Xenbase Image ID: 126096

Fig. 2. Developmentally regulated expression and subcellular localization of XLAP2 isoforms in Xenopus. a Immunoblot analysis of XLAP2 proteins during development. Positions of three major protein bands representing XLAP2 isoforms are marked together with an actin band used as a loading control. The embryonic XLAP2 proteins, viz. XLAP2ω and XLAP2γ, are the only two isoforms present up to gastrula stage, showing an accumulation of the amount of protein until stage 22 followed by a steady decrease up to stage 41 and its disappearance from stage 44 (a, right). Expression of the somatic XLAP2β isoform is first detected at the gastrula stage and increases significantly from the 28th stage. The additional protein band of 76 kDa may be a degradation product of the 86-kDa isoform. Molecular masses of reference proteins (in kDa) are marked. b–f Subcellular localization of XLAP2 protein isoforms during Xenopus development and in XTC cells. The paraffin sections from Xenopus embryos and adult tissues were probed for XLAP2 (b, c) or costained for XLAP2 (red) and either lamin B2/B3 (d) or lamin B2 (e, f; green). DNA was stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Images were via a fluorescent (b) or confocal laser scanning (c-f) microscope. Images represent the Z-stack (c) or single Z-section (b’, d-f; 0.25 μm thick). b During early development, XLAP2 proteins colocalize with mitotic chromosomes in the morula and blastula in which XLAP-86 and XLAP2-40 are expressed and with the interphase nuclei of the gastrula and stage-20 embryos in which XLAP-86, XLAP2-66 and XLAP2-40 are present (arrowheads positions of cell nuclei, arrow position of XLAP2). Inset top right of morula merged image in b Higher magnification of the boxed nucleus. The localization of XLAP2 in the nuclear envelope (NE) is rare in blastula embryos (note the fragments of NE in b’) but becomes a common feature at the gastrula stage (see well-organized membranes in b’). Embryonic samples show autofluorescence (mostly from the yolk platelets) that reveals the shape and size of each cell (e.g. easily seen in red and blue channels from morula to gastrula stage). c In the morula stage embryos, XLAP2-containing fractions are associated with mitotic chromosomes (c.1) and karyomere formation is seen around individual chromosomes (c.2). Note the position of embryonic XLAP2 in membrane-like structures on the morula chromatin (arrow), the typical cluster-like location on the chromatin (arrowhead) and the XLAP2 cluster outside the chromatin (double arrowhead). In somatic cells, XLAP2β aggregates in between separating chromatids at early anaphase (c.3) and then associates with peripheral regions of chromatin in late anaphase (c.4; arrows peripheral regions of chromatids with associated XLAP2, arrowheads chromosome core regions not yet associated with XLAP2). d–f The XLAP2 proteins colocalize with B-type lamins in stage 26-28 (d) and stage 44 (e) Xenopus embryos and in adult tissues (f). Images represent brain tissue. Bars 100 μm (b, morula), 20 μm (b, blastula – stage 20), 5 μm (c), 20 μm (d), 10 μm (b’, e, f) Image published in: Chmielewska M et al. (2011) Image downloaded from an Open Access article in PubMed Central. © The Author(s) 2011 Larger Image Printer Friendly View

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