Investigation of membrane fusion as a function of lateral membrane tension

Investigation of membrane fusion as a function of lateral membrane tension

by Torben-Tobias Kliesch
Doctoral thesis
Date of Examination:2017-06-07
Date of issue:2017-09-21
Advisor:Prof. Dr. Andreas Janshoff
Referee:Prof. Dr. Tim Salditt
Referee:Dr. Florian Rehfeldt
Referee:Prof. Dr. Peter Jomo Walla
Referee:Prof. Dr. Bert De Groot
Referee:Prof. Dr. Martin Suhm
Persistent Address: http://dx.doi.org/10.53846/goediss-6495

 

 

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Abstract

English

Membrane fusion in native systems occurs on very short time scales and it has been proposed that lateral membrane tension of the presynaptic membrane is substantially increased to facilitate fusion. Proteins of the SNARE family (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are necessary to bring the membranes of a small vesicle and a cell membrane into close contact to promote fusion. It is proposed that membrane fusion is induced by, the formation of SNARE complexes, at the active zones in the plasma membrane that are composed many of different lipids and proteins. The connection of the cytoskeleton to the plasma membrane plays a pivotal role for the generation of membrane tension and the process of vesicle fusion. Molecular dynamics simulations have shown in the past that an increase in lateral tension facilitates fusion. To mimic the natural fusion process for e.g. in neuronal cells, different model systems with artificial membranes containing the SNARE core complex were investigated. In this study the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of syntaxin 1, Synaptobrevin and SNAP 25 is addressed. With two artificial model systems the fusion of lipid membranes as a function of lateral membrane tension was investigated to seek a better understanding of fusion processes. The first model system contained giant vesicles that adhered on a functionalized glass surface. In the second model system, supported lipid bilayers were spread out of giant vesicles on a stretching device. The membrane tension of giant vesicles was adjusted through the adhesion area. Isolated patches of planar bilayers were formed from giant unilamellar vesicles and deposited on a dilatable polymeric sheet, which is part of a milli-fluidic stretching device allowing to adjust lateral tension in bilayer patches. Fusion of large unilamellar vesicles (LUVs) added to the solution was followed by fluorescence microscopy. The relative increase in fluorescence intensity, originating from the added LUVs, compared to the fluorescence intensity emitted from the giant vesicle membrane and planar bilayer patches served as a measure for fusion efficiency. It was found that fusion efficiency increases considerably with lateral tension and a threshold tension of 3.4 mN m-1 was identified at which fusion is boosted tremendously.
Keywords: membrane fusion; membrane tension; SNARE proteins; adhered giant unilamellar vesicles; vesicle fusion; supported lipid bilayers; stretching of lipid membranes; membrane area dilatation; fluorescence microscopy; neuronal membrane fusion; milli-fluidic device; PDMS; N-ethylmaleimide-sensitive-factor attachment receptors (SNAREs); lateral membrane tension; anisotropic dilatation of PDMS
 
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