Membrane fusion underlies numerous basic biological processes, including fertilization, viral entry, release of hormones, and rapid communication between neurons. However, the molecular mechanisms by which proteins catalyze the merger of lipid bilayers are not yet understood.
We are interested in how biological membranes fuse and, in particular, how synaptic vesicles fuse with the plasma membrane of neurons to mediate the release of neurotransmitters. These studies emphasize the mechanisms that control the efficiency and kinetics of synaptic transmission to ultimately influence the function of neuronal circuits. We also study the botulinum neurotoxins, a group of toxins that cause botulism by blocking neuronal exocytosis. These toxins are widely used in clinical settings and, because of their extreme toxicity, they are also potential biological weapons.
Nanomechanics of Ca2+-Triggered Membrane Fusion
One approach that we have undertaken to study synaptic vesicle exocytosis is to use purified components to reconstitute the protein machinery that mediates regulated fusion. A reduced, defined system makes it possible to probe directly the inner workings of the fusion apparatus, allowing us to understand how it operates.
Three proteins (termed SNAREs)—synaptobrevin on the vesicle membrane and SNAP-25 and syntaxin on the target membrane—directly mediate membrane fusion in vitro. We have extended these findings and shown that in the presence of another protein, synaptotagmin (syt) 1, SNARE-catalyzed membrane fusion is regulated by Ca2+. Thus, syt and SNARE proteins are the minimum protein complement for Ca2+-triggered membrane fusion. We are optimizing the minimal fusion machine, by adding back additional proteins and lipids, to enable it to operate on the millisecond timescale. We have combined this approach with biophysical and optical methods to monitor protein-protein and protein-lipid interactions during fusion and to resolve intermediates during the fusion reaction. These experiments are making it possible to understand the molecular rearrangements that underlie docking, hemifusion, fusion pore opening, and complete bilayer merger.
Assigning Functions to Synaptic Proteins
While numerous synaptic vesicle (SV) and presynaptic proteins have been systematically cloned, the functions of most of these proteins in the SV cycle remain unknown. A striking example is the first SV protein to be cloned, the tetramembrane-spanning domain protein synaptophysin (syp). Syp knockout mice were reported to have normal synaptic transmission, arguing against a function in the SV cycle. This was surprising, because syp is the most abundant SV protein by mass. Using optical approaches, we recently discovered that syp regulates SV endocytosis and, using electrophysiological approaches, we found that it also plays a key role in synaptic depression. We are expanding these studies to examine the functions of a number of related SV tetramembrane-spanning domain proteins (e.g., synaptogyrin, SCAMP, synaptoporin). Other examples of assigning functions to cloned proteins include our findings that syt 7 serves as a Ca2+ sensor for SV replenishment and that syt 4 controls the SV cycle indirectly by serving as a negative regulator of brain-derived neurotrophic factor (BDNF) secretion. Thus, we have assigned functions to the three most "ancient" isoforms of syt 1, 4, and 7, which appear to be expressed in all metazoans.
Our current work is focused on discerning the functions of a number of additional synaptic and membrane trafficking proteins (e.g., SV2, complexin, nSec1), including all 17 members of the syt family. Again, using an optical approach, we found that most isoforms of syt appear to regulate the fusion of large dense-core vesicles (LDCVs), and thus they likely control the secretion of hormones and neuropeptides (including syt 4). Consistent with this idea, we found that a number of syt isoforms are most highly expressed in the pituitary, where they regulate the secretion of myriad hormones to control virtually all aspects of mammalian physiology, and we have begun to pursue this line of investigation using mice lacking specific isoforms of syt.
In addition to synchronous release, many synapses also exhibit a slower, asynchronous component of synaptic transmission that persists for tens or hundreds of milliseconds. Asynchronous release plays an important role in numerous aspects of synaptic physiology, including persistent reverberation, which is thought to mediate aspects of memory. Our in vitro biophysical studies revealed that the Ca2+-binding protein Doc2 has kinetic properties that are consistent with asynchronous release. These findings prompted genetic and electrophysiological experiments that revealed that Doc2 is required for the slow phase of synaptic transmission. Thus, our laboratory studies neuronal membrane traffic and excitatory synaptic transmission on three different timescales: synchronous transmission (milliseconds), asynchronous transmission (tens to hundreds of milliseconds), and modulation of transmission by hormones released from LDCVs (seconds to minutes).
Our current goals include engineering Ca2+ sensors to tune selectively the kinetics of synaptic transmission, applying chemical genetic approaches to determine protein function in living neurons, and applying a tagging approach, in conjunction with mass spectrometry, to identify the cargoes within LDCVs that harbor distinct isoforms of syt.
Although the majority of our work concerns the molecules that mediate exocytosis and membrane fusion, we also work on a class of toxins whose biological function is to do the exact opposite: they block exocytosis. These are the botulinum neurotoxins (BoNTs), and they are the most potent toxins known. There are seven related toxins (A–G), and they cause botulism by entering neurons and cleaving SNARE proteins, resulting in paralysis and death. We discovered that most of the BoNTs enter neurons by binding to the luminal domains of specific synaptic vesicle proteins (syt 1 and 2, and SV2A, B, and C) that are transiently exposed to the outside of neurons during exocytosis. In effect, the toxins piggyback onto recycling vesicles and enter cells via a Trojan horse strategy. This is rather "clever," since entry of the toxin will shut down the uptake pathway, preventing further uptake of toxin into nerve terminals that have already been poisoned; entry of additional toxin is thus limited to active nerve terminals.
We have expanded this work to include structural studies of toxins bound to their receptors and to address a new idea: some of the BoNTs might be able to move within networks of neurons to exert effects distal from the initial site of uptake. This latter project has important ramifications concerning the use of these agents in human patients.
Our laboratory also studies aspects of neuronal polarity, neurite outgrowth, spontaneous SV fusion, synaptic plasticity (including long-term potentiation), and behavior. The overall goal is to move back and forth between studies of purified proteins in vitro and studies of circuit function and behavior in intact organisms. In addition, we are engaged in collaborative interactions with Meyer Jackson’s laboratory (University of Wisconsin–Madison) to continue our ongoing biophysical studies aimed at eludicating the structure and dynamics of fusion pores.
This work is supported in part by grants from the National Institutes of Health.
As of December 12, 2013