Dynamics of Cellular Rigidity Sensing on the Micron and Sub-micron Scale

This thesis describes a study of the effect of environmental cues including physical attribute of the cellular environment on cellular force and force transduction. Different mechanical parameters such as geometry and rigidity of the substrate are controlled independently and forces exerted by cells were measured. The experimental system for this study is based on fabrication of micron and submicron pillar substrates and their surface functionalization and finally measurement of forces that cells exert to these substrates. In chapter 2, the interplay between the rigidity of the substrate and the cell's force response was studied. Arrays of flexible PDMS pillars used to measure the pattern of traction force generation on matrices. Using three different pillar diameters (2, 1 and 0.5 micrometers), and three different pillar stiffnesses for each diameter, we showed that cells treat larger, fibronectin-coated pillars fundamentally differently than sub-micron pillars during initial contact formation. In the case of larger pillars, mouse embryo fibroblasts generated a constant force per unit area of about 1 nN/m2 on pillars of different stiffness by causing different displacements; whereas, the sub-micrometer pillars were displaced by about 60 nm irrespective of stiffness. In addition, micron-scale pillars are all pulled toward the center of the cell, whereas sub-micron pillars were also pulled toward each other locally. Further, the focal adhesion protein, paxillin, was concentrated at the edges of large pillars but it was focused on the tops of small pillars in a pattern analogous to the pattern on continuous substrates. Thus, we suggested that initial rigidity sensing involves measuring the force needed to produce displacements of about 60 nm in local regions (1m) of the substrate. In addition, these results suggested that, to examine the effects of substrate rigidity on cellular behavior, sub-micron pillars more closely approximate continuous substrates than do micron-scale pillars. In chapter 3, a technique was described for fabricating substrates whose rigidity can be controlled locally without altering the contact area for cell spreading. The substrates consist of elastomeric pillar arrays in which the top surface is uniform but the pillar height is changed across a sharp step. Results demonstrated the effects on cell migration and morphology at the step boundary. In chapter 4, a technique was described for the fabrication of arrays of elastomeric pillars whose top surfaces are treated with selective chemical functionalization to promote cellular adhesion in cellular force transduction experiments. The technique involves the creation of a rigid mold consisting of arrays of circular holes into which a thin layer of Au is deposited, while the top surface of the mold and the sidewalls of the holes are protected by a sacrificial layer of Cr. When an elastomer is formed in the mold, Au adheres to the tops of the molded pillars. This can then be selectively functionalized with a protein that induces cell adhesion, while the rest of the surface is treated with a repellent substance. An additional benefit is that the tops of the pillars can be fluorescently labeled for improved accuracy in force transduction measurements. The same fabrication process was used for fabrication of magnetically actuated pillars in order to be able to exert external force to cells and study the eect of localized mechanostimulation

Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied by local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micrometre elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ∼2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing

Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied via local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ~2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing

Experimental Study on Seismic Response of Underground Tunnel–Soil–Piled Structure Interaction Using Shaking Table in Loose Sand

The seismic response of structures can have a significant impact on adjacent structures’ response. Although several numerical studies have been applied in the field of tunnel–soil–pile interaction systems, there is a lack of experimental research specifically focused on the effects of this interaction on tunnel cross-section deformation and the existence of structure on encircling soil response. In this study, shaking table tests were conducted to examine the seismic response of a tunnel and the surrounding soil when an eight-story structure with piles was located in the vicinity of the tunnel. Four series of physical models were analyzed, including free-field soil (S), tunnel–soil (TS), soil-piled structure (SP), and tunnel–soil-piled structure (TSP), under sinusoidal vibration at three frequencies on loose sand. According to the results, the tunnel significantly impacts the surrounding soil response during seismic excitation with reduced acceleration at the tunnel invert and increased acceleration at the tunnel crown. In the TSP model, applied frequency affects the recorded acceleration amplitude at the tunnel invert. Although acceleration amplitude decreases at 3 Hz frequency excitation compared to the free field model, 8 Hz excitation resulted in bigger values in tunnel invert. Displacements are higher at the tunnel crown, indicating tunnel-induced soil deformation and maximum shear strain concentrated near the tunnel crown. The tunnel cross-section exhibited oval shape changes, with higher forces on the tunnel crown in the presence of piles

An investigation on the strain accumulation of the lightly EICP-cemented sands under cyclic traffic loads

Industrial production of chemical cement leads to extreme emissions of greenhouse gases. Biological or bio-inspired sustainable materials for soil treatment projects can be employed instead of chemical cement to heal the carbon cycle in the ecosystem. The enzyme-induced calcite precipitation (EICP) method is one of the novel bio-inspired technologies that can be employed in soil treatment projects to increase desired properties of soils. While the monotonic and cyclic behavior of the enzymatically treated sands has been investigated comprehensively, the strain accumulation pattern in these improved soils under cyclic traffic loads has not been evaluated yet. In this paper, confined and unconfined cyclic compression tests are applied to the enzymatically lightly cemented sands, and the effects of the different parameters on their strain accumulation pattern are investigated for the first time in the literature. This study uses two types of specimens with unconfined compression strengths (UCS) equal to 42 ​kPa and 266 ​kPa. It is shown that the treated specimens have a rate-dependent behavior where cyclic loads with low frequencies lead to more resilient and plastic strains in the specimens. The results show that by approaching the maximum applied stresses to the UCS of the specimens (by breaking more calcite bonds between sand particles), the rate dependency behavior of specimens will reduce. Investigation of the effects of the cementation level demonstrated that by increasing the amount of the precipitated calcite from 0.38% to 0.83%, accumulated plastic strains are reduced almost 95% under the same loading condition. Effects of the initial static loads, confining pressures, the number of cycles, and amplitudes of the cyclic loads are also evaluated

Decoding Information in Cell Shape

SummaryShape is an indicator of cell health. But how is the information in shape decoded? We hypothesize that decoding occurs by modulation of signaling through changes in plasma membrane curvature. Using analytical approaches and numerical simulations, we studied how elongation of cell shape affects plasma membrane signaling. Mathematical analyses reveal transient accumulation of activated receptors at regions of higher curvature with increasing cell eccentricity. This distribution of activated receptors is periodic, following the Mathieu function, and it arises from local imbalance between reaction and diffusion of soluble ligands and receptors in the plane of the membrane. Numerical simulations show that transient microdomains of activated receptors amplify signals to downstream protein kinases. For growth factor receptor pathways, increasing cell eccentricity elevates the levels of activated cytoplasmic Src and nuclear MAPK1,2. These predictions were experimentally validated by changing cellular eccentricity, showing that shape is a locus of retrievable information storage in cells