Vesicle Adhesion, Fusion, and Neurotransmitter Uptake Studied by Small-Angle X-ray Scattering
by Karlo Komorowski
Date of Examination:2021-02-15
Date of issue:2022-02-15
Advisor:Prof. Dr. Tim Salditt
Referee:Prof. Dr. Tim Salditt
Referee:Prof. Dr. Claudia Steinem
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EnglishSynaptic neurotransmission is a highly complex and crucial process in the communication between neuronal cells, and is thus directly related to the complex functions of the neural system, including information processing in the brain, sensory reactions, and learning. The last decades have considerably shaped our current understanding of the underlying processes in synaptic neurotransmission on the molecular level, and up to the level of the entire synapse. X-ray crystallography and more recently also cryo-electron microscopy have provided the structural basis to elucidate the function of many synaptic proteins. At the same time, synaptic neurotransmission also relies crucially on processes governed by biological membranes. Synaptic vesicles as small membranous organelles store neurotransmitters in the cytoplasm of the synapse and release them into the synaptic cleft in a highly controlled manner. While synaptic vesicles can be described in a very detailed picture in terms of their molecular composition, less is known about structural rearrangements involved in the dynamic processes of neurotransmitter uptake and fusion with the presynaptic plasma membrane. This work employs X-ray diffraction, in particular small-angle X-ray scattering (SAXS), for structural investigations of vesicles in view of adhesion, fusion and neurotransmitter uptake. To this end, we study model lipid vesicles, as well as vesicles with reconstituted proteins and synaptic vesicles. SAXS is a powerful non-invasive technique which enables to probe the structure of the membrane as well as vesicle size and size distribution (polydispersity) under quasi-physiological conditions in solution. Membrane adhesion and fusion in the physiological system are the result of a complex interplay between lipids, fusiogenic proteins, ions and water molecules. From a physical point of view, attractive and repulsive forces are balanced in membrane adhesion, and repulsion has to be overcome to enable fusion. Experimentally, we show that adhesion of lipid vesicles induced by divalent salts can be clearly identified by SAXS. The deduced structural parameters, most importantly the interbilayer spacing, can be modeled based on theoretical inter-membrane interaction potentials. For moderately and highly charged systems the experimental results are well in line with the predicted strong-coupling attraction, which is associated with correlated counterions confined between two bilayers in close proximity. With the availability of highly brilliant synchrotron radiation, structural dynamics, reaction kinetics, and morphological transitions can be monitored by SAXS with millisecond time-resolution. This work employs rapid turbulent mixing in a stopped-flow device coupled to SAXS to study the structural dynamics of vesicle adhesion and fusion. This allows us to observe intermediate and transient adhesion states in charged vesicles. As a complementary technique to stopped-flow, we also evaluate continuous flow mixing in microfluidics devices for vesicle SAXS. Finally, we use SAXS to study the structural changes of synaptic vesicles after neurotransmitter (glutamate) uptake, and present first results on fusion of synaptic vesicles with vesicles with reconstituted fusiogenic proteins.
Keywords: small-angle x-ray scattering; time-resolved small-angle x-ray scattering; stopped-flow mixing; microfluidics; lipid vesicles; synaptic vesicles; vesicle adhesion; vesicle fusion; neurotransmitter uptake; x-ray structure analysis of vesicles; electrostatic strong coupling