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
Although much of our work is based on mouse models and cultured neurons, we also, in a bottom-up approach, use purified components to reconstitute the protein machinery that mediates regulated fusion. A reduced, defined system makes it possible to probe directly (using, for example, fluorescent reporters) the inner workings of the fusion apparatus, allowing us to gain insights into how it operates. This system also makes it possible to control variables, such as membrane curvature, that cannot be readily addressed with cell-based models.
Three proteins (termed SNAREs)—synaptobrevin, on the vesicle membrane and SNAP-25 and syntaxin, on the target membrane—directly mediate membrane fusion between artificial vesicles in vitro, but fusion was insensitive to Ca2+. Strict regulation by Ca2+ only occurred when we added back the Ca2+-binding protein, synaptotagmin (syt 1), to these fusion reactions. Thus, syt 1 and SNARE proteins appear to serve as the minimum protein complement for Ca2+-triggered membrane fusion. We are optimizing the minimal fusion machine by adding back additional proteins and lipids, with the goal of enabling it to operate on a millisecond timescale. We have combined this approach with biophysical and optical methods to monitor the dynamic changes in protein-protein and protein-lipid interactions that mediate fusion and to resolve intermediates during the fusion reaction. A key new focus concerns the structure and dynamics of reconstituted fusion pores; in these studies we use nanodiscs and optical reporters to analyze the flux of transmitters through reconstituted fusion pores. 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
Virtually all synaptic vesicle (SV) and presynaptic proteins have been systematically cloned, but the functions of most of these proteins in the SV cycle remain unknown. A striking example is synaptophysin (syp), the first SV protein to be characterized nearly 30 years ago. Although syp is the most abundant SV protein by mass, syp knockout mice were reported to have normal synaptic transmission, arguing against a function in the SV cycle. Using optical approaches, we recently discovered that syp regulates SV endocytosis and, using electrophysiological recordings, we found that syp also plays a key role in synaptic depression. We are expanding these studies to examine the functions of a number of syp-related SV proteins (e.g., synaptogyrin, SCAMP, synaptoporin).
Other examples of assigning functions to proteins include our work on the syt family of proteins, of which there are 17 members. We recently discovered 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, functions have been assigned 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, syts). 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 the pituitary gland is a "hotbed" for the expression of numerous syt isoforms, where these proteins appear to regulate the secretion of myriad hormones to control virtually all aspects of mammalian physiology.
In addition to synchronous transmitter 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. By screening numerous neuronal Ca2+-binding proteins, we discovered that Doc2 has kinetic properties that are consistent with asynchronous release. These findings prompted genetic and electrophysiological experiments, which revealed that Doc2 is required for the slow phase of synaptic transmission, and we are in the midst of altering circuit behavior by altering the expression levels of Doc2 and by expressing mutant forms of the protein in neurons.
Hence, 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. We have also recently begun to address fundamental questions regarding polarized membrane transport and neurite outgrowth. Finally, our lab has recently intensified our efforts to determine the factors that control the frequency of spontaneous SV fusion events (minis), as elementary questions (e.g., Are minis triggered by the spontaneous mobilization of Ca2+?) remain unresolved.
Although the majority of our work concerns the presynaptic mechanisms that mediate synaptic transmission and membrane fusion, we also work on a class of toxins whose biological function is the exact opposite: they block exocytosis. These are the botulinum neurotoxins (BoNTs), 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 "clever," since entry of the toxin shuts 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. To work on this project, we are using microfluidic devices in conjunction with toxins that have been conjugated to quantum dots.
Our laboratory also studies aspects of neuronal polarity, neurite outgrowth, spontaneous SV fusion, synaptic plasticity (including long-term potentiation), and behavior. We have also begun to use patterned surfaces to form predetermined neuronal circuits to study basic questions concerning information processing. Our 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 elucidating the structure and dynamics of fusion pores in cells. We also routinely collaborate with groups that have expertise in chemistry, material sciences, and various imaging methods.
This work is supported in part by grants from the National Institutes of Health.
As of April 7, 2014