We investigated the association of signaling proteins with epidermal growth factor (EGF) receptors (EGFR) using biotinylated EGF bound to streptavidin that is covalently coupled in an ordered array of micron-sized features on silicon surfaces. Using NIH-3T3 cells stably expressing EGFR, we observe concentration of fluorescently labeled receptors and stimulated tyrosine phosphorylation that are spatially confined to the regions of immobilized EGF and quantified by cross-correlation analysis. We observe recruitment of phosphorylated paxillin to activated EGFR at these patterned features, as well as β1-containing integrins that preferentially localize to more peripheral EGF features, as quantified by radial fluorescence analysis. In addition, we detect recruitment of EGFP-Ras, MEK, and phosphorylated Erk to patterned EGF in a process that depends on F-actin and phosphoinositides. These studies reveal and quantify the coformation of multiprotein EGFR signaling complexes at the plasma membrane in response to micropatterned growth factors.

We present non-faradaic electrochemical recordings of exocytosis from populations of mast and chromaffin cells using chemoreceptive neuron MOS (CνMOS) transistors. In comparison to previous cell-FET-biosensors, the CνMOS features control (CG), sensing (SG) and floating gates (FG), allows the quiescent point to be independently controlled, is CMOS compatible and physically isolates the transistor channel from the electrolyte for stable long-term recordings. We measured exocytosis from RBL-2H3 mast cells sensitized by IgE (bound to high-affinity surface receptors FcεRI) and stimulated using the antigen DNP-BSA. Quasi-static I-V measurements reflected a slow shift in surface potential () which was dependent on extracellular calcium ([Ca]o) and buffer strength, which suggests sensitivity to protons released during exocytosis. Fluorescent imaging of dextran-labeled vesicle release showed evidence of a similar time course, while un-sensitized cells showed no response to stimulation. Transient recordings revealed fluctuations with a rapid rise and slow decay. Chromaffin cells stimulated with high KCl showed both slow shifts and extracellular action potentials exhibiting biphasic and inverted capacitive waveforms, indicative of varying ion-channel distributions across the cell-transistor junction. Our approach presents a facile method to simultaneously monitor exocytosis and ion channel activity with high temporal sensitivity without the need for redox chemistry.

We are investigating the spatiotemporal association of signaling proteins with EGF receptors (EGFR) using micro‐patterned EGF surfaces. We utilize EGF‐biotin bound to streptavidin that is covalently coupled in ordered micron sized square arrays on silicon surfaces. NIH‐3T3 cells stably over‐expressing wt EGFR are added and adhere to these patterned surfaces. We observe concentration of fluorescently labeled EGFR and stimulated tyrosine phosphorylation that are spatially confined to the patterns of immobilized EGF. We also observe recruitment of paxillin‐GFP to the receptor signaling complexes formed on EGF patterns, and this association occurs in the absence of integrin co‐clustering but depends on EGFR tyrosine kinase activity and F‐actin, which also accumulates in these patterned regions. Inhibition of Src kinase with PP2 inhibits tyrosine phosphorylation of associated paxillin but does not prevent its localization with patterned EGF. We further detect recruitment of phospholipase Cγ‐1‐GFP and phosphorylated Erk to the patterned EGF in a ligand‐dependent manner. Our ongoing studies are investigating the roles of paxillin and F‐actin in recruitment of Erk to the EGF‐mediated signaling complex. These studies highlight the utility of micro‐patterned growth factors to characterize the spatially regulated formation of signaling complexes at the plasma membrane. Supported by NIH Grant R01‐AI018306.

No abstract

labeling of an azido side-chain in an engineered G protein-coupled receptor (GPCR) with the trifunctional reagent. A sequential affinity purification utilizing the epitope-tagged receptor and the handle on the trifunctional reagent allows removal of excess label and unlabeled receptor, respectively. We will show single-molecule fluorescence images of the receptors captured by immobilized avidin.