Np egta

Embryos were prepared as previously described 41. Briefly, embryos (2–2.5 h at 25°C in Figs 1, 2, 4A–E and 5, and 15–17 h at 20°C in Figs 3 and 4F–K) were collected and dechorionated with 50% bleach (hypochloride) for 90 s, dried in a desiccation chamber for ~ 10 min, covered with halocarbon oil, and injected dorsally into the vitelline space in the dark at room temperature (~ 22°C). After injection, the embryos were incubated at room temperature in the dark for about 10 min prior to uncaging.
NP‐EGTA, AM (Invitrogen) was prepared in 1× injection solution [180 mM NaCl, 10 mM HEPES, 5 mM KCl, 1 mM MgCl₂ (pH 7.2)] 11. 2 mM NP‐EGTA, AM was injected for Ca²⁺ uncaging in epidermal cells, and 1 mM NP‐EGTA, AM was injected for Ca²⁺ uncaging in amnioserosa cells. To inhibit Rock activity, 10 mM Y‐27632 (Sigma) in water was injected.

The output of a 355 nm diode-pumped solid-state laser (DPSL-355/30, Rapp OptoElectronic) was coupled to an upright Olympus BX51W1 microscope to provide a UV spot illumination of ~10 μm in diameter. The photo-liable Ca²⁺ chelator, o-nitrophenyl ethylene glycol tetraacetic acid (NP-EGTA, Molecular Probes, catalog # N6802), pre-mixed with Ca²⁺ was loaded into pillar cells through the patch pipette. Cells were patched using pipettes with a resistance of 6–10 MΩ when measured in the bath. The intracellular solution contained (in mM): KCl (12.6), KGlu (131.4), CaCl₂ (0.7), K₂HPO₄ (8), KH₂PO₄ (2), Na₄-ATP (2), Na₄-GTP (0.2), NP-EGTA (1), and fluo-4 (0.05, Molecular Probes). The flash photolysis of NP-EGTA-Ca²⁺ complex was achieved with a train of 15–25 laser pulses of 1 ms duration delivered at 166 Hz. The total duration of the pulse train was 90–150 ms. The optimal number of pulses was determined empirically to generate maximal [Ca²⁺]_i increase but minimize photobleaching of the Ca²⁺ indicator. Simultaneous time lapse bright-field and epifluorescent imaging were performed. The L-15 medium in the bath was supplemented with 100 µM of FFA to block the gap junctions between the supporting cells. Pillar cells were held at their resting potentials: −30 to −40 mV. In the control cells, the NP-EGTA in the intracellular solution was replaced with the equivalent amount of EGTA.