Rape AD, Guo W-H, Wang Y-L

Rape AD, Guo W-H, Wang Y-L. of cells. Homeostasis, a critical biological process to stabilize whole-cell/tissue physiology against external perturbations, has been generally investigated at the cellular level and beyond6-7. Yet, it remains elusive how homeostasis, as a cell-driven biological property, arise from collective, dynamic subcellular events. Understanding homeostasis down to a subcellular level can provide unprecedented insights into the origin and regulation of cell homeostatic ALPP behaviors, dysregulation of which has been associated with RAD51 Inhibitor B02 pathophysiological conditions in developmental disorders, cardiovascular and inflammatory diseases, and malignancy8-9. Recently, mounting evidence has identified mechanical homeostasis as an important component of the overall cell homeostasis1-5, wherein the actin cytoskeleton (CSK) tension and integrin-mediated focal adhesion (FA) are two central regulators directly interacting with external biophysical stimuli to elicit downstream mechanotransductive signaling and cell homeostatic behaviors to maintain stable mechanobiological says (phenotypes)10-11 (Fig. 1a). Therefore, we selected CSK tension and FA as subcellular markers and regulators of mechanical homeostasis and analyzed how their quick, mechanosensitive dynamics at a subcellular level could collectively drive single-cell mechanical homeostasis as an emergent biological phenomenon in response to external biophysical stimulation. Open in a separate window Physique 1 Dynamics of subcellular cytoskeleton (CSK) tension and focal adhesion (FA) during single-cell mechanical homeostasis. (a) Conceptual schematic of single-cell mechanical homeostasis. Upon mechanical perturbation, a biphasic cellular response comprising an excitation phase (= 0 min, ground state) and after (= 1 min, excited state; = 30 min, homeostatic state) the onset of 8% static equibiaxial stretch. = 0 min. Data represents the mean s.e.m with = 10. = 0 min) and after (= 1 min and 30 min) cell stretch. 0.05. *, 0.05. (f) Paired subcellular CSK tension – FA size data showing correlation during mechanical homeostasis. Data RAD51 Inhibitor B02 points represent individual FAs detected at = 0 min. More than 2,500 FAs were analyzed from = 10 REF-52 fibroblasts. Data styles are plotted as moving averages (solid lines) s.e.m. Mean results obtained at = 0, 1, 10, 30 min are plotted as indicated. (g&h) Temporal evolutions of CSK tension (g) and FA size (h) for four representative single FAs (marked by color-coded arrowheads in c) during single-cell mechanical homeostasis. (i) Temporal trajectories of paired CSK tension – FA size data for the same four single FAs in g&h during single-cell homeostasis. To visualize FA dynamics and quantify CSK tension, rat embryo fibroblasts REF-52 stably expressing yellow fluorescent protein (YFP)-paxillin fusion proteins were assayed using a stretchable micropost array (SPA) cytometry to apply defined static equibiaxial cell stretches simulating external mechanical activation (Supplementary Fig. 1; Methods). Deflections of microposts underlying cells seeded around the SPA cytometry were continuously monitored using fluorescence microscopy for quantification of dynamic subcellular CSK tension (Fig. 1b,c and Supplementary Fig. 2a). Clustering of paxillin, a protein residing in FA and involved in FA assembly and disassembly12, on micropost tops was recorded simultaneously to examine subcellular FA dynamics4,13-15 (Fig. 1c, Supplementary Fig. 2b-g, and Supplementary Fig. 3). Together, with live-cell fluorescence microscopy, the SPA was capable of applying controlled equibiaxial cell stretches while simultaneously reporting dynamic responses of subcellular CSK tension and corresponding FA size (represented by paxillin fluorescence intensity) with a one-to-one spatial registration. We first examined whether individual REF-52 fibroblasts would exhibit mechanical homeostasis at a global cellular level. Before cell stretch, whole-cell summation of CSK tension and FA size of single REF-52 fibroblasts remained constant as mechanobiological ground (quasi-static) states, and they restored their respective ground-state values at the single-cell level within 30 min after the onset of 8% static equibiaxial stretch (Fig. 1d,e and Supplementary Fig. 4). Such RAD51 Inhibitor B02 homeostatic behaviors of REF-52 fibroblasts at the single-cell level for both CSK tension and FA were characterized by biphasic dynamics comprising.