Chemotactic cell trapping in controlled alternating gradient fields

摘要

Directed cell migration toward spatio-temporally varying chemo tactic stimuli requires rapid cytoskeletal reorganization. Numerous studies provide evidence that actin reorganization is controlled by intracellular redistribution of signaling molecules, such as the PI4,5P2/PI3,4,5P3 gradient. However, exploring underlying me chanisms is difficult and requires careful spatio-temporal control of external chemotactic stimuli. We designed a microfluidic setup to generate alternating chemotactic gradient fields for simulta neous multicell exposure, greatly facilitating statistical analysis. For a quantitative description of intracellular response dynamics, we apply alternating time sequences of spatially homogeneous concentration gradients across 300 μm, reorienting on timescales down to a few seconds. Dictyostelium discoideum amoebae re spond to gradient switching rates below 0.02 Hz by readapting their migration direction. For faster switching, cellular repolariza tion ceases and is completely stalled at 0.1 Hz. In this "chemotacti cally trapped" cell state, external stimuli alternate faster than intracellular feedback is capable to respond by onset of directed migration. To investigate intracellular actin cortex rearrangement during gradient switching, we correlate migratory cell response with actin repolymerization dynamics, quantified by a fluorescence distribution moment of the GFP fusion protein LimE△cc. We find two fundamentally different cell polarization types and we could reveal the role of PI3-Kinase for cellular repolarization. In the early aggregation phase, PI3-Kinase enhances the capability of D. discoideum cells to readjust their polarity in response to spatially alternating gradient fields, whereas in aggregation competent cells the effect of PI3-Kinase perturbation becomes less relevant.