Experimental observations of cell migration and pseudopodia. (

*A*) Single-cell speed distribution, with inset showing speed vs. time for four single cells. (

*B*) Average single-cell speed distribution (blue; error bars indicate SD) is well fitted by a generalized extreme value (GEV) distribution characterized by the location parameter

*m*, the scale parameter

*s*, and the shape parameter ξ. (

*C*) Population distribution of single-cell maximum path distance (MPD). (

*D*) Chamber-average directional autocorrelation (blue circles) and fit (orange line). Also shown are single-cell autocorrelations from two sample cells moving nominally straight (lines). The SD of the distribution decays from ±0.20 min close to τ

_{lag} = 0 to ± = 0.01 at τ

_{lag} = 200 min (

*SI Appendix*, Fig. S5). (

*E*–

*H*) Effect of density on collective cellular migration; dashed lines in

*F*–

*H* indicate results for isolated cells extracted from ref. (compare with

*SI Appendix*, Fig. S4). (

*E*) Examples of trajectories (compare with

*SI Appendix*, Fig. S2) and (

*F*) the corresponding average directional autocorrelations that follow the same exponential decay (

*Inset*). (

*G*) Weight

*ϕ* and persistence time τ

_{p} from least squares fits of average directional autocorrelations to

as a function of the average minimum nucleus-nucleus distance

in the chamber showing that persistence time τ

_{p} is not affected by the changing density whereas the weight factor

*ϕ* decreases due to higher collision rate. (

*H*) Location

*m*, scale

*s*, and shape ξ from least squares fits of average single-cell speed distributions to the GEV distribution remains constant across densities. (

*I*) Time periods of contact for colliding cell pairs (combined for all densities) is heavily dominated by short times, and the distribution is independent of cell density (

*SI Appendix*, Fig. S8

*A*). Also shown are the actual trajectories of two colliding cells (blue and green), with red arrows indicating direction of motion. (

*J*) Pseudopod formation angle Δα with the current direction of motion (pooled across densities) shows a clear a clear preference of pseudopod formation in the current direction of motion, although pseudopodia are observed to form at all angles. This distribution is independent of cell density (

*SI Appendix*, Fig. S8

*C*). (

*K*) Position and angle of pseudopod formation Δθ in relation to the nearest neighbor cell. At time

*t* = 0, the entire volume of the microfluidic chamber is replaced with fresh media, effectively removing any chemokine background and allowing new chemokine gradients to be established (see schematic to the right). The cells overwhelmingly move to the nearest neighbor during the first 20 min after media replacement but only mildly so (and only when the nearest neighbor is very close) after 60 min, indicating that secreted chemokines induce pseudopod formation (

*SI Appendix*).

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