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

Abstract

1 Mitochondria-cytoskeleton interactions modulate cellular 2 physiology by regulating mitochondrial transport, position-3 ing, and immobilization. However, there is very little struc-4 tural information defining mitochondria-cytoskeleton inter-5 faces in any cell type. Here, we use cryo-focused ion beam 6 milling-enabled cryo-electron tomography to image mam-7 malian sperm, where mitochondria wrap around the ciliary 8 cytoskeleton. We find that mitochondria are tethered to their 9 neighbors through inter-mitochondrial linkers and are an-10 chored to the cytoskeleton through ordered arrays on the 11 outer mitochondrial membrane. We use subtomogram aver-12 aging to resolve in-cell structures of these arrays from three 13 mammalian species, revealing they are conserved across 14 species despite variations in mitochondrial dimensions and 15 cristae organization. We find that the arrays consist of 16 boat-shaped particles anchored on a network of membrane 17 pores whose arrangement and dimensions are consistent 18 with voltage dependent anion channels. Proteomics and in-19 cell cross-linking mass spectrometry suggest that the con-20 served arrays are composed of glycerol kinase-like proteins. 21 Ordered supramolecular assemblies may serve to stabilize 22 similar contact sites in other cell types where mitochondria 23 need to be immobilized in specific subcellular environments, 24 such as in muscles and neurons. 25 sperm | mitochondria-cytoskeleton contact | cryo-electron tomography | cryo-26 FIB milling | cross-linking mass spectrometry | subtomogram averaging 27