Date of Award
Doctor of Philosophy (PhD)
Daniel A. Hammer
Neutrophils are a type of white blood cell and first responders to tissue trauma and infection. This thesis explores the role of extracellular adhesivity in dictating neutrophil phenotype with respect to cell shape, motility, mechanical force generation, and the molecular constituents involved in these processes. The principle tool employed is microcontact printing, a powerful method to spatially organize a cell's adhesive environment. We demonstrate the capacity of neutrophils to sense adhesive density on stiff substrates and differentially respond to surfaces with low and high fibronectin content. On low and moderately adhesive surfaces neutrophils assume a highly spread, uropod-absent phenotype reminiscent of keratocytes. On highly adhesive surfaces neutrophils assume the classic amoeboid morphology with an elongated cell body, narrow lamellipodium, and knob-like trailing uropod. Our work reconciles conflicting observations of these two phenotypes previously attributed solely to the underlying stiffness of substrate. The spreading and motility quantified are haptokinetic, induced through the quiescent cell's interaction with immobilized adhesive ligand alone. Function blocking antibody studies implicated the promiscuous Mac-1 integrin receptor in supporting haptokinetic migration. We elucidate the density sensing length scale by presenting high and low adhesive cues to the cells simultaneously. Through rational design of the adhesive domains we conclude that neutrophils sense density at the whole cell length scale, integrating adhesive stimuli over their entire contact interface. Adhesion density sensitivity in stiff microenvironments has applicability to the study of cancer metastasis and particularly the epithelial-to-mesenchymal transition model. We also employ the microfabricated-Post-Array-Detectors (mPADs) traction platform to measure the forces associated with neutrophil spreading. We resolve with high spatial and temporal resolution a highly coordinated protrusive wave front of pN magnitude that propagates radially outwards from the cell center. Small molecule inhibitor studies establish that spreading was not analogous to lamellipodium formation but was sensitive to perturbations of actin cortical stiffness. Lastly, we apply the principles uncovered in neutrophils to the patterning of surface-active microfluidic vesicles by tuning vesicle-substrate adhesion and repulsion at the contact interface. The generation of ordered arrays of micron scale vesicles was a first of its kind.
Henry, Steven, "Biophysics of Human Neutrophil Haptokinesis" (2015). Publicly Accessible Penn Dissertations. 1061.