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

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.


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In many cell types, mitochondria collectively form a dy-29 namic network whose members divide, fuse, and commu-  (Fig. 1a-f). 146 To investigate variations in mitochondrial width along the 147 midpiece, we first measured the width of each mitochondrion 148 at multiple points along its length. We then divided mito-149 chondria into groups based on their positions along the mid-150 piece, as measured by their distance from the head (Fig. S1). 151 The midpiece is~10 µm long in both pig and horse sperm, 152 but~20 µm long in mouse sperm, so each group represents 153~2 µm in the pig and the horse and~4 µm in the mouse. We 154 found that mouse sperm mitochondria are~1.5 times wider 155 than pig and horse sperm mitochondria overall (Fig. S1a). 156 In all three species studied, most mitochondria in the mid-  (Fig. 1k-l). Pig sperm mitochondrial 178 morphology is intermediate (Fig. 1g-h), and although the mi-179 tochondrial matrix was dense, we could identify individual 180 complexes that resembled ATP synthase on cristae of FIB-181 milled mitochondria (Fig. S2a-b), which was confirmed by 182 subtomogram averaging (Fig. S2b'). 183 Inter-species differences in cristae morphology correlate 184 with measurements of matrix volume relative to mitochon-185 drial volume (Fig. S2d). In this regard, horse sperm mito-186 chondria resemble "condensed" mitochondria, which corre-187 late with higher rates of oxidative activity in a number of 188 different cell types, including developing germ cells, neu-189 rons, and liver (Hackenbrock, 1968;De Martino et al., 1979;190 Perkins and Ellisman, 2011). Indeed, horse sperm are de-191 pendent on oxidative phosphorylation (Davila et al., 2016), 192 whereas pig (Marin et al., 2003) and mouse sperm (Mukai 193 and (a-f) Slices through Volta phase plate cryotomograms (left) and corresponding three-dimensional segmentations (right) of mitochondria from the start (a-c) or middle (d-f) of the midpiece from pig (a,d), horse (b,e), and mouse (c,f) sperm. (g-l) Slices through cryo-tomograms of FIB-milled pig (g,h), horse (i,j), and mouse (k,l) sperm midpieces. Right panels show digital zooms of the regions boxed out in the left panels. The outer mitochondrial membrane is traced in green, the inner mitochondrial membrane in yellow, and the plasma membrane in blue. Arrowheads indicate inter-mitochondrial linker complexes. Labels: nuc -nucleus, sc -segmented columns, m -mitochondria, odf -outer dense fibers, dc -distal centriole, ax -axoneme, mtd -microtubule doublets, cpa -central pair apparatus, pm -plasma membrane. Scale bars: (a-l) left panels -250 nm, (g-l) right panels -100 nm.  (Picard et al., 2015). To our knowledge, this is the first time 207 this phenomenon has been observed in mature sperm from 208 any lineage. It is particularly curious, however, that trans-209 mitochondrial cristae alignment in sperm is species-specific. 210 We found that inter-mitochondrial junctions are charac-211 terized by novel inter-mitochondrial linker complexes in all 212 three species (arrowheads in Fig. 1g-l, Fig. S2c). These   cryo-FIB milled lamellae (Fig. 2). We found that the 231 axoneme-facing surface of the OMM is characterized by 232 an ordered protein array that is absent from the plasma 233 membrane-facing surface (Fig. 2a). These arrays are present 234 Fig. 2. Ordered protein arrays on the outer mitochondrial membrane directly interact with the submitochondrial reticulum. (a) Slice through a cryo-tomogram of a FIB-milled horse sperm midpiece, showing mitochondria (mito), the submitochondrial reticulum (smr) outer dense fibers (odf), microtubule doublets (mtd), and the central pair apparatus (cpa). Note how individual complexes (like the radial spoke, rs) are visible in the raw tomogram. The ordered protein array is only found on the axoneme-facing surface (yellow) of midpiece mitochondria, and not on the plasma membrane-facing surface (red). (b,c) Slices through a cryo-tomogram of a FIB-milled horse sperm midpiece showing how the array directly interacts with the submitochondrial reticulum to anchor mitochondria to the ciliary cytoskeleton (arrowheads). In right panels, the outer mitochondrial membrane is traced in green, the inner mitochondrial membrane in yellow, and the plasma membrane in blue.  2b-c), indicating that these arrays tether mitochondria to the 241 midpiece cytoskeleton. 242 We then aligned and averaged sub-volumes containing 243 the protein arrays and the underlying OMM (Fig. 3, Ta-244 ble S1). Our averages revealed~22-nm-long two-fold-245 symmetric boat-shaped structures connected via four densi-246 ties to a porous membrane (Fig. 3, Fig. S3g-i). Each boat-247 shaped particle rises~5 nm above the membrane and consists 248 of two tilde-shaped densities arranged end-to-end. The boat-249 shaped structures form rows in which each particle is related 250 to its closest neighbors by a~10 nm translation perpendicu-251 lar to the particle long axis and a~6 nm shift along this axis, 252 yielding a center-to-center spacing of~12 nm (Fig. 3d-f).  (Fig. 3, Fig. S3). This conservation suggests that these 260 arrays are a crucial structural element of the mitochondrial 261 sheath.

