Neurons communicate with each other via chemical signals called neurotransmitters. When a neuron fires, synaptic vesicles filled with neurotransmitters fuse with a nerve cell’s plasma membrane to release their contents into the synaptic cleft. The neurotransmitters then bind to receptors on postsynaptic cells. Edwin Chapman and his team study proteins and molecular triggers that mediate this chemical release. In parallel, his team also investigates the mechanisms that mediate the release of hormones from neurons and neuroendocrine cells.
Chapman’s lab was the first to reconstitute calcium-triggered membrane fusion in vitro, and his team has pioneered studies to look at how the calcium-binding protein synaptotagmin 1 (syt1) triggers membrane fusion. They discovered that syt1 and a set of proteins that mediate vesicle-membrane fusion, called SNAREs, are the minimum complement of proteins essential for calcium triggered fusion. More recently, his laboratory showed that the exocytotic fusion pore is a hybrid structure, composed of both lipids and the transmembrane domains of SNARE proteins. The team is now focused on discerning the functions of a number of additional synaptic and membrane trafficking proteins, which have already been cloned. For example, they recently discovered that a protein named Doc2 drives the slow, asynchronous phase of synaptic transmission.
Chapman is also interested in the botulinum neurotoxins (BoNTs), which block exocytosis. BoNTs inhibit vesicle-membrane fusion by cleaving SNARE proteins, which halts fusion and neurotransmitter release. Chapman’s team discovered the receptors for most of the BoNTs, and showed that they sneak into an open vesicle when it’s exposed during exocytosis. Now, they are looking at what happens once the toxin has infiltrated the neuron, investigating the possibility of “distal effects,” or the toxin’s ability to jump from neuron to neuron in a circuit, doing damage along the way.
In recent years, Chapman has also started to explore organelle trafficking in neurons, looking at how mitochondria and lysosomes move through cells to regulate synaptic function, and to understand how neurons become so highly polarized.
Chapman Abstract Slideshow
Figure 1: Probing fusion pore structure using a nanodisc based fusion assay.
Left panel: Illustration for the nanodisc-vesicle fusion assay. Right panel: specific residues in the transmembrane domain of syntaxin (syx) 1A and synaptobrevin (syb) 2 are exposed to solvent during membrane fusion, as revealed by the substituted cysteine accessibility method.
Modified from Bao, H. et al. 2016 Nat Struct Mol Biol. 23:67-73.
Figure 2: Synaptotagmin 1 membrane interactions govern the time course of synaptic release.
A) Synaptotagmin (syt) 1 (blue) binds Ca2+ (red) and penetrates the plasma membrane; this constitutes the activated state of syt1. If this protein-Ca2+-membrane complex stays bound sufficiently long, syt1 will trigger exocytosis (A, middle). If Ca2+ dissociates prior to fusion (A, right), syt1 will either remain bound to the membrane and trigger exocytosis (A, right, small arrow) or more likely will dissociate without triggering exocytosis (A, right, large arrow). B) The syt1/7 chimera (green) increases the dwell time the sensor remains inserted in the target membrane, increasing the likelihood that the sensor-membrane complex will trigger release (B, right, large arrow). C) Conceptual time course of synaptic release. The release probability (Prelease) of terminals harboring syt1 (blue) follows the time course of the presynaptic Ca2+ transient (red). Terminals expressing the syt1/7 chimera (green) exhibit a wider window of high release probability, due to the slower disassembly kinetics of the chimera-membrane complex.
This figure is based on Evans, C.S. et al. 2015 J Neurosci. 35:11769-79.
Figure 3: Different states of syt1 regulate evoked versus spontaneous release as revealed by poly-proline linker mutants.
Top panel: Poly-proline linkers, with different numbers of proline residues, constrain the relative orientation between the C2A and C2B domains of syt1 to different angles. The C2A and C2B domains point in the same direction when connected by a 6 or 9 residue poly-proline linker (left). A 7 proline linker yields a relative inter-domain angle of approximately 120 degree (middle) while the 8 proline linker mutant exhibits an angle of -120 degree (right). Bottom panel: The periodicity of three for the inter-domain angle (dictated by the periodicity of three for poly-proline helices) was confirmed by photoinduced electron transfer (PET) quenching experiments (blue). The ability of linker mutants to penetrate membranes (green) and drive evoked synaptic transmission (red) shows a similar periodicity. Strikingly, the ability of the proline linker mutants to clamp spontaneous release (black) displays an inverse periodicity. Thus, alterations in the relative orientation of the C2A and C2B domains allow syt1 to change functional states.
Data are modified from Bai, H. et al. 2016 Nat Commun. 7:10971
Figure 4: Doc2 regulates asynchronous release.
Top panel: illustration showing Doc2 structure and the synaptic vesicle cycle. Lower left: Doc2 KO hippocampal neurons largely lack asynchronous release. Lower right: WT Doc2 translocates to the plasma membrane of PC12 cells in response to Ca2+ influx; the D297,303N Ca2+ ligand mutations in the C2B domain abolish translocation and completely disrupt the ability of Doc2 to drive slow transmission; the D218,220N Ca2+ ligand mutations in the C2A domain result in constitutive translocation and activation of the protein and result in large increases in asynchronous release.
Modified from Xue, R. et al. 2015 Proc Natl Acad Sci U S A 112:E4316-25.
Figure 5: Model depicting two distinct trafficking pathways for BoNT/A, D and TeNT.
Right: (1) These toxins can enter neurons via a synaptic vesicle dependent pathway (by binding to SV2), which leads to local effects and results in substrate cleavage throughout the cell (dashed lines), or they can enter via an alternative receptor and pathway (solid lines). After initial sorting steps, toxins entering via this latter pathway converge into the common, non-acidified, retrogradely transported organelle (2). The holotoxin is then delivered to the somato-dendritic compartment where it undergoes further sorting (3) followed by exocytosis (4). Holotoxin is then able to enter a secondary neuron (shown in red) and exert “distal effects” on upstream networks of neurons. Left: Illustration of the microfluidic devices used to study the transfer and action of the toxins on upstream neurons.