Slk19p–GFP movement in live cells. (A) Cells were visualized by epifluorescence microscopy at the indicated times. Time zero is an arbitrary starting point. Arrows indicate the origin of the third spot of Slk19–GFP staining. The starting and ending DIC images are shown. Growth of the bud indicates these cells remain viable over the recorded time period. (B) The distances between the two poles, brighter pole and center spot, and dimmer pole and center spot of Slk19-GFP fluorescence are shown for a single anaphase cell. The measurements were made twice with similar results.

Mt phenotypes of SLK19 mutants. (A) Disruption of SLK19 leads to reduced spindle Mts and increased cytoplasmic Mts. Fixed cells were treated for antitubulin immunofluorescence (Materials and Methods). Spindles are visible as a bright fluorescent bar between fainter staining cytoplasmic Mts. To compare kar3Δ and slk19Δ mutants at the same point in the cell cycle, we imposed an HU arrest for 3.5 h. HU blocks progression through S phase, but in S. cerevisiae, allows spindle assembly (Mitchison and Carter 1975). Shown is a composite image of representative cells from the same sample. Bar, 5 μm. (B) In a separate experiment, wild-type (wt), slk19Δ, and kar3Δ cells were arrested with HU for 3.5 h and immunostained with antibodies to the SPB protein, Spc90p (Rout and Kilmartin 1990). Bar, 5 μm. (C) The lengths of 80 randomly picked spindles were measured and the numbers of cytoplasmic Mts for 400 randomly picked spindles from wild-type, slk19Δ, and kar3Δ cells were counted. SEM represented by error bars.

Immunolocalization of Slk19p–GFP and Kar3p–GFP fusion proteins. (A) Cells with SLK19:GFP integrated into the genome were fixed and stained with anti-GFP, antitubulin antibodies, and DAPI. The Slk19p is located on the spindle, near the poles, and at the spindle midzone of elongating spindles. Rarely, Slk19p staining can be seen along the length of the short spindle as shown. Bar, 5 μm. (B) Cells with integrated SLK19:GFP were treated for immunoelectron microscopy with antibodies to GFP. The white arrows point to the edges of the SPB and the nuclear side is labeled N. (C) The midzone cluster of Slk19p staining is shown on an elongated anaphase spindle. White arrowheads point to the midzone staining, black arrowheads to Slk19p at the poles, and white arrows to the edges of the SPB. (D) Chromosomal association of Slk19p. A diploid cell with homozygous integrated SLK19:GFP was grown in sporulation medium for 28 h. The cells were then spheroplasted to remove the cell wall, and lysed in low ionic strength solution. The lysate was allowed to attach to glass slides, fixed with paraformaldehyde, and stained with antibodies to GFP and with DAPI. Two examples are shown. (E) Immunoelectron microscopy localization of Kar3p. Cells with integrated KAR3:GFP were treated for immunoelectron microscopy with antibodies to GFP. The black arrows point to the edges of the SPB and the nuclear side is labeled N.

Coimmunoprecipitation of CEN DNA and Slk19p–GFP. Cross-linked chromatin was prepared from strains WSY990, integrated SLK19:GFP (A); and JKY292, integrated KAR3:GFP (B), and then immunoprecipitated with no antibody (lane 2), anti-GFP (lanes 3 and 4, performed in duplicate), or anti-Cbf2p antibodies (lane 5). Total input DNA (lane 1) and coimmunoprecipitated DNA were analyzed by PCR using primers specific to CEN3, CEN16, ARS2, or MET2 (Materials and Methods). The PCR products were run on 2% agarose gels and stained with ethidium bromide. Sizes of the PCR products are shown in parentheses.

Loss of function of both KAR3 and SLK19 in the same cell leads to a severe mitotic arrest as large budded cells with a single nucleus. (A) The strain kar3Δ slk19Δ pkar3-ts was grown to log phase at 26°C and then switched to 35°C for 3 h. The cell cycle arrest was monitored by DAPI staining after 70% ethanol fixation. Arrest in G2/M was indicated by an increase in the number of cells with a large bud and a single nucleus. 300 randomly picked cells were counted. (B) The kar3Δ slk19Δ pkar3-ts cells at 35°C from A were fixed and treated for antitubulin immunofluorescence and DAPI staining. (C) In a separate experiment, kar3Δ slk19Δ pkar3-ts cells grown at 35°C for 3 h were immunostained with antibodies to the SPB protein, Spc90p (Rout and Kilmartin 1990). Bar, 5 μm.

(a) Spindle collapse in the slk19 kar3 double mutant. slk19Δ kar3Δ pkar3-ts and control cells were arrested in YEPD with 0.1 M HU for 3.5 h. The cells were then transferred to 35°C while still in HU. After 20 min at 35°C, the cells were resuspended in fresh medium without HU at 35°C, and returned to a shaking water bath at 35°C. At the selected times, cells were fixed and stained with antibodies to tubulin (white) and spindle poles (red). A, B, and C are wild-type cells; D, E and F are slk19Δ kar3Δ pkar3-ts mutants. Total time at 35°C is: 0 min, A and D; 20 min, B and E; and 100 min, C and F. (b) Cbf2p–GFP is found at the spindle midzone. A CBF2:GFP hybrid gene was integrated into a wild-type genome and the position of the fusion protein in live cells recorded. The staining was similar to that reported previously in fixed cells (Goh and Kilmartin 1993), except that additional labeling at the spindle midzone also was noted. GFP is shown in green and the corresponding DIC image on its right.

Synthetic lethality of kar3Δ slk19Δ and cin8Δ slk19Δ combinations. (A) The indicated strains were transformed with plasmids containing URA3 and CIN8 or KAR3, and plated onto YEPD and 5-FOA plates at 30°C for 2 d. (B) Benomyl, but not a KIP2 deletion, could suppress the growth defect of kar3 and slk19 mutants. The strains were plated onto YEPD plates at 26°C or 34°C (except dyn1Δ kar3Δ pkar3-ts, which requires 35°C to show an effect of benomyl) for 48 h with (Y + Ben) or without 5 μg/ml benomyl. wt, wild-type.