The cryo-EM 3D image reconstruction of isolated Lethocerus indicus Z-discs.

The Z-disk of striated muscle defines the ends of the sarcomere, which repeats many times within the muscle fiber. Here we report application of cryoelectron tomography and subtomogram averaging to Z-disks isolated from the flight muscles of the large waterbug Lethocerus indicus. We use high salt solutions to remove the myosin containing filaments and use gelsolin to remove the actin filaments of the A- and I-bands leaving only the thin filaments within the Z-disk which were then frozen for cryoelectron microscopy. The Lethocerus Z-disk structure is similar in many ways to the previously studied Z-disk of the honeybee Apis mellifera. At the corners of the unit cell are positioned trimers of paired antiparallel F-actins defining a large solvent channel, whereas at the trigonal positions are positioned F-actin trimers converging slowly towards their (+) ends defining a small solvent channel through the Z-disk. These near parallel F-actins terminate at different Z-heights within the Z-disk. The two types of solvent channel in Lethocerus are similar in size compared to those of Apis which are very different in size. Two types of α-actinin crosslinks were observed between oppositely oriented actin filaments. In one of these, the α-actinin long axis is almost parallel to the F-actins it crosslinks. In the other, the α-actinins are at a small but distinctive angle with respect to the crosslinked actin filaments. The utility of isolated Z-disks for structure determination is discussed.

Tau induces formation of α-synuclein filaments with distinct molecular conformations.

Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.