Femtojet

U2OS cells were seeded on a glass bottom dish, well size 14 mm (Cellvis) and transfected with 0.1 μg of a vector encoding for EGFP-KDEL 48 hr before the experiment. Subsequently, a mixture of Alexa Fluor 647 conjugated dextran (0.5 mg/ml, MW 11 kDa) (Thermo Fischer Scientific) and GTP or GTPγS (10 mM) in permeabilization buffer was injected into the cytoplasm of cells using an Eppendorf FemtoJet and InjectMan Ni2 microinjection unit at a Leica DMI6000B inverted microscope equipped with a 20X, 0.4 NA, Ph1 HCX PlanFluotuar LD (long distance) objective. ER dynamics in microinjected cells was imaged for 1 min at a frame rate of 1/s using a Visitron spinning disk Nikon Eclipse T1 microscope with a 100X, 1.49 CFI, Apo TIRF objective. Image analysis was performed with ImageJ. Brightness and contrast were adjusted across the images. From each cell, 1–2 ROIs of 100 μm² in the peripheral ER were selected for manual analysis of successful or unsuccessful membrane attachments.