@phdthesis{Dunsing2020,
  author    = {Dunsing, Valentin},
  title     = {Fluorescence fluctuation spectroscopy techniques to quantify molecular interactions and dynamics in complex biological systems},
  doi       = {10.25932/publishup-47849},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-478494},
  school      = {Universit{\"a}t Potsdam},
  pages     = {VII, 164, XXV},
  year      = {2020},
  abstract  = {Living cells rely on transport and interaction of biomolecules to perform their diverse functions. A powerful toolbox to study these highly dynamic processes in the native environment is provided by fluorescence fluctuation spectroscopy (FFS) techniques. In more detail, FFS takes advantage of the inherent dynamics present in biological systems, such as diffusion, to infer molecular parameters from fluctuations of the signal emitted by an ensemble of fluorescently tagged molecules. In particular, two parameters are accessible: the concentration of molecules and their transit times through the observation volume. In addition, molecular interactions can be measured by analyzing the average signal emitted per molecule - the molecular brightness - and the cross-correlation of signals detected from differently tagged species. In the present work, several FFS techniques were implemented and applied in different biological contexts. In particular, scanning fluorescence correlation spectroscopy (sFCS) was performed to measure protein dynamics and interactions at the plasma membrane (PM) of cells, and number and brightness (N\&B) analysis to spatially map molecular aggregation. To account for technical limitations and sample related artifacts, e.g. detector noise, photobleaching, or background signal, several correction schemes were explored. In addition, sFCS was combined with spectral detection and higher moment analysis of the photon count distribution to resolve multiple species at the PM. Using scanning fluorescence cross-correlation spectroscopy and cross-correlation N\&B, the interactions of amyloid precursor-like protein 1 (APLP1), a synaptic membrane protein, were investigated. It is shown for the first time directly in living cells, that APLP1 undergoes specific interactions at cell-cell contacts. It is further demonstrated that zinc ions induce formation of large APLP1 clusters that enrich at contact sites and bind to clusters on the opposing cell. Altogether, these results provide direct evidence that APLP1 is a zinc ion dependent neuronal adhesion protein. In the context of APLP1, discrepancies of oligomeric state estimates were observed, which were attributed to non-fluorescent states of the chosen red fluorescent protein (FP) tag mCardinal (mCard). Therefore, multiple FPs and their performance in FFS based measurements of protein interactions were systematically evaluated. The study revealed superior properties of monomeric enhanced green fluorescent protein (mEGFP) and mCherry2. Furthermore, a simple correction scheme allowed unbiased in situ measurements of protein oligomerization by quantifying non-fluorescent state fractions of FP tags. The procedure was experimentally confirmed for biologically relevant protein complexes consisting of up to 12 monomers. In the last part of this work, fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT) were used to characterize diffusive transport dynamics in a bacterial biofilm model. Biofilms are surface adherent bacterial communities, whose structural organization is provided by extracellular polymeric substances (EPS) that form a viscous polymer hydrogel. The presented study revealed a probe size and polymer concentration dependent (anomalous) diffusion hindrance in a reconstituted EPS matrix system caused by polymer chain entanglement at physiological concentrations. This result indicates a meshwork-like organization of the biofilm matrix that allows free diffusion of small particles, but strongly hinders diffusion of larger particles such as bacteriophages. Finally, it is shown that depolymerization of the matrix by phage derived enzymes rapidly facilitated free diffusion. In the context of phage infections, such enzymes may provide a key to evade trapping in the biofilm matrix and promote efficient infection of bacteria. In combination with phage application, matrix depolymerizing enzymes may open up novel antimicrobial strategies against multiresistant bacterial strains, as a promising, more specific alternative to conventional antibiotics.},
  language  = {en}
}