G-protein Coupled Receptor Mediated Chemoattractant Sensing and Phagocytosis

一种新的基于分子相互作用的化学物质感知模型：支持真核化学感知的信号网络是一个复杂的组合，包括受体介导的跨膜信号、蛋白质转位以及膜结合和胞液成分的差异激活/去激活。它为计算和实验相结合的分析提供了特别有趣的挑战。我们开发了一个新的详细的分子信号模型，当用来模拟对引诱剂cAMP的反应时，可以预测网柄苔藓的化学传感。这些预测，包括意想不到的空间不对称、多相、cAMP诱导的PTEN易位和PIP3的产生，通过定量单细胞显微镜实验验证，导致我们对目前化学诱导剂诱导的生化极化的标准模型提出了重大修改(Meier-Schellersheim等人，2006年)。 活体细胞成像揭示了GPCR介导的趋化物质传感网络中的一种局部控制的抑制机制：GPCRs信号介导的兴奋和抑制活性的平衡调节了Dictyostelials对cAMP的化学敏感，但抑制剂的分子性质和动力学尚不清楚。在这里，我们报告了短暂的cAMP刺激诱导PIP3反应而没有不应期，这表明GPCR介导的抑制积累和衰退缓慢。此外，我们还证明，暴露于cAMP梯度会导致抑制成分的不对称分布。梯度诱导PIP3报告基因PHCrac-GFP在cAMP源附近的细胞前部稳定积累。这些生化极化细胞的梯度迅速取消，导致G蛋白亚基迅速重新结合，PIP3磷酸酶PTEN和PHCrac-GFP恢复到刺激前的分布。有趣的是，重新施加cAMP刺激产生了明显的PHCrac-GFP易位到后面，而不是到前面，这意味着在极化细胞的前面保持着更强的抑制。我们的研究展示了一种新的时空特征，这是一种目前未知的局部作用于PI3K激活途径的抑制机制(Xu等人。2007)。 囊泡表面酪氨酸激酶调节吞噬小体的成熟：吞噬作用是一个进化保守的过程，对于寄主抵御微生物病原体和盘基网柄藻营养物质的获取至关重要。吞噬颗粒通过一条复杂的途径从吞噬小体运送到溶酶体进行降解，但吞噬小体成熟过程中涉及的分子机制尚不清楚。在这里，我们鉴定了一个新的囊泡相关受体酪氨酸激酶样蛋白VSK3，并证明了它在吞噬小体成熟过程中的新作用。VSK3位于晚期内体/溶酶体的膜上，其C末端的激酶域面向细胞质。通过基因破坏使VSK3失活会降低细胞内的吞噬速率，而VSK3的重新表达可以挽救这种吞噬速度。我们发现，VSK3在体内的功能依赖于激活域的存在和囊泡的定位。此外，我们对吞噬过程的详细观察表明，VSK3不是吞噬所必需的，而是吞噬小体与晚期内小体/溶酶体融合所必需的。由于盘状葡萄球菌和后生动物在控制吞噬作用的已知机制上有显著的相似之处，我们的发现表明，在所有吞噬细胞中，内体/溶酶体表面定位和调节的酪氨酸激酶信号可能代表着吞噬体成熟的一般控制机制(方等，2007年)。

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).