Research

We study how biological membranes fuse and separate. These events continuously take place in all cells as proteins and lipids are shuttled back and forth between different cellular compartments, as cells secrete compounds to their exterior via vesicle fusion with the plasma membrane, merge with one another, undergo division, or are infected by enveloped viruses such as influenza, Ebola, or HIV. Despite the enormous variety of fusion and fission reactions, only a few classes of proteins mediating these events have been discovered, suggesting that the principles that govern membrane fusion and fission reactions are general.

We are particularly interested in how neurotransmitter- or hormone-filled vesicles fuse with the plasma membrane to release their contents to the exterior of the cell, a process called exocytosis. The key proteins that constitute the exocytotic fusion machinery are well known, but how they interact with one another, lipids, and calcium that triggers fusion is not well understood. Release of neurotransmitters and hormones are tightly regulated, even after the initial narrow connection between the vesicular and the plasma membrane –the fusion pore– has been established. Using novel assays sensitive to single, nanometer-sized fusion pores with sub-millisecond time resolution, we study how exocytotic pores nucleate and how their dynamics are regulated.

A second, more recent, area of interest is how membranes undergo fission, a fundamental process required for, e.g. cell division and synaptic vesicle recycling. In eukaryotes, most fission reactions are catalyzed by dynamin and ESCRT-III. In contrast, very little is known about how bacteria achieve membrane fission for division and sporulation. We aim to unravel the mechanisms by which FisB, a recently reported bacterial membrane fission protein, mediates fission during sporulation. When nutrients are scarce, bacteria like B. subtilis form spores by first dividing asymmetrically to produce a larger mother cell and a smaller forespore. The mother cell engulfs the forespore in a phagocytosis-like process which ends with a fission event that requires FisB. Like its eukaryotic counterparts that each interact with a specialized lipid and oligomerize on membranes, FisB forms oligomers and binds cardiolipin (CL), a lipid whose subcellular localization changes during sporulation. Using a combination of imaging, biochemical, and biophysical approaches, we are studying FisB-CL interactions, FisB oligomerization, and CL dynamics to see how together they account for the clustering, recruitment to the fission site, and membrane fission activity of FisB.

Nucleation and dynamics of exocytotic fusion pores

Exocytosis underlies neurotransmitter and hormone release. In neurons, synaptic vesicles (SV) packaged with neurotransmitter fuse with the plasma membrane to release their content that is sensed across the synaptic cleft. This process is tightly regulated: release is stimulated by a local increase in the free calcium concentration following the arrival of an action potential. Hormones are released in a similar fashion using some of the same protein machinery, via fusion of hormone containing secretory granules (SG) with the plasma membrane. The initial connection between a SV or SG and the plasma membrane is a small pore (~1 nm wide) that can open and close in succession before either closing permanently (transient, or kiss-and-run fusion) or dilating fully. There is large variability in behavior between cell types (pore open times span ~100 ms to 10s of s) and within the same cell (some pores flicker, some dilate abruptly). Pore flickering is modulated by physiological inputs such as stimulation strength, with important consequences about what is released (only small cargo can escape through a small pore), on what time course, and how exocytosis is coupled to endocytosis. Despite the fundamental importance of fusion pores in regulating neurotransmitter and hormone release, very little is understood regarding mechanisms controlling pore nucleation and dynamics. This is mainly due to difficulties in studying fusion pores in reconstituted systems with well-defined protein and membrane components that would allow isolating the role of each component. Fusion mediated by exocytotic SNARE proteins and their regulators has been reconstituted and studied for the past 15 years. However, existing methods are not able to resolve single reconstituted fusion pores and follow pore dynamics with sufficient time resolution. We develop novel experimental approaches to enable probing mechanisms of nucleation and flickering of exocytotic fusion pores. Combining electrophysiological methods, single-particle fluorescence, microfabricated devices, and artificial bilayer technologies we develop in vitro assays that allow direct, simultaneous monitoring of single pore flickering and lipid mixing; counting protein numbers and/or probing protein-protein interactions; and controlling membrane curvature and tension. Using these assays, we study factors that govern nucleation and dynamics of SNARE-mediated fusion pores.

Membrane fission during sporulation

Prokaryotes constitute most of the biomass and the number of species on earth. They have colonized some of the most inhospitable corners of the world, as well as our bodies. The gut microbes hosted by a healthy person greatly outnumber the number of human cells that make up that person. The normal gut microbiota can be thought of as an organ because of the important metabolic and immunological roles it plays.

Recently, in collaboration with David Rudner’s laboratory at Harvard, we described the first membrane fission protein in bacteria, FisB (Doan et al., Genes & Dev. 2013). When nutrients are scarce, bacteria like B. subtilis form endospores by first dividing asymmetrically to produce a larger mother cell and a smaller forespore. The mother cell then engulfs the forespore in a phagocytosis-like process which ends with a fission event that requires FisB, a membrane anchored protein whose bulk lies outside the cytoplasm. Like its eukaryotic counterparts that each interact with a specialized lipid and oligomerize on membranes, FisB forms oligomers and binds cardiolipin (CL), a lipid whose sub-cellular localization and concentration change during sporulation. Because no other players have been implicated, our guiding hypothesis is that the combination of the unique membrane topology, FisB-CL interactions, FisB oligomerization, and CL dynamics alone can account for the clustering, recruitment to the fission site, and membrane fission activity of FisB. Using a combination of molecular biology, biochemistry, quantitative imaging, micromechanical measurements, microfabrication, and electrophysiology, we are defining factors controlling cardiolipin dynamics during sporulation, characterizing factors that govern FisB oligomerization and recruitment to the fission site, and determining how FisB remodels membranes.