TY - THES A1 - Nordblom, Noah Frieder T1 - Synthese und Evaluation von Gephyrinsonden für hochauflösende Mikroskopieverfahren T1 - Synthesis and Evaluation of Super Resolution Compatible Gephyrin Probes N2 - This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay. N2 - Neuentwickelte, hochauflösende Fluoreszenzmikroskopieverfahren sind prinzipiell geeignet, molekulare Mechanismen und zelluläre Vorgänge im niedrigen Nanometerbereich aufzulösen. Die maximal erreichbare Auflösung wird unter anderem von der eingesetzten Fluoreszenzmarkierung beeinflusst. In dieser Arbeit beschreibe ich die Synthese neuartiger, funktioneller, fluoreszierender Proben und evaluiere deren Eigenschaft Gephyrin, einen universalen Marker der neuronalen inhibitorischen Postsynapse, zu visualisieren. Hierzu wurden Peptide mittels Festphasenpeptidsyntese hergestellt und mit fluoreszierenden Farbstoffen konjugiert, die für hochauflösende Mikroskopieverfahren geeignet sind. Der Syntheseerfolg und die Reinheit der Stoffe wurde mittels massenspektrometrischer und chromatographischer Methoden bestimmt. In einem Hochdurchsatzverfahren wurden die Proben in einem zellulären Kontext untersucht, spezifisch Gephyrin zu markieren. In einem semi-automatisierten, parallelen Verfahren wurden drei ausgewählte Proben synthetisiert und deren Kolokalisation mit dem fluoreszierenden Zielprotein in transfizierten HEK-Zellen untersucht. Aus dieser Arbeit ist NN1DC hervorgegangen, eine peptidische Sonde zur Visualisierung von Gephyrin. Diese Probe weist verbesserte Färbeeigenschaften wie eine höhere Spezifität und Sensitivität, verglichen mit bisher bekannten peptidischen Gephyrinsonden, auf. Darüber hinaus kann NN1DA als hochaffiner Binder von Gephyrin zur Entwicklung neuer gephyrinbindender Moleküle in einem high-througput Verfahren genutzt werden. KW - Fluoreszenzmikroskopie KW - Peptidsynthese KW - Gephyrin KW - Inhibitorische Synapse KW - Fluorescence microscopy KW - Solid-phase peptide synthesis KW - Affinity probe KW - Inhibitory synapse Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-302300 ER - TY - JOUR A1 - Eckhardt, Manon A1 - Anders, Maria A1 - Muranyi, Walter A1 - Heilemann, Mike A1 - Krijnse-Locker, Jacomine A1 - Müller, Barbara T1 - A SNAP-Tagged Derivative of HIV-1-A Versatile Tool to Study Virus-Cell Interactions JF - PLoS ONE N2 - Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV derivative HIV(SNAP), which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIV(SNAP) represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy. KW - Human-immunodeficiency-virus KW - Fusion proteins KW - Live cells KW - Fluorescence microscopy KW - Stimulated-emission KW - Plasma-membrane KW - Living cells KW - Real-time KW - TYPE-1 KW - GAG Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133534 VL - 6 IS - 7 ER -