Skip to main content
. 2016 Feb 4;14(6):1517–1527. doi: 10.1016/j.celrep.2016.01.027

Figure 2.

Figure 2

RAB-5 and DYN-1 RNAi Knockdown Induces EFF-1 Accumulation on the Apical Plasma Membrane

(A, B, and F) Live images of EFF-1::GFP (green) and apical junctions, DLG-1::RFP (magenta) in embryos treated with control RNAi (A), rab-5 RNAi (B), and dyn-1 RNAi (F). Right panel represents the magnified region of the inset area in (A) and (B).

(A) EFF-1::GFP is in puncta in control RNAi. See also Movies S1 and S2.

(B) EFF-1::GFP in rab-5 RNAi embryo appears diffuse on the cytoplasm in smaller vesicles and associated with the apical plasma membranes and junctions. See also Figure S2G and Movies S3 and S4.

(C) Intensity profile plotted along the line (white dashed line in A) of EFF-1::GFP (green line on the plot) and DLG-1::RFP (magenta line on the plot) shows no correlation.

(D) Intensity profiles plotted along dashed line from (B) represent correlation of EFF-1::GFP and DLG-1::RFP intensities on the cell junctions, showing the enrichment of EFF-1::GFP on the apical plasma membrane.

(E) Representative stages from time-lapse images of cell fusion under rab-5 RNAi showing EFF-1::GFP/DLG-1::RFP colocalization (arrows). Cell junction undergoing fusion is marked by an arrowhead. Note that EFF-1 dissociated from the cell junction at 10 min of fusion (arrowhead). Time in min is indicated. See also Movie S3.

(F) EFF-1::GFP apical plasma membrane expression (arrows) caused by dyn-1 RNAi is shown on the embryo surface focus (upper panels) and center focus (lower panels). Embryogenesis defects and hyperfusion are visualized by DLG-1::RFP pattern (Movie S5).

(G) EFF-1 localization and embryonic defects caused by rab-5 and dyn-1 downregulation. The first bar graph represents percentage of embryos showing EFF-1 localization with plasma membrane (white), plasma membrane together with vesicular puncta (gray), and vesicular expression (black). The second bar represents the percentage of embryos presenting defective embryogenesis (light green) and hyperfusion (dark green). Most rab-5(RNAi) and dyn-1(RNAi) embryos arrest at early embryogenesis before EFF-1 expression and before the time of hypodermal fusions (elongation/morphogenesis). Most embryos that escape the early arrest show hyperfusion and EFF-1 in apical plasma membranes. Mean of three independent experiments; n ≥ 20 embryos per experiment.

The scale bars represent 10 μm in (A), (B), and (F) and 2 μm in (E).