Here's just a sampling of the projects we're working on in our research group.

Biomembranes are complex, two-dimensional fluids that regulate with high fidelity the communication between cells and their surroundings and within individual cells.  Much of this communication occurs through cell surface receptors (proteins and lipids) and ligands that are either soluble or bound to the surfaces of neighboring cells.  The resulting signaling events occur on spatial scales ranging from tens to hundreds of nanometers and are temporally well controlled. Our long-term goal is to understand quantitatively how critical molecular events in a variety of essential membrane-involved processes control biological function. Specifically, we study immunoglobulin E (IgE) receptor signaling, which initiates the allergic response in mast cells. IgE receptor signaling has been proposed to proceed through an intricate, tightly choreographed dance of specific protein-protein and protein-lipid interactions that are controlled, in part, by heterogeneities in membrane structure, and this pathway is similar to that carried out by the related T cell and B cell receptors. We develop new quantitative tools, particularly those exploiting fluorescence, to probe spatial and temporal dynamics of the molecular interactions involved in immunoreceptor signaling. Our multidisciplinary laboratory uses state-of-the-art fluorescence microscopy and spectroscopy, optical trapping and nanotechnology to manipulate and interrogate single molecules in living cells and biomimetic systems. These molecular interactions are investigated using approaches from biophysics and optics, to materials chemistry, to molecular and cellular biology and biochemistry. Questions we are investigating range from the physical chemistry of functional interfacial lipid-lipid and lipid-protein interactions to the initiation of signal transduction and exocytosis. More recently, we are investigating the roles of macromolecular crowding in bulk solution, in living cells, and on protein-membrane interactions and intermembrane interactions that mimic exocytotic processes. We also apply our experimental approaches to intermolecular interactions at biosurfaces and biosensors.

 
Crosslinking of the high affinity receptor for IgE with multivalent antigen stimulates the allergic response in mast cells and basophils. This initial event is facilitated by interactions of the receptor with specialized cholesterol-rich membrane domains. The final step in this process is the exocytotic release of histamine-containing granules.

Crosslinking of the high affinity receptor for IgE with multivalent antigen stimulates the allergic response in mast cells and basophils. This initial event is facilitated by interactions of the receptor with specialized cholesterol-rich membrane domains. The final step in this process is the exocytotic release of histamine-containing granules.

 

immunoreceptor signaling

We are interested in understanding how specialized membrane domains facilitate immunoreceptor signaling.  Specifically, we are interested in two systems: the IgE receptor signaling in the allergic response and the T cell receptor in aging.  We are focused on understanding the spatio-temporal dynamics of heterogeneous regions (called "lipid rafts"), which are enriched in cholesterol and saturated lipids, in both living cells and biomimetic membranes.  Cholesterol-rich nanodomains in the biological membrane have been postulated to participate in a variety of cellular functions; however, they are too small and transient to resolve optically.  We developed a quantitative and sensitive means of monitoring membrane nanostructure, which is based on ultrafast dynamics imaging that allows us to effectively overcome limitations of optical resolution.  What makes this project particularly exciting is that it bridges the gap between many cell biological and biochemical studies that implicated cholesterol-rich nano domains in live cell imaging and related membrane biophysics.

 
Evanescent excitation (or total internal reflection) is used to selectively excite fluorophores that are within 100 nm of a surface.  We combine evanescent excitation with imaging and fluorescence correlation spectroscopy to measure interactions that model those occurring in endocytosis and exocytosis.

Evanescent excitation (or total internal reflection) is used to selectively excite fluorophores that are within 100 nm of a surface.  We combine evanescent excitation with imaging and fluorescence correlation spectroscopy to measure interactions that model those occurring in endocytosis and exocytosis.

 

biomembrane dynamics

Our long-term goal is to interrogate and understand the molecular interactions and dynamics occurring both within and between biomembranes in vivo and in vitro.  To accomplish this goal, we have combined, on a single integrated platform, fluorescence microscopic and spectroscopic techniques (fluorescence imaging, correlation spectroscopy, anisotropy and single molecule tracking, super-resolution, evanescent excitation) with holographic optical trapping in which tens of individual traps can be uniquely and individually controlled, in real time, to manipulate and measure molecules and their dynamics.  We have expertise in constructing symmetric and asymmetric lipid bilayers and hybrid systems that can also be spatially patterned.  In addition to investigating intramembrane dynamics, we also use these approaches to investigate intermembrane interactions that mimic endocytosis and exocytosis.

 
The cellular interior is packed with organelles and macromolecules. 

The cellular interior is packed with organelles and macromolecules. 

 

macromolecular crowding

The interior of the cell is crowded with organelles and biomolecules, which contrasts with the buffered solutions that are typically used in conventional biochemical studies.  These crowded conditions impact the functions, interactions and dynamics of biomolecules.  We are investigating the physical and chemical effects of macromolecular crowding (synthetic polymers and proteins) on these protein-membrane and membrane-membrane interactions and dynamics.  This complexity requires the acquisition of single-molecule information, together with bulk studies, to understand the length- and time-scale dependence associated with crowding effects on protein association kinetics and activities.  To investigate these effects, we use ultrafast laser-based spectroscopy to measure the rotational and translational diffusion and molecular volume of a given molecule as a function of crowding agent. We are investigating these dynamics in the crowded environments of bulk solution and live cells.