First published online 29 January 2008
doi: 10.1242/jcs.022053
Journal of Cell Science 121, 496-503 (2008)
Published by The Company of Biologists 2008
Actin stress fibers transmit and focus force to activate mechanosensitive channels
Kimihide Hayakawa1,
Hitoshi Tatsumi1,2 and
Masahiro Sokabe1,2,3,*
1 Cell Mechano-sensing Project ICORP/SORST, Japan Science and Technology Agency, Nagoya University Graduate School of Medicine, 65 Tsurumai Syouwa-ku, Nagoya 468-8550, Japan
2 Department of Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai Syouwa-ku, Nagoya 468-8550, Japan
3 Department of Molecular Physiology, National Institute for Physiological Sciences, NINS, Myodaiji, Okazaki 444-8585, Japan
View larger version (86K):
[in this window]
[in a new window]
|
Fig. 1. Localized mechanical stimuli were applied to HUVECs by displacing an FN bead. (A) Schematic drawing of the experimental set-up. A HUVEC was plated on a fibronectin-conjugated elastic polyacrylamide gel substrate (blue). (B) HUVECs were stained with anti-paxillin antibodies (upper panel) and Rhodamine-phalloidin (middle panel), and sectioned in the x-z plane. These images are overlaid in the panel below. The yellow semi-circle shows the position of the FN bead. Stress fibers (yellow arrows) connect the apical focal adhesions (FAs) to the basal FAs. The bottom panel shows the x-y confocal image of the cell. A 10 µm bead promoted formation of focal adhesions on the apical surface (shown by white arrows). The pattern of FAs underneath the FN bead was scattered and irregular (arrowheads). FAs on the cell base were also imaged. Twenty confocal images (5 µm thickness) were projected to the x-y plane. (C) An example of gel deformation caused by a mechanical stimulation. An FN bead (shown by green dotted circle) on a HUVEC (white broken line denotes the cell perimeter) was displaced by a piezoelectric-driven glass pipette (shadows in the top and bottom panels). Small orange circles indicate the position of 50 nm fluorescent beads (middle panel). Insets show fluorescent beads at higher magnification (a fluorescent spot at the centre of the circle; scale bar, 5 µm). Each red arrow in the bottom panel indicates the direction and relative amplitude of fluorescent particle displacement (longest arrow corresponds to 0.76 µm displacement) when the FN bead was moved 4 µm in the direction shown in the top panel by an arrow. (D) Very small substratum deformation underneath the bead was observed in cytochalasin-D-treated cells (note red arrow in the lower right marginal region of the cell). (E) Time course of the displacement of a 50 nm bead exposed to the mechanical stimulus along with the time course of another bead that did not move because it is located far from the FN bead (>40 µm). The interval between frames was 1 second.
|
|
View larger version (85K):
[in this window]
[in a new window]
|
Fig. 2. 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).
|
|
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3. 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.
|
|
View larger version (48K):
[in this window]
[in a new window]
|
Fig. 4. 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.
|
|
View larger version (43K):
[in this window]
[in a new window]
|
Fig. 5. Activation of MS channels in the vicinity of FAs by mechanical stimulation. Time-lapse imaging of the increase in [Ca2+]i at a time resolution of 17 milliseconds (A) and a 2 millisecond snapshot image of the [Ca2+]i increase (B). (A) The FN bead was displaced by 1 µm in the direction indicated by the arrow (a). The distribution of FAs was imaged by interference reflection contrast microscopy; green spots denote the FAs (b). (c-g) A series of time-lapse images of the increase in [Ca2+]i caused by the mechanical stimulation. (h) Superimposed images of b and d (green, FAs; red, [Ca2+]i increase). The white arrows indicate FAs inside of the area of the [Ca2+]i increase. (B) DIC (a, upper) and TIRF (a, lower) images of a cell at low magnification. The area enclosed by the white dotted rectangle in a is magnified in b. Regions of high [Ca2+]i ([Ca2+]i microdomains) 2-4 milliseconds after the onset of the stimulation are shown as red spots. These spots are located in the vicinity of integrin clusters (green). The upper left panel in b shows approximately 10 [Ca2+]i microdomains. The middle left panel shows β1 integrin and the lower left panel shows an overlay of the [Ca2+]i microdomain and the integrin. The right-hand panels in b show another example of the [Ca2+]i microdomains 0-2 milliseconds after the onset of the stimulation. The graph in c shows the intensity profiles of fluo-3 and anti-β1-integrin fluorescence in the region denoted by the yellow line in the lower left panel in b.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2008
