Redox-Activated Probes Enable High-Contrast Live Imaging of Native Postsynaptic Scaffolds

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Direct visualization of postsynaptic scaffolds is crucial for understanding synaptic dynamics and plasticity.
• Current live imaging methods are limited by genetic engineering or incompatible reagents, leading to poor contrast and delivery issues.

Purpose of the Study:

• To introduce Sylives, compact synthetic fluorescent peptides for high-contrast live imaging of inhibitory and excitatory postsynaptic scaffolds in native neurons.
• To overcome limitations of existing probes and delivery strategies for accurate visualization of synaptic structures.

Main Methods:

• Development of Sylives, redox-cleavable CPP-probe conjugates designed for efficient cytosolic delivery and intracellular reduction-restored scaffold binding.
• Quantitative evaluation of Sylive uptake and off-target binding to define parameters for effective intracellular delivery.
• Validation of Sylive specificity and near-traceless uptake through comparison with transiently expressed proteins and immunolabeling.

Main Results:

• Sylives enable high-contrast live imaging of gephyrin and PSD-95 postsynaptic scaffolds in native neurons without genetic modification.
• Pre-purification of redox-cleavable conjugates prevents side-product formation, ensuring reliable delivery and restored binding.
• Defined transferrable parameters for effective intracellular delivery, achieving nanomolar probe levels versus micromolar CPP.

Conclusions:

• Sylives offer a modular platform for targeting intracellular proteins in living neurons, enabling specificity-restored labeling of endogenous postsynaptic sites.
• This approach overcomes major constraints of existing live synapse visualization techniques.
• Sylives facilitate high-contrast imaging without genetic manipulation, advancing the study of synaptic plasticity.