Understanding polarity and directional migration is a holy grail of cell biology. In a canonical polarized cell, the neutrophil, activation of a chemoattractant receptor activates """"""""frontness"""""""" and """"""""backness"""""""" via different sets of trimeric G proteins, Gi and G12/13. Signaling 3'- phosphoinositide lipids (PI3Ps), Rho GTPases, and specialized actin assemblies define and spatially separate the two opposing responses. Polarity signals are distinct from but closely related to the poorly understood """"""""compass"""""""" that senses the direction of an attractant gradient. Biochemical assays, mutant proteins, fluorescent markers, and RNAi will be applied in an experimental neutrophil model, the differentiated HL-60 cell to tackle three related questions: 1. How do signals and actin assemblies organize polarity? This laboratory has identified proteins that control frontness (including 3'-phosophoinositide kinases, Rac) and backness pathways (including Rho, myosin II). Proteins that suffice to trigger and/or maintain polarity will be identified by observing translocation of these candidate proteins, appropriately tagged with fluorescent or epitope markers, to the plasma membrane and by rapamycin-induced translocation (symmetric and asymmetric) of the same proteins. 2. What molecules compose the neutrophil's compass, and how does it direct orientation of the cell's polarity? The compass will be investigated in latrunculin-treated cells, which cannot polarize. The hypotheses are that the compass is driven by accumulation of PI3Ps, controlled by a Pixalpha- and Cdc42-dependent, F-actin-independent feedback loop, and that local concentrations of these lipids constitute the compass's output signal. 3. Do cells interpret shallow gradients by mechanisms that compare attractant concentrations in space or in time? Experiments in a recently developed microfluidic device will characterize orientation and migration of dilL-60 cells in rapidly imposed stable gradients, and will test the notion that desensitization plays a role in temporal interpretation of gradients. ? ?
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