Cells have intricately shaped membranes that are created and maintained by the interplay between proteins, lipids, and physical forces. Sorting of proteins on regions of curvature can be a key feature in understanding how cells sense mechanical signals in the surrounding environment. Curved membranes function to control protein activity, to sort proteins in space, and are the product of protein-lipid interactions. The shapes observed in vivo vary dramatically in size, from nanometers to microns, and the affinity of a protein for a curved lipid surface are shape dependent. In our work we:
- Design novel, curved lipid membranes that can be used to understand the molecular interactions with curved lipid bilayers and proteins
- Use high-resolution, spatio-temporal dynamics to understand how curved membranes sort lipids and proteins
- Assay protein function and affinity while varying membrane shape
Exocytosis: SNARE proteins and PLD
SNARE proteins catalyze membrane fusion. A complex of three SNARE proteins facilitates exocytosis by folding into a highly stable complex that brings the neurotransmitter-filled vesicle in close proximity with the plasma membrane upon Ca2+ influx. Two SNARE proteins on the plasma membrane (Syntaxin and SNAP25) form a bridge with one SNARE protein on the vesicle membrane (Synaptobrevin). We use biochemical and cellular systems to assess SNARE protein function. Specifically, we:
- Express SNARE proteins and SNARE accessory proteins in secretory cells to spatially and temporally map their location in the fusion process.
- Create mimics of neuronal membranes to measure the effect of lipid composition on SNARE protein function.
Beyond proteins, lipids play an important role in secretion. Phospholipase D (PLD) is thought to convert PC into PA and facilitate the fusion pore formation. The loss of PLD halts membrane fusion. We perform cellular and biochemical assays with PLD to determine when and where it acts during the membrane fusion process.
Single Molecule Imaging
We use total interal reflection fluorescence (TIRF) microscopy to image single molecules on the plasma membrane of live cells and in reconstituted membrane systems. TIRF preferentially illuminates the surface of the cell, making it an ideal imaging technique for measuring protein dynamics and interactions on the cell surface. We use single molecules in two ways:
- as a calibration for the number of proteins in a complex
- as a probe to measure the dynamics of individual proteins or lipids.
Single molecule techniques are used to characterize the molecular mechanism of proteins while they perform their biological function.