of Membrane Fusion
the central nervous system, regulated exocytosis is characterized
by a rapid synchronized fusion of synaptic vesicles with the
plasma membrane. This process is triggered by an influx of Ca2+
into the presynaptic cell. Like fusion in other systems, this
process involves the participation of a large number of soluble
and membrane associated protein components, including the SNAREs.
The SNAREs are membrane proteins which assemble into a coiled-coil
complex. This complex bridges the vesicle and plasma membranes
and drives the two together. Although SNAREs have been proposed
to constitute a minimal machinery required for fusion, Ca2+ does
not appear to directly bind and regulate the SNAREs. Synaptotagmin
is a membrane protein associated with the synaptic vesicle that
is believed to act as the Ca2+ sensor for neuronal exocytosis.
There is compelling evidence that Ca2+ triggers fusion by binding
to the two C2 domains of synaptotagmin.
the present time, the molecular role of synaptotagmin is not
understood. It is not known whether synaptotagmin triggers fusion
by binding to membranes, SNAREs, or both. There have been numerous
studies on soluble fragments of synaptotagmin and the SNAREs,
but relatively little data is available on the configurations
of these proteins or their interactions at the membrane surface.
Numerous genetic studies on synaptotagmin have examined the effects
of mutations on function, but virtually no information is available
on how these mutations alter the position of the C2 domains at
the membrane interface.
collaboration with Lukas
Tamm (Physiology, UVa) and Reinhard
Jahn (Max-Planck Institute, Gottingen) we currently have
an NIH funded program project grant directed at determining the
mechanism of pre-synaptic membrane fusion from a biophysical
perspective. Our role in this program project is to determine
the configuration of synaptotagmin and segments of the SNAREs
when associated with the membrane interface, and to test models
of the role of synaptotagmin in fusion. The binding of the polyphosphoinositide,
PI(4,5)P2, to these proteins will also be determined because
it is a lipid that has been shown to be critical for fusion.
The overall goal of the proposed work is to define the molecular
mechanisms that trigger fusion in the central nervous system.