Signal transduction through receptor tyrosine kinases involves the formation of signaling complexes on the plasma membrane. These complexes undergo remodeling during the activation, amplification and attenuation stages of receptor signaling. Receptor dimerization, clustering of dimers, invagination of plasma membrane and endocytosis represent local physical rearrangements that will influence the biochemical environment of the signaling complex. This study will apply new live cell microscopy methods to quantify the influence of receptor clustering and membrane topography on signaling complex remodeling. Specifically, this project will test the roles of receptor clustering and membrane curvature on signal strength, character and duration of signaling from the macrophage colony stimulating factor receptor (M-CSFR). This receptor tyrosine kinase is a simple dimeric receptor that plays a central role in regulating the activities of macrophages. The M-CSFR is activated through ligand driven dimerization followed by docking of adaptor proteins and endocytosis. Existing biochemical and proteomic data provide a framework for M-CSFR signaling, but lack the spatiotemporal resolution necessary to model the kinetics and hierarchy of single complex formation, remodeling and dissolution. Here, we will quantify these dynamics on the surface of live cells using total internal reflection fluorescence (TIRF) excitation. Single particle trackingwill monitor diffusion and dimerization of single receptors. Clustering dynamics will be imaged using fluorescent chimeric receptor and quantified by Fluorescence Resonance Energy Transfer (FRET). Composition of signaling complexes will take advantage of imaging fluorescently tagged cytoplasmic proteins whose recruitment is observed as puncta in the TIR field. New polarized TIRF methods will be implemented to monitor the orientation of a lipophilic tracer as a measure of membrane curvature. This work will define the spatiotemporal interplay between the local environment and receptor-mediated signaling. The context for this work is primary macrophages that depend on M-CSF for their survival. The results from this study will aid in our mechanistic understanding of how M-CSFR signaling influences normal macrophage function in innate immune responses, antigen presentation and removal of apoptotic cells. The function of M-CSFR in regulating macrophage biology will have impacts in disease states including inflammatory disorders, cancer and atherosclerosis.
Essential macrophage functions including immune surveillance and pathogen clearance are largely under the control of the macrophage colony stimulating factor receptor (M-CSFR). This study focuses on mechanisms of M-CSFR signaling and downregulation at the cell surface, with an emphasis on modulation imposed by the cell membrane. The proposed work is expected to provide important details of M-CSFR signaling that will have therapeutic value for many disease pathologies including osteoporosis, atherosclerosis and cancer.
|Lou, Jieqiong; Low-Nam, Shalini T; Kerkvliet, Jason G et al. (2014) Delivery of CSF-1R to the lumen of macropinosomes promotes its destruction in macrophages. J Cell Sci 127:5228-39|