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Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

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Isolation and ultrastructural characterization of squid synaptic vesicles.

Gulcin Pekkurnaz¹, Andrea Fera, Jessica Zimmerberg-Helms

¹National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892, USA.

The Biological Bulletin

|May 10, 2011

Summary

Researchers developed a new method to isolate squid synaptic vesicles, revealing their surface protein organization and identifying the vacuolar-ATPase (V-ATPase) as a key surface structure. This improves understanding of synaptic vesicle function.

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Published on: June 7, 2019

Area of Science:

Neurobiology
Cell Biology
Biochemistry

Background:

Synaptic vesicles are crucial for neurotransmission, mediating membrane fusion.
The surface organization of synaptic vesicle proteins remains poorly understood.

Purpose of the Study:

To develop an improved method for isolating squid synaptic vesicles.
To characterize the protein organization on the surface of these vesicles.

Main Methods:

Glycerol density gradient centrifugation for vesicle isolation.
Electron microscopy (including negative-stain tomography) for structural analysis.
BLAST search of expressed sequence tags for protein identification.

Main Results:

A refined isolation technique yielded highly pure, intact squid synaptic vesicles.
Electron microscopy revealed extensive surface coverage by proteins.
Stalked globular structures consistent with vacuolar-ATPase (V-ATPase) were identified on vesicle surfaces.
Identification of 10 V-ATPase subunits expressed in squid stellate ganglia.

Conclusions:

The study presents a robust method for synaptic vesicle isolation and characterization.
The V-ATPase is a prominent component of the synaptic vesicle surface.
Detailed structural insights into vesicle surface proteins were obtained, advancing our understanding of synaptic vesicle architecture.