The objective of this project is to determine the 3-D arrangement of actin filaments and auxiliary proteins in an actin anchoring network, the Z disk of striated muscle. The analysis will be based on electron microscopy, applied in combination with 3-D image reconstruction and averaging techniques. 3-D images (density maps) of native Z disks will be calculated from images of tilted thin sections; these will be analyzed to determine the arrangements of components and their interconnections. (1) The structure analysis of the entire bee flight muscle Z disk will be completed by determining the structure of the flanking regions of the Z disk which were not included in the reconstructions done in the previous grant period. The structure of the attachment site of C filament extensions of the thick filament to the Z line will be determined. (2) Structure features resolved in Z disk density maps will be identified by specific antibody labelling of bee Z disks, followed by 3-D structure analysis. Antibodies to alpha-actinin, and other Z disk component proteins will be prepared and used as labels. (3) The dynamics and function of the Z disk structure will be analyzed by determining the 3-D structure of the Z disk in different states of bee flight muscle contraction and tension. (4) The 3-D structure of the Drosophila Z disk will b determined as a first step towards a combined genetic and structural analysis of the function of the component proteins of the Z disk. (5) Pilot labelling experiments with monoclonal antibodies raised against Z disks and their component proteins will be carried out, with the eventual goal of identifying and localizing all of the component proteins of the Z disk.
