A Versatile Synaptotagmin-1 Nanobody Provides Perturbation-Free Live Synaptic Imaging And Low Linkage-Error in...

Area of Science:

• Neuroscience
• Molecular Biology
• Biophysics

Background:

• Current methods for imaging living synapses often involve overexpressing fluorescently tagged proteins, which can disrupt normal synaptic function.
• This alteration in protein stoichiometry affects the physiological state of synapses, limiting the accuracy of imaging studies.

Purpose of the Study:

• To develop a minimally invasive tool for imaging synaptic activity in living neurons.
• To overcome the limitations of traditional overexpression-based fluorescent reporters.

Main Methods:

• Development of a nanobody (NbSyt1) that binds to the calcium sensor synaptotagmin-1.
• Utilizing NbSyt1 as an intrabody (iNbSyt1) within living neurons.
• Structural analysis via crystal structure of NbSyt1 bound to Synaptotagmin-1.
• Physiological data collection to assess synaptic transmission.
• Generation of a NbSyt1-jGCaMP8 chimera for measuring presynaptic calcium (Ca2+).

Main Results:

• The intrabody NbSyt1 (iNbSyt1) was shown to be minimally invasive, with synaptic transmission remaining largely unaffected.
• Crystal structure analysis confirmed the binding of NbSyt1 to Synaptotagmin-1.
• The NbSyt1-jGCaMP8 chimera successfully measured spatially localized presynaptic Ca2+.
• The small size of NbSyt1 makes it suitable for super-resolution imaging techniques.

Conclusions:

• NbSyt1 is a versatile tool for advancing imaging in cellular and molecular neuroscience.
• This nanobody enables imaging with unprecedented precision across various spatiotemporal scales.
• NbSyt1 offers a less disruptive alternative to traditional overexpression methods for studying synaptic physiology.