A new molecular interaction-based modeling of chemoattractant sensing: Cells can orient their migration in response to small local differences in the concentration of extracellular chemicals (chemoattractants). Understanding this process (chemosesing) requires the time and position-depentent behaviors of the signaling molecules with the responding cell, making it an especially interesting challenge for both experimental and computational investigation. We have previously developed live cell imaging techniques to quantitative measure spatiotemporal dynamics of multiple signaling events of chemoattractant gradient sensing in Dictyostelium discoideum (Xu et al., 2005). Recently, we reported the development and testing of a new detailed molecular model of the chemosensing apparatus of the D. discoideum (Meier-Schellerssheim et al., 2006). Computer simulations performed using this model predicted unexpected and patterns of changes in the concentration and location of the two important intracellular signaling molecules. These predictions were experimentally verified using live cell imaging experiments, suggesting the need for modifications to the current models of eukaryotic chemosensing. The high degree of detail in our model was made possible by a new software called ?simmune?, which allows biologists to enter information about molecular interactions using a graphical interface. This new tool has helped us to translate qualitative representations of cell biological processes into quantitative, predictive models. A locally controlled inhibitory mechanism in GPCR-mediated chemoattractant sensing network revealed by live cell imaging: Activation of G-protein-coupled receptors by chemoattractants induces dissociation of the heterotrimeric G-protein, activation of Ras and PI3K, and membrane translocation of PTEN, leading to a re-distribution of PIP3. Several models have proposed spatiotemporal features of an inhibitory process for polarized cellular responses. Since the molecular components involved in the inhibition remain unknown, the temporal-spatial distribution of the inhibition has never been examined experimentally. Using live cell FRET and fluorescence imaging techniques, we designed sequential stimulation protocols to detect temporal and spatial aspects of the inhibition process in single living cells. We found that repeated transient cAMP stimuli, unlike a sustained cAMP stimulation, induced repetitive PIP3 transient responses without refractory, supporting that the receptor-mediated inhibitions rise and fall slowly. More significantly, we detected an asymmetric distribution of the inhibition process in a cell exposing to a cAMP gradient. A sustained cAMP gradient led to a stable PHCrac-GFP accumulation (indicative of PIP3 level) in the front of the cell. A sudden withdrawal of the gradient from this polarized cell led to a rapid return of G-protein subunit association, PTEN and PHCrac-GFP distribution to basal levels. Interestingly, there was a short time period during which re-activation of receptor/G-protein around the cell membrane induced a clear PHCrac-GFP translocation to the back but not to the front of the cell, indicating a stronger inhibition in the front. Our study aided by quantitative modeling reveals novel spatiotemporal features of a hidden inhibitory mechanism on PI3K signaling and also suggests the candidates for the missing components (Xu, Meier-Schellersheim and Jin, unpublished). 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 it C-terminal kinase domain facing the cytoplasm. Both inactivation of VSK3 by gene disruption as well as over-expression of VSK3 reduced the rate of phagocytosis, while over-expression of VSK3 lacking the kinase domain had no effect. Though the protein is not involved in the engulfment process, it is required for the fusion of phagosomes with late endosomes/lysosomes. These findings, along with the remarkable similarities between D. discoideum and metazoans in the known mechanisms that govern phagocytosis, suggest that regulated tyrosine kinase signaling on the surface of endosome/lysosomes may represent a general control mechanism for phagosome maturation in all phagocytes (Fang, Brzostowski, and Jin, submitted).
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