PhD Scientific Days 2021

MO_II_P: Molecular Sciences II. Posters

Development of a two-dimensional array for exploring the biophysics of protein binding to actin

Text of the abstract

Actin is the most abundant protein in the human body. It takes part in several biological processes including signal transduction, muscle contraction and it determines cell shape and motility. As a scientific hotspot, all actin’s properties are widely studied. In striated muscle tissue it binds not just with myosin, but with giant protein titin as well. The exact localisation and strength of the bond is still unexplored.
Our aim is to create a setup where we can visualise titin-actin interactions in physiological environment with great magnification. We need a stable actin structure or pattern to measure titin binding and a microscopic method which allows us to follow the binding process real time and its limit of resolution should be nanometer-scale.
A 1:1 mixture of DPPC (Dipalmitoylphosphatidylcholine) and EPC (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) lipids was hydrated in buffer (10 mM Tris, 100 mM NaCl, 3 mM CaCl2, pH 7.4) at 1 mM final lipid concentration. We created lipid vesicules using extrusion with 100 nm polycarbonate membrane. On a freshly cleaved mica surface 100 µl of lipid mixture was incubated for 30 minutes at room temperature. The temperature was raised to 55°C for 15 minutes and vesicules were ruptured to form a charged lipid bilayer. Actin filaments (at 2µM concentration) were added to the surface, after a 15-minute-long incubation actin paracrystals were formed. We chose atomic force microscopy (AFM) as a microscopical method. Non-contact mode allows us to achieve high resolution without disturbing protein-protein interactions.
We succesfully created a stabile two dimensional structure, with its help we can visualise actin filaments with great magnification. Important characteristics of the filaments could be measured like : monomer structures, thickness, subunits and conformational changes. This new setup enables us to follow titin-actin binding process and actin polimerization as well.

Funding: EFOP-3.6.3-VEKOP-16-2017-00009

University and Doctoral School

Semmelweis University, Doctoral School of Theoretical and Translational Medicine