Mechanics of tether extraction, surface protrusion, and cortical tension of human neutrophils and extension of single DNA molecules.

摘要

To reach sites of inflammation, neutrophils first roll on the endothelium before firm adhesion and trans-endothelial migration. Stable rolling requires not only molecular features of selectin-ligand bonds but mechanical properties of the cell as well, the most prominent being tether (membrane nanotube) extraction, surface protrusion and cortical tension. In previous studies, these mechanical properties were characterized with simplified in vitro micromanipulation experiments where conditions were markedly different from the in vivo situation. For example, most previous experiments were performed at room temperature and tethers were all extracted perpendicularly to the cell surface. To better understand how neutrophils behave in their native environment, in this dissertation, we studied tether extraction, surface protrusion and cortical tension at body temperature with the Micropipette Aspiration Technique (MAT) and a heated microscope chamber, and non-perpendicular tether extraction and two closely related problems---lateral motility of tether-cell junction on the cell surface and multiple tether coalescence---with the micro-cantilever technique and fluorescent microscopy. With fluorescent microscopy, we also studied tether retraction (after the extraction force suddenly drops to zero). Our results show that (1) temperature plays a significant role in regulating neutrophil mechanics, (2) neutrophil tether extraction is optimized for rolling stabilization, and (3) tether retraction is extraction-speed dependent and highly nonlinear. Besides contributing to knowledge of neutrophil mechanics, our results shed light on how neutrophils stabilize the rolling under physiological conditions and provided more realistic parameters for modeling the rolling process.;Molecular function, interaction, and deformation often involve sub-piconewton or femtonewton level forces. Yet, few techniques have the capability of imposing femtonewton level forces. A recently proposed technique, the Extended-MAT (EMAT), was shown by finite element analysis to have this capability, but it has never been validated experimentally. In this dissertation, we validated the femtonewton capability of the EMAT by using it to stretch single lambda DNA molecules. The validation established the MAT as the only technique with force application capability in three regimes: femtonewton, piconewton, and nanonewton, increasing its potential in studies of single molecule/cell biophysics.