Cryo-Electron Tomography and Sub-Tomogram Averaging of Isolated Z-Discs from Honeybee Flight Muscle

Abstract

The Z-disc is the mechanical linkage that transmits the active and passive forces developed by muscle sarcomeres. It is also the location of proteins involved in diverse other processes, such as stress sensing into signaling pathways controlling muscle growth and wasting. About 40 different proteins are now known to be in the Z-disc, some to which are transient and relocate to other destinations such as the nucleus. The main route of force transmission is through pairs of antiparallel α-actinin Z-bridges; these link the overlapped ends of thin filaments from adjacent sarcomeres. The gross structure of the Z-disc varies widely between different muscles and species. Thickness varies in proportion to the number of registers of Z-bridges. The lattice of thin filament ends is hexagonal in invertebrates, whereas in vertebrates it is tetragonal. However the structure of the Z-disc is known only in outline to ∼ 7nm resolution and the detailed layout of its components is mostly unknown. Methods to isolate Z-discs date back 50 years but such preparations have not been subjected to modern electron microscopy methods, such as cryo-EM, tomography or image processing. An advantage of isolated discs is they are thin, which obviates the need for sectioning for microscopy, which is damaging. We have prepared Z-discs from honeybee flight muscle using high salt extraction and density gradient purification. Vitrified preparations were examined in the 300 kV Krios microscope at the MRC Laboratory of Molecular Biology, Cambridge, UK. Tilt series images were recorded to +/− 70° with a Falcon II direct electron detector. 3D reconstruction and sub-tomogram averaging used Protomo software