a. Abolition of calcium flickers by streptomycin or removal of external calcium. b. Visualisation of calcium entry through single SACCs. From top to bottom: suction through the patch pipette, single-channel currents, and linescan image and line plot of local calcium transients. c. RNA interference knockdown of TRPC6, TRPM7, TRPV2 and TRPP2 assayed by Western blotting. NC, negative control RNA; SC: scrambled control RNA; Si1 and Si2, different siRNA sequences (see supplementary Table 2). d. Knockdown of TRPM7, but not TRPC6, TRPV2 or TRPP2, abolished calcium flickers. Data are expressed as mean±s.e.m.; n =12–21 cells in each group, **p < 0.01 vs. respective SC. e. TRPM7 knockdown prevented shear stress-induced calcium flickers.

a. Maps for calcium flicker ignition sites (red dots) and FAs (green). FAs were visualized by immunostaining for integrin α5 after calcium flicker acquisition. Enlarged views of calcium flickers, FAs, and their overlay are shown to the right. “N” denotes the nucleus. Scale bar: 8 µm. b. Lamella flicker prior to (left) and after (right) application of compounds that affect traction force-generating elements. c. Statistics of calcium flicker amplitude and probability (Pf). HBSS, Hepes-buffered saline solution; BDM, 2,3-butanedione monoxime (10 mM); (−)BB, (−)blebbistatin (100 µm); cytoD, cytochalasin D. Error bars represent s.e.m.; n = 10–19 in each group. **p < 0.01 vs. RGES; ^††p < 0.01 vs. HBSS. Note that flicker probability rather than amplitude was preferentially altered by varying the traction force, in contrast to the situation shown in Fig. 3.

a. Calcium flickers. In a polarised WI-38 fibroblast (insert), local calcium increases (ΣΔF) were summed over 30 consecutive images acquired at 6 s intervals. “N” marks the nucleus. Scale bar: 15 µm. b. Calcium flickers (colour overlay) in motile lamellipodia (box in a). Scale bar: 5 µm. c. Polarisation of calcium flicker activity. The image consists of 10000 front-to-rear linescans, expressed as the ratio between fluo-4 and fura-red fluorescence (R). d. Opposing gradients of calcium flicker activity (ΔR) and global calcium (R, inclusive of flicker activity). Similar results were observed in eight cells.

a. Typical calcium flicker responses to thapsigargin (TG), xestospongin C (Xec) or IP₃-BM. Linescan data of ΔF/F₀ are rendered as surface plots. b. Western blotting of IP₃Rs. See supplementary Table 2 for sequences of negative control (NC), respective scrambled control (SC), Si1 and Si2. Data are expressed as mean±s.e.m. (n=3–4). **p < 0.01 vs. respective SC. c. Flicker amplitude and probability under various experimental conditions. Pf: fractional time spent in calcium flickers; HBSS: Hepes-buffered saline solution; Rya: ryanodine (25µM, 15 min); n=10–29; *p < 0.05; **p < 0.01 vs. HBSS; ^††p < 0.01 vs. respective SC. Note that perturbing the ER calcium release mainly altered flicker amplitude without affecting flicker probability.

a. Asymmetrical lamella flicker activity induced by a PDGF gradient. Upper left, contours of the cell border at -14 (pink), 0 (purple) and 15 min (yellow). Upper right, trajectory of the centre of the cell and time course of calcium flicker production (bar graph). “SM”: signal mass of calcium flickers within the lamella. Lower panels: overlays of calcium flickers in 1-min windows (labelled 1–3 in the trajectory above). Dashed lines bisect the leading lamella into upper (α, facing the PDGF source) and lower portions (β) b. Correlation between cumulative asymmetric flicker activity (ΣSM_α-β) and displacement along the PDGF gradient (y-distance). c. Relationship between turning angle and ΣSM_α-β (at 15 min). C, control with no treatment; SP, streptomycin 200 µM; Si1, Si2 and SC: two siRNA constructs and a scrambled control; IP₃-BM 2 µM; E-A, EGTA-AM, L, 2 µM, H, 20 µM. Data are expressed as mean±s.e.m.; “n” values are shown in parentheses. *p < 0.05; **p < 0.01 vs. turning angle of control or respective SC; ^†p < 0.05; ^††p < 0.01 vs. ΣSM_α-β of control or respective SC. d. Chemotaxis of WI-38 fibroblasts. CK, chemokinesis assay with the same concentration of PDGF-BB on both sides of the well; E-A, EGTA-AM (2 µM). n=4–10, *p < 0.05; **p < 0.01 vs. control or respective SC. e. Cartoons of patterned calcium flicker activation in persistently moving (left) and turning fibroblasts (right). Calcium flicker activity during persistent movement displays a front-to-rear polarisation, opposing a rear-to-front global calcium gradient. During turning, calcium flicker activity becomes asymmetric across the leading lamella, in addition to enhanced flicker frequency and accentuated front-to-rear polarisation.