Physically-Induced Cytoskeleton Remodeling of Cells in Three-Dimensional Culture

Abstract

Characterizing how cells in three-dimensional (3D) environments or natural tissues respond to biophysical stimuli is a longstanding challenge in biology and tissue engineering. We demonstrate a strategy to monitor morphological and mechanical responses of contractile fibroblasts in a 3D environment. Cells responded to stretch through specific, cell-wide mechanisms involving staged retraction and reinforcement. Retraction responses occurred for all orientations of stress fibers and cellular protrusions relative to the stretch direction, while reinforcement responses, including extension of cellular processes and stress fiber formation, occurred predominantly in the stretch direction. A previously unreported role of F-actin clumps was observed, with clumps possibly acting as F-actin reservoirs for retraction and reinforcement responses during stretch. Responses were consistent with a model of cellular sensitivity to local physical cues. These findings suggest mechanisms for global actin cytoskeleton remodeling in non-muscle cells and provide insight into cellular responses important in pathologies such as fibrosis and hypertension.

Magnetically actuated cell-laden microscale hydrogels for probing strain-induced cell responses in three dimensions

“…This system enabled simple visualization of the hydrogel samples in real time through the objective of a fluorescence microscope and is the first high-throughput device capable of doing so. 37 The tensile tests of the μMACs were performed in situ with varied high strain levels, thus overcoming the challenges faced by existing 3D culture systems such as sample handling, diffusion barriers, specimen variability and limited strain levels induced by mechanical loading.…”

Cellular micro-environments reveal defective mechanosensing responses and elevated YAP signaling in LMNA-mutated muscle precursors

“…Biophysical attributes of cellular microenvironment might well contribute to these differences. Indeed, matrix dimensionality has a major impact on the structure and fate of cells (Cukierman et al, 2001;Fraley et al, 2010;Lee et al, 2012). For instance, the actin cap, which is anchored to the nuclear envelope through the LINC complex and becomes disrupted in LMNA cells (Lee et al, 2007), covers the top of the nucleus in 2D but not in 3D environments .…”

“…Because tissue architecture and stiffness of the cell microenvironment have a major impact on the spatial arrangement and dynamic regulation of the cytoskeleton and focal adhesion sites (Pelham and Wang, 1997;Abbott, 2003;Chiron et al, 2012;Lee et al, 2012), and thus on cell mechanosensing, myoblasts from four non-related patients exhibiting L-CMD (Quijano-Roy et al, 2008) and four controls (supplementary material Table S1) were examined in a three-dimensional (3D) soft microenvironment as well as on two-dimensional (2D) substrates of various stiffness. Our data reveal that LMNA-mutated cells (hereafter referred to as LMNA cells) present severe defects in their capacity to sense the stiffness of their environment and to respond to mechanical stress.…”

Tissue constructs: platforms for basic research and drug discovery

“…For the cellular component, the curves are more nearly linear with a relatively shallow slope, suggesting a response dominated by a small elastic response superimposed upon a constant contractile stress. Regions of negative curvature are evident in the regions of lowest and highest force, reflective of the straininduced disruption of the actin cytoskeleton that later analyses confirmed [104,105,143]. At the lowest strains, the force exerted by the ECM is very small, so that the hysteresis curve for the intact construct also shows negative curvature.…”

“…Y-27632, the Rhoassociated kinase inhibitor, can be used to specifically affect the rho/ROCK pathway [108], and ML-7 can inhibit rsfs.royalsocietypublishing.org Interface Focus 6: 20150095 myosin light chain kinase [109]. Calcium-dependent contraction can be modulated via thapsigargin, which releases internal stores of calcium, with BAPTA AM, which chelate intracellular calcium and with EGTA, which chelates calcium in the external medium [105,106,110,111].…”