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In Situ Scanning Probe Microscopy Studies of Tetanus Toxin-Membrane Interactions

Andrea L. Slade ^*, Joseph S. Schoeniger , Darryl Y. Sasaki  and Christopher M. Yip ^*

^* Department of Biochemistry, Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; Sandia National Laboratories, Biosystems Research, Livermore, California; and Sandia National Laboratories, Biomolecular Interfaces & Systems, Albuquerque, New Mexico

Correspondence: Address reprint requests to Christopher M. Yip, IBBME, Rosebrugh Bldg., University of Toronto, 4 Taddle Creek Rd., Toronto, Ontario, Canada, M5S 3G9. Tel: 416-978-7853; Fax: 416-978-4317; E-mail: christopher.yip{at}utoronto.ca.

Despite the considerable information available with regards to the structure of the clostridial neurotoxins, and their inherent threat as biological warfare agents, the mechanisms underpinning their interactions with and translocation through the cell membrane remain poorly understood. We report herein the results of an in situ scanning probe microscopy study of the interaction of tetanus toxin C-fragment (Tet C) with supported planar lipid bilayers containing the ganglioside receptor G_T1b. Our results show that Tet C preferentially binds to the surface of fluid phase domains within biphasic membranes containing G_T1b and that with an extended incubation period these interactions lead to dramatic changes in the morphology of the lipid bilayer, including the formation of 40–80 nm diameter circular cavities. Combined atomic force microscopy/total internal reflection fluorescence microscopy experiments confirmed the presence of Tet C in the membrane after extended incubation. These morphological changes were found to be dependent upon the presence of G_T1b and the solution pH.