JCS024521 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The limitations of Fast Fourier analysis to detect cell-cell coordination. (A) To detect the predominant Ca²⁺_i oscillation periods, Fura-2 Em₃₄₀/Em₃₈₀ fluorescence ratios (Fig. 2A) were band-pass filtered (dotted-line and inset) to eliminate noise and subjected to Fast Fourier Transformation (FFT). The main peak in the power spectrum (PSD=squared magnitude of the Fourier transform) corresponds to the dominant oscillation frequency from which the dominant period T_D is deduced. Our Ca²⁺ signal recording frequency was one frame every 5 or 3 seconds (200 or 333 mHz) and the frequency resolution was always greater than 3 mHz. With this resolution, our period analysis was confined to a range between 10 and 300 seconds, which was well adapted to the obtained period distribution (Fig. 1C,G). To demonstrate the potential of our period analysis, Ca²⁺ oscillations are computationally simulated and analysed in B-C. (B) Cell A (blue) initially exhibits high Ca²⁺_i oscillation frequency (T₁=50 seconds); hypothetical addition of a drug (pointed line) abruptly reduces the frequency (T₂=70 seconds). Cells B (red) and C (black) both show the inverse behaviour. FFT analysis delivers the same profile for all three cells with two main peaks corresponding to the dominant periods T_D1 and T_D2, even though cell A and cell B are clearly not coordinated. Our method discriminates between the perfect coordination of cells B and C (black data points on the diagonal) and the absence of coordination between cell A and B (grey data points and ellipse far from the diagonal). (C) Cell A (blue) gradually changes its frequency over time (from T=50 seconds to T=100 seconds) whereas cells B (red) and C (black) both exhibit the inverse behaviour. FFT analysis generates the same profile for all three cells with no clear dominant frequency peak, due to the gradual variation of frequency over time. By contrast, our method detects the perfect coordination of cells B and C (black data points on the diagonal) and the absence of coordination between cells A and B (grey data points and ellipse far from the diagonal). This discrimination between both cases is not possible with FFT analysis that does not provide information at the level of cell-cell coordination.

• Supplemental Figure S2 -

  Fig. S2. Potential of the period analysis method. (A) To demonstrate the potential of our period analysis, Ca²⁺ oscillations of two (virtual) contacting fibroblasts were computationally simulated over an observation time of 100 minutes (only 1000 seconds displayed). Fortunately, virtual cells do not bleach. The occurrence of peaks in the fluorescence ratio was randomly generated on the basis of the experimental period distribution (histogram Fig. 1G). Coordinated occurrence of peaks between cell 1 (turquoise) and cell 2 (green) was taken into account using three different correlation probabilities (P_corr). With P_corr=95%, one peak in the fluorescence profile of cell1 almost always correlated with a peak in the profile of cell 2, simulating strong coupling. With P_corr=0%, peak occurrence in the fluorescence profiles was completely independent between both cells, simulating uncoupled cells. (B) The profiles of cell 1 and cell 2 were then analysed with our method and summarised in a period scatter plot (P_corr=0%, red; P_corr=70%, green; P_corr=95%, blue). Note that ellipses and data clouds are larger and more distant from the diagonal with decreasing degrees of cell coupling. The plot also shows that our method detects differences of coupling.

• Supplemental Figure S3 -

  Fig. S3. Synchronisation of periodic Ca²⁺_i oscillations between myofibroblasts implies adherens junction coupling, cell contraction and MS channels. Spontaneous Ca²⁺_i oscillations in cultures of Fura-2-loaded myofibroblasts (A,C,E) and fibroblasts (B,D,F) were compared between contacting cells that have been pre-selected for exhibiting coordinated periods. Fura-2 Em₃₄₀/Em₃₈₀ ratios of each cell were recorded every 3-5 seconds and are displayed for two contacting cells. Different drugs were added during recording at the indicated time points. (A,B) Adherens junctions were disassembled by adding a mixture of anti-N- and anti-OB-cadherin peptides at 0.5 mg/ml for 45 minutes. (C,D) Cell contraction was inhibited by adding 1 mM BDM. (E,F) MS channels were inhibited by adding 300 µM Gd³⁺. Arrows on top of the fluorescence profiles indicate how the position of Ca²⁺_i peaks of cell 2 (grey) develops over time in respect to the matched peaks of cell 1 (black); dots show simultaneous oscillations of both cells and changing vector lengths over time demonstrate uncoupling of two cells.

• Movie 1 -

  Movie 1. Myofibroblasts and fibroblasts exhibit distinct periodic and spontaneous Ca²⁺_i oscillations. The Em₃₄₀ fluorescence images of Fura-2-loaded myofibroblasts (left) and fibroblasts (right) were recorded every 3 seconds for 7.5 minutes. Note that both cell types exhibit periodic Ca²⁺_i oscillations that appear to be more regular and coordinated in myofibroblasts. Scale bar: 100 µm.

• Movie 2 -

  Movie 2. Ca²⁺ dependent contraction of myofibroblasts. Treatment of myofibroblasts cultured on wrinkling silicone substrates with the Ca²⁺ ionophore A23187 causes cell contraction within 10 minutes. Contraction is visible from enhanced formation of wrinkles in the deformable silicone surface. Phase-contrast images were recorded every minute for 70 minutes; frame width is 400 µm.

• Movie 3 -

  Movie 3. Myofibroblasts and fibroblasts exhibit different kinetics of intercellular Ca²⁺_i wave propagation. Myofibroblasts (top) and fibroblasts (bottom) were grown between 50 µm-wide non-adhesive lines and loaded with Fura-2 Em₃₄₀ fluorescence images were recorded every second for 50 seconds; total frame height is 100 µm. The first cells in the row are locally stimulated with a micropipette at the same time for both types of cells (indicated by *), which elicits a Ca²⁺_i wave that propagates intercellularly over the neighbouring cells. Ca²⁺_i wave propagation over several cells is slower in myofibroblasts than in fibroblasts; this is due to a significant delay of Ca²⁺_i wave spreading at cell-cell junctions, indicated by arrows.