Phase separation of biomimetic membranes:
Influence of glycosphingolipid structure and substrate adhesion
by Jeremias Sibold
Date of Examination:2019-10-14
Date of issue:2019-12-11
Advisor:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Jörg Enderlein
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Abstract
English
The bacterium enterohemorrhagic Escherichia coli (EHEC) is one of the main causes of food borne illness. The toxin of this bacteria is shiga toxin (STx) which inhibits the protein biosynthesis of cells. Approaching the cells B-subunit of STx (STxB) binds to the receptor lipid globotriaosyl ceramide (Gb3) which is heterogeneous distributed in the outer membrane of human cells. Such a cellular membrane is itself very heterogeneous and has regions of higher and lower ordering which influences Gb3 distribution. Furthermore, the cellular membrane is heterogeneously connected either to the cytoskeleton in the inside or to the surfaces on the outside of the cell, inducing different adhesional properties in the cell membrane (adhered membranes and non-adhered membranes). As postulated in a theory of Lipowsky et al.[1] this is assumed to influence ordering and lipid distribution in lipid membranes. In this work, the phase distribution of the Gb3 is analyzed in biomimetic model membranes as a function of the creamide backbone harboring different fatty acids. Fatty acid labeled Gb3s showed unnatural phase preference of STxB Gb3 binding. Fluorescently head group labeled Gb3s were investigated to study the influence of different fatty acids in liquid ordered (lo)/liquid disordered (ld) phase-separated giant unilamellar vesicles. Gb3s with the saturated fatty acid is preferentially distributed in the lo phase, while the Gb3s with the unsaturated fatty acid prefer the ld phase. An a-hydroxylation at the fatty acid had no effect of the distribution of Gb3. In contrast different sphingomyelins (SMs) influence the partitioning of Gb3. The influence of heterogeneous membrane adhesion to biomimic membranes was analyzed using pore-spanning membranes (PSMs) on SiOx functionalized substrates. The heterogeneous membrane adhesion influences the measured phase diagram. A membrane composed of DOPC/SM/cholesterol (Chol) (1:1:2) is still phase-separated with the ld phase in the solid supported PSM (s-PSM) and the lo phase in the freestanding PSM (f-PSM). Phase diagrams of homogeneous membrane adhesion were reported to have no phase separation at the same Chol content which verifies the theory of Lipowsky et al. for the analyzed lipid compositions.
Keywords: lipid membrane; phase separation; pore-spanning membrane; shiga toxin; globotriaosyl ceramide