MICROSCOPY Vol.45▶No.1 2010
â– Lectures

3D Structural Analysis of Eukaryotic Flagella by Electron Cryo-tomography

Takashi Ishikawa

ETH Zurich

Abstract: Recently electron cryo-tomography provides new possibilities of structural analysis of biological molecules and organelles in vitro and in vivo. Frozen specimen is continuously tilted in the transmission electron microscope. Micrographs are taken at various tilt angles and backprojected into 3D reconstruction. Here in this article we will review our recent knowledge on the structure of outer and inner dynein arms, which is responsible for bending motion and its regulation, by making sliding among nine microtubule doublets in eukaryotic flagella using electron cryo-tomography. Three outer arm dyneins and eight inner arm dyneins have been identified by biochemistry, while physiological analysis of mutants characterized the outer dynein arm as a force generator and the inner dynein arm as a regulator of wave forms. 3D structural analysis by electron cryo-tomography revealed the location and conformation of dyneins in flagella. Three outer arm dyneins stack vertically, while eight inner arm dyneins array horizontally. All the dyneins have N-terminal tail domains at the distal side of flagella and head rings at the proximal side. Interestingly composition of inner arm dyneins is not identical among nine microtubule doublets from green algae Chlamydomonas. This might indicate the mechanism of planar asymmetrical bending motion of Chlamydomonas flagella.

Key words: Electron tomography, cryo-EM, 3D reconstruction, flagella, dynein