FIGURE 5:

LST-4 and DYN-1 depend on each other for their association to phagosomal surfaces. (A and B) The interaction between DYN-1 and LST-4 was detected in the yeast two-hybrid assay (A) and the coimmunoprecipitation assay (B). Protein interactions were detected in (A) by X-Gal assays and in (B) by immunoblotting Flag-tagged LST-4 that was coimmuniprecipitated with HA-tagged proteins. CED-1C (the cytoplasmic domain of CED-1), CED-12, and CED-6 are negative controls to demonstrate the specificity of LST-4/DYN-1 interaction. (C–E) Time-lapse recording of the recruitment of DYN-1::GFP onto C2 phagosomes in a wild-type embryo (C), a snx-1(tm847) mutant embryo (D), and a lst-4(tm2423) mutant embryo (E). “0 min”: the time point when the engulfment was just completed. Arrowheads mark DYN-1::GFP puncta associating with phagosomes. Scale bars: 2 μm. (F) Quantification of different dynamic DYN-1::GFP localization patterns on phagosomes by monitoring multiple C1, C2, and C3 phagosomes over time. At least 16 phagosomes were monitored for each genotype. (G and H) Time-lapse images showing the dynamic recruitment of LST-4(d)::GFP onto a C3 phagosome in a wild-type embryo (G) or the lack of it in a dyn-1(en9) mutant embryo (H). “0 min”: the time point when the engulfment was just completed. Arrows in (H) indicate the C3 phagosome. Arrowheads in (H) indicate cytoplasmic LST(d)::GFP(+) puncta, which are surrounding but not associating with the C3 phagosome. The cell boundary of the engulfing cell for the C3 cell corpse is outlined in (H[a]). (H[j]) The DIC images corresponding to (H[a]) that indicates the position of the C3 phagosome. Scale bars: 4 μm.