3D STRUCTURE OF ACTIVATED AXONEMES STUDIED BY CRYO-ELECTRON TOMOGRAPHY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Eukaryotic cilia and flagella are highly conserved motile systems built on a microtubule-based scaffold called the axoneme. The complexity of this organelle and size of its major motor protein, dynein, have made it difficult to understand the molecular mechanisms that underlie flagellar beating. We are using electron tomography of frozen-hydrated flagella to solve this problem. 3-D reconstructions have enabled us to identify and localize single macromolecules in axonemes. Many of our results confirm previous descriptions of axonemes, but novel features have already been revealed. Outer dynein arms have been extracted from tomograms of a bent flagellum and classified into structural groups, using single particle averaging and Eigenvector-Eigenvalue analysis. This approach has identified three groups of dynein structures along a single doublet microtubule. The sites within the axoneme from which each of these three arm structures have come correlate with positions along the flagellar bend that correspond to straight, curving, and curved parts of the axoneme. These results indicate that the groups identified by objective methods might represent different conformational states of the dynein complex. However, two problems have emerged: 1) the thickness of the ice-layer in which the flagella are embedded is a resolution-limiting factor, so it is important to minimize it, so long as the flagella are not compressed. Indeed, the majority of frozen-hydrated flagella in our early preparations were somewhat compressed, but we have solved this problem by using a plunge-freezer with improved control of blotting prior to freezing. 2) The ¿missing wedge¿ is to date an inevitable artifact of cryo-electron tomographic reconstructions; it causes anisotropic distortions in the tomograms. Thus, more sophisticated image processing tools, e.g. for 3-D particle alignment, classification and ¿missing-wedge¿ correction must be developed. Though current data are encouraging, they must be improved to be truly informative about dynein¿s mechanism of action.