Figure 1.
Fixed and stained images of the C2C12 myoblast cytoskeleton.
Control cells are show in (A) and cells treated with anti-cytoskeletal inhibitors for 60 min are shown in (B) Cyt-D, (C) nocodazole, (D) ML-7, (E) Y-27632 and (F) blebbistatin. The actin and MT cytoskeleton are shown at the top and middle row respectively. A merged image of the actin (red), MT (green) and nucleus (blue) are in the bottom row. Scale bar = 20 µm and applies to all images.
Figure 2.
Time-lapse IRM imaging on inhibitor treated C2C12 cells.
IRM time-lapse imaging on C2C12 cells before, during and after a 60 min exposure to Cyt-D and nocodazole in comparison to control cells (scale bar = 20 µm and applies to all images). Cells treated with Cyt-D and nocodazole display decrease in the dark regions of the cell over the course of 60 mins. The control cells exhibit no change in the low intensity regions of the cell. ML-7 and Y-27632 treatments (data not shown) displayed similar trends as those shown for the Cyt-D and nocodazole treatments. Scale bar = 20 µm and applies to all images.
Figure 3.
Computational analysis of cell contact area in IRM images.
(A) Raw IRM image of a cell adhered to a glass substrate (scale bar = 25 µm, and applies to all). (B) The same image after applying a FFT band-pass filter to create a uniform intensity background. (C) The image after applying a background subtraction/lightening filter to isolate sites of close cellular contact from the background. (D) The image is then thresholded to form a binary image where regions of close contact (corresponding to grey values lower than the background intensity in (C)) are set to zero intensity. Artifacts arising from debris are manually removed and then the zero intensity pixels are summed to provide a measure of cell CCA. (E–G) The effect of different user specified thresholds (shown in red) yield different values of the (H) absolute CCA as a function of time. (I) Normalizing the data yields the change in CCA and all three curves fall on one another. (J) Time-lapse imaging of C2C12 cells during exposure to EDTA. All cells showed a consistent decrease in contact area of about 75% and that CCA is calcium dependent.
Figure 4.
Dynamic changes in C2C12 close contact area over 60 mins.
Cells were treated with various inhibitors and IRM images were acquired every 15 mins for 60 mins. (A) Control, (B) Cyt-D, (C) nocodazole, (D) Y-27632A, (E) ML-7 and (F) blebbistatin treated cells (n = 3 cells for each condition). In each case, except for the control, a significant decrease in contact area occurs after 60 mins of exposure to each inhibitor. However, there was no statistically significant dependence on the type of inhibitor used. On average, the ΔCCA was ∼13% for each condition, except for the control.
Table 1.
CCA, Cell Height and Cell Area after 60 min of exposure to various inhibitors.
Figure 5.
Flow chamber adhesion assay and changes in cell adhesion strength.
(A) In face flow chamber dimensions: L = 2 cm and W = 1 mm. (B) SU-8 master used to fabricate flow chamber with a height of 160 µm. (C) Water with red dye shows the flow chamber working principle with bent stainless steel needle attached to the inlets and outlets allowing media to be pumped through the chamber. (D) Typical adhesion assay where cells were exposed to shear stress over a period of 30 min. Cells were counted before and after flow to quantify changes in cell-substrate adhesion strength (scale bar = 500 µm). (E) Bar graph showing the ratio of cells remaining on the glass substrate after exposition to a shear stress of 65 Dyne/cm2 over a period of 30 min. There is no significant difference between the control and Y27632 treatment (p>0.1). All other treatments yield a significant decrease relative to the control (p<0.04).
Table 2.
Changes in CCA, Cell Area, Adhesion, FAs and ß1-integrin for control cells and cells exposed to various inhibitors for 60 min (↓ = decrease, NC = no change from the control, D = diffuse, PL = point-like, NP = not present and P = present).
Figure 6.
Overlay of IRM imaging and vinculin expression after 60 mins.
Simultaneous IRM and fluorescence imaging of live cells expressing Vinculin-EGFP after exposure to inhibitors for 60 min. (A) Control (scale bar = 20 µm and applies to all), (B) Cyt-D, (C) nocodazole, (D) Y-27632A, (E) ML-7 and (F) blebbistatin treated cells. Cells treated with nocodazole, Y27632 and ML7 posses smaller but point-like FA structures, similar to the control. Vinculin becomes diffuse after treatment with CytD and blebbistatin.
Figure 7.
Fixed and stained images of ß1-integrin and the membrane of C2C12 cells.
Cells were exposed to inhibitors for 60 min and then fixed and stained for the presence of β1-integrin (greyscale and red in the merge) and the plasma membrane (green in the merge). LSCM images were acquired at the cell-substrate interface. (A) Control (scale bar = 20 µm and applies to all), (B) Cyt-D, (C) nocodazole, (D) Y-27632A, (E) ML-7 and (F) blebbistatin treated cells. In all cases, integrin-ß1 is well distributed over the cell contact area. However, after 60 min of CytD treatment a significant decrease in integrin-ß1 was observed, correlating to a significant decrease in cell adhesion strength (Fig. 5).
Figure 8.
Fixed and stained images of fibronectin and the membrane of C2C12 cells.
Cells were exposed to inhibitors for 60 min and then fixed and stained for the presence of fibronectin (Fn, greyscale and green in the merge) and the plasma membrane (red in the merge). LSCM images were acquired at the cell-substrate interface. (A) Control (scale bar = 20 µm and applies to all), (B) Cyt-D, (C) nocodazole, (D) Y-27632A, (E) ML-7 and (F) blebbistatin treated cells. In all cases, cell-secreted fibronectin was distributed over the substrate and found in higher concentrations underneath the cell body.