Mechanical properties of cytoskeleton proteins studied in living cells by combining optical tweezers and deformability cytometry.

The topic of this thesis focuses on studying the mechanical properties of the living cell plasma membrane and the mechanical associations of the plasma membrane with the underlying cytoskeleton. The mechanical properties of the cell components, cell plasma membrane and cytoskeleton, as well as membrane-cytoskeleton associations, determine the mechanical properties of the whole cell, important for cellular shape changing behavior and mechanical signal transduction in living cells. Examples of biological processes involving cellular shape changes are deformation of erythrocytes in capillaries, cell division, phagocytosis, pseudopodium and dendritic spine formation, and electromotility of the outer hair cells. The study of cell membrane mechanics accomplished during my PhD activity is based on the use of two most advanced technological approaches to investigate both local membrane deformation by dual laser optical tweezers (DLOT, (Bianco et al., 2011)) and global cell deformation by real-time florescence deformability cytometry (RT-FDC). This dissertation is divided into two main parts. In the first part I present my contribution to the development and application of a system able to study the mechanical properties of plasma membrane of the Human Embryonic Kidney (HEK293) cells in the adherent state. The DLOT was first applied to investigate the dynamics of the formation/retraction of membrane nanotubes (tethers). In physiological conditions, several parameters of plasma membrane and its interaction with the underlying cytoskeleton (tether formation and elongation, tether radius, Threshold force for the membrane tether elongation and its viscoelastic nature, the tether diameter, the bending modulus, the membrane-cytoskeleton adhesion energy) were determined and compared with those present in literature. The information on cell membrane mechanics was integrated with indentation measurements by using the DLOT for applying local deformations in force feedback in the range experienced by the membrane of macrophages during phagocytosis.The mean value of the elastic modulus (Young’s modulus) derived from indentation experiments was 32 ± 8 Pa (mean ± SD), significantly lower than that obtained with AFM measurements. The difference confirms data found in previous work (Coceano et al., 2016) and indicates that my approach, though limited by its intrinsic compliance to lower time resolution, is unique in resolving the load – dependent dynamics of the cytoskeletal rearrangement triggered by a force step. In the second part I present experiments conducted during the one-year Ph-D period spent at the University of Greifswald, under the supervision of Dr. Oliver Otto. The experiments were aimed at characterizing the mechanical properties of the HEK293 cells in suspension and how they are influenced by hypoxic stress. Mechanical parameters from control experiments under physiological condition are compared to those obtained in the presence of an increased subpopulation of cells in apoptotic/necrotic state using a high-throughput system, Real-Time Deformability Cytometry (RT-DC (Otto et al., 2015)), which allows to study a high number of cells in short time (1000 cells/s). In combination with fluorescence-based flow cytometry (RT-FDC, (Rosendahl et al., 2018)), this technology permits to analyze treated fluorescent cells and discriminate between subpopulations. The mechanical parameters considered are cell size, elastic modulus and cell deformation. Hypoxia stressed cells were studied at different incubation times and the dependence from hypoxia of the mechanical parameters were determined. After 12h of oxygen deprivation (12h-hyp) cell stiffness is increased and cell deformation is reduced. An overall increase in concentration of the main cytoskeletal proteins (ß-actin, α/-tubulin, vinculin and talin-1), determined by Western blots, was found to accompany mechanical – structural modification by hypoxia. The successful application of the protocols developed here for the definition of local and global mechanical properties of the cell membrane and the associated underlying cytoskeleton of HEK293 line, opens the possibility of new investigations on the effects on the relevant cell mechanical parameters of different physical and chemical interventions (temperature, pH and buffer ionic composition), different physiological conditions (metabolic stress as hypoxia) modification of the membrane composition, as cholesterol content, or effect of disruption or mutation of membrane-linked cytoskeleton proteins. In this respect the methodology established in this thesis represents a new powerful tool for the investigation o membrane-cytoskeleton structure-function in health and disease.