Cell membrane tethers generate mechanical force in response to electrical stimulation

Abstract

Living cells maintain a huge transmembrane electric field across their membranes. This electric field exerts a force on the membrane because the membrane surfaces are highly charged. We have measured electromechanical force generation by cell membranes using optically trapped beads to detach the plasma membrane from the cytoskeleton and form long thin cylinders (tethers). Hyperpolarizing potentials increased and depolarizing potentials decreased the force required to pull a tether. The membrane tether force in response to sinusoidal voltage signals was a function of holding potential, tether diameter, and tether length. Membrane electromechanical force production can occur at speeds exceeding those of ATP-based protein motors. By harnessing the energy in the transmembrane electric field, cell membranes may contribute to processes as diverse as outer hair cell electromotility, ion channel gating, and transport.

Figure 1

Experimental setup. Schematic of membrane tether formation with optical tweezer and electrical stimulation. Both constant and sinusoidal electrical stimuli are delivered to the cell through the whole-cell tight-seal patch electrode. Electromechanical force (F_EM) was quantified by measuring the bead displacements along the tether axis using the quadrant photodetector (QPD).

Figure 2

Photomicrographs of OHC (top) and HEK (bottom) cells connected to trapped beads by membrane tethers. The HEK tether is visible but OHC tethers were too narrow to be resolved. Scale bar = 5 μm.

Figure 3

Representative tether force profiles plotted against time for tethers from two HEK cells at −60 mV (black trace) and 60 mV (red trace) holding potentials. Membrane tethers were pulled at a constant rate of 1 μm/s to a 30-μm length; note that the force profiles begin (time zero) when the tethers have reached a length of 10 μm. For a population of cells held at –60 mV (n = 12), the peak force (value at the end of elongation) was 24.5 ± 2.8 pN (mean ± SE) and the steady-state force was 16.4 ± 4.1 pN. Another population was depolarized to 60 mV (n = 9), the peak force was 18.4 ± 3.2 pN, and the steady-state force was 8.7 ± 2.9 pN. The force values at the two holding potentials are significantly different (p < 0.001, unpaired Student's t-test).

Figure 4

Effect of changing the transmembrane electric field on tether force generation (F_EM) by 30-μm-long tethers. (A) F_EM generated by membrane tethers (top two traces) in response to 1 Hz sinusoidal stimulation (bottom trace). (B) Representative F_EM of OHC (blue), HEK (black), and HEK + 10 mM salicylate (green) tethers is shown in response to a sinusoidal voltage signal (±20 mV, 2.5 kHz) riding on a –60 mV holding potential. The traces have been normalized to the OHC peak value (0.49 pN—an underestimate, see Supporting Material). The diameter of the HEK tether is larger than the OHC tether (see Fig. 2).

Figure 5

Effect of changing the holding potential on F_EM. (A) Average F_EM and standard error (SE) for two populations of HEK cell tethers stimulated at 1 kHz, one population in normal saline, and the other in 10 mM salicylate, as well as average F_EM for OHC tethers stimulated at 6 Hz. (B) F_EM as a function of holding potential for an OHC tether at 6 Hz and 1 kHz. The forces in panels A and B have been normalized to the value obtained at zero mV holding potential.

Figure 6

Effect of tether diameter on F_EM. (A) HEK tether force profile as the tether approached mechanical steady state (F_eq) for three different tether lengths. (Insets) Photomicrographs of the tether from another non-voltage-clamped HEK cell taken after steady-state forces were reached at 10 μm (left inset) and 30 μm (right inset). (B) The F_EM was measured continuously while the tether was elongated with the same electrical stimulus as in panel A. The three F_EM values measured at 10, 20, and 30 μm in panel A are plotted in red (normalized to the F_EM at 10 μm).

Figure 7

Tether cable properties affect F_EM. (A) The monotonically increasing tether force is plotted as a continuous function of tether length and the normalized F_EM (♦) is plotted every 1.8 μm between 10 μm and 60 μm. (B) F_EM generated by an HEK cell tether at different lengths and frequencies. The results are normalized to the F_EM amplitude at the 10-μm length except for the 2.5 kHz values, which are normalized to the amplitude at 20 μm. The F_EM amplitude for 2.5 kHz was below the noise floor at the 10-μm and 50-μm lengths.