Fig. 5. Effect of confining SMCs in a line using micropatterning.

(A) When SMCs were patterned in lines, (B) the probability of finding SMCs with high time periods decreased and the variance of the time periods from aligned cells was significantly smaller than confluent cells (F test, P < 0.001). Matrix stiffness still affected the oscillation period (soft, N = 8; stiff, N = 6; Mann-Whitney rank sum test). To probe the limits of the SMC cluster’s collective matrix sensing abilities, we simulated localized ECM stiffening by (C) patterning SMCs in lines spanning a dual-stiffness PDMS substrate (E = 13 kPa highlighted in green, left, and E = 0.3 kPa, right). (D) The Ca²⁺ oscillation time period of cells along lines is plotted as a function of the distance from the stiff matrix. Binning cells in 400-μm intervals, only those over 800 μm from stiff matrix had different Ca²⁺ oscillation time periods from cells directly in contact (t test, N = 4). Scale bars, 250 μm.