G-protein Coupled Receptor Mediated Chemoattractant Sens

一种新的基于分子相互作用的化学引诱剂传感模型：细胞可以根据细胞外化学物质（化学引诱剂）浓度的微小局部差异来定向迁移。理解这一过程（化学反应）需要信号分子与响应细胞的时间和位置依赖行为，这使得它成为实验和计算研究的一个特别有趣的挑战。我们之前已经开发了活细胞成像技术来定量测量Dictyostelium disideum中化学引诱剂梯度感应的多个信号事件的时空动态（Xu et al., 2005）。最近，我们报道了一种新的详细分子模型的开发和测试（Meier-Schellerssheim et al., 2006）。使用该模型进行的计算机模拟预测了两种重要细胞内信号分子的浓度和位置的意外变化和模式。这些预测通过活细胞成像实验得到了验证，这表明需要对当前真核化学传感模型进行修改。我们模型中的高度细节是由一种名为simune的新软件实现的。，它允许生物学家使用图形界面输入分子相互作用的信息。这个新工具帮助我们将细胞生物学过程的定性表征转化为定量的预测模型。

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).