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Science beyond Barriers

abberior instruments

Neurobiology, Technology

2023
bioRxiv

A versatile Synaptotagmin-1 nanobody provides perturbation-free live synaptic imaging and low linkage-error in super-resolution microscopy

Authors:

Karine Queiroz Zetune Villa Real, Nikolaos Mougios, Ronja Rehm, Shama Sograte-Idrissi, László Albert, Amir Mohammad Rahimi, Manuel Maidorn, Jannik Hentze, Markel Martínez-Carranza, Hassan Hosseini, Kim-Ann Saal, Nazar Oleksiievets, Matthias Prigge, Roman Tsukanov, Pål Stenmark, View ORCID ProfileEugenio F. Fornasiero, Felipe Opazo

Keywords:

Nanobodies, Live Cell Imaging, linkage error

Abstract:

Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy changes the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here we introduce a nanobody that binds the calcium sensor synaptotagmin-1 (NbSyt1). This nanobody functions in living neurons as an intrabody (iNbSyt1) and is minimally invasive, leaving synaptic transmission almost unaffected, as demonstrated by the crystal structure of the NbSyt1 bound to synaptotagmin-1 and by our physiological data. Its single-domain nature enables the generation of protein-based fluorescent reporters, as we showcase here by measuring spatially-localized presynaptic Ca2+ with an NbSyt1-jGCaMP8 chimera. Moreover, its small size makes the NbSyt1 ideal for various super-resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience at a higher precision than possible in the past, over multiple spatiotemporal scales.

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