Force transduction and strain dynamics in actin stress fibres in response to nanonewton forces

Abstract

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.

Investigating cell mechanics with atomic force microscopy

“…By altering tip geometry or chemistry, a multitude of both local and whole-cell studies can be performed on living cells in their native environments (figure 3). Studies of this nature have been shown to induce a rapid response of cells through shape change, remodelling of the cytoskeleton and calcium signalling, which all depend on frequency, duration, magnitude and location of applied force [9,29,37,102]. In the following sections, we discuss the role of AFM in measuring the mechanical properties of cells, particularly cell stiffness and viscous properties.…”

“…Details on these methods [106] as well as Young's moduli of a variety of cell types are listed in [35]. Apparent stiffness of mammalian cells, as measured with AFM, typically ranges between 1 and 100's of kPa [9,107,108]. Apparent cell stiffness is highly correlated with the stiffness of the actin cytoskeleton, the structure and mechanics of which are directly influenced by mechanical forces including tension/compression [109,110], hydrostatic and osmotic forces [31,99].…”

<i>In Situ</i> Observation and Measurement of Actin Stress Fiber Deformation in Stretched Osteoblast like Cell

“…Guolla et al also have reported the strain dynamics in actin stress fibers in response to applied external force [10]. They conducted simultaneous observation of atomic force microscopy (AFM) and fluorescent microscopy, and evaluated the precise strain dynamics in actin stress fibers.…”

Physical confinement signals regulate the organization of stem cells in three dimensions

“…D1306). A full protocol has been published previously [27]. Samples were then mounted using Vectashield (Vector Labs) and a no.…”