Signal amplification and membrane recruitment

In the eukaryotic chemosensory signaling circuit, attractant signals sensed by cell-surface receptors are amplified by a positive feedback loop at the leading edge of the cell. The feedback loop, in turn, generates second messenger signals that recruit dozens of proteins to the leading edge, where these proteins stimulate actin and membrane remodeling that drives the leading edge up the attractant gradient.

The Falke group is identifying the second messenger signals generated at the leading edge, and the molecular mechanisms by which these signals orchestrate one of the most dramatic protein redistributions in cell biology. Traditionally, the signaling lipid PIP3 has been considered the only relevant second messenger at the leading edge, but recent live cell studies by the group have revealed that normal leading edge structure and function also requires a localized Ca(II) signal. Together, these PIP3 and Ca(II) signals (and perhaps others) recruit PH- and C2-domain proteins, respectively, to the leading edge membrane.

Current work is targeting the molecular mechanisms underlying (i) the rapid recruitment of master kinases to the leading edge membrane, (ii) their activation on the membrane surface, and (iii) their interactions with lipids and proteins essential for substrate binding and the formation of multi-protein signaling complexes. The targeted master kinases include PKCalpha, PI3Kalpha, and PDK1. Structure-based EPR spectroscopy and EPR-guided molecular dynamics simulations are being used to determine the structures and dynamics of protein-membrane complexes. In addition, bulk FRET measurements are elucidating the equilibrium and kinetic parameters of the membrane docking reactions. Moreover, an innovative single molecule fluorescence method developed by the group is revealing, for the first time, the surface diffusion behaviors and interactions of membrane-docked signaling proteins. Together, these diverse approaches will provide new insights into the molecular basis of macrophage chemosensing at the leading edge membrane during the primary immune response and inflammation. More broadly, each of the master kinases plays central roles in other signaling pathways and in multiple human cancers.