TRAFFIC OF SYNAPTIC VESICLES IN NEURONS AND ENDOCRINE CELLS 
PiETRo De Camilu, M.D., Investigator 
Dr. De Camilli's research is aimed at elucidating 
mechanisms of membrane traffic in neurons and en- 
docrine cells, with special emphasis on the traffic of 
synaptic vesicles (SVs). SVs are specialized organ- 
elles by which neurons secrete nonpeptide neuro- 
transmitters at synapses. They are small secretory 
vesicles, highly homogeneous in size, that are clus- 
tered at nerve terminals in close proximity to release 
sites. They undergo exo-endocytotic recycling in 
nerve endings and, at each cycle, are reloaded with 
neurotransmitter content. The laboratory is focused 
on the elucidation of the biogenesis and exo- 
endocytotic recycling of SVs. (Some aspects of this 
work are carried out in collaboration with the labo- 
ratories of Dr. Reinhard Jahn [HHMI, Yale Univer- 
sity] and Dr. Thomas Siidhof [HHMI, University of 
Texas Southwestern Medical Center at Dallas].) 
A central working hypothesis underlying Dr. De 
Camilli's work is that SVs are closely related to or- 
ganelles found in endocrine cells and more distantly 
related to organelles found in all cells. The elucida- 
tion of molecular mechanisms involved in the life 
cycle of SVs may be of general relevance for the un- 
derstanding of vesicular traffic in all eukaryotic 
cells. 
A second focus of research in Dr. De Camilli's lab- 
oratory is the role of autoimmunity directed against 
SV proteins in human diseases. 
Synaptic-like Microvesicles 
of Endocrine Cells 
Recent work had shown that peptide-secreting 
endocrine cells contain a population of recycling 
microvesicles (synaptic-like microvesicles, SLMVs) 
that share important similarities with neuronal SVs. 
Many of the major membrane proteins of SVs are also 
present in the membrane of SLMVs. During the 
past year the properties of SLMVs were further 
investigated. 
One line of research addressed the question of 
whether the recycling of membrane components of 
SLMVs involves the same endosomal intermediates 
that participate in the recycling of plasmalemma re- 
ceptors (transferrin receptors). Results have sug- 
gested that membrane proteins of SVs and transferrin 
receptors transit through the same endosomes but 
are then sorted into distinct vesicular carriers. The 
relevance of these findings to the pathway of SV re- 
cycling in neurons is being investigated. In neurons, 
transferrin receptors are excluded from axons. Yet 
the mechanisms of endosomal sorting that operate 
in endocrine cells may also operate in nerve end- 
ings. (This work is supported in part by the National 
Institutes of Health.) 
The possible function of SLMVs in neurotransmit- 
ter storage and uptake was studied, using pancreatic 
/? cells as the model system. It was shown that 
SLMVs of these cells have a transporter for GABA 
(7-aminobutyric acid) in their membranes. The 
pharmacological properties of this transporter are 
very similar to those of the transporter present in SVs 
of GABA-secreting neurons. 
An Assay to Monitor Exo-endocytosis of SVs 
Am important step toward the elucidation of mo- 
lecular mechanisms of SV exocytosis is the develop- 
ment of exocytotic assays independent of the mea- 
surement of neurotransmitter release. These assays 
will allow the monitoring of SV exocytosis in the 
absence of a synapse and irrespective of experimen- 
tal manipulations that may affect the loading of SVs 
with neurotransmitters. An exocytotic assay based 
on antibodies directed against the luminal domain 
of the synaptic vesicle protein synaptotagmin was 
developed. 
This assay has already permitted the determina- 
tion that SVs undergo exo-endocytosis and recycling 
in the processes of developing hippocampal neu- 
rons in culture before the formation of synaptic con- 
tacts. The recycling that takes place in these imma- 
ture neurons is very active, suggesting that the 
formation of a synapse correlates with a down- 
regulation of the rate of constitutive recycling. The 
regulation of SV exocytosis in developing neurons is 
being investigated. 
Molecular Mechanisms of Exocytosis 
The possibility of using yeast genetics to identify 
some of the proteins involved in SV exocytosis was 
explored. This experimental approach is based on 
the hypothesis that at least some basic feature of the 
exocytotic process may have been conserved in evo- 
lution, irrespective of whether the exocytotic event 
is regulated. A variety of temperature-sensitive yeast 
mutants defective in vesicle exocytosis {sec mu- 
tants), and therefore in grovvTih, have been isolated 
in recent years. To identify proteins involved in the 
final states of the secretory pathway in mammalian 
cells, including neurons, Dr. De Camilli and his co- 
workers (in collaboration with the laboratory of Dr. 
Peter Novick, Yale University) searched mammalian 
gene products that rescue sec mutations. Rat brain 
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