Fig. 4. Association of Smad2-Smad4 heterodimers with nuclear membranes in interphase cells and with chromatin in mitotic cells.

(A) Cells derived from an embryo expressing VC155-Smad2 and VNm9-Smad4 cultured in the absence of Activin. There are weak strands of BiFC fluorescence in the cytoplasm and around the nucleus. Blue staining derives from coexpressed ECFP lineage tracer that tends to accumulate slightly in the nucleus. (B) Strands of BiFC nuclear fluorescence in untreated animal pole cells are colocalised with nuclear membranes. Left-hand panel shows staining due to ECFP tagged emerin, middle panel shows BiFC fluorescence, and right-hand panel shows the merged image. (C) Cells identical to that in (A) but which have been treated with Activin. Note strong nuclear fluorescence. (D) Three images of a mitotic Xenopus animal pole blastomere derived from an embryo injected at the one cell stage with RNA encoding ECFP tagged Histone H2B (specific fluorescence visible in left-hand panel) and RNA encoding VNm9-Smad4 and VC155-Smad2 (specific fluorescence visible in centre panel). Note that Histone and BiFC fluorescence co-localise (right-hand panel). (E) Smad complexes are associated with chromatin during mitosis. The cell illustrated is derived from an embryo injected with BiFC constructs designed to reveal heteromeric interactions between molecules of Smad2Δexon3 and Smad4. Images are taken at intervals of 1.5 minutes. Note fluorescence associated with chromatin. Cells co-express an ECFP-GPI membrane marker. (F) Localisation of Smad-BiFC complexes to nuclear membranes and chromosomes is specific. Injection of RNA encoding the complementary VENUS fragments VC155 and VNm9 (200 pg of each, representing a 20 fold molar excess over concentrations used in Smad-BiFC experiments) reveals weak fluorescence in the cytoplasm and at the periphery of cells, but not in the nuclei or on chromosomes. Higher levels of fluorescence at the periphery of cells may be due to displacement of material by yolk platelets.