Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras.

Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells.

T cell triggering through T-cell antigen receptors (TCRs) results in spatial assembly of the receptors on multiple length scales. This assembly is mediated by the T cell actin cytoskeleton, which reorganizes in response to TCR phosphorylation and then induces the coalescence of TCRs into microclusters, followed by their unification into a micrometer-scale structure. The exact outcomes of the association of TCRs with a dynamic and fluctuating actin network across these length scales are not well characterized, but it is clear that weak and transient interactions at the single-molecule level sum to yield significant receptor rearrangements at the plasma membrane.

EphA2 receptor activation by monomeric Ephrin-A1 on supported membranes.

The receptor tyrosine kinase EphA2 interacts with its glycosylphosphatidylinositol (GPI)-linked ephrin-A1 ligand in a juxtacrine configuration. The soluble ephrin-A1 protein, without its GPI membrane linker, fails to activate EphA2. However, preclustered ephrin-A1 protein is active in solution and has been frequently used to trigger the EphA2 receptor.

T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters.

T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell.

Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling.

Physical inputs, both internal and external to a cell, can directly alter the spatial organization of cell surface receptors and their associated functions. Here we describe a protocol that combines solid-state nanolithography and supported lipid membrane techniques to trigger and manipulate specific receptors on the surface of living cells and to develop an understanding of the interplay between spatial organization and receptor function. While existing protein-patterning techniques are capable of presenting cells with well-defined clusters of protein, this protocol uniquely allows for the control of the spatial organization of laterally fluid receptor-ligand complex at an intermembrane junction.

Restriction of receptor movement alters cellular response: physical force sensing by EphA2.

Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed.

Synthesis, characterization, and spectroscopy of model molybdopterin complexes.

The preparation and characterization of new model complexes for the molybdenum cofactor are reported. The new models are distinctive for the inclusion of pterin-substituted dithiolene chelates and have the formulation Tp(*)MoX(pterin-R-dithiolene) (Tp(*)=tris(3,5,-dimethylpyrazolyl)borate), X=O, S, R=aryl. Syntheses of Mo(4+) and (5+) complexes of two pterin-dithiolene derivatives as both oxo and sulfido compounds, and improved syntheses for pterinyl alkynes and [Et(4)N][Tp(*)Mo(IV)(S)S(4)] reagents are described.