Project Gallery
Structures of Vesicle Fusion Proteins
Our approach to understanding the molecular basis for neurotransmission consists of a combination of structural and biophysical studies. Structural information about the most important complexes between the individual molecular components is first obtained by x-ray crystallography or electron cryo-microscopy. This information provides the framework for investigations targeted at the dynamic aspects of the system, using single-molecule techniques.
Single Molecule Studies of Vesicle Fusion Proteins
We use single-molecule fluorescence microscopy to observe fluorescent-dye labelled individual SNARE complexes, synaptotagmin, other associated proteins, and vesicles. We monitor the interactions of the proteins and the fusion of single vesicles by illuminating the interaction region with laser light and observing the fluorescent signatures of dyes covalently attached at selected locations on the surface of proteins involved in calcium triggered synaptic vesicle fusion.
Single Vesicle Reconstitution of Vesicle Fusion
We have achieved the long-standing goal to establish an in vitro single vesicle-vesicle system with reconstituted synaptic proteins that produces a rapid burst of content release upon injection of Ca2+. Donor vesicles mimic synaptic vesicles, and acceptor vesicles mimic the plasma membrane in the active zone. Prior to Ca2+ injection, the system is in a state of single interacting pairs of donor and acceptor vesicles, and fusion events are rare. Our system differentiates between membrane docking, membrane lipid exchange, and complete fusion (i.e., pore formation) upon Ca2+ injection, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Initially, we included neuronal SNARE, synaptotagmin 1, and complexin, and we are currently investigating the function of other proteins with our system, including Munc18 and NSF/SNAP.
Synaptic Adhesion
Establishment of neural connections is critical for proper brain function, and errors in the process are thought to be associated with autism and other disorders. Nerve impulses are triggered when a presynaptic neuron releases a chemical neurotransmitter into the synapse that is recognized by the postsynaptic neuron. Neurexin and neuroligin, respectively, are presynaptic and postsynaptic connector proteins that extend outside of the cells where they are produced and contact one another to form a physical link across the synapse.
Mechanism of Clostridial Neurotoxins
Botulinum neurotoxins (BoNTs) are produced by clostridia and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng / kg, they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. We are studying the mechanism of action of BoNTs, especially receptor recognition and substrate interactions.
AAA ATPases and Associated Proteins
Fusion of opposing membranes results in the formation of cis-SNARE complexes that are disassembled for recycling and reactivation by the joint action of SNAP and NSF. NSF is a hexamer and belongs to the AAA (ATPases associated with cellular activities) family of proteins. We are also pursuing structural and functional studies of p97 (also called VCP, valosin-containing protein; VAT in Archaebacteria; and cdc48 in yeast), a distant homolog of NSF. p97 has been implicated in variety of functions, including the extraction of misfolded luminal and membrane proteins from the ER for cytosolic degradation (ERAD).
Sec6/8 Complex
While SNARE proteins localize specifically throughout the cell, the pairing of these proteins is unlikely to be a major determinant of the specificity of the membrane compartment organization, since SNAREs from different cellular compartments can form complexes in vitro. An initial tethering step has been postulated as the critical determinant in the specificity of membrane fusion. Multimeric protein complexes that are involved in most intracellular trafficking events are essential for this tethering. The tethering complex for exocytosis at the plasma membrane is referred to as the sec6/8 complex, or exocyst, in yeast.
Endocytosis
The supply of synaptic vesicles in the presynaptic terminal is limited. With continuous stimulation of the neuron, the available pool would be rapidly depleted. Possible transport of vesicles from the neuronal cell body would be too slow to maintain fast synaptic transmission in the terminal. Instead, the synaptic vesicle membrane that fuses with the plasma membrane is recycled via clathrin-mediated exocytosis.
Small G Proteins
Small G proteins and their effectors involved in vesicle trafficking.
Post-Synaptic Density
The release of neurotransmitters triggers a cascade of events in the postsynaptic neuron. The first stage is the binding of the neurotransmitter to a cell surface receptor. The second stage involves an increase of second messengers, the opening or closing of ion channels, or the recruitment of cytoplasmic proteins. The third stage involves the activation of enzymes, typically protein kinases or phosphatases, which mediate the biological response. Thus, the initial interaction of a ligand with a receptor results in amplification of the signal by means of a cascade of responses. These processes are in part regulated by sub-cellular localization and clustering of the cell-surface receptors. Recent studies suggest a major role of protein-binding modules in this process. PSD-95 (PostSynaptic Density-95) is a member of the membrane-associated guanylate kinase (MAGUK) superfamily. In excitatory synapses, PSD-95 clusters glutamate ionotropic receptors and ion channels at specific sites in the postsynaptic membrane and organizes downstream signaling molecules and cytoskeletal components.
High Resolution Structures
Structures solved at high resolution and structures solved using highly accurate experimental phases.
Protein Design
Structures of repacking mutants of E. Coli Rop.
Various
Other structures solved in the laboratory.