against CD18 or with 30 mM sodium azide (NaN3) had no effect on tether formation (Fig. 5). Tether Formation on Platelet Monolayers To study whether observable membrane tethers were formed between neutrophils and monolayers of platelets, neutrophils were perfused over a subconfluent monolayer of immobilized platelets at a wall shear rate of (150 s21). When islands of platelets were present, neutrophils rolled across them. However, when there were gaps in the plate- let surface coverage, neutrophils often formed membrane tethers as they translated from one island to the next. Fig. 6 shows a neutrophil that has been rolling on platelets and subsequently forms a series of membrane tethers as it translates between the gaps between upstream and down- stream islands of platelets. Neutrophil Rolling on Purified P-selectin–coated Surfaces To demonstrate that the tether membrane originates from the neutrophil (not the platelet), and that P-selectin/ PSGL-1 interactions supports tether formation, we investi- gated whether membrane tethers were formed when neu- trophils rolled on P-selectin–coated surfaces. Tethering in this system was easier to visualize by DIC microscopy since the tethers formed in close proximity to the glass sur- face of the flow chamber. When neutrophils were perfused over P-selectin–coated surfaces (z10 sites/mm2, n 5 3) at a wall shear rate of 150 s21, they rolled over the surface and formed thin membrane tethers similar to those observed with platelets (Fig. 7 a). The neutrophil displayed a jerky motion of stops and starts as it rolled on the P-selectin sur- face concomitant with the growth of a membrane tether bonded to the surface. On purified P-selectin, distinct pauses in the neutrophil motion were detected (Fig. 7 c), the majority of which were ,1 s, which is consistent with the known lifetime of the P-selectin/PSGL-1 bond. In many recordings, the breaking of the tether occurred at the terminal site of attachment with the surface, and the tether rapidly snapped back to the neutrophil (Fig. 7 a, t 5 3.967 s). At the instant that the tether was released from the surface, the neutrophil experienced a sudden rotation forward that was driven by the shear forces (Fig. 7, b and c). To test whether b2-integrins were involved in the formation of these long-lived tethers, neutrophils were treated with mAb IB4 against CD18. Blockade of CD18 did not alter the ability of neutrophils to form tethers Figure 6. Neutrophil tether formation between islands of plate- lets. A neutrophil formed a series of membrane tethers as it translated between gaps in upstream and downstream islands of platelets. As the neutrophil translated from platelet island A to island B, a membrane tether was pulled (t 5 0–1.03 s). The neu- trophil remained relatively stationary on platelet island B while tethered at island A (t 5 1.03–1.73 s). When the tether broke, the neutrophil rolled over and paused on island B (t 5 1.73–4.53 s). The neutrophil formed a new tether as it translated away from is- land B to island C (t 5 4.53–4.83 s). The neutrophil paused on is- land C (t 5 4.83–7.73 s) before it released and translated down- stream to island D (t 5 7.73 to 9.1 s). The tether finally broke at t 5 10.13 s. The Journal of Cell Biology, Volume 149, 2000 724