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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 co-expressed 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 of
ECFP-tagged emerin, middle panel shows BiFC fluorescence, and right-hand panel
shows the merged image. (C) Cells identical to those in A but treated
with Activin. Note strong nuclear fluorescence. Arrows indicate spots referred
to in text. (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 colocalise
(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 Smad2
exon3 and Smad4. Images were 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. Arrows indicate mitotic chromatin. Higher
levels of fluorescence at the periphery of cells may be due to displacement of
material by yolk platelets.
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