%0 Generic
%A Wiegand, Tina
%D 2019
%F heidok:24163
%R 10.11588/heidok.00024163
%T Receptor-mediated Forces for Cell Sensing of Extracellular  Ligands and for Virus Particle Uptake
%U https://archiv.ub.uni-heidelberg.de/volltextserver/24163/
%X Mechanical forces between cells ensure organic development and  homeostasis, but they are also associated with diseases such as cancer  or viral infections. The absolute forces in nature can be as high as  1.5 kN, which allows the mantis shrimp to smash oysters, down to a  few pN transduced by single cellular receptors. However, these small  forces are strong enough for cells to probe their local environment.  While the mechanism, which enables cells to investigate the stiffness  of their surrounding, is already well elucidated, information on how  cells sense spatial distribution of ligands is missing.  In this thesis I established a method, which allows to measure cellular  traction forces on elastic substrates with varying nano-spacing  of extracellular ligands. In contrast to previous studies on stiff substrates,  adhesion complexes and tractions were larger for longer distances  between extracellular adhesion sites. This can be theoretically  explained by the force load on individual integrin receptors, which  has to exceed a certain threshold value to promote adhesion growth  through conformational changes in a protein of the “clutch complex”.  In order to experimentally access the force load per integrin heterodimer,  I combined molecular tension fluorescence microscopy  (MTFM) with traction force microscopy (TFM). For the first time, I  could assess a homogeneous distribution of forces > 19pN underneath  the adhesion area of cells on soft substrates. Simultaneously,  macroscopic tractions up to 2.7 kPa were observed at the cell edges.  Applying stronger tension probes and analyzing tractions in the zdirection  will help to cross-validate the results obtained from these  two state-of-the-art methods in biomechanics as a next step.  In the second part I investigated the mechanical parameters of  virus particle uptake by cells. Many intracellular pathogens, such as  mammalian reoviruses as employed in this thesis, mimic extracellular  motives to interact with host cells and initiate their internalization.  This leads to the assumption that host cells sense this specific ligand  presentation, engage the endocytic machinery and generate forces,  which are able to overcome the bending and tension energy of their  plasma membrane.  I demonstrated that these forces exerted on single reovirus particles  on the basolateral side of cells are strong enough to break down the  biotin-NeutrAvidin bond used for virus immobilization on stiff and  soft substrates. I quantified the forces to exceed 40pN by kinetic analysis  of the tearing of viruses from these surfaces and single MTFM  with covalently immobilized reoviruses.  The herein presented methods are powerful tools to study forces exerted  by individual receptors as well as on single particles e.g. during  endocytosis. The involvement of the actin cytoskeleton, specific receptors  or molecules of the endocytic machinery was examined. Inhibition  of the ligand-receptor interactions between reoviruses and cells  did not significantly change the rate of virus uptake. Interestingly,  bare nanoparticles of comparable diameter lacking specific binding  sites were torn off at a similar rate and thus with the same forces  as viruses. Hence, specific receptors seem to be dispensable for virus  particle uptake.