Rapid signal transduction in living cells is a unique feature of mechanotransduction.

It is widely postulated that mechanotransduction is initiated at the local force-membrane interface by inducing local conformational changes of proteins, similar to soluble ligand-induced signal transduction. However, all published reports are limited in time scale to address this fundamental issue. Using a FRET-based cytosolic Src reporter in a living cell, we quantified changes of Src activities as a local stress via activated integrins was applied. The stress induced rapid (<0.3 s) activation of Src at remote cytoplasmic sites, which depends on the cytoskeletal prestress. In contrast, there was no Src activation within 12 s of soluble epidermal growth factor (EGF) stimulation. A 1.8-Pa stress over a focal adhesion activated Src to the same extent as 0.4 ng/ml EGF at long times (minutes), and the energy levels for mechanical stimulation and chemical stimulation were comparable. The effect of both stress and EGF was less than additive. Nanometer-scale cytoskeletal deformation analyses revealed that the strong activation sites of Src by stress colocalized with large deformation sites of microtubules, suggesting that microtubules are essential structures for transmitting stresses to activate cytoplasmic proteins. These results demonstrate that rapid signal transduction via the prestressed cytoskeleton is a unique feature of mechanotransduction.

Is cell rheology governed by nonequilibrium-to-equilibrium transition of noncovalent bonds?

A living cell deforms or flows in response to mechanical stresses. A recent report shows that dynamic mechanics of living cells depends on the timescale of mechanical loading, in contrast to the prevailing view of some authors that cell rheology is timescale-free. Yet the molecular basis that governs this timescale-dependent behavior is elusive. Using molecular dynamics simulations of protein-protein noncovalent interactions, we show that multipower laws originate from a nonequilibrium-to-equilibrium transition: when the loading rate is faster than the transition rate, the power-law exponent alpha(1) is weak; when the loading rate is slower than the transition rate, the exponent alpha(2) is strong. The model predictions are confirmed in both embryonic stem cells and differentiated cells. Embryonic stem cells are less stiff, more fluidlike, and exhibit greater alpha(1) than their differentiated counterparts. By introducing a near-equilibrium frequency f(eq), we show that all data collapse into two power laws separated by f/f(eq), which is unity. These findings suggest that the timescale-dependent rheology in living cells originates from the nonequilibrium-to-equilibrium transition of the dynamic response of distinct, force-driven molecular processes.

Plectin contributes to mechanical properties of living cells.

Plectin is a 500-kDa cross-linking protein that plays important roles in a number of cell functions including migration and wound healing. We set out to characterize the role of plectin in mechanical properties of living cells. Plectin(-/-) cells were less stiff than plectin(+/+) cells, but the slopes of the two power laws in response to loading frequencies (0.002-1,000 Hz) were similar. Plectin(-/-) cells lost the capacity to propagate mechanical stresses to long distances in the cytoplasm; traction forces in plectin(-/-) cells were only half of those in plectin(+/+) cells, suggesting that plectin deficiency compromised prestress generation, which, in turn, resulted in the inhibition of long distance stress propagation. Both plectin(+/+) and plectin(-/-) cells exhibited nonlinear stress-strain relationships. However, plectin(+/+) cells, but not plectin(-/-) cells, further stiffened in response to lysophosphatidic acid (LPA). Dynamic fluorescence resonance energy transfer analysis revealed that RhoA GTPase proteins were activated in plectin(+/+) cells but not in plectin(-/-) cells after treatment with LPA. Expression in plectin(-/-) cells of constitutively active RhoA (RhoA-V14) but not a dominant negative mutant of RhoA (RhoA-N19) or an empty vector restored the long distance force propagation behavior, suggesting that plectin is important in normal functions of RhoA. Our findings underscore the importance of plectin for mechanical properties, stress propagation, and prestress of living cells, thereby influencing their biological functions.