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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02899

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

¹ Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA
² Department of Chemical Engineering, University of Florida, Gainsville, FL 32011, USA
³ Howard Hughes Medical Institute graduate training program, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA

View larger version (51K): [in a new window]   Fig. 1. Ballistic injection and tracking of nanoparticles embedded in the cytoplasm of adherent cells. (A) Schematic of the method of ballistic injection used to efficiently transfer nanoparticles to the cytoplasm of adherent cells. (1) Macrocarriers coated with 100 nm fluorescent nanoparticles are placed into a hepta adapter. (2) Pressurized helium gas (2200 psi) flows through the adapter, and (3) accelerate the macrocarriers toward a stopping screen. (4) Macrocarriers are stopped by the screen and (5) nanoparticles cross the plasma membrane and penetrate the cytoplasm of adherent cells. Typical mean squared displacements (MSD) of nanoparticles in unsheared (B) and sheared (C) serum-starved Swiss 3T3 fibroblasts. Ensemble average MSD is overlaid for unsheared (red) and sheared (blue) fibroblasts. The insets show typical trajectories of the centroids of nanoparticles imbedded in the cytoplasm of cells before (B) and after (C) the application of 40 minutes of shear flow (shear stress, 9.4 dyn/cm2). Bars in insets, 50 nm.

View larger version (25K): [in a new window]   Fig. 2. Micromechanical response of serum-starved cells to shear flow. (A) Phase-contrast (top) and immunofluorescence (bottom) micrographs of Swiss 3T3 fibroblasts before (left) and after (right) shear flow. Actin (green) and vinculin (red) structures were overlaid with a micrograph of injected nanoparticles, which were colored according to the local value of the cytoplasmic deformability (evaluated at time scale of 0.1 seconds). The nanoparticles were enlarged to aid visualization. Blue denotes the least deformable (stiffest) regions of the cytoplasm; red denotes the most deformable (softest) regions. The inset is a magnified view of focal adhesions at the ends of actin stress fibers. Bar, 20 µm. (B) Shear compliances (i.e. deformability) of the cytoplasm of Swiss 3T3 fibroblasts before (red) and after 40 minutes of shear flow (blue) (n=6). Values represent mean ± s.e.m. (C) Frequency-dependent viscous and elastic moduli of cytoplasm, G'() (circles) and G''() (squares). (D) Frequency-dependent phase angle of cytoplasm, ()=tan-1 (G''/G'), calculated from viscoelastic moduli shown in panel C. From a rheological standpoint, a phase angle of 90° describes the rheological behavior of a liquid; a phase angle of 0° describes the rheological behavior of a stiff solid.

View larger version (57K): [in a new window]   Fig. 3. Inhibition of actomyosin interaction or Rho-kinase diminishes the mechanical response of cells to shear flow. Cells were pretreated with BDM (20 mM) for 30 minutes to inhibit actomyosin interactions, and were either left unsheared (A) or sheared (B) for 40 minutes. Phase-contrast micrographs show minor changes in cell area and shape. Bar, 20 µm. Mean cellular compliances of unsheared fibroblasts (n=3) were similar after 40 minutes of BDM treatment. BDM treatment and shear flow caused fibroblasts to stiffen, but considerably less compared with control conditions, as shown by mean cellular compliances of sheared fibroblasts (n=4). Cells were also pretreated with specific Rho-kinase inhibitor Y-27632 (30 µM) for 30 minutes and were either left unsheared (C) or sheared (D) for 40 minutes. Phase-contrast micrographs show minor changes in cell area and shape. Bar, 20 µm. Mean cellular compliances of unsheared fibroblasts (n=3) were similar after 40 minutes of drug treatment. However, mean cellular compliances of sheared fibroblasts (n=4) show that cells did not stiffen in response to shear flow, and even became softer. All values represent mean ± s.e.m.

View larger version (45K): [in a new window]   Fig. 4. Contractility and stress fiber/focal adhesion formation are needed for micromechanical response of cells to shear flow. (A) Immunofluorescence of actin (green) and vinculin (red) of unsheared (top) and sheared (bottom) Swiss 3T3 fibroblasts. Control fibroblasts exhibit extended stress fibers and large focal adhesions (Inset, bottom). The formation of these cytoskeleton structures was abrogated after cell treatment with inhibitors BDM (middle) or Y-27632 (right). Bar, 20 µm. (B) Comparison of plateau elastic moduli. Only untreated cells exhibited a significant increase in elasticity when subjected to fluid shear stress. This stiffness increase is reduced with BDM treatment, whereas a slight decrease in elasticity is observed with Y-27632 treatment. (C) Relaxation times of the cytoskeleton for each condition, which were normalized by each respective initial (unsheared) value. (D) Intracellular shear viscosity values for each condition, which were normalized by the initial value of the shear viscosity of unsheared cells. For (B) and (D), one-way ANOVA tests yielded P<0.0001; stars denote P values from two-tailed t-tests within conditions (see Materials and Methods). All values represent mean ± s.e.m.

View larger version (20K): [in a new window]   Fig. 5. Proposed signaling pathway describing the mechanical response of cytoplasm in cells subjected to shear flow. (A) Color-coded (see gradient legend bar) cell for each condition indicates changes in intracellular viscoelasticity with respect to unsheared conditions. Upon application of mechanical shear flow, activation of Rho causes the downstream activation of ROCK (ROK/Rho-kinase), LIM kinases, and mDia. Such activation results in (1) increased F-actin formation; (2) actomyosin contractility; (3) focal adhesion formation, and ultimately, significant intracellular cytoplasmic stiffening of Swiss 3T3 fibroblasts. Drug treatment experiments are shown in green. (B) Comparison of chemical and mechanical stimulation on intracellular mechanics. Biomechanical stimulus (shear flow) causes a sustained dramatic stiffening response whereas a biochemical stimulus (LPA) causes a transient intermediate stiffening response. Such an intermediate response is recovered in mechanical stimulation when actomyosin interactions are inhibited. One-way ANOVA test of shear data and previous LPA treatment data (Kole et al., 2004a) yielded P<0.0001. Stars denote P values from two-tailed t-tests within conditions (see Materials and Methods). (C) Illustration of twitch phenomenon in muscle cells versus sustained tetanus that occurs when there is insufficient time for relaxation, which is analogous to the difference in intracellular mechanics measured in biochemical versus biomechanical-stimulated cells.

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