Institute of Anatomy, University of Bern, Bern, Switzerland.
Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark.
EMBO Rep. 2023 May 4;24(5):e55719. doi: 10.15252/embr.202255719. Epub 2023 Mar 6.
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.
突触小泡 (SV) 与质膜 (PM) 的融合经过中间步骤，但这些中间步骤仍未得到很好的解析。持续的高或低胞吐活性对中间步骤的影响尚不清楚。我们使用喷雾混合式快速冷冻 cryo-electron 断层扫描技术，在接近天然的样本中以纳米分辨率观察突触刺激后的事件。我们的数据表明，在刺激后立即进入的阶段，称为早期融合，PM 和 SV 膜曲率发生变化，形成一个点接触。下一阶段 - 晚期融合 - 显示融合孔的打开和 SV 的塌陷。在早期融合中，近端连接的 SV 与 PM 形成额外的连接，并增加 SV 连接器的数量。在晚期融合阶段，PM 近端的 SV 失去它们的连接，使它们能够向 PM 移动。两种 SNAP-25 突变，一种阻止，一种抑制自发释放，导致连接器丢失。抑制突变导致膜近端的多连接 SV 丢失。总的来说，连接的形成和连接器的溶解是由刺激触发的，并对自发融合速率的操纵作出反应。这些形态学观察结果可能对应于 SV 从一个功能池向另一个功能池的转变。
