Figure 5. Membrane tension increases during protrusion.

A) Schematic outline of membrane tension measurement experiment. The tension in the plasma membrane can be measured by pulling a thin tube of membrane from the cell surface with an adhesive polystyrene bead in an optical trap. Increases in membrane tension result in higher pulling forces on the bead. We hypothesized that cell spreading, induced by uniform fMLP addition, should cause the membrane tension to increase. As a control, we flow in buffer, which does not induce spreading and should not increase membrane tension.

B) Pulling force over time for a representative cell. For primary human neutrophils, the tube was first pulled to a length of ~2 microns (pull 1, arrow, light green bar) and held there briefly (hold 1, light blue bar). The tube was then extended to a length of ~10 microns (pull 2, arrow, dark green bar) and held there (hold 2, dark blue bar) before fMLP (arrow) was flowed in. The colored bars denote the time period over which the forces were averaged for the graph in D; these regions were selected to avoid sudden force jumps. Addition of fMLP caused the cell to spread and the pulling force to increase dramatically (red bar). The inset graph shows the increase in spread area (green) and the increase in tether force (blue), both of which were normalized to the total area or force increase that occurred during the response. Brightfield images of the cell, with the outline superimposed in yellow, are shown below. The tether position, determined with a fluorescent membrane dye (DiI), is also superimposed in yellow.

C) Pulling force over time for individual cells following buffer addition or fMLP stimulation. The left panel shows the force traces of tubes held at constant length as buffer is flowed through the chamber to control for the effects of flow on the force measurements. The right panel shows the force traces of tubes held at constant length as the cells were stimulated by flowing fMLP through the chamber. In both panels, flow begins at the beginning of each trace.

D) Pulling force at different stages of the experiment. The graph shows the forces at different times during the experiment (denoted by the colored bars in B) for the eight fMLP-stimulated cells depicted in C. Each black dot represents the force measurement of an individual cell. The large and small maroon bars indicate mean force values and standard errors, respectively. After fMLP addition, the cell spreads and the force increases dramatically (p = 0.0006) and briefly plateaus (post-spread, red bar in B) before the tube detaches from the bead.