Local application of stress gives rise to cell-scale intracellular flow.

A. Schematic diagram of the experiment. Collagen-coated fluorescent beads are bound to the cell surface. An AFM cantilever is brought into contact with one side of the cell and bead movement in the z-direction is imaged over time. B. Representative image showing a combined phase contrast and fluorescence image of the cell. Top panels: left: The AFM cantilever appears as a dark shadow on the left of the image. The bead is visualized by fluorescence. When the plane of focus is moved higher than the cell, a halo of fluorescence centered on the bead appears (right). The diameter of the halo of the bead reports on the distance between the bead and the plane of focus (see SI, Fig S1A). Variations in halo radius report on changes in height caused by indentation. The distance between the bead and the AFM tip is indicated by a red line. Middle panels: left: Profile of a cell before indentation. A cell-impermeable fluorescent indicator has been added to the medium and the cell appears dark. The AFM cantilever was imaged by reflectance and appears bright. Right: Representative image of the halo before indentation. Bottom panels: left: Profile of the same cell as in the middle panel during indentation by an AFM cantilever. Right: Halo of the same bead as in the middle panel during indentation. Scale bar= 10μm. C. Change in bead height as a function of time for a total of 12 beads on 7 cells. Inset shows normalized displacement of three beads at different distances from the AFM tip highlighting the slower response in the second phase for more distant beads. (inset) The color code in the inset is the same as the main figure. The part highlighted by a solid black line in each curve is used as a definition of poroelastic relaxation time scale (see SI methods, Fig S2). D. Characteristic relaxation time τ of the second phase as a scatter plot for control cells (7 cells) and cells treated with sucrose (6 cells). E. Comparison of relaxation times for control and sucrose treated cells. In the box plot, the red line is the median, the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points that are not outliers. Data points appear as black dots.

Intracellular fluid propagation in response to pressure gradients.

A. Schematic diagram of the experiment. Collagen-coated fluorescent beads (yellow) are bound to the cell surface of a cell in fluidic communication with a micropipette. At time t=0s, pressure is increased within the pipette leading to injection of fluid into the cell (dark blue). After a time lag δt, the fluid propagation front reaches a bead in δx resulting in an increase of its height by δz. B. Top: Representative image showing a combined DIC and fluorescence image of a cell. The micropipette appears at the top of the image and a fluidic connection is established. The distance from the pipette tip to each bead is indicated by red lines. Scale bar=10μm. Bottom: Defocused image of the fluorescent beads tethered to the cell surface before (left) and after (right) propagation of the fluid flow through the cell. C. Temporal evolution of height for beads situated at distances listed in Figure legend. The time at which microinjection starts is t=0 s (indicated by the arrow). The beads respond to injection with a time lag that increases with increasing bead-pipette distance. The dashed black line corresponds to a 0.1 displacement of the beads from their initial position. This threshold is used to calculate the time lag δt between injection and bead response (see SI methods). D. Bead displacement as a function of distance from the pipette after t=2s. E. Time lag δt as a function of distance from the pipette. F. Velocity as a function of estimated pressure gradient.

The cytoplasm of metaphase cells displays a porous behavior.

A. Schematic diagram of the experiment examining the change in diameter of metaphase HeLa cells due to application of a step increase in pressure through the micropipette. To detect the arrival of fluid flow at the cell periphery following application of a step pressure in the micropipette, we monitored changes in cell diameter. An increase in cell diameter was detectable after a time delay δt that depended on the amplitude of the pressure step. B. DIC images of representative experiments. The step change in pressure was applied at t=0s. Top: equatorial plane of cells blocked in metaphase at t=0s. The micropipette tip is out of the plane of focus. Scale bar=10um µ. Bottom: cells at t=2.6s after pressure application. The initial diameter is indicated by the red dashed circle. C. Temporal evolution of the relative diameter of metaphase HeLa cells subjected to different pressure steps. Error bars represent standard deviations. N=3 cells per pressure. The timing of pressure application is indicated by a vertical black line. The pressure corresponding to each curve is indicated on the graph. D. Time lag δt between pressure application and the onset of diameter increase as a function of the amplitude of the pressure step. The red dashed line indicates a hyperbolic fit. Whiskers indicate the standard deviation. N=3 cells per pressure. E. Velocity as a function of estimated pressure gradient. The dashed red line indicates a linear fit. Whiskers indicate the standard deviation. N=3 experiments per data point.

A poroelastic cytoplasm enables the emergence of steady state pressure gradients.

