A Rotor Driven by the Torque Stored in Braided DNA Molecules

Abstract

Neutrophil recruitment to the sites of inflammation involves selectin-mediated rolling followed by chemokine-induced activation and beta2 integrin-mediated arrest. PSGL-1, a ligand for endothelial P-selectin is presented on the tips of neutrophil microvilli. It has been predicted that P-selectin-PSGL-1 bonds form when the microvillus tip approaches the P-selectin expressing substrate to within 70 nm, but this prediction has not been tested experimentally. A PDMS based microfluidic device with a glass substrate coated with P-selectin/ICAM-1 was perfused with blood from an anesthetized mouse expressing green fluorescent protein (GFP) in neutrophils. Rolling interactions were studied at wall shear stress of 6-8 dynes/cm2 using TIRF microscopy which provides high resolution in z-direction. The contact zones of rolling neutrophils were revealed as footprints which were 3-6 mm in diameter, about twice as large as what would be expected for spherical cells. Following bond formation, microvilli in the footprint undergo compression, approaching the substrate to within 25 nm near the center of the cell. At the trailing edge, the P-selectin-PSGL-1 bonds stretch to a length of 125-150 nm before they dissociate. Adding the chemokine CXCL1 to the substrate induced neutrophil arrest and formation of single, long, branched tethers that stretch for up to 10 mm behind the arrested cells. The closest contact between the arrested neutrophil and the substrate is always found in front of the cell center and covers 1-3 mm2. Its distance from the substrate (44 nm) corresponds to the length of the ICAM-1-LFA-1 bond. These results identify the molecular and cellular dimensions of rolling neutrophils and provide a framework for the biomechanical analysis of this fundamental process.