(gw z 1,600 s21), Chen and Springer observed neutrophil pull back of 1.4 mm upon instantaneous reduction of flow to 1 dyne/cm2. Given these limits at low and high flow, the observed average tether length of z6 mm in our study (Fig. 4 c) for shear rates between 100 and 250 s21 repre- sented a plateau to suggest that membrane tethering is most operative in this narrow range of shear rates. After neutrophil tether breakage, the flowing neutrophil traveled in a plane closer to the wall. The height above the wall and the shear force on the cell, Fs were estimated at each wall shear stress using the results of Goldman et al. (1967). By assuming a single bond at the end of the fully grown tether and using the experimentally determined tether lengths (,), we estimated the force on the bond to be Fb 5 86 pN at gw 5 100 s21 (average bond life of 630 6 380 ms, n 5 9) and Fb 5 172 pN at gw 5 250 s21 (average bond life of 133 6 51 ms, n 5 10). These bond strengths and lifetimes represent the first such estimates for the platelet P-selectin and neutrophil PSGL-1 bond pair, un- der conditions where the receptors reside in their respec- tive cellular membranes with a definitive single point at- tachment between the two cells, and a direct measurement of the length of the lever arm for the force analysis. Our observation that z32% of neutrophils interacting with platelets formed tethers agrees with Shao’s sugges- tion (Shao et al., 1998) that there may be heterogeneity in the association of the PSGL-1 ligand with the cytoskele- ton. Shao et al. (1998) observed two different kinds of be- havior, microvillus extension or tether formation, when a given force was applied to the microvilli of the neutrophil. The fact that the same force can result in two types of be- havior suggests that they depend on their membrane–cyto- skeleton association strength. Microvilli with a strong as- sociation between membrane and cytoskeleton can only be stretched a short distance (z1–2 mm), whereas mi- crovilli with a weak association can be pulled out to form tethers. The average force required to extract an integral glycoprotein from the lipid bilayer of a cell has been esti- mated to be 100 pN (Bell, 1978). However, Evans et al. (1991) measured a force of 10–20 pN for glycophorin A extraction from a red cell membrane, whereas Shao and Hochmuth (1999) measured the forces of 25–45 pN for L-selectin extraction, 60–130 pN for b2 integrin extraction, and 35–85 pN for CD45 extraction from neutrophils. How- ever, this was a slow process requiring 1–2 s for L-selectin, 1–4 s for b2 integrin, and 1–11 s for CD45 extraction. For tethering events that lasted under 1 s, we measured bond forces of .60 pN. This appears to agree with Shao (Shao et al., 1998; Shao and Hochmuth, 1999) and Alon et al. (1995) who have hypothesized that the cytoplasmic do- mains of selectins and their counterreceptors, in this case PSGL-1, help anchor these molecules to the cytoskeleton so that they can resist extraction. When neutrophils interacted with surfaces coated with P-selectin under laminar flow conditions, they rolled with a characteristic jerky motion while forming observable membrane tethers to the surface. Thus, neutrophil PSGL-1 bonding with P-selectin can facilitate the formation of membrane tethers under venous flow conditions. In con- trast to the tethers formed by flowing neutrophils with sin- gle platelets, membrane tethers formed by neutrophils rolling on P-selectin were of longer length and longer du- ration (Figs. 7 and 8). These longer lengths and durations were not due to b2-integrin bonding since treatment of neutrophils with anti-CD18 did not inhibit tether forma- tion. More likely they appear to be due to multiple binding interactions in the cell-surface contact area that would re- duce the stress on the tether as well as the close proximity of the tether to the glass, which allowed multiple bonding of the tether to the absorbed P-selectin (as seen in Figs. 7 and 8). During neutrophil tethering to P-selectin–coated sur- faces, it appeared that detached membrane tethers were occasionally deposited on the surface. These deposited tethers may contain PSGL-1 and L-selectin to facilitate neutrophil (or platelet) interactions. Similarly, membrane tethers, if sufficiently long, may serve as a template for the formation of linear strings of neutrophils, a multicellular structure previously described by others (Alon et al. 1996; Walcheck et al., 1996; Lim et al., 1998). This tether-depen- dent mechanism of string formation would be distinct from neutrophil collision–assisted deposition via L-selec- tin, which would be expected to produce branched struc- tures on the surface, not linear structures. Adhesion of free-flowing neutrophils to activated endo- thelium or platelets is a key step in inflammation and thrombotic processes. The dynamics of selectin bonding determines whether a flowing neutrophil will adhere or detach. We present data that over 32% of neutrophil– platelet interactions under flow result in the formation of elongated membrane tethers. Similarly, we also demon- strated that neutrophils form elongated tethers when roll- ing on P-selectin–coated surfaces or platelet monolayers. The formation of these tethers is physiologically signifi- cant in that they shield the bond from full loading by changing the length and angle of the lever arm. Tether for- mation reduces the force on the bond by a factor of z2 (Shao et al., 1998) with an expected reduction of koff by a factor of z2 for Fb , 150 pN (Smith et al., 1999). Given the reduction of stress on the bond due to the tethers and the consequent enhancement of the cellular interaction time, these tethered neutrophils are the ones with the greatest likelihood of transitioning to firm arrest in vivo. In addition, these tethers also may provide a mechanism by which neutrophils can translate between gaps between platelets and remain on or near the surface in close prox- imity to localized activating agents. By directly visualizing the formation of membrane tethers by neutrophils as they interact with platelets and P-selectin–coated surfaces, we have confirmed a widely postulated structure in cell–cell adhesion. The formation of these structures influences the dynamics of cell rolling as well as its biomechanical analy- sis and may have important implications for the rolling and adhesion of neutrophils in vivo. The authors thank Steve Rogers for his valuable help with P-selectin site density determination. This work was supported by a National Institutes of Health grant (HL56621). Dr. D.W. Schmidtke is a postdoctoral fellow of the Southeast- ern Pennsylvania American Heart Association (grant no. 9920392U) and Dr. S.L. Diamond is an Established Investigator of the National American Heart Association (grant no. 99-40027). Submitted: 26 June 1999 Revised: 7 March 2000 Accepted: 16 March 2000 The Journal of Cell Biology, Volume 149, 2000 728