Actin binding to lipid-inserted alpha-actinin

• Published in: Biophysical journal 1993-11, Vol.65 (5), p.1878-1885
• Main Authors: Fritz, M., Zimmermann, R.M., Bärmann, M., Gaub, H.E.
• Format: Article
• Language: English
• Subjects: Actinin - chemistry; Actinin - metabolism; Actins - chemistry; Actins - metabolism; Adsorption; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Biophysical Phenomena; Biophysics; Contractile proteins; Fundamental and applied biological sciences. Psychology; Holoproteins; In Vitro Techniques; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Models, Biological; Protein Binding; Proteins; Solubility; Water
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(93)81252-9

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.