Isolation of integrin-based adhesion complexes.
The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry.
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Matthew C
MC
Jones
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Jonathan D
JD
Humphries
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|Present address: Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Adam
A
Byron
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Angélique
A
Millon-Frémillon
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Joseph
J
Robertson
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Nikki R
NR
Paul
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Daniel H J
DHJ
Ng
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Janet A
JA
Askari
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.|All authors made equal contributions to method development or protocol preparation.
Martin J
MJ
Humphries
D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D003584:Cytological Techniques / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005353:Fibronectins / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000302:isolation & purification* / Q000378:metabolism*; D020014:K562 Cells; D008297:Male; D008863:Microspheres; D040901:Proteomics
D005353:Fibronectins; D016023:Integrins
adhesion complexes; affinity purification; cell adhesion; extracellular matrix; integrins; ventral membranes