Show simple item record

Modulation of lateral membrane tension and SNARE-mediated single vesicle fusion on pore spanning membranes

dc.contributor.advisorSteinem, Claudia Prof. Dr.
dc.contributor.authorKuhlmann, Jan Wilhelm
dc.publisherNiedersächsische Staats- und Universitätsbibliothek Göttingende
dc.titleModulation of lateral membrane tension and SNARE-mediated single vesicle fusion on pore spanning membranesde
dc.contributor.refereeSteinem, Claudia Prof. Dr.
dc.description.abstractengSynaptic vesicle fusion is driven by soluble N-ethylmaleimide-sensitive- factor attachment receptors (SNAREs) but is also regulated by physical properties of the membrane like curvature and tension. Using artificial SNARE containing prestressed pore-spanning membranes (PSMs) comprised of supported membranes (s-PSM) and freestanding membranes (f-PSM), the influence of lateral tension on SNARE-induced fusion, as well as dynamic aspects of vesicle docking and fusion on the two distinct membrane parts can be investigated. PSMs were prepared on porous substrates functionalized with binary self-assembled monolayers (SAM) composed of 6-mercaptohexanol (6-MH) and cholesterylpolyethylen-oxythiol (CPEO3). Varying the amount of CPEO3 in the SAM allowed to modulate the lateral tension in the PSM. The tensions ranged from 1.4 mN/ m for 0 mol% CPEO3 to 4.5 mN/m for 35 mol% CPEO3 in the SAM. With increasing lateral tension, i.e. higher CPEO3 surface concentrations, the membrane stability decreased resulting in rupturing of the freestanding membranes. To investigate the influence of the lateral tension on SNARE-mediated fusion and to compare fusion on s-PSM and f-PSM, vesicular-SNARE proteoliposomes were fused with target-SNARE containing PSMs. The PSMs were prepared on pure 6-MH SAMs as the membranes composed of a physiological lipid mixture, which was essential for efficient fusion, were unstable on SAMs containing CPEO3. Docking of the liposomes could be observed on the s-PSM and f-PSM but was two times more likely on the s-PSM. The s-PSM docked liposomes were completely immobile, while those on the f-PSMs were mobile with a mean diffusion coefficient of D = 0.42 µm2/s. In comparison to a single SNARE transmembrane domain, the diffusivity of the f-PSM docked vesicles were eight times slower indicating a close contact of the membranes induced by tight SNARE-complexes. The immobile s-PSM docked vesicles probably arise from interactions between the SNAREs and the support. The docking time distributions on s-PSMs and f-PSMs were both in the range of seconds indicating that the higher mobility of the vesicles and the lateral membrane tension in the f-PSM do not significantly alter the fusion kinetics. The fusion kinetics were also unaffected by the size of the fusing vesicle as small (90 nm) and large (240nm) vesicles exhibited comparable docking times. The shape of the docking time distributions indicated a two-step process, which can be either interpreted with the assembly of two SNARE complexes or with a kinetic
dc.contributor.coRefereeSalditt, Tim Prof. Dr.
dc.subject.englipid bilayerde
dc.subject.engfluorescence microscopyde
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de
dc.identifier.ppn1004916477 1000147673

Files in this item


This item appears in the following Collection(s)

Show simple item record