A. Schematic representation of quasi two dimensional slab of poroelastic material with a deformable upper surface. The material is parameterized by its elasticity E, its poroelastic and diffusion constant D. The bottom surface is uniformly tethered to an impermeable infinitely stiff material. The top surface is subjected to an indentation δ applied by a spherical indenter of radius R. The surface profile after deformation is indicated by the dashed black line. B. Schematic representation of a two-dimensional slab of poroelastic material (grey). The cytoplasmic material is parameterized by its elasticity E, its poroelastic diffusion constant D, and its pressure Pin. One part of the surface is permeable and fluid is injected into the cell through this region at a pressure Papp. The bottom surface is uniformly tethered to an impermeable infinitely stiff material. The surface profile after fluid injection is indicated by the dashed grey line. C. Experimental (grey) and predicted steady-state vertical displacement of beads tethered to the cell surface in response to localized indentation. D. Vertical displacement of beads in response to fluid microinjection after 2s as a function of distance from the micropipette. Grey data points indicate experiments and blue data points the simulation. The red line indicates the trendline. E. Time lags of the onset of vertical displacement as a function of distance from the micropipette. Grey data points indicate experiments and blue data points the simulation. F. Schematic diagram of depressurization experiment. A two-dimensional slab of poroelastic material (grey) surrounded by a less permeable thin layer (yellow) representing the cortex. The cytoplasmic material is parameterized by its elasticity E2, its poroelastic diffusion constant D2, and its pressure Pin. The outer layer is parameterized by E1, D1, and Pin. One part of the surface is permeable and fluid is released from the cell through this region to atmosphere pressure Patm. At t=0s, the cell is subjected to a suction Pin through the micropipette. G. Pressure distribution immediately after depressurization (top) and at steady state (bottom). H. Intracellular pressure as a function of distance from the micropipette for a range of internal pressures Pin. I. Intracellular pressure profiles normalised to the pressure at x=30u mµ from the micropipette. J. Intracellular pressure distribution as a function of distance from the micropipette for different membrane-cortex poroelastic diffusion constants D1.

Whole cell patch clamp and pressure release give rise to an outflow of fluid from the cell.

A. Differential interference contrast image of a typical experiment. The top cell is subjected to pressure release, while the bottom cell is not. Scale bar=5µm. B. Temporal evolution of CMFDA cytoplasmic fluorescence intensity in control cells and cells subjected to depressurization. The solid lines indicate the average and the whiskers the standard deviation. N=5 experiments were averaged for each condition. C. Fluorescence intensity of the cells in A prior to and after 575s of depressurization. Scale bar=5µm.

Cells can accommodate sustained intracellular pressure gradients.

A. Intracellular pressure in Filamin-deficient blebbing M2 melanoma cells and HeLa cells. B. Schematic diagram of the pressure release experiment. At time t=0s, a fluidic communication is established between a cell and a micropipette, resulting in a small suction pressure at the tip of the pipette. This leads to the establishment of a pressure gradient within the cell. As blebs are pressure driven protrusions, they are used as pressure gauges to report on the effect of pressure release. C. Computational prediction of the pressure profile in response to depressurization. The dashed line represents the pressure below which blebbing cannot occur based on the pressure measured in control M2 cells and cells treated with the Rho-kinase inhibitor Y27632. D. Representative pressure release experiments in M2 cells. Top row: DIC images of a representative experiment. Pressure is released at the pipette tip in a blebbing cell at t=0s and maintained constant thereafter. The location of the pipette tip is indicated by a white arrow. A second cell in the field of view serves as a negative control. Scale bar=10μm. Bottom row: Fluorescence images of the F-actin cytoskeleton in blebbing cells during a pressure release experiment. The location of the micropipette is indicated by the black arrow. Scale bar=10μm. E. Pressure release experiments in interphase (top) and metaphase (bottom) HeLa cells. Pressure is locally released in the cells at t=0s through a micropipette. The presence of intracellular pressure is detected by the emergence of blebs in response to partial depolymerization of the F-actin cytoskeleton by latrunculin treatment (t=80s and t=150s). Scale bar=10μm. F. Pressure release experiments in interphase (top) and metaphase (bottom) HeLa cells treated with the Rho-kinase inhibitor Y27632. G. Laser ablation of the cortex of metaphase HeLa cells expressing GFP-actin. The target region for laser ablation is indicated by the red circle in the before images and by a red arrow in the after images. Control cells, cells treated with the inhibitor of contractility Y27632, and cells in which a suction was applied through a pipette are shown.