The directed migration of cells, or chemotaxis, is essential not only for the normal physiology of embryos and adults, but also for cancer metastasis and the pathology of inflammatory disorders such as asthma, arthritis, and vascular disease. Significant gains in our knowledge of this process are imminent. A network of parallel signaling pathways that mediate chemotaxis is emerging and, recently, it has been shown that this biochemical system displays excitability that may drive the extension of pseudopodia. It is proposed that an adaptive signal generated by chemoattractant receptors and associated G-proteins biases the excitable network to direct cell migration. How does the topology of the network lead to excitability? How do G-proteins link to the network and what is the inhibitory mechanism that causes adaptation? Studies of Dictyostelium amoebae are designed to address these central questions, with the results assessed for generality in human neutrophils. An understanding of these chemotactic systems will enable the design of strategies to inhibit aberrant migration events and alter the course of disease. Three closely related areas of investigation are planned. First, tools to measure and control the temporal and spatial activation of Ras proteins, Tor complex 2, and protein kinases B (PKBs) in living cells will be constructed. With these, critical feedback loops that give rise to excitability will be defined by activating specific points in the network and monitoring responses at others. The mechanism of activation of TorC2 by RasC, will be explored by identifying regulatory subunits and determining changes in the structure of the complex upon activation. Corresponding studies will explore the role of the Ras-TorC2 paradigm in HL-60 neutrophils. Second, the functions of twelve signaling and cytoskeletal proteins that undergo rapid chemoattractant-induced, PKB-mediated phosphorylation will be assessed by genetic suppression. A complex of one PKB substrate, Ras exchange factor GEFs, and principal investigator 3-kinase will be purified and evaluated for a possible involvement in a feedback loop. Other studies will examine the regulation of PakA by PKBs and Rac proteins and determine how these components alter cytoskeletal activity by identifying relevant substrates of PakA. In addition, the role of a novel PH- and WD40-domain protein, not a PKB substrate, in the formation of the "back" of polarized cells will be investigated using gene disruption and mislocalization of the protein to ectopic regions of the cell. Third, the links between G-proteins and the downstream signaling network will be determined and the inhibitory process, essential for directional sensing, that acts at the level of this connection will be identified. To accomplish this, proteins that directly bind to or genetically interact with the G12 will be identified and disrupted, and a cell-free system in which activated G12 drives activation of RasC and TorC2 will be created and explored.
Many diseases involve the migration of cells to distant sites in the body. For example, cancer becomes lethal when cells migrate, or metastasize, from the original tumor;in asthma, immune cells infiltrate into the lungs;and in multiple sclerosis glial cells accumulate in regions of the brain. If we can learn how cells know where to migrate, we may be able to control their behavior and modify the consequences of these and many other diseases.
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