Contact-dependent cells, such as fibroblasts and endothelial cells, continually generate internal traction forces via integrin-mediated adhesion to extracellular matrix (Ridley et al., 2003
). In contrast, leukocytes in the vascular compartment must arrest on inflamed endothelium. This suddenly exposes them to high external forces from flowing blood, which requires leukocytes to resist this detaching force. We investigated the cellular and molecular responses of adherent leukocytes subjected to hydrodynamic forces. Our data reveal a mechanism that allows leukocytes to sense and resist these forces (). Exposure of blood leukocytes adherent via high affinity VLA-4 to an external force stretches microvilli and triggers localized accumulation of active Rap1 and PI3K, which regulate Rac-dependent actin polymerization. As a result, F-actin–enriched anchors form and provide the tensile strength necessary to reinforce and resist a detaching force.
Figure 10. Schematic illustration of leukocyte anchor formation and stabilization mechanism in response to hydrodynamic force. Leukocytes attached to VCAM-1 via high affinity VLA-4 stretch microvilli in response to fluid flow. Tension generated through integrins (more ...)
PSGL-1–mediated rolling results in a rapid elongation of microvilli and microridges and the formation of tethers (Schmidtke and Diamond, 2000
; Ley et al., 2007
). Tethers form as a result of separation of the lipid bilayer from the cortical cytoskeleton (Ramachandran et al., 2004
), and tether formation has been associated with lower rolling velocities. In part, this may be because each tether acts as a fulcrum that promotes cell rotation, which changes the moment of force, and decreases the strain experienced by PSGL-1–selectin bonds (Schmidtke and Diamond, 2000
). Recently, total internal reflectance fluorescence microscopy was used to visualize the formation of membrane tethers that extended as much as 16 µm upstream of rolling neutrophils and counteracted hydrodynamic forces (Sundd et al., 2010
). However, unlike anchors, tethers are transient structures consistent with the transient nature of PSGL-1–selectin bonds. In contrast, high affinity integrin bonds are more stable and maintain prolonged contact with activated endothelium. This leads to anchor formation. When we inhibited Rap1, PI3K, Rac, or actin polymerization, the cell body was displaced from the initial site of adhesion. Moreover, anchors were destabilized, and F-actin was extruded from the cortical cytoskeleton, yet integrin-mediated adhesive contacts persisted (, , , and ).
Rapid accumulation of F-actin and anchor formation was observed in real time when fluid flow was applied to adherent cells (). Anchors were formed upon stretching of an upstream microvillus or microridge. Anchors were maintained for as long as cells were exposed to a hydrodynamic force (). The length and height of anchors can act as lever arms dissipating forces transmitted by integrin bonds, as previously described for selectin-mediated rolling (Alon et al., 1995
). Anchor dimensions were proportionally similar in human monocytes and larger U937 cells, suggesting a potentially universal structural response in leukocytes to defined hydrodynamic forces ( and Fig. S3, b and c).
The contribution of tensile forces to outside-in integrin signaling initiated by ligand binding and integrin clustering is poorly understood. We did not observe significant changes in cell morphology or in the distribution of F-actin when leukocytes with high affinity integrins were adhered to VCAM-1–coated surfaces under static conditions ( and Videos 4 and 5). Only after the application of fluid flow did F-actin accumulation and the formation of anchors occur. VLA-4 located at the tips of anchors bound to VCAM-1 on the adhesion surface, yet we observed F-actin accumulation throughout anchors and at their insertion in to the cortical cytoskeleton. This suggests that tension transmitted throughout anchors initiates signaling ultimately leading to actin polymerization. Potential mechanosensory molecules include adaptor proteins, such as p130cas (Sawada et al., 2006
) stretch-activated receptors and/or channels (Matthews et al., 2006
Consistent with a previous study (Sawada et al., 2001
), we found that force induced active Rap1 accumulation at sites of anchor insertion, which was critical for adhesion stabilization (). The role of Rap1 in leukocyte arrest is controversial. Mn2+
-induced T lymphocyte adhesion to ICAM-1 or VCAM-1 has been reported to be both dependent (de Bruyn et al., 2002
) and independent (Ghandour et al., 2007
) of Rap1. In the context of chemokine-induced arrest mediated by VLA-4, Rap1 was shown to be dispensable (Ghandour et al., 2007
). The reasons for this discrepancy remain unclear, but it is possible that differences in expression or activation of Rap1 GEFs and GTPase-activating proteins are responsible.
Previous studies indicate that Rap1 signaling functions to activate PI3K (Fukuyama et al., 2006
; Kortholt et al., 2010
). It is possible that amplification loops might exist that regulate their activity. In Dictyostelium discoideum
, activated Rap1 binds and activates PI3K, which in turn is critical for Rac1 activation, cell adhesion, and subsequent pseudopod formation (Kortholt et al., 2010
). In E-cadherin–mediated cell–cell adhesion, PI3K was required for Rap1-dependent activation of Rac via Vav2, a Rac-GEF; in addition, overexpression of constitutively active Rap1 could not overcome the effects of PI3K inhibition, suggesting that PI3K is downstream of Rap1 (Fukuyama et al., 2006
). Whether PI3K regulates Rap1-GEFs is not currently well understood.
Previous studies have demonstrated the activation of PI3K in cells exposed to tensile forces (Suzuma et al., 2002
; Katsumi et al., 2005
). In our study, leukocyte structural adaptations in response to fluid flow are dependent on force-induced activation of PI3K and localization of its product, PIP3
, within anchors (). PI3K activation was also required for Rac-dependent F-actin accumulation in anchors (). This finding is consistent with the established role of PI3K as a regulator of actin polymerization (Vanhaesebroeck and Waterfield, 1999
; Pollard et al., 2000
). The reinforcement of tension-bearing structures by actin polymerization is critical for adaptation of cells to external forces.