Interaction of the cytoskeletal component vinculin with bilayer structures analyzed with a photoactivatable phospholipid.
The cytoskeletal component vinculin has been proposed to act as an actin-plasma membrane linker. In order to demonstrate a possible direct interaction of vinculin with bilayers, photolabeling with a phospholipid generating a highly reactive carbene was used. This phosphatidylcholine analogue (1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[3H] 9-oxaundecanoyl]-sn-glycero-3-phosphocholine), with the photoactivatable diazirine group on its apolar portion, has been shown to label selectively membrane-embedded domains of membrane proteins. Vinculin is significantly labeled upon incubation and photolysis with liposomes containing trace amounts of this photoactivatable phospholipid, but only when the liposomes also contain acidic phospholipids. Labeling of vinculin is markedly increased (5-17-fold) by all acidic phospholipids tested so far (30%, w/w), compared to labeling in neutral phospholipids. Labeling is high at low ionic strength, but significant vinculin labeling can still be observed at physiological salt concentrations and acidic phospholipid content of the membrane. Our results provide evidence that vinculin inserts into the hydrophobic part of the bilayer by interacting with acidic phospholipids. A similar interaction may be of importance in vivo.
D000199:Actins / Q000378:metabolism; D000818:Animals; D001389:Azirines / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D003599:Cytoskeleton / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D010782:Photolysis; D013329:Structure-Activity Relationship; D016596:Vinculin
D000199:Actins; D001389:Azirines; D008051:Lipid Bilayers; D008081:Liposomes; D009124:Muscle Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; C048903:1-palmitoyl-2-(10-(4-((trifluoro-methyl)diazirinyl)phenyl)-9-oxaundecanoyl)-sn-glycero-3-phosphocholine; D016596:Vinculin