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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 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 signaling complexes, and (v) sequential information flow between the protein components of the membrane-based signaling 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, interactions, and regulatory mechanisms 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.  Many other biophysical and biochemical methods are also employed as needed to answer key questions.  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.

In the signaling circuit that initiates phagocytosis, cell-surface receptors trigger phagocytosis when they recognize and bind motifs characteristic of pathogens, or the antibodies coating pathogens, or damaged tissue.  Phagocytosis and engulfment yields an internalized phagosome, which then undergoes three stages of development.  The early stage is 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.  Next, during the middle stage of phagosome development Class III PI3-kinase is strongly activated and the resulting intense PI3P signal activates the production of reactive oxygen species (ROS) that kill the trapped pathogens within the phagosome.  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 development of specific antibodies against the pathogen via adaptive immunity.    

The Falke group is currently investigating the molecular mechanisms of signal transduction on the phagosome membrane during its three stages of development.  Questions being addressed include (i) 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 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.  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 biophysical and biochemical tools as needed to answer key biological questions. 

The resulting progress towards a molecular understanding of macrophage chemotaxis and phagocytosis will advance basic signaling 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.