Confocal microscopy is known for its capability to produce exceptional 3D images, even in living tissue. At the same time, it is a powerful spectroscopic tool, facilitating fluores- cence methods such as Fluorescence Correlation Spectroscopy (FCS) or single-molecule Förster Resonance Energy Transfer (FRET). It is heavily used to investigate a wide range of biological problems. This holds true especially for protein properties such as ligand binding, complex formation, conformational changes, or the intracellular distribution of the species in question. In this work, I will describe the assembly of two instruments: The first is a multi- parameter fluorescence detection (MFD) setup. It is a purely spectroscopic tool that offers the capability to characterize a fluorescent molecule, delivering information like fluorescence lifetime, anisotropy or the speed of its diffusion in free solution. When the molecule of interest is labelled with two fluorophores, additional information, like the energy transfer in-between them, becomes accessible and the correct distance between these two fluorophores can be calculated. If the two fluorophores are attached to different molecules, the MFD setup can detect interactions of these molecules in the range from pM up to μM with the help of Fluorescence Cross-Correlation Spectroscopy (FCCS). The second instrument, a stimulated emission depletion setup, combines some of the mentioned techniques, like FCS, with the superior image capability of a confocal micro- scope. One particular problem of fluorescent microscopes, though, is that image resolution is always restricted to the diffraction limit of the wavelength of the laser light. The STED setup utilizes the effect of stimulated emission in order to circumvent the diffraction bar- rier and allows images with a three-fold resolution increase, down to 75nm. These two setups will be used for several applications: The first will be centered around the molecular conformation of proteins, which are sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. I will apply single-pair FRET to a small 29 amino acid long model peptide to investigate unfolding mechanisms of different unfolding reagents from the Hofmeister series, like sodium perchlorate or guanidinium chloride. The results show that certain salts, which are commonly summarized as denaturing agents, achieve the unfolding by either collapsing the molecule to a compressed state or swelling it to a denatured state. 7 The second application of the MFD setup is the investigation of the enhanced green fluorescent protein (EGFP). Although highly used in biochemistry and biophysics, for example to read out the expression level of genes, it is still not fully known what percentage of EGFP is fluorescent. This lack of knowledge makes it nearly impossible to make quantitative statements. With the help of FCCS, it is shown that the folding efficiencies range from 40 − 90%, depending on the environment of the fluorescent protein and which particular mutant is used. In the third application, the focus will be shifted to nucleation- and polymerization- behavior of actin. The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell di- vision. In order to compare results of confocal spectroscopy methods with well-established bulk essays, we successfully ported the standard bulk essay to the confocal microscope, allowing for the first time to follow the decrease of monomer concentration and appear- ance of small filaments. Also, the formation of dimers or other small oligomers below the critical concentration is proven for the first time, using FCCS. The last application will utilize the STED setup in order to carry out the first steps towards the investigation of the nucleation and branching behavior of actin in cooperation with the actin related protein 2/3 (ARP2/3). This protein complex preferentially attaches to actin filaments that are located at the leading edge of a cell and forms branched filamentous structures. The exact conditions under which this process occurs are not well characterized. This part of the work will deal with the steps that are necessary to follow the polymerization process on the STED setup.

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Targeting the cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. Whereas drugs which address microtubule CSK such as vinca alkaloids or taxanes are well established in the clinic, compounds binding to the actin CSK are still far away from their therapeutical application. One reason might be the lacking knowledge on their mode of cytotoxicity and moreover their tumor specific mechanism of action. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity by actin targeting in different breast cancer cells, namely MCF7 and MDA-MB-231. Chondramide inhibits actin filament assembly and dynamics shown by a fluorescence-based analysis (FRAP) in whole cells and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases (-9 and -3). Detailed analysis revealed, that Chondramide induces apoptosis by enhancing the occurrence of mitochondrial permeability transition (MPT). Known MPT-modulators were found to be affected by Chondramide: Hexokinase II (HkII) bound to the voltage dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad was recruited to the mitochondria. Importantly, PKCε, a prosurvival serine/threonine kinase possessing an actin-binding site and known to regulate the HkII/VDAC interaction as well as Bad phosphoylation was identified as the link between actin CSK and apoptosis induction. PKCε which was found overexpressed in breast cancer cells accumulated in actin bundles induced by Chondramide and lost its activity. The second goal of our work was to inform on a potential tumor specific action of actin binding agents such as Chondramide. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide induced apoptosis and notably express very low level of PKCε we claim that trapping PKCε via Chondramide induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCε, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKCε signaling shown in a xenograft breast cancer model. For the actin targeting compound Doliculide we could show that Doliculide impairs the dynamics of the actin CSK similar to Chondramide. Moreover, it reduces the proliferation rate and migration of cancer cells and also leads to the induction of apoptosis, thus Doliculide is also an interesting lead structure for further preclinical investigations.

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