FIGURE 3:
Lgd binds to CHMP7 at the inner nuclear membrane. (A) Representative images of four-cell stage embryos coexpressing a GFP fusion to Lgd and an mCherry fusion to histone H2B were imaged live using confocal microscopy in the presence and absence of CHMP4 (n = 5 animals each). Zoomed regions of nuclei are also shown. Bar, 10 μm; inset bar, 2 μm. (B, D) Representative images of embryos (zoomed to visualize nuclei) coexpressing a GFP fusion to Lgd and an mCherry fusion to histone H2B during nuclear envelope breakdown (B) and nuclear envelope reformation (D), in the presence (top) and absence (bottom) of CHMP7. Bars, 2 μm. (C) Linescan analysis showing the fluorescence intensity of GFP-Lgd along the dashed lines shown in panel B in the presence and absence of CHMP7. (E) GST and GST fusions to CHMP7 (full-length or fragments) bound to glutathione agarose were incubated with full-length recombinant Lgd, and after extensive washing, proteins were eluted and separated by SDS–PAGE followed by staining using Coomassie (n = 3). Note that Lgd associates with the CHMP7 carboxyl-terminal domain (CHMP7C), but not its amino-terminal domain (CHMP7N).