FIG. 5.

Fine timing of MCAP chromosome staining. (A) P19 cells were stained with anti-MCAP antibody, Hoechst 33342, and anti-β-tubulin antibody. Arrows in prophase indicate condensing chromosomes. At this stage, MCAP distribution is uniform over the entire nucleus. In prometaphase, centrioles move toward the opposite poles (arrowheads), the nuclear membrane breaks down, and chromosome condensation increases (arrow). At this stage, chromosomes begins to be stained with MCAP antibody (arrow). During metaphase, MCAP is found entirely on fully condensed chromosomes that were assemble on the metaphase plate. MCAP remains on chromosomes in anaphase, when they are pulled apart in two daughter cells. The bar corresponds to 3.5 μm. (B) Colocalization analysis with phospho-histone H3. P19 cells were stained with antibody to phospho-histone H3, MCAP, or Hoechst 33342. In G₂ and prophase, phospho-histone H3 localizes on the pericentric heterochromatin regions, which condense early and are seen as large dots (arrows in G₂ and prophase). MCAP is uniformly distributed over the nucleus at these stages. When cells reach prometaphase and move from metaphase to anaphase, phospho-histone H3 staining spreads over the entire, fully condensed chromosomes, overlapping MCAP staining (arrows).