Minimal F-actin cytoskeletal system for planar supported phospholipid bilayers.
Preferential binding of F-actin to lipid bilayers containing ponticulin was investigated on both planar supported bilayers and on a cholesterol-based tethering system. The transmembrane protein ponticulin in Dictyostelium discoideum is known to provide a direct link between the actin cytoskeleton and the cell membrane ( Wuestehube, L. J. ; Luna, E. J. J. Cell Biol. 1987, 105, 1741- 1751 ). Purification of ponticulin has allowed an in vitro model of the F-actin cytoskeletal scaffold system to be formed and investigated by AFM, epi-fluorescence microscopy, surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D). Single filament features of F-actin bound to the ponticulin containing lipid bilayer are shown by AFM to have a pitch of 37.3 +/- 1.1 nm and a filament height of 7.0 +/- 1.6 nm. The complementary techniques of QCM-D and SPR were used to obtain dissociation constants for the interaction of F-actin with ponticulin containing bilayers, giving 10.5 +/- 1.7 microM for a physisorbed bilayer and 10.8 +/- 3.6 microM for a tethered bilayer, respectively.
Barfoot
Richard J
RJ
School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT, United Kingdom.
Sheikh
Khizar H
KH
Johnson
Benjamin R G
BR
Colyer
John
J
Miles
Robert E
RE
Jeuken
Lars J C
LJ
Bushby
Richard J
RJ
Evans
Stephen D
SD
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry*; D004023:Dictyostelium / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008840:Microfilament Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018625:Microscopy, Atomic Force; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D020349:Surface Plasmon Resonance
D000199:Actins; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D008051:Lipid Bilayers; D008840:Microfilament Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids