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Structure and dynamics of artificial lipid membranes containing the glycosphingolipid Gb3

Ole Mathis Schütte
Doctoral thesis
Date of Examination: 2015-07-16
Date of issue: 2015-09-07
Advisor: Steinem, Claudia Prof. Dr.
Referee: Steinem, Claudia Prof. Dr.
Referee: Diederichsen, Ulf Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0023-960D-7

 

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Abstract

English

The lateral structure of the plasma membrane of mammalian cells is governed by the underlying actin cytoskeleton and the complex composition of the membrane. Using phase-separated membranes of the raft-like mixture DOPC/ Sphingomyelin/Cholesterol 40:40:20 allows to study the membrane structure in a chemically well defined model system. Solid supported membranes were prepared and the phase separation into an ordered a disordered phase was investigated by fluorescence and atomic force microscopy. The membranes contained 5 mol% of one of the naturally occurring derivatives of the receptor glycosphingolipid Gb3 of the Shiga Toxin B-subunit (STxB) with either a saturated, an unsaturated, a α-hydroxylated or one of the diastereoisomers of the α-hydroxylated and unsaturated fatty acid bound to its ceramide backbone. Depending on the Gb3 species in the membrane the lipid distribution between the phases varies. STxB binds only to the ordered phase and its lateral organization vastly differs. Protein cluster formation leads to an interphase lipid rearrangement and sterically more demanding Gb3 derivatives induce a homogeneous coverage of the ordered phase with STxB. In giant unilamellar vesicles, containing a α-hydroxylated and unsaturated Gb3 derivative, STxB binding induced the formation of tubular membrane invaginations which are the toxin’s mechanism of endocytosis. The results show that the fatty acid of Gb3 strongly influences the lateral membrane organization and STxB binding, indicating distinct biological functions of the molecules. To mimic the role of the cytoskeleton, phase-separated membranes were prepared on porous substrates with different pore diameters and porosities. The combination of solid supported and freestanding membrane areas induces the formation of small lipid domains. Contact to the solid support immobilizes them, while domains fully located in the freestanding membrane area are moving. To describe the diffusion of the domains, a theoretical model was developed. Analysis of the movement in con- junction with topographic data, gathered by scanning ion-conductance microscopy, identifies a curved membrane region at the pore border as a major determinant of domain diffusion. STxB binding to the membranes leads to lipid redistribution between the solid supported and freestanding membrane areas, resulting in an altered morphology of the domains. The dynamics of lipid domains and protein clusters show that porous substrates compartmentalize the membrane in a similar way as proposed for the cytoskeleton underlying the plasma membrane.
Keywords: lipid domains; fluorescence microscopy; scanning probe microscopy; pore-spanning lipid bilayers; diffusion; glycolipids; Shiga toxin
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