Yeast Dam1p is required to maintain spindle integrity during mitosis and interacts with the Mps1p kinase

Abstract

We have identified a mutant allele of the DAM1 gene in a screen for mutations that are lethal in combination with the mps1-1 mutation. MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint. Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel. The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p. DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex. We report here that cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature. Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function. We have observed that a Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis.

Figure 1

Characterization of the asynchronously growing diploid dam1-1 strain at the permissive temperature (25°C) and after shift to the nonpermissive (37°C) temperature for 2.5 h. (A) Flow cytometric analysis to measure DNA content. 1C and 2C, DNA content (propidium iodide fluorescence) before and after DNA replication, respectively. Each histogram represents 5000 cells. (B) Immunofluorescence staining of DNA (DAPI) and tubulin (α-Tubulin) in cells grown at 25 and 37°C, as described in MATERIALS AND METHODS.

Figure 2

Electron micrograph of the asynchronously growing diploid dam1-1 strain after shift to the nonpermissive (37°C) temperature for 2.5 h. Arrows denote spindle pole bodies. Bar, 0.5 μm. Of spindles examined from 13 large-budded cells, 11 were discontinuous as shown here, and 2 appeared normal.

Figure 3

Characterization of synchronized wild-type and dam1-1 haploid strains after shift to the nonpermissive (37°C) temperature. (A) Flow cytometric analysis to measure DNA content, as in Figure 1. Asynch, asynchronously growing cells at 25°C; 0, 2.2, and 3.3, hours at 37°C after removal of α-factor, %LB, percentage of cells with bud size more than half the size of the mother cell; 200–400 cells were counted. (B) Immunofluorescence staining of DNA (DAPI) and tubulin (α-Tubulin) in wild-type and dam1-1 cells grown at 37°C for 2.2 h (a and b) or 3.3 h (c). Of spindles from large-budded dam1-1 cells with two separated DAPI-staining regions at 2.2 h, 90% are discontinuous or abnormally thin, as in b and c, 6% are otherwise abnormal (e.g., not associated with DNA), and 4% appear normal (n = 200).

Figure 4

Budding indices of wild-type and dam1-1 cells after release from α-factor arrest to the nonpermissive temperature represented as a bar graph. Each sample represents 200–400 cells examined.

Figure 5

DNA content of wild-type, dam1-1, and dam1-1mad1null cells. Left panels (0 h), flow cytometric data of cells at α-factor arrest; right panels (3 h), flow cytometric data (performed as in Figures 1 and 3) of cells 3 h after release from α-factor to the nonpermissive temperature. Thirty-four percent (n = 400 cells) of dam1-1, mad1Δ cells were multibudded at this time point compared with 0% for both wild-type and dam1-1 cells.

Figure 6

Summary of genetic and two-hybrid interactions involving DAM1. Jagged lines denote two-hybrid interactions, and straight lines denote synthetic lethal interactions. The CIN8-MPS1 data are from Geiser et al. (1997). The STU1-MPS1 data are from Luca and Winey (personal communication). The DAM1-DUO1 data are from Hofman et al. (1998).

Figure 7

Localization of myc-Dam1p in wild-type yeast cells using immunofluorescence microscopy. DAPI, DNA staining; SPC42-GFP, autofluorescence of a GFP-tagged version of Spc42p; α-MYC, anti-myc 1° antibody plus Texas Red anti-mouse 2° antibody; MERGED, merging of fluorescence of SPC42-GFP (green) and α-MYC (red). (a) Two unbudded cells. (b and c) Budded cells with short spindles before anaphase B elongation. (d) Two focal planes of a large budded cell in late anaphase. Bar, 1 μm.

Figure 8

Transition of myc-Dam1p localization from one to two foci. Bar graph of spindle lengths in cells with the following patterns of myc-Dam1p staining. GROUP 1, myc-Dam1p localization as an uninterrupted bar between the SPBs (n = 19; average = 1.5 μm; SD = 0.3); GROUP 2, myc-Dam1p localization as two staining regions adjacent to the SPBs (n = 13; average = 2.4 μm; SD = 0.3). Spindles were measured using Slidebook software with an error of ±0.1 μm.

Figure 9

Colocalization of myc-Dam1p with microtubules using immunofluorescence microscopy. (A) DAPI, DNA staining; α-MYC, anti-myc 1° antibody plus FITC anti-mouse 2° antibody; α-Tub1-GFP, anti-tubulin 1° antibody plus Texas Red anti-rat 2° antibody. The granular staining using Texas Red is present in controls lacking primary antibody and is considered background. MERGED, merging of α-MYC (green) and α-Tub1p fluorescence (red). (a) Unbudded cell. (b and c) Budded cells with short spindles before anaphase B elongation. (d and e) Large budded cells in late anaphase. (B) myc-Dam1p localization after treatment of cells with microtubule-depolymerizing agents. DIC (differential interference contrast) shows cell outline. MERGED, merging of DAPI (blue), α-MYC (green), and α-Tub1p (red) fluorescence. The left panel shows residual colocalization (yellow) of myc-Dam1p (green) and tubulin (red), and the right panel shows punctate nuclear myc-Dam1p staining (green) in the absence of microtubules (red). Bar, 1 μm.