Quantitative analyses of chemotactic motility
Over the last few years we have focused on the mechanisms underlying chemotactic motility in developed Dictyostelium cells. Chemotaxis, which is characterized by directed movement of cells up a chemical gradient, is a key component in a multitude of biological processes, including neuronal patterning, wound healing, embryogenesis, and angiogenesis. We are using an integrated approach to studying chemotaxis to cAMP in the model organism Dictyostelium discoideum where many of the key signaling components have been shown to function in manners similar to their homologs in mammalian cells. Using biochemical and advanced genetic techniques available in this system many of these components have been placed in physiological pathways. In some cases, multicomponent complexes and protein-protein interaction networks have been mapped and the pathways sufficiently worked out that we can write an almost complete flow diagram from receptor activation to a downstream cellular behavior. However, the mechanisms of directional sensing, signal amplification, establishment and stabilization of a leading edge, as well as the cellular basis for adhesion and motility are still poorly understood.
We are using microfluidic devices to quantitatively control the stimuli presented to cells. Live cell imaging with a spinning disk laser-confocal microscope gives temporal resolution of 100 ms and spatial resolution of 0.3 microns. Chemotactically relevant proteins such as the cAMP receptor, activated RAS, and F-actin can be localized within the cells be fusing them to fluorescent markers (GFP, RFP etc.). Computer assisted analyses of cell motility and subcellular localization of critical components are used to constrain theoretical models of chemotactic motility. In turn, the modeling refines the experiments to test crucial predictions.
We have found that developed Dictyostelium cells can respond to shallow gradients that generate only 1% difference in the concentration of cAMP across a 10 micron cell as long as the local concentration is above 10 nM (Song et al., 2006; Fuller et al., 2010). The signal is amplified by activation of RAS which is found almost exclusively at the tips of anterior pseudopods (Skoge et al., 2010). These results have been mathematically analyzed to gnerate a computer simulation of chemotactic cells (Hu et al. 2010; Hecht et al., 2010). We are now exploring the mechanism for amplification of the cAMP signal and its transduction into pseudopod extension and substratum adhesion.
Bill Loomis received his Ph.D. from the Massachusetts Institute of Technology. He received an NIH Senior Research Scientist Fellowship and was named an American Cancer Society Scholar. Professor Loomis is a Fellow of the American Association for the Advancement of Science.