G-protein Coupled Receptor Mediated Chemoattractant Sensing and Phagocytosis

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A new molecular interaction-based modeling of chemoattractant sensing: The signaling network underlying eukaryotic chemisensing is a complex combination of receptor-mediated trans-membrane signals, protein translocations, and differential activation/deactivation of membrane-bound and cytosolic components. It provides particularly interesting challenges for a combined computational and experimental analysis. We developed a novel detailed molecular signaling model that, when used to simulate the response to the attractant cAMP, made predictions about Dictyostelium chemosensing. These predictions, including the unexpected existence of spatially asymmetrical, multiphasic, cAMP-induced PTEN translocation and PIP3 generation, were experimentally verified by quantitative single- cell microscopy leading us to propose significant modifications to the current standard model for chemoattractant-induced biochemical polarization (Meier-Schellersheim et al., 2006). A locally controlled inhibitory mechanism in GPCR-mediated chemoattractant sensing network revealed by live cell imaging: A balance of excitatory and inhibitory activities mediated by GPCR signaling regulates chemosensing to cAMP in Dictyostelium, but the molecular nature and kinetics of inhibitors are still unknown. Here, we report that transient cAMP stimulations induced PIP3 responses without a refractory period, suggesting that GPCR-mediated inhibition accumulates and decays slowly. Moreover, we demonstrate that exposure to cAMP gradients leads to asymmetric distribution of the inhibitory components. The gradients induced a stable accumulation of the PIP3 reporter PHCrac-GFP in the front of cells near the cAMP source. A rapid withdrawal of the gradient from these biochemically polarized cells led to a rapid re-association of G-protein subunits, and the return of the PIP3 phosphatase PTEN and PHCrac-GFP to their pre-stimulus distribution. Interestingly, re-applying cAMP stimulation produced a clear PHCrac-GFP translocation to the back but not to the front, implicating the maintenance of a stronger inhibition in the front of a polarized cell. Our study demonstrates a novel spatiotemporal feature of a currently unknown inhibitory mechanisms acting locally on the PI3K activation pathway (Xu et al. 2007). A vesicle surface tyrosine kinase regulates phagosome maturation: Phagocytosis is an evolutionarily conserved process that is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we have identified a novel vesicle associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum and demonstrated its novel role in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, our detailed observations of the phagocytic process indicate that VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Because of the remarkable similarities between D. discoideum and metazoans in the known mechanisms that govern phagocytosis, our findings suggest that localized and regulated tyrosine kinase signaling on the surface of endosome/lysosomes may represent a general control mechanism for phagosome maturation in all phagocytes (Fang et al., 2007).