Research Interests
The central goal of the Falke lab is a molecular understanding of cellular signaling on membrane surfaces, specifically the lipid signaling pathways听that control macrophage chemotaxis听and phagocytosis听in the innate immune response. Macrophages and other white blood cells possess a remarkable chemosensory pathway that enables these first responders听to follow chemical trails to sites of infection, inflammation, and tissue damage. Upon arrival, the phagocytosis pathway triggers the engulfment听and destruction of invading bacteria, viruses, and damaged cells. The regulatory hubs of these pathways are lipid kinases (PI-3-kinases or PI3Ks) that phosphorylate substrate phosphatidylinositol (PI) lipids at the 3-position of the inositol sugar headgroup, yielding the essential signaling lipids PI-3,4,5-trisphosphate (PIP3) in the chemosensory pathway, and PI-3-phosphate (PI3P) in phagocytosis. More broadly, PIP3听and PI3P signals are essential in all cell types where they control multiple cell processes,听and their dysregulation triggers a diverse听array of pathologies including cancer. Understanding the molecular mechanisms of these signals is crucial for optimal therapeutic targeting.
The Falke lab seeks to elucidate the mechanisms by which PIP3听and PI3P signaling pathways are regulated by听native signals, disease-linked mutations, and potential听therapeutic drugs. The group employs听a unique approach combining complementary in vitro single molecule and live cell methods听to probe the switching of signaling proteins between their 'on' and 'off' signaling states, as well as sequential information transfer between signalng proteins in a working biological circuit. The approach begins with single molecule TIRF studies of a reconstituted, multi-protein听circuit on a supported lipid bilayer mimicking the native membrane. 听These single molecule studies elucidate the regulatory mechanisms underlying signal transduction. 听Subsequently, the hypothesized mechanisms are tested by fluorescence听imaging studies in live macrophages, thereby revealing which mechanisms are most important in the听cellular context. 听Projects are available to carry out single molecule studies of reconstituted signaling circuits, or model testing via cell imaging in live macrophages, or both. 听The lab also employs a broad听array of other听biophysical and biochemical tools as needed to address key biomedical questions.
Summary of Falke Lab Accomplishments
More about Macrophage听Chemotaxis and Phagocytosis
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Signal transduction mechanisms in macrophage chemotaxis
In the chemosensory signaling circuit that controls macrophage chemotaxis, 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, controls second messenger signals that recruit dozens of proteins to the leading edge membrane, where these proteins form the signaling circuit that drives actin and membrane remodeling to push the leading edge up the attractant gradient.
The Falke group is investigating the molecular mechanisms underlying the assembly and operation of the chemosensory circuit on the leading edge membrane. Traditionally, the signaling lipid PIP3 has been considered the only relevant second messenger at the leading edge, but live cell studies by the group revealed that a localized Ca(II) signal is also an essential component of the leading edge postitive feedback loop. Together, these PIP3 and Ca(II) signals recruit PH- and C2-domain proteins, respectively, to the leading edge membrane, where they serve as upstream regulators or downstream effectors of the Class I PI3K听lipid kinase that serves as the regulatory hub of the leading edge chemosensory circuit.
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, (iii) their interactions with target and substrate lipids, (iv) their interactions with other membrane proteins to form听signaling complexes, and (v) sequential information flow between the protein components of the membrane-based signaling听circuit. The targeted master kinases include PKCalpha, PI3Kalpha, PDK1, and AKT1/PKB. An innovative single molecule fluorescence method developed by the group is revealing, for the first time, the surface diffusion,听interactions, and regulatory mechanisms听of the circuit components reconstituted on a supported lipid bilayer resembling the native membrane. Subsequent live cell imaging studies test the resulting mechanistic models in the native context of the macrophage leading edge. 听Many other biophysical and biochemical methods are also employed as needed to answer key questions. 听Together, these diverse approaches are providing new insights into the molecular basis of macrophage chemosensing at the leading edge membrane during the primary immune response to infection, inflammation, and tissue damage. More broadly, each of the master kinases plays central roles in other signaling pathways and in multiple human cancers.
