Tracking Synaptic Vesicles in Live Neurons Using Single-Molecule Super-resolution Microscopy.

Area of Science:

• Neuroscience
• Cell Biology
• Biophysics

Background:

• Neurotransmitter release depends on synaptic vesicle (SV) fusion at presynaptic terminals.
• Mechanisms of SV clustering and dynamic organization into distinct pools remain poorly understood.
• Traditional studies relied on ultrastructural analysis, limiting dynamic insights.

Purpose of the Study:

• To investigate the nanoscale dynamic organization of SVs in live neurons.
• To explore SV clustering and the formation of distinct SV pools.
• To correlate SV dynamics with neuronal activity.

Main Methods:

• Single-particle tracking photoactivated localization microscopy (sptPALM) for total SV pool mobility and clustering.
• Universal Point Accumulation Imaging in Nanoscale Topography (uPAINT) for plasma membrane protein dynamics.
• Dual-pulse subdiffractional Tracking of Internalized Molecules (DsdTIM) for reserve and recycling SV pools.
• Electrical field stimulation to mimic physiological neuronal depolarization.

Main Results:

• Resolved mobility and clustering of the total SV pool using sptPALM.
• Tracked mobility of SV proteins on the plasma membrane with uPAINT.
• Simultaneously monitored reserve and recycling SV pools with DsdTIM.
• Observed SV dynamics in response to physiological stimulation.

Conclusions:

• Advanced optical and super-resolution microscopy techniques enable the study of SV dynamics in live neurons.
• These methods provide insights into the nanoscale organization and trafficking of SVs.
• Understanding SV dynamics is key to deciphering mechanisms of neurotransmission.