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Falke Group: Research Interests and Accomplishments

The broad goal of the Falke group is a molecular understanding of biological signaling pathways, using chemosensory pathways as a model. Motile cells possess chemosensory pathways that control cell movement, enabling migration up or down gradients of chemical attractants and repellents, respectively. A chemosensory pathway is a biological integrated circuit in which receptors detect specific attractants or repellents and trigger circuit activaiton, then downstream kinases and other enzymes generate output and feedback signals that control directed cell migration and sensory adaptation. Current studies in the Falke group focus on two distinct types of chemosensory circuits that (i) bacteria use to find their way to a food source or wound, and (ii) human macrophages and white blood cells use to track down a bacterial infection or tissue damage.

More specifically, the Falke group is using a structure-based approach to probe the switching of receptors and signaling proteins between their 'on' and 'off' signaling states at the molecular level in the working biological circuit. The approach involves the development of novel techniques to analyze the structural dynamics of switch proteins, as well as the molecular docking events regulated by their on-off switching. Rather than studying isolated pathway components, the group generally studies reconstituted complexes and networks of signaling proteins much as they are found in the cell. Of particular interest are signaling and on-off switching events at membrane surfaces, which play a central role in regulating chemsensory and most other biological signaling circuits and, in medical applications, are the targets of most pharmaceuticals. The group employs biophysical, biochemical, and molecular biological methods including: FRET, bulk and single molecule fluorescence, EPR, X-ray crystallography, mass spec, site-directed mutagenesis, cysteine chemistry, molecular modeling, and intracellular fluorescence microscopy.

Below please find information on both projects, and a list of selected lab accomplishments.

 

Project 1.

Cell-surface receptors and kinase regulation in bacterial chemosensing. In the bacterial chemosensory pathway, all of the cytoplasmic components assemble onto an lattice of transmembrane chemoreceptors to generate an ultrastable, ultrasensitive signaling array. This array is one of the simplest, yet highly efficient, biological integrated circuits.

The Falke group is currently investigating the molecular mechanisms of on-off switching of components within the chemosensory array, especially the mechanisms by which transmembrane receptors bind specific attractants and regulate the output kinase activity. This kinase activity controls the swimming motor and cellular migration up attractant gradients. The known structures of the pathway components greatly facilitate molecular analysis of their on-off switching mechanisms.

The group is developing an array of biochemical, chemical and spectroscopic approaches capable of analyzing structural dynamics during on-off switching in the functional, membrane-bound, signaling array. Specific goals include elucidation of the mechanisms by which attractant and adaptation signals are transmitted through the transmembrane receptors to the kinase, and how these receptor on-off signals switch the kinase on and off. The resulting molecular mechanisms are likely conserved throughout the diverse family of 2-component pathways, which control most cellular processes in bacteria. Such broad relevance ensures that mechanistic advances will have significant impacts on the field of signaling biology, as well as on the development of new classes of (i) broad-spectrum antibiotics, (ii) chemotaxis inhibitors that block wound seeking and infection, and (iii) ultrasensitive, ultrastable biosensors. Moreover, many of the tools developed in this simple, bacterial system can ultimately be applied to eukaryotic systems.

  Figure 1, Receptor Project
Figure2, Receptor Project

Figure 3, Receptor Project

Summary Figure, Project 1



Project 2.

Signal amplification and membrane recruitment in macrophage chemosensing. 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.

  Figure 4, Calcium Project
Figure 5, Calcium Project

Figure 6, Calcium Project

 

Summary Figure, Project 2


Selected Falke Group Accomplishments (see "Publications" for reference)

(2017) Discovery via Single Molecule Analysis that Calmodulin Inhibits the Activation of Ras, PI3K, and PIP3 Signaling: A Postulated Mechanism for Creating Temporal Pulses of PIP3 Formation (Buckles, Ziemba, Masson, Williams & Falke)

(2017) Elucidation via Single Molecule Analysis of the Molecular Mechanism by which the Oncogenic Small GTPase Ras Activates the Oncogenic Lipd Kinase PI3K and thereby Amplifies PIP3 Signals, (Buckles, Ziemba, Masson, Williams & Falke)

(2016) Reconstitution of the Amplification Circuit That Couples Ca and PIP3 Signals at the Leukocyte Leading Edge, Including PKC, (Ziemba, Buckles, Swisher, Burke, Masson, Williams & Falke)

(2014) Engineered Disulfide Bonds that Further Enhance the Kinetic Stability of the Bacterial Chemosensory Array (Ziemba, Pilling, Calleja, Larijani & Falke)

(2014) Discovery of a New, Predominant Intermediate in the Activation Mechanism of Protein Kinase C Bound to its Target Membrane (Ziemba, Li, Landgraf, Knight, Voth & Falke)

(2013) Elucidation of a Novel Dimer-to-Monomer Activation Mechanism for the PH domain of PDK1 (Ziemba, Pilling, Calleja, Larijani & Falke)

(2013) Discovery that Bound Lipid and Protein Keels In the Bilayer Make Additive Contributions to the Total Friction of Peripheral Proteins Undergoing Lateral Diffusion (Ziemba & Falke)

