RT Journal Article
SR Electronic
T1 PHluorin-conjugated secondary nanobodies as a tool for measuring synaptic vesicle exo- and endocytosis
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2024.09.23.614406
DO 10.1101/2024.09.23.614406
A1 Georgiev, Svilen V.
A1 Rizzoli, Silvio O.
YR 2024
UL http://biorxiv.org/content/early/2024/09/23/2024.09.23.614406.abstract
AB Neuronal communication relies on synaptic vesicle recycling, which has long been investigated by live imaging approaches. Synapto-pHluorins, genetically encoded reporters that incorporate a pH-sensitive variant of GFP within the lumen of the synaptic vesicle, have been especially popular. However, they require genetic manipulation, implying that a tool combining their excellent reporter properties with the ease of use of classical immunolabeling would be desirable. We introduce this tool here, relying on primary antibodies against the luminal domain of synaptotagmin 1, decorated with secondary single-domain antibodies (nanobodies) carrying a pHluorin moiety. The application of the antibodies and nanobodies to cultured neurons results in labeling their recycling vesicles, without the need for any additional manipulations. The labeled vesicles respond to stimulation, in the expected fashion, and the pHluorin signals enable the quantification of both exo- and endocytosis. We conclude that pHluorin-conjugated secondary nanobodies are a convenient tool for the analysis of vesicle recycling.Competing Interest StatementThe authors have declared no competing interest.