Supplemental Figures

Materials and methods for Immunoelectron microscopy Samples for immunoelectron microscopy were prepared as described (Cooke et al., 1997). In short, the cells were extracted in PHEM (60 mM PIPES, pH 6.9, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2) containing 0.1% TritonX-100 for 1 min at room temperature before fixation with 0.1% glutaraldehyde for 10 min, and incubated with anti-Kid antibody followed by incubation with FluoroNanogold (Nanoprobe Inc.). Cells were post-fixed with 2% glutaraldehyde for 15 min, silver enhanced, and processed for electron microscopy as previously described (Suzuki and Hirosawa, 1994).

This article contains the following supporting material:

• Supplemental Figures 1-3 - Supplemental figure 1.

  Nocodazole treatment dose not rescue the short spindle phenotype A, HeLa cells stained for Kid (green in Merge panel), α-tubulin (red in Merge panel) and DNA (blue in Merge panel) after transfection with or without siRNA-Kid in the presence of 100nM nocodazole. B, Inter-centrosome distances in siRNA-Kid treated HeLa cells transfected with RNAi-refractory Kid mutant constructs. Cells were fixed and stained with antibodies against Kid and γ-tubulin, and the pole-to-pole distance was measured. Twenty metaphase cells were scored in each of three independent experiments. Error bars represent the standard deviation. *P<0.0005.

  Supplemental figure 2.

  Kid localized to the spindle microtubule bundles in prometaphase cells. To examine the localization of Kid in prometaphase cells in greater detail, we carried out immunoelectron microscopic analysis. Kid was labeled in prometaphase HeLa cells with 1.4-nm gold particles, silver enhanced, and observed by electron microscopy. A and C, Low magnification views of prometaphase HeLa cells. B, D and E, High magnification views of the area within the squares shown in A and C. The arrowheads in E indicate the Kid signals along an MT-bundle. The asterisks in A and C indicate a centrosome. Arrows in B and D indicate kinetochores. Bars represent 1 μm (A, C and F) and 500 nm (B, D and E). In prometaphase HeLa cells, particles indicative of Kid were found along spindle MTs as well as on chromosomes (A-E). At higher magnifications of chromosome regions, Kid signals were present on MT bundles growing from kinetochores (B and D), and also found along the MT-bundles (D and E). These Kid signals were almost never observed if the cells were stained using preimmune serum (F).

  Supplemental figure 3.

  The coiled-coil region is required for oligomerization of Kid. Lysates from HEK293T cells co-expressing GST-tagged Kid and truncation mutants of Flag-tagged Kid (Flag-Kid-WT, Flag-Kid-delDB, or Flag-Kid-delC, as indicated in Figure 5A), were subjected to GST-pull down followed by immunoblotting (IB) with anti-FLAG or anti-GST. Protein expression was confirmed by immunoblotting analysis of total lysate. The result that Kid-WT interacts with Kid-delDB but not with Kid-delC, suggests that the Kid’s coiled-coil region is required for oligomerization of Kid.