Morphofunctional changes at the active zone during synaptic vesicle exocytosis.

Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

CryoVesNet: A dedicated framework for synaptic vesicle segmentation in cryo-electron tomograms.

Cryo-electron tomography (cryo-ET) has the potential to reveal cell structure down to atomic resolution. Nevertheless, cellular cryo-ET data is highly complex, requiring image segmentation for visualization and quantification of subcellular structures. Due to noise and anisotropic resolution in cryo-ET data, automatic segmentation based on classical computer vision approaches usually does not perform satisfactorily. Communication between neurons relies on neurotransmitter-filled synaptic vesicle (SV) exocytosis. Cryo-ET study of the spatial organization of SVs and their interconnections allows a better understanding of the mechanisms of exocytosis regulation. Accurate SV segmentation is a prerequisite to obtaining a faithful connectivity representation. Hundreds of SVs are present in a synapse, and their manual segmentation is a bottleneck. We addressed this by designing a workflow consisting of a convolutional network followed by post-processing steps. Alongside, we provide an interactive tool for accurately segmenting spherical vesicles. Our pipeline can in principle segment spherical vesicles in any cell type as well as extracellular and in vitro spherical vesicles.