Abstract
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.
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Data availability
The tomograms and subtomogram averages have been deposited into the EMDB under accession code EMDB-41124 and the raw data deposited in EMPIAR under accession code EMPIAR-11594.
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Acknowledgements
This research was supported by NIH grants R01 GM30598 and R35 GM139616 to KAT. The Titan was partially funded from NIH grant S10 RR025080 with funds for the DE-20 provided by S10 OD018142. ZH and HR were funded by American Heart Association predoctoral fellowships 15PRE25090150 and 20PRE35120273 respectively. We thank Dr. Belinda Bullard for her comments on an earlier version of the manuscript and Dr. Alimohammed Hojjatian for his assistance in the data upload. We dedicate this paper to the late Prof. John Trinick who stimulated our interest in studying the invertebrate Z-disk structure.
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FAY performed the reconstruction and wrote the paper CS produced an earlier reconstruction and wrote the paper DWT isolated the Z-disks and prepared cryoEM specimens ZH collected the reconstruction data HR Supervised subtomogram processing KAT supervised the work and wrote the paper.
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Yeganeh, F.A., Summerill, C., Hu, Z. et al. The cryo-EM 3D image reconstruction of isolated Lethocerus indicus Z-discs. J Muscle Res Cell Motil 44, 271–286 (2023). https://doi.org/10.1007/s10974-023-09657-1
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DOI: https://doi.org/10.1007/s10974-023-09657-1