Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - Summary of RNAi phenotypes of five mitotic kinesins studied in this paper

  Schematic representation of the spindle phenotypes observed after RNAi of five kinesins in S2 cells (Goshima and Vale, 2003). Klp61F RNAi leads to mitotic arrest with monopolar spindles. Cells depleted of Klp10A, a microtubule depolymerase, often produce monopolar spindles with longer astral microtubules. However, monopolar spindles are eventually converted to bipolar spindle by chromatin-mediated, acentrosomal pole formation. Anaphase and cytokinesis takes place in this asymmetric spindle. RNAi of Klp67A, another type of microtubule destabilizer, leads to monopolar spindle formation with longer kinetochore microtubules and to bipolar conversion. However, unlike the case of Klp10A, chromosomes are not congressed in the bipolar spindle, and anaphase entry is inhibited. RNAi of Ncd, a minus-end directed motor, causes defocusing of microtubules at the pole and produces multi-polar spindles. Anaphase chromosome segregation and cytokinesis take place in the multiple spindles. Finally, central spindle bundling during cytokinesis is impaired after Pav RNAi. Quantitation of each phenotype is also described in Goshima and Vale (2003).

• Supplemental Figure 2 - Rescue of spindle phenotypes by expression of GFP-tagged kinesins

  (A) Rescue assay for Ncd-GFP cell line. (left) Immunoblot demonstrates specific knockdown of endogenous Ncd by its UTR RNAi, and expression of Ncd-GFP. (right) Metaphase spindles with unfocused poles or multiple poles were frequently seen after RNAi, and this defect was rescued by moderate expression of Ncd-GFP that localizes to the spindle, indicating that Ncd-GFP could functionally replace endogenous Ncd (see also Table 1). (B) Rescue assay for Klp10A-GFP cell line. (left) Immunoblot demonstrates specific knockdown of endogenous Klp10A by its UTR RNAi, and expression of Klp10A-GFP. (right) Almost all of the bipolar spindles have abundant and clearly longer microtubules after RNAi, and moderate expression of Klp10A-GFP, which targeted to spindle poles and centromeres, rescued the defect, indicating that Klp10A-GFP could functionally replace endogenous Klp10A for spindle formation (see also Table 1). (C) Rescue assay for Klp67A-GFP cell line. (left) Immunoblot demonstrates specific knockdown of endogenous Klp67A by its UTR RNAi, and expression of Klp67A-GFP. Asterisks indicate contamination bands detected by anti-Klp67A serum. (right) Almost all of the mitotic cells had long pre-anaphase spindles with curly interpolar microtubules, and expression of Klp67A-GFP, which targeted to kinetochores, rescued the defect, indicating that Klp67A-GFP could functionally replace endogenous Klp67A (see also Table 1). (D) Rescue assay for Pav-GFP cell line. (left) Phase contrast images. Upon UTR RNAi treatment, the majority of the control cells (no Pav-GFP transfection) became larger than normal because of the cytokinesis failure, whereas many normal-sized cells were detected when RNAi was applied for Pav-GFP cell lines. (right) Strong correlation was found between Pav-GFP expression and successful cytokinesis (detected as interphase cells with single nucleus), indicating that Pav-GFP could functionally replace endogenous Pav for cytokinesis (see also Table 1). Red; tubulin. Green; GFP. Blue; DNA. Bars, 10 μm.

• Supplemental Figure 3 - Live cell localization of moderately expressed kinesin-GFP in the presence of endogenous kinesins

  Localization of five GFP-tagged mitotic kinesins in interphase (left), metaphase (middle) and late anaphase (right). Cells stably expressing kinesin-GFP (white) were put on a Con-A plate to spread, and images were taken without fixation by wide-field microscope. DNA was stained by Hoechst 33342 (blue). Localization pattern of each kinesin-GFP was identical to those observed after RNAi knockdown of endogenous proteins (compare with Figure 2). Bar, 10 μm.

• Supplemental Figure 4 - Spindle localization of Klp61F-GFP and Ncd-GFP depends upon the presence of microtubules

  Mitotic Klp61F-GFP (A) and Ncd-GFP (B) localization in the presence or absence of colchicine, a microtubule inhibitor. Spindle localization of these kinesins were undetected in the absence of microtubules. Instead, whole cell diffusion or "nuclear" like accumulation was detected. The basis of the "nuclear" like localization after NEB is unclear. However, similar accumulation was also seen for GFP-tubulin (not shown), suggesting that NEB might not occur completely in this cell line. Red; tubulin. Green; GFP. Blue; DNA. Bars, 10 μm.

• Supplemental Figure 5 - Localization of endogenous Ncd and Klp67A by immunofluoresence using specific antibodies

  (A)Immunostaining of Ncd by anti-Ncd polyclonal antibody for the cells treated with (right) or without (left) RNAi. Identical exposure time and image processing were applied to each sample. Int; interphase. M; mitosis. Bar, 10 μm. Nuclear accumulation in interphase and spindle/centrosome enrichment in metaphase of endogenous Ncd was confirmed. Blue; DNA. Green; Ncd. (B) Immunostaining of Klp67A by anti-Klp67A polyclonal antibody for the cells treated with (right) or without (left) RNAi. Nuclear accumulation in interphase (a), kinetochore localization during metaphase (b) and early anaphase (c), and central spindle enrichment during anaphase (c, d) were confirmed for endogenous Klp67A. (b') represents an enlarged image of (b), and weak spindle localization of Klp67A is also detected during metaphase. Bars, 10 μm (a-d) and 2 μm (b').

• Supplemental Figure 6 - Distinct localization of Klp67A and Klp10A at the kinetochore in metaphase

  Immunostaining of (A) Cid, a centromere specific histone H3 variant (Henikoff et al. , 2000), and (B) Klp10A, an inner-centromere kinesin (Rogers et al., 2004), in metaphase chromosomes (blue) in a Klp67A-GFP expressing cell line. Klp67A-GFP (green) was located slightly (Cid) and far (Klp10A) peripheral to known inner-kinetochore markers (red) in the outer region of kinetochores. Bars, 1 μm.

• Supplemental Movie 1 - Klp67A-GFP dynamics from metaphase to telophase. Images were taken every 5 sec by spinning-disk confocal microscope.
• Supplemental Movie 2 - Klp10A-GFP dynamics in prophase and prometaphase. Images were taken every 5 sec by spinning-disk confocal microscope.
• Supplemental Movie 3 - Klp10A-GFP dynamics in metaphase and anaphase. Images were taken every 2 sec by spinning-disk confocal microscope.
• Supplemental Movie 4 - GFP-tubulin dynamics from metaphase to late anaphase in an untreated cell. Images were taken every 5 sec by spinning-disk confocal microscope.
• Supplemental Movie 5 - GFP-tubulin dynamics from metaphase to late anaphase in a Klp10A RNAi cell. Images were taken every 5 sec by spinning-disk confocal microscope.
• Supplemental Movie 6 - GFP-tubulin dynamics from early to late anaphase in a BubR1 RNAi cell. Images were taken every 5 sec by spinning-disk confocal microscope.
• Supplemental Movie 7 - GFP-tubulin dynamics from mid to late anaphase in a double Klp67A/BubR1 RNAi cell. Images were taken every 5 sec by spinning-disk confocal microscope.
• Supplemental Table