Cell Surface Deformation during an Action Potential

Abstract

The excitation of many cells and tissues is associated with cell mechanical changes. The evidence presented herein corroborates that single cells deform during an action potential. It is demonstrated that excitation of plant cells (Chara braunii internodes) is accompanied by out-of-plane displacements of the cell surface in the micrometer range (∼1-10 μm). The onset of cellular deformation coincides with the depolarization phase of the action potential. The mechanical pulse: 1) propagates with the same velocity as the electrical pulse (within experimental accuracy, ∼10 mm s^-1), 2) is reversible, 3) in most cases is of biphasic nature (109 out of 152 experiments), and 4) is presumably independent of actin-myosin-motility. The existence of transient mechanical changes in the cell cortex is confirmed by micropipette aspiration experiments. A theoretical analysis demonstrates that this observation can be explained by a reversible change in the mechanical properties of the cell surface (transmembrane pressure, surface tension, and bending rigidity). Taken together, these findings contribute to the ongoing debate about the physical nature of cellular excitability.

Figure 1

Geometry of the aspiration model (not to scale; in the calculations R/R_p ∼ 10²). To see this figure in color, go online.

Figure 2

Cell surface deflection during an AP. (a) In Chara, the cytoplasm (cp) is marginalized by the tonoplast (to)-covered vacuole (vac). The cellular cortex consists of the cell wall (cw), cell membrane (cm), cortical cytoskeleton (cc), chloroplasts (chlo), and subcortical actin bundles (ab) (see (42)). (b) When turgor was reduced by increasing the extracellular osmolarity, cm separated from cw. Deflections (dashed arrow) of the projection edge of the protoplast surface (prot) were tracked by light microscopy. (c) Upon excitation of an AP, the cell surface underwent a biphasic, reversible deflection (stimulus indicated by arrow; top trace, membrane potential; bottom trace, kymograph of surface deflection). (d) Given here is the membrane potential pulse (black) and out-of-plane displacement of the cell surface (red); note: an initial inward movement is followed by expansion. To see this figure in color, go online.

Figure 3

Effect of cytochalasin D on surface deflection during an AP. (Top) Shown here is the displacement of the cell surface upon excitation of an AP in artificial pond water (APW) and (bottom) in a different cell that had been incubated with APW + 50 μM cytochalasin D. (Arrow) Stimulus is indicated. Vertical scale bars represent 20 μm.

Figure 4

Cell mechanical changes during an AP. (a) Shown here is aspiration of Chara cell membrane into a micropipette (membrane projection indicated by m, protoplast surface by p). Note: the cell membrane is peeled off the dense array of chloroplasts (also see Movie S3). (b) During an AP, the membrane underwent a reversible cycle of motion into and out of the pipette at constant suction pressure. Aspirated membrane cap is indicated by a dashed arrow. (c) Suction pressure (Δp) before stimulation of an AP was clamped at 0 < Δp < Δp_cap (see text for definition of Δp_cap). Shown here is the membrane potential record (top) and aspirated length (L_p; bottom) during an AP. (d) Shown here is the initial phase of membrane motion into the pipette (n = 6 experiments in N = 4 cells; individual traces (black) and average (red)). See text for additional data and statistics. Unlabeled scale bars represent 10 μm. To see this figure in color, go online.

Figure 5

(a) The energy E as a function of aspiration length L_p for three values of the surface tension reveal that the weakly aspired state (L_p = 0) can be stable, critical, or unstable. Other parameters were held constant: κ = 10⁻¹⁹ J, Δp = 3N/m², R_p = 10 μm. For convenience, the energy was scaled to zero at an aspiration length of zero (L_p = 0). In addition, it was normalized by the mean thermal energy at room temperature (20°C), to indicate that the elastic energy stored in the surface is considerably larger. (b) Shown here is the instability line in the Δp−σ phase space. Weakly aspirated states are located below the line. The estimated resting state of the cell is depicted by the gray ellipse (its width reflects the experimental error in the measurement of σ_rest, whereas its height reflects the experimental error in the aspiration pressure that sets the weakly aspirated state). Decreasing κ effectively shifts the instability line in the direction of the arrow. The three states studied in (a) are depicted as small diamonds in (b).