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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.
