Fig. 5. Focal adhesion–independent collective migration occurs without persistent retrograde flows.

(A) Schematic representation of cell treadmilling (left) and translation of the whole cluster (right). (B) Representative tracks of nuclei in the cluster reference frame. Median section of a histone 2B (H2B)-RFP–expressing HT29-MTX cluster, migrating to the right in a PEG-coated microchannel over 11 hours. The image is the first time point. (C) Representative example of nuclei tracks in the lab reference frame. Median section of an H2B-RFP–expressing HT29-MTX cluster, migrating in a PEG-coated microchannel over 10 hours. Scale bar, 50 μm. (D) Superimposition of the maps of individual nuclei displacements for n = 22 clusters (from seven independent experiments). Nuclei are tracked in median sections of H2B-RFP–expressing HT29-MTX clusters migrating in PEG-coated microchannels (25 min to 11 hours). Blue boxes, lateral nuclei defined for further nuclei speeds analysis (15 μm thickness at the contact with the channel walls). (E) Frequency distribution of the x component of the mean velocity of every lateral nucleus. Same clusters as in (D). (F) Median section showing instantaneous myosin flow velocity vectors superimposed on the raw image and detected by particle image velocimetry (PIV) of a representative HT29-MTX cluster–expressing mTurquoise-MLC and migrating in a PEG-coated channel. Time point, T = 58 min. Blue boxes, contact zone defined for further myosin flows analysis (2 μm thickness at the contact with the channel walls). (G) PIV map of myosin flow velocity vectors of median sections of clusters, averaged over time (25 min to 7.4 hours) and clusters (n = 10 migrating clusters from four independent experiments). (H) Frequency of velocities (x component) of myosin flow velocity vectors at the contact obtained from PIV maps of median sections. Same clusters as in (G).