Coordinated protrusion–retraction cycles and unique linear adhesions are associated with 1D migration. (A–C) Coordination between leading edge protrusion (red), cell body movement (green), and tail retraction (blue) in directionally migrating cells on 2D (A), in 3D matrix (B), and along a 1D line (C). In A, protrusion is reduced (filled arrowhead) until the tail retracts. As retraction begins (open arrowhead), protrusion resumes. Both 3D matrix and 1D migration display efficient protrusion–retraction cycles. (D) Confocal images showing localization of FAK, phospho-FAK397, α5-integrin, and activated β1-integrin during 1D fibrillar migration and a 2D control. (E, top) Adhesions containing GFP-vinculin using TIRF (green) are shown. Kymograph analysis (middle) of GFP-vinculin adhesions during 1D migration. Red boxes (bottom) show leading and trailing edges. Red, yellow, and green lines indicate slopes of vinculin movement within adhesions at the leading edge, under the cell body, and at the tail, respectively. Dashed lines indicate the cell’s initial position. (F) Widefield image of vinculin (top) shows elongated leading lamella of rapidly migrating cells. TIRF reveals vinculin in a front to rear gradient spanning leading to trailing edge. (G) Line scan (dashed line in F) of the cell in F shows the gradient pattern (green) and close proximity of adhesions to the leading edge (LE). Arrows indicate the direction of migration. Bars, 10 µm.
