Walczak CE , Verma S , Mitchison TJ .

F32 GM016100 NIGMS NIH HHS

	Figure 2. XCTK2 contains a COOH-terminal motor domain and binds microtubules. (A) A schematic representation of the predicted structure of XCTK2. XCTK2 contains an NH2-terminal globular domain, a central α-helical stalk and a COOH-terminal motor domain. (B and C) Immunoblots of microtubule pelleting assays in Xenopus egg extracts. (B) Xenopus egg high speed supernatants (lane 1) or AMP-PNP microtubule pellets (lane 2) were probed with anti-CTP1 antibody. The slight band shift is due to the high amounts of protein present in the high speed supernatant that alter the migration of XCTK2. (C) Microtubules were polymerized in mitotic high speed supernatants of Xenopus egg extracts and pelleted in the absence of ATP and the presence of AMP-PNP (lanes 1 and 2). The microtubule pellet, with associated proteins, was resuspended and extracted with various nucleotides and salt to generate supernatants (S) and pellets (P) of each extraction condition: 2 mM Mg–ATP (lanes 3 and 4); 0.5 M NaCl (lanes 5 and 6); 1 M NaCl (lanes 7 and 8), or 2 mM Mg–ATP with 0.5 M NaCl (lanes 9 and 10). The blots were probed with anti-XCTK2 antibody.
	Figure 3. Immunolocalization of XCTK2 in Xenopus tissue culture cells. The cells were stained with 1 μg/ml anti-XCTK2 followed by FITC-conjugated goat anti–rabbit secondary antibody. DNA was visualized by staining with 0.05 μg/ml propidium iodide. Images were recorded on a laser scanning confocal microscope. Interphase (I); prophase (P); prometaphase (PM); metaphase (M); anaphase (A); telophase (T). Bar, 20 μm.
	Figure 4. XCTK2 is important for mitotic spindle assembly. Mitotic spindles were assembled in the presence of either (A) 95 μg/ml control IgG, (B) 95 μg/ml anti-CTP1, or (C) 95 μg/ml anti-XCTK2. Spindles were assembled in CSF extracts that contained rhodamine-labeled tubulin and antibody for 60 min and then sedimented onto coverslips. In the presence of antibodies that inhibit XCTK2 function, there is a decrease in the percentage of bipolar spindles that formed. Six random structures were photographed from five fields of view, and they were compiled into the field of view that is shown. To compare between panels, count the number of bipolar spindles versus half spindles in each panel. Rhodamine-labeled microtubules are shown. Bar, 20 μm. (D) Quantitation of XCTK2 immunoinhibition. The percentage of half spindles, bipolar spindles, and aster-like structures that formed per nuclei were quantitated. The data are represented as the percentage of bipolar spindles that formed, normalized to the IgG control addition as 100%. The bars represent mean ± 1 SD (n = 1,020 nuclei for IgG addition [four experiments]; n = 965 nuclei for anti-CTP1 addition [four experiments]; n = 1,007 nuclei for anti-XCTK2 addition [four experiments]).
	Figure 5. Anti-XCTK2 antibodies cause relocalization of XCTK2. Mitotic spindles were assembled in the presence of rhodamine-labeled tubulin in cycled CSF extracts for 60 min and then sedimented onto coverslips. (Top) Spindles were assembled, sedimented, fixed, and then stained with antiXCTK2 antibodies, followed by FITC-conjugated goat anti–rabbit secondary antibody. DNA was visualized by staining with Hoechst 33528. XCTK2 stains the spindle with an enrichment toward spindle poles. (Bottom) Spindles were assembled in the presence of anti-XCTK2 antibody, sedimented, fixed, and then stained with FITC-conjugated goat anti–rabbit secondary antibody. DNA was visualized by staining with Hoechst 33528. XCTK2 is now concentrated at spindle poles after assembly in the presence of anti-XCTK2 antibodies. Bar, 20 μm.
	Figure 6. Purification of XCTK2. XCTK2 was expressed in insect Sf-9 cells using the baculovirus expression system. Lysates were made of infected Sf-9 cells, centrifuged and filtered through an HPLC syringe filter, run on a SP-Sepharose column (Hi-TrapTM; Pharmacia Fine Chemicals) and eluted with a linear 100–500 mM NaCl gradient. The peak fractions were pooled, concentrated, and run on a Superose 6 gel filtration column. The peak fractions were pooled, sucrose was added to 10% final wt/vol, and they were frozen in 15 μl aliquots at −80°C until use in the experiments described. Fractions from the purification were run on 10% SDS-PAGE and visualized by staining with Coomassie. (Ld) SPSepharose column load. (FT) SP-Sepharose column flow-through. (SP) Pool of peak XCTK2 containing fractions from SP-Sepharose column. (Sup6) Pool of peak XCTK2 containing fractions from Superose 6 column. Molecular mass markers are shown on the left side of the gel.
	Figure 7. Addition of excess XCTK2 stimulates mitotic spindle assembly. Mitotic spindles were assembled in the presence of either (A) control buffer, (B) ∼10 μg/ml XCTK2, or (C) ∼10 μg/ml XCTK2 that had been heat killed for 5 min at 70°C before use. Spindles were assembled in CSF extracts that contained rhodamine-labeled tubulin and buffer or XCTK2 protein for 30 min and then sedimented onto coverslips. In the presence of excess XCTK2 protein, there is an increase in the percentage of bipolar spindles that formed. Six random structures were photographed from four to six fields of view, and they were compiled into the field of view that is shown. Rhodamine-labeled microtubules are shown. (D–F) Quantitation of excess XCTK2 addition. Spindles were assembled in CSF extracts in the presence of control buffer (black bar), ∼10 μg/ml XCTK2 (hatched bar), or ∼10 μg/ml heat-killed XCTK2 (cross-hatched bar) for 15–60 min and then sedimented onto coverslips. The percentage of asters, directed asters, and half spindles were quantitated at the 15 min time point, and the percentage of half spindles, bipolar spindles, and other structures that formed per nuclei were quantitated at the 30 and 60 min time points. The data are represented as mean ± 1 SD of four independent experiments (n ⩾ 800 nuclei for IgG addition; n ⩾ 800 nuclei for anti-CTP1 addition; n ⩾ 800 nuclei for anti-XCTK2 addition for each time point). Bar, 20 μm.
	Figure 8. Immunoprecipitation of XCTK2 from egg extracts. Immunoprecipitation was performed using control rabbit IgG (lane 1), affinitypurified anti-CTP1 (lane 2), or affinity-purified anti-XCTK2 (lane 3). Antibody was bound to protein A beads and then incubated in Xenopus egg high speed supernatants that contained ∼10 μg/ ml XCTK2. The protein A complexes were washed, separated by 7.5% SDS-PAGE, and visualized by staining with Coomassie.
	Figure 9. Model for XCTK2 function in spindle assembly. XCTK2 (lollipops) is required to bundle microtubules in each half spindle. A certain amount of bundling of microtubules is necessary for efficient fusion of half spindles to occur (top diagram). In the presence of anti-XCTK2 antibodies (lower diagram), the protein becomes mislocalized toward the spindle poles, and the spindles splay and are incapable of forming bipolar spindles.

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