Separation-of-function slk19 alleles show consistent interactions with BIM1 or CLA4 deletions. (A) Haploid strains lacking BIM1, containing a deletion allele of slk19 or spo12Δ for a FEAR⁻ control and also carrying pRK44 [YCp–SLK19∷URA3] were streaked to single colonies on YPAD and replica plated onto 5-FOA–containing plates. Strains: slk19Δ, MATa (dKH274-1.4b) and MATα (dKH274-1.9d), slk19–ΔF, MATa (dKH268-7.7d) and MATα (dKH268-2.8b), slk19–ΔA, MATa (dKH262-6.4b) and MATα (dKH262-6.10b), and spo12Δ, MATa (dKH280-5.3b) and MATα (dKH280-5.7a). (B) Haploid cla4Δ strains carrying pRK44 [YCp-SLK19∷URA3] were grown to stationary phase in YPAD medium, serially diluted 10-fold in water, spotted onto 5-FOA medium, and grown at 30°. Strains: SLK19 (TKH223) and slk19Δ (dKH272-3.3a). (C) Ura⁻ (therefore they do not contain the SLK19 expressing plasmid), cla4Δ strains also containing a slk19 partial deletion (or spo12Δ, FEAR⁻ control) were isolated from tetrad dissection. These strains were serially diluted 10-fold in water, spotted onto YPAD medium, and allowed to grow at 34° or 24°. Strains: SLK19 (TKH223), slk19–ΔA (dKH260-5.1a), slk19–ΔF (dKH266-6.2c), and spo12Δ (dKH278-5.3b).

slk19 separation-of-function mutants display disrupted anaphase spindle elongation independent of FEAR defects. (A) Example of WT elongation: an otherwise wild-type strain expressing GFP–TUB1 (dKH160-1.2a) was arrested in α-factor, trapped in a microfluidic plate from CellASIC, and imaged every 30 sec while flowing complete medium through the chamber. Yellow dots in the left column were added to mark the location of SPBs. Bar, 5 μm. (B) A cartoon representing a WT trace, highlighting the four observed stages; M, metaphase; F, fast phase; P, paused state; S, slow phase. (C) Individual elongation traces were aligned at anaphase initiation (time = 0 min) and the average distance determined at each timepoint. (D) Comparison of time spent in anaphase and final length of the spindle. Anaphase was deemed completed at the timepoint prior to the rapid movement of SPBs that occurs when the spindle breaks and disassembly begins. (E) The rate of elongation during three 4-minute windows, corresponding to early, mid, or late anaphase, were determined from individual elongation traces and then averaged. Strains and number of cells counted: SLK19 (dKH257-1.2d), n = 25; slk19Δ (dKH256-5.5a), n = 40; spo12Δ (dKH282-5.10c), n = 40; slk19–ΔA (dKH283-1.2b), n = 40; and slk19–ΔG (dKH255-7.6a), n = 100. [D and E, bars show standard deviation (SD) **P < 0.01 and ***P < 0.001 when compared to SLK19 using a two-tailed unpaired t-test.] (F) Anaphase elongation rates of slk19 spo12Δ double mutants. Strains: SLK19 (dKH295-1.9b), n = 32; slk19Δ (dKH296-1.1a), n = 43; slk19–ΔA (dKH297-1.19c), n = 36; and slk19–ΔG (dKH299-1.16d), n = 40.

slk19 C-terminal mutants have a disrupted ipMT distribution. Cells expressing GFP–TUB1 were trapped under an agarose pad following release from an α-factor arrest, and a Z-series of 15 images, 0.25 μm apart, was obtained with a spinning-disk confocal microscope. Relative intensity was determined starting with a sum projection of the stack and using Metamorph's linescan tool to measure the intensity of a 5-pixel column at each point along the spindle. (A) Examples of sum projections of observed spindles and the corresponding intensity traces are shown. Arrows mark the GFP intensity peaks denoting the SPBs, and the length between the poles is given. (B) Each spindle was divided into 24 bins, split into half, and then spindles of lengths 4–5.9 μm, 6–7.9 μm, and 8–9.9 μm were averaged. Graphs show relative intensity and standard error of the mean (SEM) for each bin. Strains used and number of half spindles (4 μm), (6 μm), (8 μm); SLK19 (dKH160-1.2a), n = 68, 80, and 64; slk19Δ (TKH198), n = 40, 62, and 26; spo12Δ (TKH206), n = 74, 66, and 30; bim1Δ (dKH291-2.7c), n = 42, 78, and 74; slk19–ΔG (TKH194), n = 16, 20, and 14; and slk19–ΔA (TKH196), n = 60, 78, and 84.

