G-protein Coupled Receptor Mediated Chemoattractant Sens

一种新的基于分子相互作用的化学引诱物传感建模：细胞可以根据细胞外化学物质（化学引诱物）浓度的微小局部差异定向迁移。理解这一过程（chemosesing）需要信号分子与响应细胞的时间和位置依赖性行为，使其成为实验和计算研究的一个特别有趣的挑战。我们先前已经开发了活细胞成像技术来定量测量盘基网柄藓中化学引诱物梯度感测的多个信号传导事件的时空动态（Xu et al.，2005年）。最近，我们报道了一个新的详细的分子模型的化学传感器的D。盘状体（Meier-Schellerssheim等，2006年）。使用该模型进行的计算机模拟预测了两种重要的细胞内信号分子的浓度和位置的意外变化和变化模式。这些预测进行了实验验证，使用活细胞成像实验，这表明需要修改目前的真核生物化学传感模型。在我们的模型中的高度细节是由一个新的软件称为？simmune？，它允许生物学家使用图形界面输入有关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示转化为定量的预测模型。 通过活细胞成像揭示的GPCR介导的化学引诱物传感网络中的局部控制的抑制机制：化学引诱物对G蛋白偶联受体的激活诱导异源三聚体G蛋白的解离、Ras和PI 3 K的激活以及PTEN的膜易位，导致PIP 3的重新分布。几个模型提出了极化细胞反应的抑制过程的时空特征。由于参与抑制的分子组分仍然未知，抑制的时空分布从未被实验研究。使用活细胞FRET和荧光成像技术，我们设计了顺序刺激方案，以检测在单个活细胞的抑制过程的时间和空间方面。我们发现，与持续cAMP刺激不同，重复的瞬时cAMP刺激诱导重复的PIP 3瞬时反应，而无难治性，支持受体介导的抑制缓慢上升和下降。更重要的是，我们检测到一个不对称分布的抑制过程中的细胞暴露于cAMP梯度。持续的cAMP梯度导致细胞前部稳定的PHCrac-GFP积累（指示PIP 3水平）。从该极化细胞中突然撤回梯度导致G蛋白亚基缔合、PTEN和PHCrac-GFP分布快速返回到基础水平。有趣的是，在细胞膜周围的受体/G蛋白的再活化诱导明显的PHCrac-GFP易位到细胞的后部而不是前部的短时间段，表明在前部的较强抑制。我们的研究在定量建模的帮助下揭示了PI 3 K信号传导的隐藏抑制机制的新时空特征，并提出了缺失组分的候选者（Xu，Meier-Schellersheim和Jin，未发表）。 囊泡表面酪氨酸激酶调节吞噬体成熟：吞噬作用是一个进化上保守的过程，对于宿主防御微生物病原体和获得盘基网柄藻中的营养物质至关重要。被吞噬的颗粒通过复杂的途径从吞噬体递送到溶酶体以进行降解，但是吞噬体成熟过程中涉及的分子机制还没有很好地理解。在此，我们在D. discoideum，并证明了其在吞噬体成熟的新作用。VSK 3位于晚期内体/溶酶体的膜上，其C-末端激酶结构域面向细胞质。通过基因破坏使VSK 3失活以及VSK 3的过表达都降低了吞噬率，而缺乏激酶结构域的VSK 3的过表达没有影响。虽然该蛋白不参与吞噬过程，但它是吞噬体与晚期内体/溶酶体融合所必需的。这些发现，沿着与D.盘状体和后生动物在已知的控制吞噬作用的机制中的作用表明，内体/溶酶体表面上调节的酪氨酸激酶信号传导可能代表了所有吞噬细胞中吞噬体成熟的一般控制机制（Fang，Brzostowski和Jin，提交）。

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).