Figure 7. Tether formation during neutrophil rolling on P-selectin. (a) A single 14.6- mm tether was pulled over 4 s and recoiled fully back to the cell body (gw 5 150 s21, P-selec- tin site density of z10 sites/ mm2). The instantaneous tether length (b) and cell cen- troid position (c) for three tethering events. The tether- ing event in a is shown as tether 3 in b and c. Distinct pausing in neutrophil rolling was observed in concert with the growth of membrane teth- ers, whereas the breaking of the tether (dashed line in b and c) caused a sudden move- ment forward. on P-selectin–coated surfaces (data not shown). Clearly, P-selectin/PGSL-1 bonds were being formed and broken at the cell-surface contact area to cause pausing, as well as at the end of the tether to cause leaps forward when the tether was released. The force loading is difficult to model because of an uncertainty in contact area loading and bonding in the presence of a tether, which could also be multiply bonded to the surface (Fig. 8). The aver- age tether length observed was 8.9 6 8.8 mm (n 5 31), av- erage tether duration was 3.79 6 3.32 s (n 5 29), and the average tether growth rate was 1.7 6 0.9 mm/s (n 5 29). In contrast to tethering with platelets, where the neutrophil was translating z0.3 mm above the surface, the neutrophil is attached to the surface by P-selectin bond(s) in the con- tact area as well as by bond(s) of the tether with the sur- face. This accounts for the long tether lifetime and slow tether growth rate observed with P-selectin–coated sur- faces. Multiple Point Attachments of Tethers on P-selectin–coated Surfaces Frame-by-frame analysis revealed several unique features of the interaction between these membrane tethers and the P-selectin–coated surface never before directly visual- ized. Tethers were not always straight, but at times were kinked with sharp angles, which is indicative of multiple bonding events with the surface along the length of the tether. As the tether grew, these kinks were straightened as the linkages with the surface along the length of the tether were stressed and broken (Fig. 8 a). Also, tethers did not always recoil completely back to the neutrophil body when the attachment at the tip of the tether was bro- ken. Fig. 8 b shows a tether growth sequence illustrating a tether with two kinks at points 2 and 3 (Fig. 8 b, t 5 0 s) that loses its attachment at the original tethering site at point 1. Instead of the tether immediately snapping back its full-length to the cell body, it recoiled only to the posi- tion of nearest attachment at point 2 (t 5 0.033 s). The crooked tether continued to grow as the neutrophil rolled for the next second (t 5 0.033–1.033 s) at which time the bonding at point 3 was stressed and the attachment re- leased at point 3 while remaining tethered at point 2 (t 5 1.067 s). The angle of the tether from the surface to the neutrophil body can be appreciated in the last panel of Fig. 8 b at t 5 1.067 s, where only the lower half of the tether is in the DIC focal plane. Adjustment of the focal plane al- lowed visualization of the upper part of the tether. The long duration of the neutrophil tethers on P-selectin– coated surfaces over many seconds is accounted for by the Schmidtke and Diamond Neutrophil Tethering under Flow 725