Kif2b expression in cultured cells. (A) Total cell extract from untransfected U2OS cells (U2OS) or U2OS cells transfected with the plasmid encoding GFP-Kif2b (GFP-K2b U2OS) were separated by size by SDS-PAGE and blotted with GFP-specific antibodies (α-GFP) and the Kif2b polyclonal serum (α-Kif2b). (B) Extracts prepared from ∼10⁷ mitotic HeLa cells were precipitated with either preimmune serum (Pre-Imm IP) or Kif2b polyclonal serum (Kif2b IP) and separated into soluble (Supe) and antibody-bound bead (Beads) fractions. The entire pellet fraction, and only ∼1% of the supernatant and total cell extract fractions were separated by size by SDS-PAGE and blotted with Kif2b polyclonal serum. Positions of β-galactosidase (116) and phosphorylase B (97) are shown in kiloDaltons. (C) U2OS cells were transfected with the plasmid encoding GFP-Kif2b, fixed, and stained with DAPI and either preimmune serum or Kif2b polyclonal serum. Scale bar, 5 μm. (D) Fluorescence intensity for EGFP, MCAK, and Kif2B are reported as a ratio of tubulin fluorescence of transfected cells to that of untransfected cells within the same field of view (dark blue bars). The average GFP fluorescence in the transfected cells was measured for each transfected cell and arbitrarily normalized as an indication of the level of expressed transfected protein (light blue bars). Error bars, SDs for each dataset. The microtubule polymer level in the Kif2b transfected cells was significantly lower than that of EGFP control cells (p < 0.01), indicating that Kif2b is a microtubule depolymerizing kinesin. (E) Fluorescent intensities of tubulin polymer content in mitotic U2OS cells that were untreated, treated with 100 μM monastrol (+mon), or transfected with MCAK-, Kif2a-, and/or Kif2b-specific siRNA (Kif2b). Error bars, SDs.

Localization of GFP-Kif2b. (A) Human U2OS cells expressing GFP-Kif2b during interphase and at various stages of mitosis as indicated. Cells were stained for microtubules in red, DNA in blue, and GFP-Kif2b in green. (B) A prometaphase cell just after nuclear envelope breakdown is stained for DNA in blue, the kinetochore protein Hec1 in red, and GFP-Kif2b in green. The inset in the lower right corner is a 4× magnification of a kinetochore pair (white box). Scale bar, 5 μm.

Kif2b is essential for spindle bipolarity. (A) Untreated U2OS or U2OS cells transfected with Kif2b-specific siRNA were fixed and stained for microtubules in green and DNA in blue. Scale bar, 5 μm. (B) Quantification of the percentage of mitotic cells with bipolar spindles after transfection with a control siRNA (untreated) or Kif2b-specific siRNA. Error bars, SD. (C) Total cell extracts prepared from GFP-Kif2b expressing U2OS cells that were either untreated or transfected with Kif2b-specific siRNA were separated by size by SDS-PAGE and blotted with antibodies specific to GFP and actin as indicated.

Chromosome velocities are suppressed in Kif2b-deficient cells. (A) U20S cells were treated with monastrol or transfected with Kif2a- or Kif2b-specific siRNA as indicated and imaged by time-lapse DIC microscopy during mitosis. Times are shown in seconds. White arrows identify individual chromosomes over time, and the asterisk in the Kif2b RNAi cell denotes the position of a centrosome as judged by GFP-tubulin distribution (not shown). (B) The position of two individual chromosomes relative to the spindle pole were tracked over time in mitotic cells treated with monastrol or transfected with Kif2a- or Kif2b-specific siRNA as indicated.

Kif2b-deficient cells form stable kinetochore microtubule attachments. U2OS cells were either untreated or transfected with Kif2b-specific siRNA as indicated. Cells were extracted in buffer containing 1 mM calcium for 4 min before fixation to induce depolymerization of nonstable microtubules. Microtubules are shown in green, DNA in blue, and kinetochores (Hec1 staining) in red. The insets in the top right corners of each panel are 3× magnifications of kinetochore pairs (white box). Scale bar, 5 μm.

Cytokinesis is impaired in Kif2b-deficient cells. Time-lapse DIC microscopy of Kif2b-deficient cells with either monopolar (A) or bipolar (B) spindles failing to complete cytokinesis. Times are in hours:minutes.

Kif2a, Kif2b, and MCAK have distinct functions during mitosis. (A) Percentage of mitotic cells with bipolar spindles in cells transfected with MCAK-, Nuf2-, or Kif2a- and Kif2b-specific siRNA are represented by black bars. Percentage of mitotic cells with bipolar spindles in cells transfected with Kif2a- (dark blue bars) or Kif2b-specific (light blue bars) alone or in combination with other treatments including transfection with MCAK- or Nuf2-specific siRNA, treatment with 100 nm nocodazole (100 nm Noc), or injection with antibodies specific to NuMA and HSET (N/H Ab inj). (B) U2OS cells were fixed and stained for microtubules in green and DNA in blue. Cells were either untreated or transfected with MCAK-, Kif2a-, or Kif2b-specific siRNA. Subsequent to transfection, cells were either fixed without injection (uninjected) or injected with NuMA- and HSET-specific antibodies (NuMA/HSET Ab inj). Scale bar, 5 μm.

Phylogenetic comparison of kinesin-13 genes. Amino acid sequences from three kinesin-13 proteins encoded by fruit fly (Dm), mouse (Mm), and human (Hs) are compared using Clustal X. The sequence of yeast Kip3 is used as an outgroup to root the tree. Line lengths are not to scale.