Figure 1.
Increasing the E-modulus of silicone substrates increases [Ca2+]i oscillation frequency.
SCMF were cultured on FN-coated silicone substrates produced with E-moduli of 5, 15, and 50 kPa for 2 days. A) Cells were immunostained for α-SMA (red), F-actin (green) and nuclei (blue). Scale bar = 50 μm. B) Representative fluorescence ratios (Em340/Em380) were recorded over time on Fura-2-loaded cells of each stiffness group. C) The dominant periods of regular oscillations were determined and pooled into a histogram that was fitted following a generalized extreme value distribution. D) [Ca2+]i period distribution fits of 5 kPa, 15 kPa and 50 kPa groups are displayed and maxima highlighted with dotted lines (nexp≥14, ncells≥44). E) Period distribution fit maxima were translated into oscillation frequency (peaks/min) and expressed as a function of the Young's E-modulus of silicone substrates.
Figure 2.
The E-modulus of collagen gels modulates [Ca2+]i oscillation frequency.
SCMF were grown in gels of 1.0, 1.5, 2.0, or 2.5 mg/ml collagen for 2 days. A) Confocal reflection microscopy imaging of cell-free gels demonstrates collagen fiber density. B) The Young's E-modulus of cell-free gels was measured using a micro-indentation approach. C) Representative fluorescence ratios (F/F0) were recorded over time on Fluo-4-loaded cells grown in collagen gels. D) [Ca2+]i period distribution fits are displayed and maxima highlighted with dotted lines (nexp = 18–41, ncells = 60–93). E) Period distribution fit maxima were translated into oscillation frequency (peaks/min) and expressed as a function of the Young's E-modulus of collagen gels. F) Myofibroblasts in collagen gels were stained after 2 days for F-actin (green) and collagen ECM was overlaid with confocal reflection imaging (red). Scale bars = 50 μm. The collagen density was measured by applying an integrated density function to confocal images of gels either in G) cell-free regions of the gels or H) in the vicinity of SCMF. I) [Ca2+]i oscillatory frequencies are expressed as a function of measured collagen densities (Fig. 2I). (n = 3; mean±SD, **p≤0.01).
Figure 3.
Decreasing cell adhesion decreases [Ca2+]i oscillation frequency.
SCMF were grown for 2 days on glass coverslips, coated with PLL at 5.0 μg/cm2, 0.5 μg/cm2, or with FN. A) Cells were immunostained for F-actin (red), vinculin (green) and nuclei (blue). Scale bar = 50 μm. B) Representative fluorescence ratios (Em340/Em380) were recorded over time on Fura-2-loaded cells. C) [Ca2+]i period distribution fits are displayed and maxima highlighted with dotted lines (nexp = 29–34, ncells = 68-93). D) SCMF area (n = 3; mean±SD) and E) the length of vinculin-positive focal adhesions were quantified from fluorescence staining (n = 3; mean±SEM, *p≤0.05, ***p≤0.001), F) Period distribution fit maxima were translated into oscillation frequency (peaks/min) and expressed as a function of the mean SCMF focal adhesion lengths on differently adhesive substrates.
Figure 4.
Restricting cell size decreases [Ca2+]i frequency.
A) SCMF were grown on microcontact-printed FN square islets of 100–10,000 μm2, immunostained for F-actin, and imaged by confocal microscopy. A composite was produced by stitching images from different cells on the same substrate, containing all square sizes. Scale bar = 500 μm. B) Cells spreading on FN islets of 2,500 and 4,900 μm2 were immunostained for F-actin (red), vinculin (green), and FN (blue). Scale bar = 250 μm. C) Representative fluorescence ratios (Em340/Em380) were recorded over time on Fura-2-loaded cells, stimulated with increasing concentrations of endothelin-1 (ET-1). D) Distribution fits of [Ca2+]i oscillation periods are displayed for cells grown on 2,500 and 4,900 μm2 islands (nexp = 18–25, ncells = 24–29) and treated with 50 nM ET-1.
Figure 5.
Disrupting actin stress fibers decreases [Ca2+]i oscillation frequency.
SCMF were grown for 2 days on FN-coated coverslips. A) Cells fixed before (left panel) and 30 min after Cytochalasin D treatment were immunostained for F-actin (red), vinculin (green) and nuclei (blue). Scale bar = 50 μm. B) Representative fluorescence ratio (Em340/Em380) of a Fura-2-loaded cell over time is shown. Fluorescence was recorded for 15 min before and 15 min after 30 min treatment with Cytochalasin D (15 μM) or vehicle (DMSO) only (C). D) [Ca2+]i oscillation period was calculated before Cytochalasin D treatment and plotted against the period after treatment for the same cell. Any point above the diagonal indicates a period decrease after addition of the drug (nexp = 9, ncells = 25).