Mechanical stimulation evoked a transient increase in [Ca²⁺]_i and induced whole-cell inward current. (A) Displacement of the FN bead (1 μm for 100 milliseconds) evoked an increase in [Ca²⁺]_i within 53 milliseconds from the start of stimulation. The red circle in the DIC image denotes the FN bead and the white dotted line shows the perimeter of the HUVEC. The black arrow indicates the direction of displacement. (B) Increase in [Ca²⁺]_i with sequential displacement of the FN bead for 0.15, 0.7 and 0.9 μm. Typical data are shown in the figure. The increase in the signal intensity by 0.5-0.7 μm displacement of FN beads was significantly larger that of 0.15-0.2 μm displacement (P<0.05; n=5).

Experimental set-up for patch-clamp recordings and high-speed TIRF microscopy, and inward currents induced by mechanical-stimuli. (A) A whole-cell patch-clamp recording was made from a cell subjected to localized mechanical stimulation. The displacement of the FN bead was monitored with photodiodes during the experiment using an IR YAG laser. Blue laser light (473 nm) was introduced into the objective lens to produce an evanescent illumination for the measurement of [Ca²⁺]_i and was controlled with a high-speed shutter (results are shown in Fig. 5B). (B) Displacement of the bead induced an inward current. 20 μM Gd³⁺ reduced the amplitude of the inward current. The inward current partially recovered after the removal of Gd³⁺ (right trace). Arrows denote the displacement of the FN bead.

Activation of MS channels by applying mechanical force to beads attached to actin stress fibers. (A) Phalloidin-conjugated 40 nm fluorescent beads were microinjected into HUVECs. These beads bound to the actin stress fibers and were trapped by laser optical tweezers. The movement of the trapping point was monitored with two photodiodes. Whole-cell patch-clamp recordings were made from the same cell. (B) Phalloidin-conjugated green fluorescent beads were located along the actin stress fibers (red). The cell was fixed 30 minutes after microinjection and stained with Rhodamine-phalloidin in this case. (C) A transient inward current was induced when the optical tweezers transiently passed an aggregate of phalloidin coated beads (shown by the two arrows in a). No current was induced when the same experiment was made with control beads (b). (D) The same mechanical stimulation with optical tweezers increased the [Ca²⁺]_i. The asterisks in the indicate the site of the aggregate of phalloidin-conjugated beads. The white broken line shows an outline of the HUVEC.