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

<div><p>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.</p> </div

Three types of cytoskeletal response were observed following stretch (A, with two examples shown).

<p>The first time point imaged in each case was approximately two minutes after stretch, allowing sufficient time for stretch-induced cytoskeletal depolymerization to occur. The normalized fibrosity metric measures changes in the total length of features in an image that are within the thickness range of stress fibers (“fibrosity”). These three classes of response were significantly different relative to an unstretched control (B). Each curve represents the average of at least three cells that were imaged at both 5% and 10% stretch; error bars are standard error.</p

Model predictions of whether or not sectors of stress fibers in a circular cell would depolymerize following ETC stretch.

<p>Symbols represent the degree of stretch in stress fibers in individual sectors that would exist after the application of stretch to the ETC if depolymerization of stress fibers did not occur. Depolymerization does occur when stretch in a sector lies outside of a prescribed range, and the red symbols represent sectors that were predicted to depolymerize. For very low variation of stress fiber pre-stretch (top panel), only sectors nearly parallel (0°) to or perpendicular (90°) to the direction of ETC stretch were predicted to depolymerize. For higher variance in pre-stretch values (bottom two panels), the direction-dependence of depolymerization became weaker.</p