In the last decade, the in vivo biophysical research techniques such as live imaging, single molecule analysis and other methods have been developed. However the knowledges from structural biology are limited to the in vitro level. Therefore we need to know the structure of proteins which have function in the cells. In other words, we have to unveil the 3D structures of protein in vivo and atomic resolution. This is very difficult because of there complexity and large molecular weight.
We adopted the cryo-ET (cryo-electron tomography) technique to reveal the intracellular architecture at the molecular resolution. We focused on the formation mechanisms of filopodia. They are involved in many important roles such as cell migration, cell motility and so on.
We were successful to observe the ultrastructures and get the 3D volumes of filopodia with cryo-ET. In filopodia, actin filaments are bundled tightly and parallel. They are bundled by fascin which the famous actin filaments bundling protein. They bind to actin filaments in 36 nm period and this is equal to the half pitch of actin filaments. Moreover we using helical averaging technique to intracellular actin filaments and get the their molecular resolution 3D model and the actin filaments bundling model by fascin are observed.
The next step for our research is revealing architectures at the atomic resolution with 3D subtomogram averaging technique and EM-labeling techniques with metallothionein. This technique will give us more high quality 3D model of intracellular structures and new insights for filopodia formation mechanisms.