Actin binding of ezrin is activated by specific recognition of PIP2-functionalized lipid bilayers.
In a quantitative manner, we investigated the mechanism of switching ezrin from the dormant to the active, F-actin binding state by recognition of PIP 2. For this purpose, we established a novel in vitro model mimicking ezrin-mediated membrane-cytoskeleton attachment and compared the F-actin binding capability of ezrin that either had been coupled via a His tag to a lipid bilayer displaying Ni-NTA or had been bound to supported membranes containing PIP 2. Epifluorescence and colloidal probe microscopy (CPM) were employed to demonstrate ezrin's conformational switch into an active conformation capable of binding F-actin. Epifluorescence images revealed attachment of fluorescently labeled F-actin solely to PIP 2-bound ezrin. For the first time, colloidal spheres equipped with an artificial cytoskeleton composed of firmly attached F-actin filaments were used to measure quantitatively the maximal adhesion forces and the work of adhesion of the ezrin-F-actin interface. We found that the work of adhesion between PIP 2-bound ezrin and F-actin is substantially larger than that measured between F-actin and ezrin bound to the membrane via the His tag. Collectively, these data indicate that activation of ezrin can occur as a consequence of PIP 2 binding and does not require additional cofactors.
Janke
Matthias
M
Institute of Physical Chemistry, University of Mainz, Welder Weg 11, 55128 Mainz, Germany.
Herrig
Alexander
A
Austermann
Judith
J
Gerke
Volker
V
Steinem
Claudia
C
Janshoff
Andreas
A
D000199:Actins / Q000378:metabolism*; D003102:Colloids / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D018625:Microscopy, Atomic Force; D009829:Oleic Acids / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D011487:Protein Conformation; D013386:Succinates / Q000737:chemistry; D000099149:Ezrin
D000199:Actins; D003102:Colloids; D003598:Cytoskeletal Proteins; D008051:Lipid Bilayers; D008239:Lysine; D009829:Oleic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D013386:Succinates; D000099149:Ezrin; C476561:N(alpha),N(alpha)-bis(carboxymethyl)-N(epsilon)-(1,2-dioleyl-sn-glycero-3-succinyl)-L-lysine