Figure 6

Mitotic defects in transfected HeLa and COS-7 cells expressing C -terminal ASPM GFP fusion proteins. A. Organisation of the 3477 amino acid human ASPM protein as described in the legend to Figure 1. C-terminal fragments expressed as fusion proteins with GFP are indicated D1-D3. Position of the C-terminal mutation (IVS25+1G > T) is indicated by an arrow. B. Panels a, b; HeLa cells expressing ASPM-D1-GFP immunostained to reveal GFP (green), α-tubulin (red) and DAPI (blue) then examined by confocal microscopy. Aggregates of fusion protein are indicated by arrows. Microtubules are disorganized and unfocused (arrowheads) and spindle poles cannot be identified. Scale bars = 5 μm. Panel c; HeLa cells expressing ASPM-D1-GFP immunostained to reveal GFP (green), γ-tubulin (red) and DAPI (blue). These abnormal cells possessed two spindle poles (arrows) and condensed DNA, suggesting mitotic arrest. Scale bar = 10 μm. Panels d-f; COS-7 cells expressing ASPM-D1-GFP immunostained to reveal GFP (green), α-tubulin (red) and DAPI (blue). Transfected cells were large and binucleate (panels e-f) or possessed abnormal lobular nuclei (panel d). Peripheral microtubule arrays or bundles were present (panels d-e, arrows). Panel f shows a binucleate cell containing a midbody (arrow). Scale bar = 20 μm. C. Aberrant cytokinesis in living COS-7 cells expressing ASPM-D2-GFP. Panel a; a binucleate COS-7 cell expressing ASPM-D2-GFP. Small aggregates of fusion protein can be seen. Panels b-d; COS-7 cells co-expressing ASPM-D2-GFP and EB3-GFP, used as a marker of microtubule organisation. Binucleate cells containing EB3-GFP positive midbodies (panels b and c, white arrows) or no midbody and a loose microtubule array (panel d, black arrow) are shown. Black arrow in panel c indicates the position of an incomplete and unusually asymmetrical cleavage furrow. White arrows in panel d indicate the position of centrosomes associated with the two nuclei, suggesting chromosomal segregation has occurred but cytokinesis has failed. In all panels asterisks indicate the location of nuclear structures. Images are shown in inverted grayscale for clarity. Scale bar = 10 μm.