Chemotactic cell trapping in controlled alternating gradient fields

Abstract

Directed cell migration toward spatio-temporally varying chemotactic 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 mechanisms is difficult and requires careful spatio-temporal control of external chemotactic stimuli. We designed a microfluidic setup to generate alternating chemotactic gradient fields for simultaneous 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 respond to gradient switching rates below 0.02 Hz by readapting their migration direction. For faster switching, cellular repolarization ceases and is completely stalled at 0.1 Hz. In this "chemotactically 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 LimEΔcc. 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.eukaryotic chemotaxis | pseudopod-based motility | gradient sensing | flow chamber

Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

“…In the absence of stimuli, however, the typical time interval between successive pseudopods is between 10 and 20 s on average (38). Together with our findings, this may indicate that the cytoskeletal machinery selectively amplifies internal signals that lead to the formation of pseudopods with a characteristic period of 10-20 s (to establish directional responses, more time is needed as could be shown earlier by exposing chemotactic cells to gradient fields of changing direction) (39).…”

Theoretical model for cell migration with gradient sensing and shape deformation

“…By relating our results to the experimental result mentioned above, the case of Δ D 1 (s) > 0 seems more probable, implying that, in Dictyostelium cells, the limit of the gradient sensing ability may not be given by the MLE for both steepness and direction. Together with the above discussion on the interdependence of s and λ, this implication on the sensing ability seems to be consistent with the highly elongated shapes of Dictyostelium cells under a cAMP gradient [24]. A mechanism that biases the internal polarity to the cell shape orientation may also be possible, considering the experimental fact that the non-uniform distribution of some intracellular processes tends to orient to the leading edge of the cell.…”

Cellular memory in eukaryotic chemotaxis

“…During chemotaxis, cells polarize in the gradient direction and can reverse their polarity when the gradient is changed (22)(23)(24). In uniform chemoattractant, cells undergo a random walk with a persistence time of ∼3-10 min (25)(26)(27)(28).…”