Fig. 2

A low-affinity GFP binder partially tethers extracellular GFP to cell membranes in zebrafish embryos. (a) Schematic of the localization assay. GFP binders were expressed in zebrafish embryos by microinjecting 100 pg of the corresponding mRNAs at the one-cell stage. After 3.5 h of embryonic development, GFP and a fluorescent dextran were injected extracellularly followed by confocal microscopy to determine the localization of GFP, the GFP binder (mCherry), and dextran (Cascade Blue). The panel on the right illustrates the localization of the three fluorescent signals shown in panel b. (b) Without GFP binders, GFP is distributed homogeneously in the extracellular space. In embryos expressing the weak GFP binder, GFP can be detected both on cell membranes and in the extracellular space. In the presence of the morphotrap, the majority of GFP localizes to cell membranes. Scale bars correspond to 50 μm. (c) A mask was created from the extracellular dextran signal and used to extract the GFP signal in cell-free areas within a circular region of interest (ROI, white). The graph shows measurements of extracellular GFP normalized to total GFP in the ROI from single embryos (black dots). Red lines indicate mean values. The scale bar corresponds to 50 μm.