title: Isolation and ultrastructural characterization of squid synaptic vesicles.
abstract: Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.
authors.affiliation: National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892, USA.
authors.forename: Gulcin
authors.initials: G
authors.lastname: Pekkurnaz
authors.forename: Andrea
authors.initials: A
authors.lastname: Fera
authors.forename: Jessica
authors.initials: J
authors.lastname: Zimmerberg-Helms
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Degiorgis
authors.forename: Ludmila
authors.initials: L
authors.lastname: Bezrukov
authors.forename: Paul S
authors.initials: PS
authors.lastname: Blank
authors.forename: Julia
authors.initials: J
authors.lastname: Mazar
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Reese
authors.forename: Joshua
authors.initials: J
authors.lastname: Zimmerberg
mesh_terms: D000818:Animals; D001695:Biology / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods; D049832:Decapodiformes / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods; D009899:Optic Lobe, Nonmammalian / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000648:ultrastructure