Supplemental Material

This article contains the following supporting material:

• Video 1 - The dynamic localization of DdINCENP during mitosis. Time-lapse video of GFP-DdINCENP fluorescence in a mitotic wild type cell. DdINCENP was localized on the centromeres before it transferred to the spindle poles and spindle at the onset of anaphase. Frames were acquired every 15 s and are shown at 2 frames/s. Also see Figure 1.
• Video 2 - DdINCENP localized at the cleavage furrow during cytokinesis. Time-lapse video of GFP-DdINCENP fluorescence in a wild type cell during cytokinesis. DdINCENP began to transfer to the cortex region of the cleavage furrow at the beginning of the cytokinesis and was highly concentrated on the cytoplasmic bridge during late cytokinesis. Frames were acquired every 15 s and are shown at 2 frames/s. Also see Figure 2.
• Video 3 - The depletion of DdINCENP sometimes caused mitotic arrest at metaphase. Time-lapse video of GFP-Histone 2B fluorescence in a mitotic DdINCENP null cell in metaphase. The mutant cell was not able to enter anaphase during the filming period (9 minutes). In Dictyostelium cells, the average time from early prophase to the start of anaphase is only about 5 minutes. Frames were acquired every 15 s and are shown at 2 frames/s.
• Video 4 - The depletion of DdINCENP caused chromosome segregation defect. Time-lapse video of GFP-Histone 2B fluorescence in a mitotic DdINCENP null cell starting from metaphase. Although the mutant cell was able to enter anaphase, it had lagging chromosomes during the segregation process (compared with Video 5). Frames were acquired every 15 s and are shown at 2 frames/s. Also see Figure 4.
• Video 5 - The chromosome segregation in wild type cells. Time-lapse video of GFP-Histone 2B fluorescence in a mitotic wild type cell. The cell segregated the chromosomes completely in less than 2 minutes. Frames were acquired every 15 s and are shown at 2 frames/s. Also see Figure 4.
• Video 6 - The localization of DdINCENP in the cleavage furrow was affected by the absence of myosin II. Fluorescence imaging of GFP-DdINCENP in a myosin heavy chain null cell undergoing traction-mediated cytokinesis (cytokinesis B). This Z-series video starts at a focal plane near the top of the cell and moves toward the cell-substrate contact. GFP-DdINCENP formed a very narrow band at the equator of the cell most prominent at the top of the cell and absent at the bottom of the cell. This distribution contrasts sharply with the broad cortical localization of DdINCENP in the cleavage furrow of wild-type cells (see Video 2). Optical sections were imaged every 0.5 μm.
• Video 7 - The localization of DdINCENP in the cleavage furrow was affected by the absence of myosin II. The same cell shown in Video 6 was subsequently imaged by time-lapse video microscopy. The very narrow band of GFP-DdINCENP at the top of the myosin null cell is seen to contract following the cleavage furrow. Frames were acquired every 15 s and are shown at 2 frames/s.
• Figure 1 - Domain organization of INCENP proteins and alignment of the IN-Box domain.

  (A) Diagram indicating the relative size of INCENP proteins from several organisms. The shaded box near the C-terminus of all INCENPs represents the conserved IN-Box domain. The hatched portions indicate regions with a high likelihood to form a coiled-coil domain as shown by the Protean Software (Lasergene). (B) Alignment of the IN-Box domain of INCENPs from different organisms. Residues highlighted in red indicate those that are identical with the Dictyostelium DdINCENP sequence. Residues highlighted in blue indicate those that are conserved in at least 4 species other than Dictyostelium. Asterisks indicate the Serine residues phosphorylated by Aurora B (Bishop and Schumacher, 2002)

• Figure 2 - DdINCENP Co-immunoprecipitates with DdAurora kinase.

  DdAurora kinase was immunoprecipitated with anti-DdAurora rabbit antisera from whole cell extracts following standard protocols. A mock immmunoprecipitation was carried out without the antisera as control. The immunoprecipitates were analyzed by Western blot analysis probed with an anti-DdINCENP antiserum. The first two lanes show whole cell extracts from wild-type and DdINCENP null cells. The asterisk indicates the position of a background nonspecific band. The western blot shows that DdINCENP coprecipitates with DdAurora.

• Figure 3 - Overexpression of DdCP224, a TOGp/XMAP215 homolog, only partially rescues the growth defect of DdINCENP null cells.

  AX2 (diamond), DdINCENP null (square) and DdINCENP null expressing DdCP224-GFP (triangle) cells were placed in suspension cultures and their cell titers monitored daily. Although overexpression of DdCP224-GFP made the DdINCENP null cells grow faster, these cells still lagged behind the wild type (AX2) cells in growth.

• Figure 4 - The mitotic spindle localization of DdINCENP is not dependent on Myosin II.

  Fluorescent microscopy images of GFP-DdINCENP expressed in myosin II heavy chain null cells. DNA is shown in blue and GFP-DdINCENP is shown in green. As in wild type cells (see Figure 1), DdINCENP localizes in the middle of chromosome congregation during metaphase (A), and is found on both central spindle and spindle pole bodies at anaphase (B). Bar = 5 μm.