SNARE-mediated membrane fusion on pore-spanning membranes – several fusion pathways analyzed by single-vesicle content release
von Peter Mühlenbrock
Datum der mündl. Prüfung:2020-12-18
Betreuer:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Tim Salditt
EnglischThe fusion of neurotransmitter filled vesicles with the presynaptic membrane is the key step in the neuronal signaling cascade and is mediated by soluble N-ethylmaleimide-sensitive factor attachment receptor proteins (SNAREs). The interaction of the three SNARE proteins synaptobrevin 2 (syb 2), syntaxin 1A, and SNAP25 (synaptosomal associated protein of 25 kDa) is pivotal to overcome the energy barrier that leads to merging of the opposing lipid bilayers and results in the transfer of neurotransmitters across the presynaptic membrane and into the synaptic cleft. To investigate this fundamental process, pore-spanning membranes (PSMs) were utilized in this work as a model system of the presynaptic membrane. PSMs are continuous lipid bilayers with solid supported parts (s-PSM) as well as freestanding membranes spanning large aqueous compartments (f-PSM). Thus they are suitable to monitor the process of content transfer through a fusion pore of added vesicles filled with a water soluble dye by means of fluorescence microscopy. Simultaneous imaging of lipid dye diffusion from the PSM into the vesicular membrane via a fusion stalk was used to quantify different fusion pathways. The surface of porous substrates with pore diameters of 1.2 m or 3.5 m was hydrophilized with a self-assembled monolayer of 6-mercapto-1-hexanol formed on gold and PSMs were then produced by spreading of acceptor complex containing giant unilamellar vesicles. Differences in densities of target SNAREs inside PSMs were noticeable in variations of docking efficiencies of syb 2 containing, sulforhodamine B (SRB) filled liposomes to the PSMs. The fusion pore formation was then directly visualized by imaging the transfer of SRB from inside the vesicle into the space underneath the f-PSM. This process proved to be very rapid and distinguishable from rarely occurring burst events. Furthermore, it mainly occurred simultaneously with lipid diffusion from the PSM into the vesicular membrane and resulted predominantly in a full release of vesicular content. Additionally, this main fusion pathway was more likely for smaller vesicles and included a rapid and full collapse of the fusing vesicle into the PSM and thus shows distinct features of full-collapse fusion observed in vivo. Apart from this, the premature closing of the fusion pore could lead to a stable three-dimensional postfusion structure that was often times accompanied by a partial SRB release. From this state the fusion pore could open again leading to the complex fusion behavior of a flickering fusion pore. In summary, the results of this study show that the diverse fusion pathways observed in vivo likely are an intrinsic property of the minimal fusion machinery and not caused by the interplay of additional proteins.
Keywords: SNAREs; fusion; single-vesicle; fusion pathways; content release