Fig. 2

Analysis of protrusion orientation during single mesendoderm cell migration. a Cell migration and protrusion formation analysis procedure, from single mesendoderm cell transplantation to automatic protrusion analyzer (APA). b Left: Control cells displaying blebs (black arrowheads) and actin-rich protrusions (white arrowheads). Right: Corresponding cell outlines after APA processing, where the different protrusion types and the centers of mass (CoM) of cells and protrusions have been labeled. Scale bar = 10 μm. c Exemplary cell trajectory displaying unit vectors pointing from the cell CoM to the blebs CoM. Blebs are classified as forming towards the front if they form in the local direction of cell displacement. d Time lapse of a control mesendoderm cell transplanted in an MZoep host displaying run and tumbles during migration. White line: trajectory of the CoM of the cell; white arrowheads: actin-rich protrusion; black arrowheads: blebs. Scale bar = 10 μm. Time in min:sec. e Frequency ratio of the formation of blebs and actin-rich protrusions during tumble versus run phases. The data points colored in blue correspond to cells where the reorientation events are associated with the formation of a new actin-rich protrusion at the leading edge. Note that the bleb frequency also includes the false negatives not detected by APA (Additional file 4: Figure S2). f Orientation of actin-rich protrusion and bleb formation in run and tumble phases. Arbitrary units (AU) are used for actin-rich protrusions as they are weighted with the total intensity of the Lifeact signal. The arrows below the diagrams indicate the local direction of cell migration. The overall orientation of each protrusion type was quantified using the polar order parameter (POP, see Additional file 1: Supplementary Methods for details). Mean ± SEM. In b and d cells express Lifeact-GFP (green) and Dextran-Alexa 594 (red). Number of cells in (e, f) = 11. Number of blebs in (f) = 349. Statistical significance by one-sided t-test (e) or by non-overlapping SEM of the POP (f) (Additional file 7: Figure S3D)