Preparation of isolated cortices

Isolated cortices were prepared following the methods previously established in the laboratory^{36, 46}. Briefly, embryos at the desired stage were transferred to calcium-free seawater then placed at high density on coverslips coated with protamine (1 mg/ml) to which they adhere within 1 min. After two washes with cortex isolation medium (cortex isolation medium (CIM): 0.8 M glucose, 0.1 M KC1, 2 mM MgCl2, 5 mM EGTA, 10 mM MOPS buffer, pH 7), a stream of CIM is sprayed gently with a Pasteur pipette, shearing off the embryos but leaving attached to the glass imprints of the adherent membrane and associated cortical structures. The coverslips are washed rapidly in CIM then placed in cold methanol for fixation, then rehydrated in phosphate-buffered saline and processed for immunofluorescence by standard procedures used for whole embryos. ER of isolated cortices was labeled with the addition of 0.2 µg/ml DiO C6(3) (Invitrogen) for 1 min. following fixation and immunolabelling^{37, 46}.