Fluorescence based studies on neuronal processes
by Sabrina Schröder
Date of Examination:2014-10-17
Date of issue:2015-07-22
Advisor:Prof. Dr. Peter Jomo Walla
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Jörg Enderlein
Persistent Address:
http://dx.doi.org/10.53846/goediss-5193
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Abstract
English
Synaptotagmin-1 is a calcium dependent trigger of the SNARE-fusion machinery that ultimately upon stimulation executes signal transmission between neurons. However, the actual mechanistic function and the role of different calcium binding domains of synaptotagmin-1 still remain unclear. The original motivation for this study was therefore based on interesting findings and suggestions of three previous publications addressing the role of synaptotagmin-1 in exocytosis [121, 119, 83]. In one of these studies, it has been proposed that the triggering function of synaptotagmin-1 is based on a membrane distance regulation mechanism [119]. In addition, it has been shown that cis-membrane binding of synaptotagmin-1 is favoured over trans-binding [121], but that fusion nonetheless can be enhanced by ATP [83]. It remains unclear whether ATP prevents cis-binding directly or acts at a later stage by just pushing the equilibrium further towards the fused state. Furthermore, the role of synaptotagmin-1 in exocytotic priming and triggering is still elusive [49]. Here, two models have been proposed with each of them accounting for conflicting findings that have been made in the past [49].The authors of the above mentioned articles used different fluorescence techniques like fluorescence anisotropy [83], Förster-resonanceenergy- transfer [119] and Fluorescence-cross-correlation-spectroscopy (FCCS) [121]. The present study aims to find further evidence for or against a distance regulation function of synaptotagmin-1 with the help of additional FCCS studies combined with fluorescence lifetime analysis. In fact, the results presented in this thesis demonstrate that synaptotagmin-1 can act as a distance regulator when bound to PiP2 by synchronous alterations in tethering and membrane distance shortening. Here, synchronous tethering to an opposing lipid bilayer is furthermore achieved by the C2B domain of synaptotagmin-1. Calcium ions, which are necessary for tethering, trigger this PiP2 dependent distance regulation function. Despite the cooperative behaviour of synaptotagmin-1 towards binding Ca2+ in the presence of PiP2 [118], a reduction of the intermembrane distance occurs proportional to tethering. However, high PiP2 concentrations increase the base level of tethering. Moreover, the distance regulation function of synaptotagmin-1 is circumvented by the presence of syntaxin 1A. In addition, a new approach to study tethering and fusion processes by optical trapping is introduced. This method reveals the first way of studying exocytotic processes at plasma membrane (PLM) analogues without introducingany artifacts like an enhanced surface tension. It is demonstrated that optical trapping of large unilamellar vesicles (d= 110 nm) is possible when filled with 2 M sucrose. Here, the combination of a CW and a TP laser beam, both operating at 800 nm, permits for super-position of the trapping center with the TPE-volume. Hence, trapping and FCS can be combined, permitting to study processes in real-time. A conceivable application arising from the combination of both laser beams is to study retention times of liposomal tethering which may be analyzed in terms of adsorption isotherms. Furthermore, fusion to a single LUV can be monitored on a min timescale.
Keywords: FCS
Schlagwörter: FCS