SummaryIt is becoming clear that mechanical stimuli are crucial factors in regulating the biology of the cell, but the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells transiently expressing actin-EGFP with controlled forces (0-20 nN) in order to investigate the structural response of the cell. Two clear force-dependent responses were observed: a short-term (seconds) local deformation of actin stress fibres and a long-term (minutes) force-induced remodelling of stress fibres at cell edges, far from the point of contact. By photobleaching markers along stress fibres we were also able to quantify strain dynamics occurring along the fibres throughout the cell. The results reveal that the cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres in resting cells that indicate localized contraction and stretch dynamics. The application of mechanical force results in the activation of myosin contractile activity reflected in an ,50% increase in strain fluctuations. This approach has allowed us to directly observe the activation of myosin in response to mechanical force and the effects of cytoskeletal crosslinking on local deformation and strain dynamics. The results demonstrate that force application does not result in simplistic isotropic deformation of the cytoarchitecture, but rather a complex and localized response that is highly dependent on an intact microtubule network. Direct visualization of force-propagation and stress fibre strain dynamics have revealed several crucial phenomena that take place and ultimately govern the downstream response of a cell to a mechanical stimulus.
The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.
It is becoming evident that physical forces in the microenvironment play a key role in regulating many important aspects of cell biology. However, although mechanical cues are known to have clear effects over the long-term (days), the short-term (seconds to minutes) cellular responses to mechanical stimuli are less well characterized. In our recent study, we exposed committed fibroblast cells to well controlled nanoscale forces while simultaneously imaging force transduction through the actin cytoskeleton. One of the earliest responses of a cell to physical force is rapid deformation of the cytoskeleton, taking place over the course of seconds. We were able to directly visualize deformation, force-propagation and strain dynamics in actin stress fibers in response to a relatively simple mechanical stimulus. Moreover, these dynamics were also dependent on myosin-driven contractility and the presence of an intact microtubule cytoskeleton. Interestingly, although stem cells are sensitive to mechanical cues, they do not display the same degree of stress fiber organization as observed in committed cells indicating the possibility of alternative sensing and mechanotransduction mechanisms.
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