Figure 1: HL60 cells migrating in confined environments possess an F-actin-rich leading edge.

In all panels, the front of the cell was taken as the origin of the x axis. (a) Microfluidic device for studying interstitial migration. The microfluidic device comprised four inlets (1–4) and one outlet. Cells were introduced into the device using inlet 2. Inlets 1 and 3 are used to circulate control medium (HBSS+BSA) and medium containing chemoattractant (fMLP), respectively. Both streams were brought into contact briefly (grey arrow) to equilibrate pressure before being separated for the creation of linear gradients (open arrow). Downstream, transversal channels with a 5 μm × 5 μm cross-section separated control stream from chemoattractant stream and interstitial cell migration was imaged in these (see b). (b-top) A linear gradient of chemoattractant was created between the control stream (grey, left) and the chemoattractant stream (green, right). This stimulated chemotaxis and migrating cells completely occluded the transversal channels (b-bottom, Supplementary Fig. S1B), a property that allowed selective treatment of the leading edge with drug (Supplementary Fig. S1C–E). (c) Orthogonal views of actin distribution in a live HL60 cell migrating in a microchannel visualized with GFP-actin. Scale bar=6 μm. (d) Perspective view of the cell in (c). The leading edge was surrounded by an interfacial region highly enriched in actin (red arrow). The nucleus and the uropod are indicated by the white and blue arrows, respectively. (e) Steady-state actin fluorescence intensity distribution in the leading edge I_SS(x,y) in a plane midway through the channel height. Two regions could be distinguished on the basis of their fluorescence intensity: an adherent F-actin network situated at the interface between the cell and the channel walls (black boxed area) and a ‘free’ F-actin network situated in the inner part of the leading edge (grey boxed area). The dotted white line delineates the cell contour. (f) Net rate of change in actin density τ in the leading edge for the cell shown in e. Warm colours indicate increases in actin density and cold colours indicate decreases. The dotted line indicates the cell contour. (g) Net change in actin density τ as a function of distance from the cell front for the cell in e. The profile was calculated by averaging data in the boxed region in f. The greyed area indicates regions close to the front membrane.