Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow.

Cells in vivo are constantly subjected to mechanical shear stresses that play important regulatory roles in various physiological and pathological processes. Cytoskeletal reorganizations that occur in response to shear flow have been studied extensively, but whether the cytoplasm of an adherent cell adapts its mechanical properties to respond to shear is largely unknown. Here we develop a new method where fluorescent nanoparticles are ballistically injected into the cells to probe, with high resolution, possible local viscoelastic changes in the cytoplasm of individual cells subjected to fluid flow. This new assay, ballistic intracellular nanorheology (BIN), reveals that shear flow induces a dramatic sustained 25-fold increase in cytoplasmic viscosity in serum-starved Swiss 3T3 fibroblasts. By contrast, cells stimulated with the actin contractile agonist LPA show highly transient stiffening of much lower amplitude, despite the formation of similar cytoskeletal structures. Shear-induced cytoplasmic stiffening is attenuated by inhibiting actomyosin interactions and is entirely eliminated by specific Rho-kinase (ROCK) inhibition. Together, these results show that biochemical and biophysical stimuli may elicit the formation of qualitatively similar cytoskeleton structures (i.e. stress fibers and focal adhesions), but induces quantitatively different micromechanical responses. Our results suggest that when an adherent cell is subjected to shear stresses, its first order of action is to prevent detachment from its substratum by greatly stiffening its cytoplasm through enhanced actin assembly and Rho-kinase mediated contractility.