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Our averages revealed that the OMM underlying the pro-263 tein arrays is studded with~3-4 nm pores arranged in a 264 pseudo-lattice with a center-to-center spacing of~5 nm.  (Table S2). Furthermore, the lattice dimensions in 273 our averages closely match those of VDAC in purified Neu-274 rospora OMM (Guo and Mannella, 1993;Mannella, 1998). 275 The lattice can be modeled by fitting multiple copies of the     (Table S2), as would be expected for proteins forming exten-317 sive arrays. Assuming an average protein density of~1.43 318 g/cm 3 (Quillin and Matthews, 2000), which corresponds to 319~0 .861 Da/Å 3 , we estimate that each boat-shaped particle in 320 the array has a molecular weight of~250 kDa. SPATA19 is 321 a small protein with an estimated molecular weight of~18 322 kDa. To fit into our EM densities, it must either be present  To build a GK-VDAC model based on our subtomogram 330 average, we used rigid-body fitting to place two GK dimers 331 end-to-end into a boat-shaped density (Fig. S4b, Fig. 4b).

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These fits defined a clear orientation for GK, with the N-333 termini pointing upwards and the C-terminal helices facing 334 the OMM (Fig. 4b). To validate our fits, we mapped the 335 cross-linked lysines onto the resulting model (Fig. 4c). All  (Fig. 2b-c).

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In this study, we used cryo-FIB milling-enabled cryo-352 ET to image the sperm mitochondrial sheath in three mam-353 malian species. Our data reveal that overall mitochondrial di-354 mensions are remarkably consistent in sperm from the same 355 species (Fig. 1, S1). This contrasts with other mitochondria-        Table S1. 561 Alignment strategies for these complexes were designed 562 to take advantage of their defined orientations relative to the 563 membrane plane. Particles were picked manually and their 564 initial orientations were defined using stalkInit. Initial refer-565 ences were either a randomly chosen particle (for ladder-like 566 arrays) or an average of all particles after roughly aligning 567 them based on their initial orientations (for ATP synthase).

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Independent alignments using independent initial references 569 were performed for datasets from different species. Align-570 ments allowed for large rotational search ranges around the 571 particle long axis (defined as the y-axis, perpendicular to the 572 membrane plane), with limited search ranges around the x-573 and z-axes (the membrane plane).

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All initial alignments were performed without symmetry.       The crystal structure of VDAC2 from zebrafish (PDB 4BUM) is shown in grey, fitted into the cryo-ET averaged map (green). (b) Two copies of a crystal structure of GK (pink and blue) from Trypanosoma brucei (PDB 5GN6) fitted into the cryo-ET averaged map (grey). On the right, the GK crystal structure is shown filtered to 30Å resolution.