes.
Focal adhesion complexes (FACs) containing integrin beta 1, talin, vinculin, talin, alpha-actinin, and paxillin formed within 15 min when round cells bound magnetic microbeads coated with integrin ligands, such as fibronectin or RGD-containing peptide, but not when coated with acetylated-low density lipoprotein. Newly formed FACs were isolated and collected for biochemical analysis using a combination of detergent extraction, sonication, dounce homogenization, and magnetic pelleting. Isolated bead complexes were greatly enriched for all FAC proteins when compared with either the whole cytoskeleton or basal cell membranes whereas actin (a general cytoskeletal marker) was relatively depleted. This method which permits isolation of intact FACs within minutes following integrin ligation should facilitate analysis of both FAC assembly and the molecular basis of integrin signaling.
Plopper
G
G
Department of Surgery, Children's Hospital, Boston, MA 02115.
Ingber
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DE
D000185:Actinin / Q000302:isolation & purification / Q000378:metabolism; D000199:Actins / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002196:Capillaries; D002417:Cattle; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D019012:Integrin beta1; D016023:Integrins / Q000302:isolation & purification / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D016608:Talin / Q000302:isolation & purification / Q000378:metabolism; D016596:Vinculin / Q000302:isolation & purification / Q000378:metabolism
D000199:Actins; D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D019012:Integrin beta1; D016023:Integrins; D051419:Paxillin; D010750:Phosphoproteins; D016608:Talin; D000185:Actinin; D016596:Vinculin