Actin binding to lipid-inserted alpha-actinin.
The interaction of alpha-actinin with lipid films and actin filaments was investigated. First alpha-actinin was incorporated in lipid films at the air/water interface. Injection of alpha-actinin into the subphase of a lipid monolayer led to a significant increase of the surface pressure only for lipid films consisting of a mixture of a negatively charged lipid with a high proportion of diacylglycerol. These alpha-actinin-containing films were transferred onto silanized quartz slides. Photobleaching experiments in the evanescent field allowed quantification of the lateral number density of the lipid-bound alpha-actinin. In combination with the area increase from the monolayer experiments, the photobleaching measurements suggest that alpha-actinin is incorporated into the lipid film in such a way that actin binding sites are accessible from the bulk phase. Binding experiments confirmed that the alpha-actinin selectively binds actin filaments in this configuration. We also showed that, in contrast to actin filaments which are adsorbed directly onto planar surfaces, the alpha-actinin-bound actin filaments are recognized and cleaved by the actin-severing protein gelsolin. Thus we have constructed an in vitro system which opens new ways for investigations of membrane-associated actin-binding proteins and of the physical behavior of actin filaments in the close neighborhood to membranes.
Fritz
M
M
Physiksdepartment der Technischen Universität München, Garching, Germany.
Zimmermann
R M
RM
Bärmann
M
M
Gaub
H E
HE
D000185:Actinin / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000327:Adsorption; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D012995:Solubility; D014867:Water
D000199:Actins; D008051:Lipid Bilayers; D014867:Water; D000185:Actinin