slk19Δ kar3Δ synthetic lethality is independent of the FEAR pathway. (A) Cells were grown to stationary phase in YPAD. Tenfold serial dilutions in water were spotted onto 5-FOA to test for growth in the absence of the plasmid MR820 [YCp–KAR3∷URA3]. Strains: slk19Δ (TRK27), kar3Δ (TRS107), slk19Δ kar3Δ (DRK212.2D), spo12Δ (DRK215.1D), and spo12Δ kar3Δ (DRK215.4). (B) slk19Δ/slk19Δ strains were sporulated and the number of tetrad and dyad asci determined with a light microscope (n > 100 cells). Strain slk19Δ/slk19Δ (DRK9) was transformed with empty vector pRS424, pRK50 (P_IME2–SLK19), or pRK52 (P_IME2–SPO12). (C) Cells were grown to stationary phase in YP–GAL, serially diluted 10-fold in water, and spotted onto 5-FOA–GAL medium to test for growth in the absence of the plasmid MR820. Strains: slk19Δ (TRK27), kar3Δ (TRS107), slk19Δ kar3Δ (DRK212.2D), and slk19Δ kar3Δ, P_GAL1–SPO12 (TRK201).

Only the N-terminal region of Slk19p is necessary for FEAR signaling. (A) A diagram depicting each of five predicted coiled-coil regions (denoted B through F) individually deleted from the endogenous locus, along with the N-terminal globular region (deletion A). (B and C) Homozygous diploid slk19 deletion strains were induced to sporulate and the ratio of tetrads to dyads scored using light microscopy (n > 100 cells). (C) Strains were grown in YP-acetate prior to sporulation. Strains: (B) (mixed parents; dc48-9.1c and dc49-7.1c derivatives) SLK19/SLK19 (DKH60), slk19–ΔA/slk19–ΔA (DKH54), slk19–ΔB/slk19–ΔB (DKH55), slk19–ΔC/slk19–ΔC (DKH56), slk19–ΔD/slk19–ΔD (DKH57), slk19–ΔE/slk19–ΔE (DKH58), and slk19–ΔF/slk19–ΔF (DKH59); (C) (both parents dc49-7.1c derivatives) SLK19/SLK19 (DD812), slk19–ΔG/slk19–ΔG (DD802), and slk19Δ/slk19Δ (DD810).

Multiple coiled-coil regions of Slk19p are required for viability in strains lacking KAR3. Haploid strains lacking KAR3, containing a deletion allele of SLK19, and also carrying the plasmid MR820 [YCp–KAR3∷URA3] were first grown in YPAD, serially diluted 10-fold in water, and then spotted onto 5-FOA medium. Below is a diagram of Slk19p showing the regions essential for viability in a KAR3 deletion strain. KAR3 strains: wild type (DC49-7.1c) and slk19Δ (TRK28). KAR3 deletion strains: SLK19 (dKH46-2.1a), slk19–ΔA (dKH53-1.1c), slk19–ΔB (dKH42-1.7c), slk19–ΔC (dKH43-1.7d), slk19–ΔD (dKH61-1.5d), slk19–ΔE (dKH44-1.6a), slk19–ΔF (dKH45-1.10d), slk19–ΔG (dKH75-1.8d), and slk19–GFP (dKH186-2.1c).

Known Slk19–GFP localizations do not require the presence of the A region, but do depend on the G region. (A) Strains expressing SPC42–mCherry and a SLK19 allele with a C-terminal GFP fusion were arrested in G1 with α-factor and then released by washing with SC media. Cells were concentrated, put onto a cover slip, and covered with an agarose pad. Multiple Z-plane images were obtained as the cell progressed through mitosis; single planes from example cell cycle stages are shown. Strains: SLK19–GFP (dKH202-2.1b), slk19–ΔA–GFP (dKH204-2.4b), and slk19–ΔG–GFP (dKH203-1.3a). (B) Using strains expressing SLK19–GFP and mCHERRY–TUB1, cells were prepared as above, maximum intensity projections are shown. Strains: SPO12 (dKH305-2.20a) and spo12Δ (dKH305-2.20c). Bars, 2 μm.

slk19Δ mutants have fewer ipMTs extending through the midzone. (A) Tubulin intensity of anaphase spindles as a function of spindle length. Data are the same as those shown in another format in Figure 7B. (B) Modeling tubulin density in slk19Δ spindles. Modeling software was used to simulate features of anaphase spindles that would yield tubulin distributions like those observed experimentally with SLK19 and slk19Δ strains (Gardner et al. 2007, 2008). The tubulin distribution that would be produced by the simulated spindles was compared to the observed distributions. The distribution of tubulin in SLK19 and slk19Δ strains was obtained by measuring spindles of 5–9 μm in length (average length 6.6 μm for each strain) using SPBs labeled with Spc42–DsRed as the endpoints. Strains and number of half spindles measured: SLK19 (dKH300-1-1d), n = 34; slk19Δ (dKH303-1.6a), n = 56. (C) Example animations produced by the modeling software used to create the simulated distributions in B. (D) A model of the defects observed in slk19–ΔG mutant spindles including shorter ipMTs from the onset of anaphase and an inability to coordinate growth of the spindle with growth of ipMTs.