Kaoru Mitsuoka

Double fertilization in angiosperms is completed by the fusion of 2 sperm cells with the egg and central cells. While sperm membrane proteins such as GEX2, DMP8/9, and GCS1/HAP2 have been identified as key regulators of gamete attachment and fusion, their specific ultrastructural roles within the embryo sac have remained unclear owing to the technical limitations of live-cell imaging based on fluorescent markers. Here, we optimized high-pressure freezing and freeze-substitution protocols for Arabidopsis pistils and performed 3-dimensional reconstructions using scanning transmission electron microscopy (STEM) and array tomography. We identified an electron-dense, partially thickened structure at the apical region of the egg cell before pollination. This structure disappeared following wild-type fertilization, suggesting it serves as a specialized “docking site” to accommodate pollen-tube contents and facilitate subsequent gamete interactions. Comparative analysis of fertilization mutants revealed distinct functional stages in the gamete-fusion process. In gex2 mutants, sperm cells failed to establish robust connections when positioned at the lateral side of the egg cell, confirming the requirement of GEX2 for initial attachment. In contrast, dmp8 dmp9 double mutant sperm cells facing each other were observed, exhibiting a more distant position from the egg surface, suggesting that DMP8/9 is involved in maintaining stable membrane adhesion before fusion. Furthermore, although gcs1 sperm cells maintained intimate contact and displayed membrane thinning, they failed to undergo final fusion. Our findings demonstrate that STEM and array tomography are powerful tools for visualizing gamete dynamics, providing an integrated model of the transition from stable adhesion to membrane fusion.

Mammalian mitochondrial ATP synthase typically organizes into rows of V-shaped dimers that impose significant membrane curvature essential for mitochondrial cristae formation. Using gentle, column-free purification combined with single-particle cryo-electron microscopy, we have identified a previously unrecognized planar dimeric form of bovine ATP synthase exhibiting minimal membrane bending. This planar dimer is characterized structurally by anti-parallel arrangement of two ATP synthase complexes linked by a straight conformation of inhibitory factor 1 (IF1), a sharp contrast to the kinked IF1 observed in tetrameric assemblies. Molecular dynamics simulations confirm that transitioning between straight and kinked IF1 conformations occurs without significant energetic barriers. The planar dimer also displays distinct peripheral stalk positioning relative to its adjacent α subunit. These structural divergences suggest a specialized function and a distinct localization for planar ATP synthase dimers, providing structural support for a division of labor within mitochondrial ATP synthase populations.

ATP synthases are rotary molecular machines that use the proton motive force to rotate the central rotor complex relative to the surrounding stator apparatus, thereby coupling the ATP synthesis. We reconstituted the V/A-ATPase into liposomes and performed structural analysis using cryo-EM under conditions where the proton motive force was applied in the presence of ADP and Pi. ATP molecules were bound at two of the three catalytic sites of V/A-ATPase, confirming that the structure represents a state adopted during ATP synthesis. In this structure, the catalytic site closes upon binding of ADP and Pi through an induced fit mechanism. Multiple structures were obtained where the membrane-embedded rotor ring was in a different position relative to the stator. By comparing these structures, we found that torsion occurs in both the central rotor and the peripheral stator during 31° rotation of rotor ring. These structural snapshots of V/A-ATPase provide crucial insights into the mechanism of rotary catalysis of ATP synthesis.

Mammalian mitochondrial ATP synthase typically organizes into rows of V-shaped dimers that impose significant membrane curvature essential for mitochondrial cristae formation. Using gentle, column-free purification combined with single-particle cryo-electron microscopy, we have identified a previously unrecognized planar dimeric form of bovine ATP synthase exhibiting minimal membrane bending. This planar dimer is characterized structurally by antiparallel arrangement of two ATP synthase complexes linked by a straight conformation of the inhibitory factor 1 (IF1), a sharp contrast to the kinked IF1 observed in tetrameric assemblies. Molecular dynamics simulations confirm that transitioning between straight and kinked IF1 conformations occurs without significant energetic barriers. The planar dimer also displays distinct peripheral stalk positioning relative to its adjacent α subunit. These structural divergences suggest a specialized functional role and localization distinct from the canonical sharply membrane bending ATP synthase oligomers, providing structural support for a model of membrane curvature-driven division of labor within mitochondrial ATP synthase populations.

ATP synthases play a crucial role in energy production by utilizing the proton motive force (pmf) across the membrane to rotate their membrane-embedded rotor c-ring, and thus driving ATP synthesis in the hydrophilic catalytic hexamer. However, the mechanism of how pmf converts into c-ring rotation remains unclear. This study presents a 2.8 Å cryo-EM structure of the V_o domain of V/A-ATPase from Thermus thermophilus, revealing precise orientations of glutamate (Glu) residues in the c₁₂-ring. Three Glu residues face a water channel, with one forming a salt bridge with the Arginine in the stator (a/Arg). Molecular dynamics (MD) simulations show that protonation of specific Glu residues triggers unidirectional Brownian motion of the c₁₂-ring towards ATP synthesis. When the key Glu remains unprotonated, the salt bridge persists, blocking rotation. These findings suggest that asymmetry in the protonation of c/Glu residues biases c₁₂-ring movement, facilitating rotation and ATP synthesis.

ParMs generally exist on low copy number plasmids where they contribute to plasmid segregation and stable inheritance. We carried out bioinformatics analysis, which indicated that ParM genes are not only confined to plasmids but are also occasionally found on genomes. Here we report the discovery and characterization of two chromosome encoded ParMs (cParMs) from the genomes of Desulfitobacterium hafniense (Dh-cParM1) and Clostridium botulinum (Cb-cParM). Both cParMs form filaments, exhibit nucleotide hydrolysis, and possess characteristic ParM subunit structures. Dh-cParM1 forms single and tightly coupled double filaments and is highly conserved on the chromosomes of five of six Desulfitobacterium species. Interestingly, these bacteria have not been reported to harbour plasmids. Cb-cParM possesses unique properties. Its filaments were stable after nucleotide hydrolysis and Pi release, and its ParR, (Cb-cParR) did not affect the initial stage of Cb-cParM polymerization but displayed properties of a depolymerization factor for mature filaments. These results indicate functional, polymerizing ParMs can be encoded on genomes, suggesting that ParM roles may extend to other functions beyond plasmid segregation.