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Signal transduction mechanisms in macrophage phagocytosis
In the signaling circuit that initiates phagocytosis, cell-surface receptors trigger phagocytosis when they听recognize and bind motifs characteristic of pathogens, or the antibodies coating pathogens, or damaged tissue. 听Phagocytosis and engulfment yields an internalized听phagosome, which then undergoes three stages of development. 听The early stage is听characterized by simultaneous Class I and Class III PI3-kinase lipid signals (PIP3 and PI3P, respectively) present on the phagosome membrane, which recruit specific signaling proteins to the membrane surface. 听Next, during the middle stage of phagosome development Class III PI3-kinase is strongly听activated and the resulting intense PI3P signal activates the production of reactive oxygen species (ROS) that kill the trapped pathogens within the phagosome. 听Finally, during the late stage of phagosome development the Class III PI3-kinase activity decreases and the phagosome is prepared for fusion with the lysosome, which breaks down pathogens into fragments used to guide the听development of specific antibodies against the pathogen via adaptive immunity. 听听听
The Falke group is currently investigating the molecular mechanisms of signal transduction on the phagosome membrane during its three stages of development. 听Questions being addressed include (i)听regulation of the Class III PI3K-kinase and PI3P production by the G protein Rab5, (ii) activation of master kinases by simultaneous PIP3 and PI3P signals in the early stage phagosome, (iii) strong activation of the听Class III PI3K lipid kinase and PI3P production by Rubicon and Rab7 in the middle stage, and (iv) downregulation of the lipid kinase and PI3P signaling in the late stage phagosome.听听As described for studies of the chemotaxis pathway, the lab elucidates molecular mechanisms of regulation using single molecule TIRFM studies of reconstituted signaling reactions in vitro, then employs live-cell imaging studies to test the resulting mechanistic models in the native macrophage context, and also incorporates other听biophysical and biochemical tools as needed to answer key biological questions.听
The resulting progress towards a molecular understanding of macrophage chemotaxis and phagocytosis will advance basic signaling听biology and immunology. Moreover, a better mechanistic understanding of pathway dysregulation by mutations and modulation by drugs may lead to new personalized therapies for an array of inflammatory, innate- and auto-immune disorders including allergy, arthritis, asthma, ataxia, celiac, and neurodegeneration.
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Signaling reactions on the macrophage leading edge membrane (PMID 17911247, 29715315)
Signaling reactions on the macrophage phagosome membrane (Falke Lab, unpublished)
Single molecule assay for Class I PI3-kinase and PIP3 production (PMID 27119641, 29211993)
Single molecule assay for Class III PI3-kinase and PI3P production (PMID 33137306)
Selected Falke Group Accomplishments
- (2021) Discovery that Conventional PKC Can Competitively听Inhibit PDK1 Phosphoactivation of AKT1 on a Target Membrane, As Revealed by Single Molecule Analysis (Gordon, Ziemba & Falke)
- (2021) Development of a UV Deconvolution Method to Analyze the听Protein Concentration, Nucleotide Stoichiometry, and Purity of Ras-Guanine Nucleotide Complexes (Swisher, Hannan, Cordaro, Erbse, & Falke)
- (2020)听Elucidation via Single Molecule Analysis of a Novel Dual听Molecular Mechanism by which the Small GTPase Rab5 Recruits and Allosterically Activates Class III PI3K and PI3P Production, (Buckles, Ziemba, Masson, Williams & Falke)
- (2020) Development of the First Single Molecule Assay for Class III PI3K Lipid Kinase Activity and PI3P Production on a Reconstituted Phagosome-Like Bilayer. 听(Buckles, Burke, Ohashi, Tremmel, Gordon, Williams & Falke)
- (2018) Hypothesis Testing in Live Macrophages Reveals that Ca-PKC, but not Ca-Calmodulin, Regulates the MARCKS-PI3K-PIP3 Circuit at the Leading Edge Membrane听听(Buckles, Ziemba, Masson, Williams & Falke)
- (2017) Discovery via Single Molecule Analysis that Ca-Calmodulin (Ca-CaM)听Stimulates PIP3 Lipid Signaling In Vitro听via a Ca-CaM-MARCKS-PI3K-PIP3 Activation Module (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) Discovery Via Single Molecule Analysis in vitro听that Ca Signals Stimulate PIP3 Lipid Signaling via a Ca-PKC-MARCKS-PI3K-PIP3 Activation Module听听(Ziemba, Swisher, Burke, Masson, Williams & Falke)
- (2016) Development of the First Single Molecule Assay for Class I PI3K Lipid Kinase Activity and PIP3 Production on a Reconstituted Plasma-Menbrane-Like Bilayer. 听(Ziemba, 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 (PKC) 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 脨 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)