Material Properties and Mechanosensing in Cells and Organs Under Extreme Forces

Abstract

Cellular and tissue biomechanics is a multidisciplinary field that focuses on understanding mechanical forces that govern the behaviors of biological systems on microscopic scales. This PhD thesis explores three distinct systems: bacterial peptidoglycan networks, the actin cytoskeleton of eukaryotic cells, and the multicellular mechanosensory chordotonal LCh5 organ of \textit{Drosophila} larvae. By integrating knowledge across these various scales, we developed a comprehensive picture of how organisms sense and react to mechanical stimuli, with potential implications in fields ranging from biotechnology to medicine.The first segment of this thesis focuses on bacterial cell wall mechanics. Bacterial cell walls contain high internal turgor pressures and also continuously expand while the bacteria grow. The load-bearing part of the bacterial cell wall, the peptidoglycan layer, is a covalently cross-linked polymer network made of rigid glycan strands cross-linked by flexible peptides. To understand the mechanical properties of PG networks, we developed coarse-grained simulations of a square patch of the \textit{E. coli} PG network, considering key molecular structural parameters including how glycans are arranged, the extent of peptide cross-linking, and the distribution of glycan lengths. Our model also mimicked isotropic pre-strain observed under non-zero turgor pressure by applying equal strains at all the edges of the patch. Our analysis established the stress-strain relationship in both the axial and circumferential directions, to be compared with experimental data, and identified the parameters determining stress ratios. We observed non-affine deformation, the formation of force chains, and stress stiffening. Additionally, our simulations match experimental observations regarding pore size distributions. Our simulations showed that a small degree of angular order in the glycan chains is sufficient to explain the anisotropic mechanical properties of PG. The second segment of this thesis is focused on the elastic characteristics of the chordotonal LCh5 organ of \textit{Drosophila} larvae. LCh5 acts as a stretch mechanoreceptor that senses muscle contractions and offers proprioceptive feedback during larval locomotion. One unique aspect of LCh5 is the exceptional stretchability of the cap cells, the molecular origin of which is not understood. Combining laser ablation with micropipette force spectroscopy, we directly investigated the mechanical behaviors of LCh5. We found that the extracellular matrix protein Prc is a primary elastic element storing a substantial resting tension in the larval LCh5 organs. Elastic recoil after laser ablation demonstrated that LCh5 organs are 2.04 pre-strained in their average configuration in third-instar larvae, which was reduced to 1.06 in the absence of Prc-deficient mutants. Using micropipette force spectroscopy, we could also quantify LCh5's pretension in vivo, of 1.25 $\mu$N, which was reduced to 0.30 $\mu$N in prc mutants. Measuring elastic response under cyclic strains indicated a softening effect in LCh5, with Prc null mutants exhibiting a more substantial Mullins effect than control larvae. Differential shear modulus measurements showed a slope of 1 for WTs but a slope of 0.5 for Prc null mutants, emphasizing Prc's importance in maintaining LCh5's elastic properties. The last segment of this thesis describes a novel FRET-based actin-binding-domain tension sensor (ABD-TS), designed to study force propagation within a cell’s actin cytoskeleton. ABD-TS are engineered with actin-binding domains of F-tractin on both ends to link the sensors to the actin cytoskeleton. When the ABD-TS is under tension, the two fluorophores are pulled apart and the FRET signal decreases. To be able to exert controlled external forces on cells expressing the ABD-TS, we used two micromechanical approaches: magnetic tweezers, and glass microneedles. Under a confocal microscope, we imaged cell deformations and quantified the FRET index while either exerting calibrated magnetic forces on bound magnetic beads or moving microneedles by a piezo actuator. Our preliminary results showed a FRET signal decrease under external mechanical micromanipulation after bleed-through corrections.