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Fig. 3. GFP-MCPH1(S) and GFP-MCPH1(L) have different subcellular localisations during mitosis. (A,B), GFP-MCPH1(S) and GFP-MCPH(L) isoforms expressed in blastoderm stage embryos followed from interphase to subsequent interphase, show different localisations during mitosis. Time in minutes is shown at the top right of each panel. Bar, 10 µm. (A) After nuclear envelope breakdown GFP-MCPH1(S) is localised at the centrosomes, and faintly on the spindle (inset t=2:16, time in minutes:seconds). At later stages of mitosis, increased fluorescence is seen at the spindle poles, either representing increased protein at the spindle poles, or accumulation on decondensing chromosomes (t=2:42, arrow). (B) GFP-MCPH(L) is localised to the interphase nucleus, and this localisation is lost at the onset of mitosis. Several foci of GFP-MCPH1(L) then appear (t=2:38, representative nucleus, circled). These foci then appear to separate (t=3:03), and the fluorescent signal then expands to become decondensing. (C) In fixed blastoderm embryos nuclear GFP-MCPH1(S) staining is lost during prophase as DNA becomes histone H3 phosphorylated. (D) Anaphase and (E) telophase: GFP-MCPH1(S) reappears in decondensing telophase nuclei with loss of phosphoH3 staining. DNA (DAPI, blue), anti-GFP antibody (green), anti-phospho-histone H3 (red) in C,D and E. Bar, 10 µm.

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