@phdthesis{Petrich2023,
  author    = {Petrich, Annett},
  title     = {Quantitative fluorescence microscopy methods to investigate molecular interactions and dynamics in living cells},
  doi       = {10.25932/publishup-61180},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-611800},
  school      = {Universit{\"a}t Potsdam},
  pages     = {244},
  year      = {2023},
  abstract  = {Biomolecules such as proteins and lipids have vital roles in numerous cellular functions, including biomolecule transport, protein functions, cellular homeostasis and biomembrane integrity. Traditional biochemistry methods do not provide precise information about cellular biomolecule distribution and behavior under native environmental conditions since they are not transferable to live cell samples. Consequently, this can lead to inaccuracies in quantifying biomolecule interactions due to potential complexities arising from the heterogeneity of native biomembranes. To overcome these limitations, minimal invasive microscopic techniques, such as fluorescence fluctuation spectroscopy (FFS) in combination with fluorescence proteins (FPs) and fluorescence lipid analogs, have been developed. FFS techniques and membrane property sensors enable the quantification of various parameters, including concentration, dynamics, oligomerization, and interaction of biomolecules in live cell samples. In this work, several FFS approaches and membrane property sensors were implemented and employed to examine biological processes of diverse context. Multi-color scanning fluorescence fluctuation spectroscopy (sFCS) was used the examine protein oligomerization, protein-protein interactions (PPIs) and protein dynamics at the cellular plasma membrane (PM). Additionally, two-color number and brightness (N\&B) analysis was extended with the cross-correlation analysis in order to quantify hetero-interactions of proteins in the PM with very slow motion, which would not accessible with sFCS due strong initial photobleaching. Furthermore, two semi-automatic analysis pipelines were designed: spectral F{\"o}rster resonance energy transfer (FRET) analysis to study changes in membrane charge at the inner leaflet of the PM, and spectral generalized polarization (GP) imaging and spectral phasor analysis to monitor changes in membrane fluidity and order. An important parameter for studying PPIs is molecular brightness, which directly determines oligomerization and can be extracted from FFS data. However, FPs often display complex photophysical transitions, including dark states. Therefore, it is crucial to characterize FPs for their dark-states to ensure reliable oligomerization measurements. In this study, N\&B and sFCS analysis were applied to determine photophysical properties of novel green FPs under different conditions (i.e., excitation power and pH) in living cells. The results showed that the new FPs, mGreenLantern (mGL) and Gamillus, exhibited the highest molecular brightness at the cost of lower photostability. The well-established monomeric enhanced green fluorescent protein (mEGFP) remained the best option to investigate PPIs at lower pH, while mGL was best suited for neutral pH, and Gamillus for high pH. These findings provide guidance for selecting an appropriate FP to quantify PPIs via FFS under different environmental conditions. Next, several biophysical fluorescence microscopy approaches (i.e., sFCS, GP imaging, membrane charge FRET) were employed to monitor changes in lipid-lipid-packing in biomembranes in different biological context. Lipid metabolism in cancer cells is known to support rapid proliferation and metastasis. Therefore, targeting lipid synthesis or membrane integrity holds immense promise as an anticancer strategy. However, the mechanism of action of the novel agent erufosine (EPC3) on membrane stability is not fully under stood. The present work revealed that EPC3 reduces lipid packing and composition as well as increased membrane fluidity and dynamic, hence, modifies lipid-lipid-interaction. These effects on membrane integrity were likely triggered by modulations in lipid metabolism and membrane organization. In the case of influenza A virus (IAV) infection, regulation of lipid metabolism is crucial for multiple steps in IAV replication and is related to the pathogenicity of IAV. Here, it is shown for the first time that IAV infection triggers a local enrichment of negatively charged lipids at the inner leaflet of the PM, which decreases membrane fluidity and dynamic, as well as increases lipid packing at the assembly site in living cells. This suggests that IAV alters lipid-lipid interactions and organization at the PM. Overall, this work highlights the potential of biophysical techniques as a screening platform for studying membrane properties in living cells at the single-cell level. Finally, this study addressed remaining questions about the early stage of IAV assembly. The recruitment of matrix protein 1 (M1) and its interaction with other viral surface proteins, hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2), has been a subject of debate due to conflicting results. In this study, different FFS approaches were performed in transfected cells to investigate interactions between IAV proteins themselves and host factors at the PM. FFS measurements revealed that M2 interacts strongly with M1, leading to the translocation of M1 to the PM. This interaction likely took place along the non-canonical pathway, as evidenced by the detection of an interaction between M2 and the host factor LC3-II, leading to the recruitment of LC3-II to the PM. Moreover, weaker interaction was observed between HA and membrane-bound M1, and no interaction between NA and M1. Interestingly, higher oligomeric states of M1 were only detectable in infected cells. These results indicate that M2 initiates virion assembly by recruiting M1 to the PM, which may serve as a platform for further interactions with viral proteins and host factors.},
  language  = {en}
}