The long term goal of this grant is to understand and model the signal transduction processes that control human cell polarization, migration and chemotaxis. These fundamental cell functions play key roles in development, immune defense and wound healing. A better understanding of these processes will provide valuable information for identifying potential drug targets for immune diseases, cancer, injury and other conditions. We have already developed a dual-positive feedback loop working model for cell polarization and a local coupling model for chemotaxis and our experimental plan is focused on testing whether these models are correct or whether alternative models have to be considered. We will pursue our goals by using a microscopy-based strategy and we will make use of fluorescent biosensors and image analysis of lamellipodia extension, cell migration and chemotaxis using a leukocyte and an endothelial cell model. Our main strategy is to use perturbations that may interfere with cell polarization and chemotaxis and then monitor possible changes in the activity gradients of Rac, CDC42, and Rho as well as in the lipid second messengers PI(45)P2 and PI(345)P3. Our main approach to rapidly activate and inhibit the main proposed intermediate signaling steps is based on a heterodimerization method that we developed to rapidly activate or inhibit Rac, CDC42, Rho, and PI3K as well as of other intermediates in the proposed positive feedback loops. We will also make use of a set of over 2000 interference RNAs that we generated against signaling and actin regulatory proteins and which we already used to identify more than 150 putative signaling proteins that regulate cell migration. We will also be using these siRNAs for perturbing polarization and chemotaxis processes. Our studies on polarization will focus on a working model that a dual positive feedback loop is needed for robust cell polarization. Our chemotaxis studies will focus on the model that mammalian cells primarily sense chemoattractant with their front and steer towards chemoattractant by a stochastic turning process. We are testing the hypothesis that the sensing of chemoattractant is achieved by coupling local receptor stimuli to local extensions of lamellipods and small turns in the direction of migration. We will test the validity of these and other potential models by comparing the experimental data from the perturbation studies with the predictions of model calculations. ? ? ?
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