Imaging and model analysis of molecular function in cells

The detailed molecular functions of purified proteins have been investigated deeply in the biophysical and biochemical works. However, physiological conditions in the cell environment are very different from those in in vitro assays of purified proteins where the experiments are performed; for example, the ion composition is different in the cells and the assays cells have adaptor proteins and cytoskeletal networks that are not present in the assays. Therefore, it is crucial to measure the molecular functions of motor proteins in cells to understand the molecular mechanisms of vesicle transport by motor proteins. We planed to measure the force and movements generated by myosin, dynein, and kinesin in living cells to understand the molecular mechanisms of movement within cells. In the previous works, the force of vesicles driven by motor proteins was measured the conventional optical trapping. The refractive index and size of the vesicles had not determined accurately in the previous works. We constructed apparatus to be able to measure the force independent of these factors. The momentum changes of photon was directly measured from the angle of scattered light and number of photons.

Tumor was exposed by dissecting the skin and a epicedium, so far. Many biological systems especially the immune system were activated by the dissection. To avoid the dissection, we developed the method of non-invasive imaging. The auricle was selected for non-invasive imaging because of thin and short hair. The auricle was illuminated by high power laser for short time. We investigated the motility of vesicle in neutrophil, a kind of white blood cell, in mice. Neutrophil is activated by the inflammation of TPA and goes out of blood vessel to cure the inflammation. 1 day after the inflammation, many of neutrophil go out of the blood vessel. We observed clearly the movement of vesicle containing antibody-quantum dot. The shape of neutrophil becomes long and front domain may pull the cell. The velocity of the vesicle was changed very much from 0.3 to 3.1 um/s. Surprisingly, the velocity of 3 um/s is about three times of velocity of dynein and kinesin.

Publications

Y. Toyoshima, H. Higuchi, “Motile and Enzymatic properties of native dynein molecules” in Handbook of Dynein. K. Hirose and LA Amos ed. (2012).