Multimodal imaging of synaptic vesicles with a single probe

• Published in: Cell reports methods 2022-04, Vol.2 (4), p.100199, Article 100199
• Main Authors: An, Seong J., Stagi, Massimiliano, Gould, Travis J., Wu, Yumei, Mlodzianoski, Michael, Rivera-Molina, Felix, Toomre, Derek, Strittmatter, Stephen M., De Camilli, Pietro, Bewersdorf, Joerg, Zenisek, David
• Format: Article
• Language: English
• Subjects: C2 domain; electron microscopy; Multimodal Imaging; PALM; STED; super-resolution; synapse; synaptic vesicles; Synaptic Vesicles - chemistry; TIRF
• ISSN: 2667-2375; 2667-2375
• 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 A2 (cPLA2) 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. [Display omitted] •Purified cPLA2 C2 domain labels recycling membranes by binding to the cell surface•Ca2+ dependence of membrane binding enables efficient removal of uninternalized probe•C2 domain can be conjugated to a variety of useful dyes and reporter proteins•Synaptic vesicles can be imaged by disparate microscopy methods using the C2 domain Currently, imaging probes for synaptic vesicles have unique strengths and weaknesses, which not only limit their utility but also confine their application to certain types of imaging approaches. We therefore developed a method of labeling synaptic vesicles that has the flexibility to be applied across many microscopy modalities. Our probe is based on the cytosolic phospholipase A2 (cPLA2) C2 domain, which reversibly binds membranes in a Ca2+-dependent manner. Through conjugation to different dyes, enzymes, and fluorescent proteins, the purified cPLA2 C2 domain can serve as a versatile probe for synaptic vesicles and other recycling membranes. An et al. demonstrate that the purified cPLA2 C2 domain can label recycling membranes in neurons and non-neuronal cells. By conjugating the C2 domain to various tags, the authors visualize synaptic vesicles by disparate microscopy methods, including electron microscopy and super-resolution techniques, such as STED microscopy and FPALM.