Tether extraction from endothelial cells with the micropipette aspiration technique

Neutrophil rolling on the endothelium is a dynamic process regulated by rapid receptor-ligand kinetics and mechanical properties of the interacting cells. Tether (long membrane nanotube) extraction from the rolling leukocyte has been well characterized and believed to stabilize this rolling process. However, it remains to be investigated whether tethers can also be extracted from the endothelium and the potential effects on leukocyte rolling stability as a consequence.; In the present study, single and double membrane tethers are extracted from endothelial cells with the micropipette aspiration technique utilizing antibody-coated beads as force transducers. Moreover, simultaneous tethers (two tethers, one from the endothelial cell and the other from the leukocyte with the receptor-ligand bond in the middle) are extracted utilizing leukocytes as force transducers, and surface-attached endothelial cells as substrates. The existence of simultaneous tethers is confirmed by agreement between experimental and theoretical values for the constitutive parameters, observation by florescence microscopy and observation of the neutrophil contribution to the composite tether by cytochalasin-D treatment.; The constitutive relations determined above are utilized in a model of a rolling cell to predict the force drop and tether length as a function of time. Simultaneous tethers (compared to single tethers from the neutrophil) are found to cause a higher force drop on the receptor-ligand bond facilitated by a much higher contribution of the endothelial cell to the composite tether length relative to the neutrophil. This is the first study that hypothesizes and proves the extraction and mechanism of simultaneous tethers (owing to a ∼ 3-4 fold lower viscosity) in stabilizing the rolling process to a much higher extent than neutrophil tethers alone by decreasing the force and increasing the lifetime of the receptor-ligand bond in accordance with the Bell model.; Additionally, the effects of cholesterol depletion and enrichment on tether extraction from endothelial cells and neutrophils are quantified. The importance of cholesterol in regulating membrane fluidity and adhesion to the underlying cytoskeleton is elucidated. Overall, this dissertation substantially improves the current understanding of the regulation of leukocyte rolling by introducing the endothelial cell tether into the broader picture.