Credentials: Investigator, Howard Hughes Medical Institute
Position title: Ricardo Miledi Professor of Neuroscience
Phone: (608) 263-1762
9557 Wi Institute Medical Research
1111 Highland Ave
Madison, WI 53705
Membrane Biology & Protein Trafficking; Physiology
Our laboratory studies membrane trafficking and fusion in neurons; our over-arching goal is to understand presynaptic aspects of synaptic transmission and plasticity. To address these questions, our laboratory is divided into three related and highly interactive subgroups:
Nanomechanics of membrane fusion
This subgroup uses a variety of technologies and approaches to understand how proteins catalyze the fusion of lipid bilayers. This includes a nanodisc-black lipid membrane electrophysiology system that allows us to study single recombinant fusion pores with µsecond time resolution. This work has led to new insights concerning the function of SNARE proteins, which form the core of the presynaptic fusion machine, and synaptotagmin (syt) 1, which we have shown – via chemical genetics and other approaches – operates as a Ca2+ sensor that triggers rapid synaptic vesicle exocytosis. We are currently adding-back numerous additional factors, with the goal of reconstituting fusion machines that operate on physiological time scales. We also study membrane fusion using proteolipsomes, including giant unilamellar vesicles that allow us to monitor membrane deformations via light microscopy. We also use single molecule fluorescence, atomic force microscopy, and DNA nanostructures/origami (to reconstitute fusion pores for cryo-electron microscopy and single particle analysis, among other applications), to address the structure and dynamics of the membrane fusion machinery.
Neuronal cell biology
The main focus of this subgroup is to determine the function of each of the seventeen isoforms of synaptotagmin (syt). We found that a number of isoforms regulate dense core vesicle exocytosis to modulate synaptic transmission, while others are targeted to distinct destinations, including: Golgi, lysosomes, endosomes, and the plasma membrane. In some cases, the same isoform is present in more than one compartment and subserves more than one function in the same cell. We are currently developing new HaloTag/Halo-ligand approaches to identify the organelles that are marked by each syt isoform. This subgroup also seeks to understand: a) how various syt isoforms are sorted within neurons, b) the life cycle of synaptic vesicle proteins as they are created and destroyed, c) how synaptic vesicles themselves are created, and d) the physical properties of these tiny organelles. They also work to assign functions to orphaned synaptic vesicle proteins. This work has resulted in numerous discoveries; e.g. using the RUSH system and a new generation of Halo ligands to study the itinerary of a syt isoform, we recently discovered a new membrane trafficking pathway in mammalian neurons. We also carry out tool development, including new methods to acutely disrupt integral membrane proteins.
Synaptic transmission and plasticity
This subgroup uses electrophysiological and optical approaches (including iGluSnFR [an optical sensor for glutamate release], high speed Ca2+ imaging, etc.) to understand the molecular mechanisms that mediate spontaneous, synchronous, and asynchronous neurotransmitter release in cultured neurons and brain slices. We and others have identified the Ca2+ sensors that mediate these modes of exocytosis and have used this information to tune the properties of synaptic communication. We also study how large dense core vesicle exocytosis converges on synaptic vesicle release, to modulate aspects of synaptic transmission. Another major focus is on short-term synaptic plasticity, including paired pulse facilitation, augmentation, and synaptic depression; these phenomena are regulated by C2-domain proteins in the syt and Doc2 protein families. Numerous aspects of plasticity appear to involve activity-dependent changes in synaptic vesicle docking. As a result, we are extending our imaging efforts to include ‘zap-and-freeze’ electron microscopy, to obtain snapshots of the synaptic vesicle cycle with msec time resolution. Finally, we are developing optical approaches to address the idea of ‘sub-quantal’ neurotransmitter release via ‘kiss-and-run’ exocytosis; this is a controversial topic that has important ramifications concerning how the post-synapse responds to neurotransmitters.
Membrane fusion, synaptic vesicle, fusion pore, neuronal cell biology, synaptic transmission, synaptic plasticity