In-cell structures of a conserved supramolecular array at the mitochondria-cytoskeleton interface in mammalian sperm

Abstract

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

Molecular characterization of a complex of apoptosis-inducing factor 1 with cytochrome c oxidase of the mitochondrial respiratory chain

“…Cross-linked mitochondria were solubilized with digitonin (9 g/g protein) for 30 to 60 min on ice. Proteins were denatured and purified as described previously ( 61 ). Briefly, denatured proteins were resuspended and digested overnight at 37 °C with Lys-C followed by trypsin.…”

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology

“…This approach so far has successfully been applied to intact organelles with large transport systems located on the membrane, including isolated nuclei and mitochondria (Liu et al, 2017;Schweppe et al, 2017;Fasci et al, 2018). Beyond organelles, in situ XL-MS has been applied to biopolymers (Klykov et al, 2020), tissues (Chavez et al, 2018), thylakoid membranes (Albanese et al, 2020), and mammalian sperm cells (Leung et al, 2021). In the case of other types of intact cells, the reports in the literature remain controversial because all currently available and most widely used crosslinking reagents, except for disuccinimidyl suberate (DSS), carry charges, preventing their passage through the membrane.…”

“…(D) Cross-section of a cellular tomogram with membrane-bound chemosensory arrays and unidentified associated densities highlighted in yellow and purple, respectively. In this case, XL-MS along with quantitative shotgun MS data generated in parallel would help to identify unassigned densities, protein complex orientation, and higher-order protein organization through protein self-links (analogous to Leung et al, 2021). The cross-sections through the cellular tomograms are taken from the publicly available Atlas of Bacterial and Archaeal Cell Structure (https://www.…”

“…The most important impact that XL-MS information may have on cryo-ET data in the near future is to provide insights into the biological function of large macromolecular assemblies. Leung et al, 2021 show the potential for MS-based methods to supplement cryo-ET data in terms of protein assignment and localization, resulting in valuable biological insights. First, based on low-resolution cryo-ET density maps, periodicity of the arrays, and pore sizes, it was possible to identify the components of repeating protein arrays in tomograms as voltage-dependent anion channels (VDACs).…”

VDAC Genes Expression and Regulation in Mammals

“…Cryo-electron tomography images of mammalian sperms resolved the structure of mitochondrial wrap around the flagellum cytoskeleton and revealed a network of proteins, including VDAC2 and VDAC3. Their role is essential for maintaining the interaction between mitochondria and cytoskeleton and to stabilize sperms during motility and activation (Leung et al, 2021 ).…”