Abstract
Neuronal synapses are among the most scrutinized of cellular systems, serving as a model for all membrane trafficking studies. Despite this, synaptic biology has proven difficult to interrogate directly in situ due to the small size and dynamic nature of central synapses and the molecules within them. Here we determine the spatial and temporal interaction status of presynaptic proteins, imaging large cohorts of single molecules inside active synapses. Measuring rapid interaction dynamics during synaptic depolarization identified the small number of syntaxin1a and munc18-1 protein molecules required to support synaptic vesicle exocytosis. After vesicle fusion and subsequent SNARE complex disassembly, a prompt switch in syntaxin1a and munc18-1-binding mode, regulated by charge alteration on the syntaxin1a N-terminal, sequesters monomeric syntaxin1a from other disassembled fusion complex components, preventing ectopic SNARE complex formation, readying the synapse for subsequent rounds of neurotransmission.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
Botulinum Toxins / pharmacology
-
Botulinum Toxins, Type A / pharmacology
-
Cerebral Cortex / cytology
-
Cerebral Cortex / drug effects
-
Cerebral Cortex / metabolism
-
Embryo, Mammalian
-
Exocytosis / genetics*
-
Gene Expression Regulation
-
Genes, Reporter
-
Green Fluorescent Proteins / genetics
-
Green Fluorescent Proteins / metabolism
-
Luminescent Proteins / genetics
-
Luminescent Proteins / metabolism
-
Membrane Fusion
-
Molecular Imaging
-
Munc18 Proteins / genetics
-
Munc18 Proteins / metabolism*
-
Neurons / cytology
-
Neurons / drug effects
-
Neurons / metabolism
-
Primary Cell Culture
-
Protein Binding
-
Protein Transport
-
Rats
-
Rats, Sprague-Dawley
-
Red Fluorescent Protein
-
SNARE Proteins / genetics
-
SNARE Proteins / metabolism
-
Synapses / drug effects
-
Synapses / metabolism*
-
Synapses / ultrastructure
-
Synaptic Transmission
-
Synaptic Vesicles / drug effects
-
Synaptic Vesicles / metabolism*
-
Synaptic Vesicles / ultrastructure
-
Syntaxin 1 / genetics
-
Syntaxin 1 / metabolism*
Substances
-
Luminescent Proteins
-
Munc18 Proteins
-
SNARE Proteins
-
Stxbp1 protein, rat
-
Syntaxin 1
-
enhanced green fluorescent protein
-
Green Fluorescent Proteins
-
Botulinum Toxins
-
Botulinum Toxins, Type A
-
botulinum toxin type C