Effect of cell shape on the orientation of the mitotic spindle.

The angle of mitotic spindle at metaphase was assessed in HeLa (GFP-H2B/RFP-tubulin or GFP-centrin-2/RFP-tubulin) cells cultured on the different platforms. To determine the orientation of the spindle parallel to the substrate plane, cells were imaged for microwell and cell shape (transmission channel, blue), DNA (green) and tubulin (red) after culture in (A) square microwells or (B) circular microwells; bars: 10 µm. Cells cultured (C) on 2D substrates or (E) within circular microwells possessed a random orientation of the mitotic spindle. (D) Conversely, a high proportion of the cells cultured in square microwells aligned the spindle along the long axis of the cell. To determine the orientation of the spindle perpendicular to the substrate plane, z stacks were reconstructed to obtain the xz view of the cell. Representative images are shown of cells with a (F) parallel versus (G) tilted orientation of the mitotic spindle; bars: 10 µm. (H) The angle of the spindle was dependent on whether cells were cultured on 2D homogenously coated substrates (black bars), or within square (light grey bars) or circular (dark grey bars) microwells. A higher percentage of cells cultured in circular microwells exhibited tilting of the mitotic spindle, in comparison to cells cultured in square microwells and on 2D substrates. (I) Furthermore, on average greater tilting was observed after cells were cultured in the circular microwells, while the mitotic spindle was aligned nearly parallel to the substrate in cells cultured in 2D or square microwells. Values represent spindle angle in degrees ± SEM. Key: *** p