(2013) Elucidation of the Structure and Function of Two Essential Protein-Protein Contacts in the Functional, Bacterial Chemosensory Array (Piasta, Natale, Duplantis, Ulliman, Slivka, Crane & Falke)

(2012) Initial Evidence that the Ultrastability of the Bacterial Chemosensory Array Requires a High Degree of Array Order (Slivka & Falke)

(2012) First Experimental Determination of a PH Domain Membrane Docking Geometry by EPR Depth Parameter Measurements (Chen, Ziemba & Falke)

(2012) Development of a Single-Molecule Method for Detecting the Formation of Signaling Protein Complexes on Membrane Surfaces (Ziemba, Knight & Falke)

(2011) Demonstration that the Sentry Glutamate is a Widespread Feature of PIP3-Specific Binding Sites in PH domains (Pilling, Landgraf & Falke)

(2011) Development of One-Sample Method for Bulk Fret Measurements, Known As OSFRET (Erbse & Falke)

(2010) First systematic study showing that tightly bound lipids make additive contributions to the bilayer friction of peripheral membrane proteins during lateral diffusion (Knight, Lerner, Velazquez, Pastor & Falke)

(2009) Discovery that the conserved cytoplasmic domain of bacterial chemoreceptors transmits signals through its long four-helix bundle via a novel yin-yang mechanism (Swain & Falke)

(2009) Discovery that the bacterial chemosensory signaling complex is ultrastable (Erbse & Falke)

(2009) Development of a novel single-molecule method to probe the protein-lipid interactions and surface dynamics of membrane-bound proteins (Knight & Falke)

(2008) Elucidation of the molecular mechanism underlying a highly oncogenic mutation in AKT1 PH domain known to cause multiple human cancers (Landgraf, Pilling & Falke)

(2008) Determination of the distinct membrane docking geometries of PKC-alpha C2 domain in two different lipid binding states (Landgraf, Malmberg & Falke)

(2007) Discovery that a localized Ca(II) influx is an essential component of the positive feedback loop at the macrophage leading edge (Evans & Falke)

(2007) Chemical structure determination that the conserved HAMP signal conversion domain of bacterial chemoreceptors is a parallel 4-helix bundle (Swain & Falke)

(2007, 2006) Demonstration that PIP2 is a third essential target lipid of PKC-alpha (Evans, Corbin, Landgraf & Falke)

(2006) Chemical mapping of four protein interactions sites on the surface of the bacterial chemosensory kinase CheA (Miller, Kohout & Falke)

(2005) Discovery of a conserved, essential Gly hinge in the cytoplasmic 4-helix bundle of bacterial chemoreceptors (Coleman, Bass & Falke)

(2005) Elucidation of the electrostatic mechanism underlying adaptation site signaling in bacterial chemoreceptors (Starrettt & Falke)

(2004) EPR determination of the highest resolution membrane docking geometry currently available the C2 domain of cytosolic phospholipase A2 (Malmberg & Falke)

(2004) Development of an electrostatic method to drive piston displacements of transmembrane helices (Miller & Falke)

(2004) Discovery that GRP1 PH domain uses an electrostatic search mechanism to rapidly find its rare target lipid PIP3 (Corbin & Falke)

(2003) Chemical mapping of the protein interaction sites on the surface of bacterial chemoreceptors (Mehan & Falke)

(2003) Demonstration that covalent adaptation introduces multiple sub-states into the on-off switching behavior of the receptor-CheA signaling complex (Bornhorst & Falke)

(1999) Chemical determination of the 4-helix bundle architecture of bacterial chemoreceptor cytoplasmic domains (Bass, Butler, Danielson & Falke)

(1997) Elucidation of the Ca(II)-signaling cycle for the membrane-docking C2 domain of cytosolic phospholipase A2, the Ca(II) sensor of inflammation (Nalefski & Falke)

(1997) Development of a novel FRET assay for monitoring the equilibrium and kinetic parameters of protein-membrane docking reactions (Nalefski & Falke)

(1996) Discovery that the amino acid at the gateway position of EF-hand sites controls the Ca(II) on-off kinetics (Drake & Falke)

(1996) Determination of the effects of protein stabilizing agents on long-range backbone motions in proteins via disulfide trapping (Butler & Falke)

(1996) Discovery that the transmembrane signal of bacterial chemoreceptors is transmitted by a piston displacement of the signaling helix (Chervitz & Falke)

(1995) Engineering reversible, lock-on and lock-off disulfide bonds that covalently trap the signaling states of bacterial chemoreceptors (Chervitz & Falke)

(1994) Use of 19F NMR to probe conformational changes in a receptor (Danielson & Falke)

(1993) Use of 19F NMR to probe conformational changes in a signaling protein (Drake & Falke)

(1992) Detection and trajectory analysis of thermal backbone motions in a folded, aqueous protein by a novel disulfide trapping method (Careaga & Falke)

(1991) Use of 19F NMR to probe conformational changes in a binding protein (Luck & Falke)