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Interactions of FCHo2 with lipid membranes

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Chwastek,  Grzegorz
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Chwastek, G. (2013). Interactions of FCHo2 with lipid membranes. PhD Thesis, Technische Universität, Dresden.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0015-7EE8-C
Abstract
Endocytosis is one of the most fundamental mechanisms by which the cell communicates with its surrounding. Specific signals are transduced through the cell membrane by a complex interplay between proteins and lipids. Clathrin depended endocytosis is one of the most important signalling pathways which leads to budding of the plasmalemma and a formation of endosomes. The FCHo2 is an essential protein at the initial stage of the this process. It is a membrane binding protein containing BAR (BIN, Amphiphysin, Rvs) domain which is responsible for membrane binding. Although numerous valuable studies on BAR proteins were published recently, the mechanistic description of BAR domain functionality is missing. In the present work, we applied in vitro systems in order to gain knowledge about the molecular basis of the activity of the FCHo2 BAR domain. In our studies, we used supported lipid bilayers (SLBs) and lipid monolayers as s model membrane system. The experiments were carried out with a minimal number of components including the purified FCHo2 BAR domain. Using SLBs, we showed that the BAR domain can bind to entirely flat bilayers.We also demonstrated that these interactions depend on the negatively charged lipid species incorporated in the membrane.We designed an assay which allows to quantify the membrane tubulation. We found out that the interaction of the FCHo2 BAR domain with the lipid membrane is concentration dependent. We showed that an area of the bilayer deformed by the protein depends on the BAR domain concentration. In order to study the relation between the mobility of the lipids and the activity of FCHo2 BAR domain, we designed a small-volume monolayer trough. The design of this micro-chamber allows for the implementation of the light microscopy. We demonstrated that the measured lipid diusion in the monolayer by our new approach is in agreement with the literature data. We carried out fluorescence correlation spectroscopy (FCS) experiments at dierent density of lipids at the water-air interface. We showed that the FCHo2 BAR domain binding anity is proportional to the mean molecular area (MMA). We additionally demonstrated that the increased protein binding is correlated with the higher lipid mobility in the monolayer. Additionally, by curing out high-speed atomic force microscopy (hsAFM) we acquired the structural information about FCHo2 BAR domains orientation at the membrane with a high spatio-temporal resolution. Obtained data indicate the BAR domains interact with each other by many dierent contact sites what results in a variety of protein orientations in a protein assemble.