Branimir Lukic

Awarded for BME Diploma Thesis

Branimir Lukic was born in Zagreb in 1979 and finished elementary and secondary school in Pozega. He followed undergraduate studies in physics at Faculty of Science at University of Zagreb and finished in 2002 with diploma work in biophysics. Currently, he is a graduate student at Swiss Federal Institute of Technology in Lausanne. His interests include mechanics on nanoscale, cell mechanics and techniques of atomic and photonic force microscopies. Since 2000, he is also involved in educational activities at Visnjan Observatory targeted at elementary and high school student population, with aim to introduce them to scientific research. In 2003 , Lukic received the Annual Prize for Best Diploma Thesis, awarded by the Croatian Medical and Biological Engineering Society for his thesis:

"Determination of the limits of mechanical stress in microtubules"

Three types of protein polymers, thin and weak acting filaments, thick and stiff microtubules and intermediate filaments, build a cytoskeleton - a meshwork of proteins present in every eucariotic cell. Very complex mechanical behavior of the cell is the result of interplay between these polymers of very different mechanical properties, which are further influenced by other proteins or drugs. Several important processes depend on them, like mitosis, transport of organelles, organisation of contents in the cell, and cell movements.

However, knowledge of their mechanical behavior is still incomplete. Only recently it has been shown that cylindrical microtubules are not isotropic polymers, structurally, energetically or mechanically. The principal aim in this thesis was to see what kind of behavior microtubules show under large stresses and strains. Such answers could provide a clue to some processes in the cell. For example, microtubules are usually connected to organelle centrosome, but in cells like neurons, many are free and are accumulated in the axon. Do they break away from the centrosome mechanically and what kind of forces would be needed for it?

Atomic force microscopy (AFM) was used in this thesis due to its ability to measure forces at the nanoscale. Microtubules (25 nm in diameter) are first deposited on a porous surface, and they occasionally lie over holes that are around 200 nm in diameter. AFM tip is then brought into contact and microtubules are pressed in the middle. By measuring both applied force and vertical deflection of the tube, one can estimate bending modulus. Experiment is done in real time, with AFM tip connected to a special interface - nanomanipulator. This haptic device, conceptually similar to a computer joystick, allows real-time positioning of the AFM tip over the sample while letting the user feel the force exerted on the AFM tip. Scaling of forces and movements is in the order of 10⁶-10⁷. The whole experiment is similar to a macroscopic case when a bar is supported on two ends and pressed in the middle.

All in all, five microtubules were deformed, some of them several times. Fracture was not observed up to forces of 0.6 nN, and they seem to be linearly elastic material up to stresses of 13 MPa. Linearity was checked by comparison of values of elastic modulus with those at smaller deformations, and elasticity by deflection of microtubules before and after deformation. These values are similar to those obtained for actin filaments, and still well below theoretical limit. But very high deformations (in one case, 66 nm in vertical deflection for a microtubule with suspended length of 125 nm) are quite surprising, given that no visible effect on the microtubule, no change in linearity and no fracture of the structure were observed. This could maybe point to an unknown biological function.