Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
Cell Rep Methods. 2022 Apr 25;2(4):100199. doi: 10.1016/j.crmeth.2022.100199.
A complete understanding of synaptic-vesicle recycling requires the use of multiple microscopy methods to obtain complementary information. However, many currently available probes are limited to a specific microscopy modality, which necessitates the use of multiple probes and labeling paradigms. Given the complexity of vesicle populations and recycling pathways, having new single-vesicle probes that could be used for multiple microscopy techniques would complement existing sets of tools for studying vesicle function. Here, we present a probe based on the membrane-binding C2 domain of cytosolic phospholipase A (cPLA) that fulfills this need. By conjugating the C2 domain with different detectable tags, we demonstrate that a single, modular probe can allow synaptic vesicles to be imaged at multiple levels of spatial and temporal resolution. Moreover, as a general endocytic marker, the C2 domain may also be used to study membrane recycling in many cell types.
要全面了解突触囊泡的再循环，需要使用多种显微镜方法来获取互补信息。然而，许多现有的探针仅限于特定的显微镜模式，这需要使用多种探针和标记范式。鉴于囊泡群体和再循环途径的复杂性，拥有新的可用于多种显微镜技术的单个囊泡探针将补充现有的研究囊泡功能的工具集。在这里，我们提出了一种基于胞质磷脂酶 A (cPLA) 的膜结合 C2 结构域的探针，满足了这一需求。通过将 C2 结构域与不同的可检测标记物连接，我们证明了单个模块化探针可以在多个时空分辨率水平上对突触囊泡进行成像。此外，作为一种通用的内吞标记物，C2 结构域也可用于研究许多细胞类型中的膜再循环。
