ABSTRACT
The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/metabolism , Helminth Proteins/genetics , Helminth Proteins/metabolism , Monomeric Clathrin Assembly Proteins , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Synaptic Vesicles/metabolism , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Animals , Caenorhabditis elegans/genetics , Clathrin/biosynthesis , Endocytosis , Homozygote , Intracellular Membranes/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Mutation , Nervous System/metabolism , Neurotransmitter Agents/metabolism , Phosphoproteins/genetics , Protein Isoforms , R-SNARE Proteins , Sequence Homology, Amino Acid , Synaptic Vesicles/ultrastructure , VertebratesABSTRACT
The fluorescent probe FM1-43 has been used extensively for imaging vesicle recycling; however, high nonspecific adsorption resulting in elevated background levels has precluded its use in certain tissues, notably brain slices. We have found that a sulfobutylated derivative of beta-cyclodextrin (ADVASEP-7) has a higher affinity for FM1-43 than the plasma membrane. ADVASEP-7 was used as a carrier to remove FM1-43 nonspecifically bound to the outer leaflet of the plasma membrane or extracellular molecules, significantly reducing background staining. This has enabled us to visualize synaptic vesicle recycling in the nematode C. elegans, intact lamprey spinal cord, and rat brain slices.
