Cryo-Electron Tomography Unveils Synaptic Vesicle Mysteries

“Synaptic vesicles are of paramount importance in brain function and they have been studied for decades. However, previous reports have described the molecular composition of an ‘average’ synaptic vesicle. Our study was able to image individual vesicles at molecular resolution,” says Kudryashev, corresponding author on the paper.

Synaptic versicles – sphere-like structures that store and release neurotransmitters such as dopamine and serotonin, are found in the presynaptic terminals of neurons. They play a crucial role in helping to transmit signals between neurons. The largest protein on their surface is a flower-shaped molecule called V-ATPase. Sitting next to it, is another small protein called synaptophysin. Researchers have thought that the two proteins interact, but no one had directly imaged them until this year, says Kravčenko.

“This study represents one of the first direct visualizations showing the localization of these two proteins on synaptic vesicle membranes,” she explains. Two other reports were published in the journals “Nature” and “Science” earlier this year.

The authors also imaged the locations of partially assembled and empty clathrin cages– lattice-like structures that play a pivotal role in recycling SVs back into cells – inside neurons. These cages were found closer to the cell membrane than had been previously shown, which could be a sign of an energy-efficient mechanism via which SVs are recycled, says Kravčenko, “but we need additional experiments to prove it.”

There are very few images of empty clathrin cages in the literature, she adds. “We show for the first time that empty cages are located closer to the cell membrane. This observation hints at a functional role for the empty cages found in neurons.”

Cryo-Electron Tomography in action

Cryo-electron tomography is an imaging technique that takes 2D images of cryogenically frozen samples at multiple tilt angles to reconstruct three-dimensional volumes of biological samples. It is most often used to study how macromolecules, cellular organelles, or cells are spatially organized, providing structural and contextual insights at sub-nanometer resolution.

Unlike other methods to analyze protein structure such as mass spectrometry or cryo-electron microscopy, cryo-electron tomography enables researchers to observe proteins in their native environment. Other techniques that require many more steps to process samples often lose important structural information.

“Our method preserves the vesicles in their native state, enabling us to image a broad variety of proteins on their surface,” says Kravčenko. Cryo-electron tomography enabled the researchers to show the spatial organization of proteins and unveiled a persistent association between V-ATPase and synaptophysin, suggesting that the interaction has an important function.

Implications for Neurological Research

The identified interactions between V-ATPase and synaptophysin offer insights into the molecular mechanisms underlying some neurological disorders. Specifically, those that involve dysfunction in synaptic vesicle recycling and neurotransmission, says Kravčenko.

“Now that we know that these two proteins interact, the information can be used for diagnostics, or to develop treatments for diseases associated with abnormal neurodevelopment.”

Reference: Kravčenko U, Ruwolt M, Kroll J, et al. Molecular architecture of synaptic vesicles. PNAS. 2024. doi: 10.1073/pnas.2407375121

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