Research Program
Cell Surface Interactions

Objectives
Cells spatially control their responses to external stimuli, leading to targeted responses. The importance of the spatial localization is reflected by the sophisticated structures employed by cells to restrict and compartmentalize many biomolecular interactions. The focus of this project is to visualize 2D compartmentalization (domains or interaction zones within plasma membrane), 3D compartmentalization (intracellular membrane trafficking) as well as molecular-scale dynamics and interactions. Patterned surfaces allow fluorescence visualization on the micron scale, whereas electron microscopy, zero mode waveguides (ZMW) and new optical systems to be developed enable examination of biomolecular distributions and dynamics on ~100 nm length scale. With IgE receptors on mast cells as our prototypic system we seek to elucidate mechanisms controlling the spatial distribution of signaling events that are initiated by immune receptors.

Methods
Micro- and nanofabrication including photolithography with parylene lift-off or ZMWs; patterned lipid bilayers and proteins; genetically encoded fluorescent constructs and immunofluorescence detection within living cells; optical and electron microscopic imaging; fluorescence confocal microscopy; total internal reflection fluorescence microscopy; fluorescence correlation spectroscopy; correlation analysis. Structural rearrangements related to cellular activities (e.g., kinase-mediated phosphorylation, Ca²⁺ mobilization, degranulation) that can be affected by siRNA knockdown of participating proteins.

Summary
Surfaces patterned with antigens on the micron scale are utilized for examination of spatially localized events in receptor-mediated signal transduction and cellular responses. Our previous studies revealed participation of the cytoskeleton in co-redistribution of membrane and signaling components with antigen-clustered receptors. Adhesion to the substrate also involves cytoskeletal interactions with integrins that are mediated by focal adhesion and other complexes. Patterned bilayers containing antigens were used to spatially separate these interactions based on integrin preference for extracellular matrix proteins that adhere to silicon oxide surface, at the same time that receptors were clustered by the antigen in the bilayers. These studies revealed receptor interaction, independent of integrins, with focal adhesion proteins (vinculin, talin, and paxillin) but not ERM proteins (ezrin, moesin) [Figures 1 and 2]. SiRNA knockdown revealed that paxillin is involved in negative regulation of signaling that is initiated by clustered receptors.

The ZMWs and related nanoapertures limit excitation of fluorescence to the submicron scale  and have proven to be very effective for examining dynamics of cell membranes at this high level of resolution. We previously demonstrated that plasma membranes from live cells penetrate these nanostructures, and we found that cellular exploration of the nanoapertures depends heavily on actin filaments but not on microtubules, suggesting that cells extend filopodia-like membranous extensions that elongate by the polymerization of actin filaments. The presence of actin filaments within ZMWs was confirmed by monitoring the fluorescence from transiently-expressed GFP-tagged actin as it polymerized within the structures and entered the excited volume. Correlation spectroscopy showed that the dynamics of filapodia exploration is similar for a membrane probe (DiIC₁₂) and cytoskeletal probe (GFP-actin): ~25 sec occupancy time and that this does not change with cell activation [Figure 3].

Accomplishments

• Patterned bilayers, show that  focal adhesion proteins (but not ERM proteins) co-redistribute with clustered IgE-receptors, independently of integrins
• Functional evaluation with siRNA cells shows that focal adhesion protein  paxillin negatively regulates receptor-mediated phosphorylation of signaling proteins with downstream impact.
• Dynamic exploration by cells of nanoapertures quantified with correlation spectroscopy, and occupancy time found to be similar for membrane and cytoskeletal probes.