Molecular Mechanical Characters Of Microtubule System, Studied By Optical Tweezers

Microtubules (MTs), one of the major components of the cytoskeleton in eukaryotic cells, play vital roles in many biological processes. One of the most important functions of MTs is to generate mechanical force to power various movements of cells. Study of the mechanical characters of MT benefits our understanding MT functions and the mechanism of force generation. Because of the advantages of non-contact and non-intrusive manipulation with living objects, and measuring displacement in nanometer scale and force in pico-Newton scale, optical tweezers have been now widely applied in biological studies. In this dissertation, a dual-optical tweezers system was used to study the molecular mechanical characters of microtubule system, including the breaking force of fluorescent labeled microtubule, the process of the photosensitive breakage, the elasticity of MT, the mechanisms of the photosensitive breakage, and the unbinding force between microtubule associate proteins (MAPs) and MTs.The biological system practicable for optical tweezers studies was first set up. Tubulin was purified from porcine brain and labeled by rhodamine and biotin. MTs were assembled in vitro and stabilized by taxol.During experiments, we found fluorescence labeled MTs broke under excitation light. To resolve this problem, we investigated the mechanism of MT cleavage in detail. The results showed that fluorescent dye and excitation light were both necessary to the photosensitive breakage. The stronger the light intensity and the higher the dye concentration, the faster broke up the MTs. It is the reactive oxygen species (ROS) produced by the interaction between the dyes and their excitation light led to the MT breakage. Free radicals, one kind of ROS, were the main destroyers. Ascorbic acid restrained the fluorescent MT breakage effectively.The operation system was then set up, including the sample cells and microsphere for the manipulation. Various sample cells were made according to different experiments. Flow cell and temperature-control flow cell were also made for replacement of the buffer and controlling temperature. Optical tweezers 'caught' single MT through microshperes which were coated with strepavidin to bind to the MT. And the mechanical force was measured by the optical tweezers.Fluorescence labeled MTs were elongated about 20% in average when stretched by optical tweezers. When some protofilaments of the MT broke up, the residual protofilaments was stretched even longer. This result indicated the elastic property of fluorescence labeled MTs. The persistence length of fluorescence labeled MT was 3 orders of magnitude shorter than that of MTs without fluorescence labeled. It was affected by taxol and excitation light. The breaking force obtained by the optical tweezers experiments was majorly around 3 pN, which was much smaller than that without fluorescence label MTs. The interaction force between tubulins was weakened by the excitation light.Stairs-like steps occurred during the recording of the breakage process. It showed that the broken of protofilaments of the MT was not simultaneous but step by step, suggesting protofilaments were not attacked by free radicals correlatively and uniformly, but independently. It also inferred that the interactions between protofilaments are very weak.In addition, we measured the unbinding force between AtMAP65-l, a MAP from plant, and MT. MTs were bundled by AtMAP65-l, and the force to separate a MT from the bundle was measured by optical tweezers. According to the results, the force might be 7.5 pN or 15 pN.The researches offer new approach and idea for studying mechanical characters of the cytoskeleton and other biological macromolecules.