Figure 1

Organisation of the ASPM protein and its cellular distribution during mitosis. A. Structure of the 3477 amino acid human ASPM protein. The region corresponding to the microtubule-binding domain of Drosophila Asp [24] is shown in white. The calponin homology domains (aa920-1261) are in light grey; ASNP repeats are dark grey boxes (aa316-342, 366-400); 81 IQ domains (aa1267-3225) are shown as vertical stripes; the armadillo repeat-like domain (aa3294-3327) is a black box; and the C-terminal region is depicted by diagonal stripes. The location of potential nuclear localization sequences are indicated by asterisks and the location of the peptides used to raise polyclonal antibodies, by dashed arrows. B-F. Analysis of ASPM distribution following immunostaining. HeLa cells were fixed and stained with antibodies specific for ASPM (green), anti-α-tubulin (red) and with DAPI (blue) to identify nuclei. Panels B-E utilised Anti-ASPM 216-1, whilst anti-ASPM 279-3 was used in panel F. B. ASPM is predominantly nuclear in interphase cells. Scale bar = 10 μm. C. ASPM is localised to the spindle poles during metaphase. Scale bar = 5 μm. D. A globular distribution of ASPM (green) is seen around the γ-tubulin (red) immunopositive core of metaphase HeLa cell spindle poles. DNA (DAPI staining) is shown in blue (confocal image). Scale bar = 2.5 μm. E. Single 0.5 μm confocal section through the centre of a telophase HeLa cell immunostained to reveal ASPM (green) and α-tubulin (red). In addition to broad spindle pole-associated labelling (arrowhead), ASPM also localizes to the minus ends of central spindle microtubules (arrow). Scale bar = 2.5 μm. F. A late telophase fibroblast immunostained with anti-ASPM 279-3 (green), anti-α-tubulin (red) and with DAPI (blue). ASPM is predominantly localized at the midzone of the central spindle. Scale bar = 10 μm. G. Immunoblotting of cell lysates of COS7 cells (lane 1), U2OS cells (lane 2), primary fibroblasts (lane 3) and HeLa cells (lane 4) with anti-ASPM 217-2 antibody. This identifies a protein of approximately 410 kDa in each lane. A blot stained with anti-β-actin is shown as loading control.