Sun Rong, Zhou Qiangjun
Department of Cell and Developmental Biology, Vanderbilt Brain Institute, Center for Structural Biology, Vanderbilt Kennedy Center, Vanderbilt University, Nashville, TN, USA.
Bio Protoc. 2025 Sep 5;15(17):e5429. doi: 10.21769/BioProtoc.5429.
Understanding the nanoscale organization and molecular rearrangement of synaptic components is critical for elucidating the mechanisms of synaptic transmission and plasticity. Traditional synaptosome isolation protocols involve multiple centrifugation and resuspension steps, which may cause structural damage or alter the synaptosomal fraction, compromising their suitability for cryo-electron tomography (cryo-ET). Here, we present an ultrafast isolation method optimized for cryo-ET that yields two types of synaptosomal fractions: synaptosomes and synaptoneurosomes. This streamlined protocol preserves intact postsynaptic membranes apposed to presynaptic active zones and produces thin, high-quality samples suitable for in situ structural studies. The entire procedure, from tissue homogenization to vitrification, takes less than 15 min, offering a significant advantage for high-resolution cryo-ET analysis of synaptic architecture. Key features • Ultrafast synaptic terminal isolation from tissue homogenization to vitrification completed within 15 min. • Retention of postsynaptic membranes with synaptic receptors and postsynaptic density (PSD) proteins. • The thickness of the samples is suitable for in situ cryo-ET analysis. • Enables cryo-ET studies of synaptic structures and postsynaptic membrane proteins such as AMPA receptors.
了解突触成分的纳米级组织和分子重排对于阐明突触传递和可塑性机制至关重要。传统的突触体分离方案涉及多个离心和重悬步骤，这可能会导致结构损伤或改变突触体组分，从而影响其对冷冻电子断层扫描（cryo-ET）的适用性。在此，我们提出一种针对cryo-ET优化的超快分离方法，该方法可产生两种类型的突触体组分：突触体和突触神经体。这种简化的方案保留了与突触前活性区相对的完整突触后膜，并产生适合原位结构研究的薄的高质量样本。从组织匀浆到玻璃化的整个过程耗时不到15分钟，为突触结构的高分辨率cryo-ET分析提供了显著优势。关键特性 • 从组织匀浆到玻璃化的超快突触末端分离在15分钟内完成。 • 保留带有突触受体和突触后致密物（PSD）蛋白的突触后膜。 • 样本厚度适合原位cryo-ET分析。 • 能够对突触结构和突触后膜蛋白（如AMPA受体）进行cryo-ET研究。
