A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast

Abstract

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.

Keywords: Cdc15; Dbf2; MEN; Mob1; Nud1; mitotic exit; spindle position checkpoint.

Figure 1

The nud1-A308T allele suppresses the growth defect of GAL-BFA1 cells. (A) The mitotic exit network components and regulators are shown. Proteins that inhibit mitotic exit are depicted in red boxes, while activators are colored as grey boxes. The majority of the components assemble on the SPBs. The cellular locations of components that are not on this structure are indicated in parentheses underneath the protein names. See text for details. (B) Wild-type (AS3), GAL-BFA1 (AS5), and GAL-BFA1 nud1-A308T (AS53) strains were grown in YEP + Raffinose (YEPR) overnight and 10-fold serial dilutions were spotted on YEPR (left) or YEPR + Galactose (YEPRG; right). The plates were incubated at 25 °C for 3 days.

Figure 2

nud1-A308T partially bypasses the spindle assembly checkpoint (SAC). Log phase wild-type (AS3), NUD1-3V5 (AS326), bub2Δ (AS112), and nud1-A308T (AS387) cells were arrested in the presence of alpha-factor pheromone (5 ug/mL) for a total of 3 h at 25 °C. Cells were then released into medium containing nocodazole (15 ug/mL) and samples were taken at the indicated times for budding analysis. After 120 min and 240 min, 7.5 ug/mL nocodazole was re-added to maintain the metaphase block. The percentage of budded (squares) and re-budded (triangles) cells observed at each time point in wild-type (A), NUD1-3V5 (B), bub2Δ (C), and nud1-A308T-3V5 (D) cells was plotted (n = 100).

Figure 3

The nud1-A308T allele causes bypass of the Spindle Position Checkpoint (SPoC). Log phase wild-type (AS417) and nud1-A308T (AS474) cells containing both dyn1Δ and GFP-TUB1 were grown at 14 °C for 25 h to induce spindle mispositioning. Samples were collected and the cells were fixed with paraformaldehyde and stained with DAPI prior to imaging. (A) Representative images of a cell undergoing a proper anaphase (normal), arrested in anaphase with a mispositioned spindle (arrested), and with multiple nuclei and spindles indicating SPoC bypass (bypassed) are shown. (B) The percentage of cells from each strain with arrested or bypassed morphology was plotted (n > 500).

Figure 4

Mutants containing nud1-A308T have a shortened anaphase duration. (A) and (B): wild-type (AS138) and nud1-A308T (AS303) cells were grown to log phase and arrested in 5 ug/mL alpha-factor pheromone for 2.5 h. The cells were then released into medium lacking pheromone and samples were taken at the indicated times for tubulin immunofluorescence. The percentage of cells with metaphase (circles) or anaphase (squares) morphology in wild-type (A) and nud1-A308T (B) strains was quantified and plotted (n = 100). (C) and (D): Wild-type (AS476) and nud1-A308T (AS477) cells harboring Tub1-mCherry and Dbf2-eGFP fusions were grown to log phase in SC + glucose medium and living cells growing on an agar pad were imaged every 4 min for a total of 4 h. (C) The start and completion of anaphase was marked and the number of cells with anaphase duration of 16, 20, 24, or 28 min in each strain was plotted (n > 19). The average duration of anaphase in each strain is indicated above the graph. (D) A representative wild-type and nud1-A308T cell is shown.

Figure 5

The nud1-A308T allele does not promote association of Tem1 to SPBs. Wild-type (AS15), GAL-BFA1 (AS79), and GAL-BFA1 nud1-A308T (AS296) cells all harboring a Tem1-GFP fusion were grown to log phase at 30 °C in YEPR; 2% galactose was then added to induce the overexpression of BFA1 for a total of 3 h. Samples were taken just prior to galactose induction and after 3 h induction and the cells were fixed in paraformaldehyde prior to imaging. (A) Representative composite images of anaphase cells for each strain prior to galactose addition, and following a 3 h galactose induction are shown. The DNA stained with DAPI is shown in blue and Tem1-GFP is in green. Arrowhead indicates Tem1-GFP localization to the SPB. (B) The percentage of anaphase cells with Tem1-GFP localized to one or both SPBs in the absence of galactose (black) and 3 h (gray) after galactose addition for each strain was plotted. (n > 100 cells).

Figure 6

Cdc15 is recruited to SPBs in all cell cycle phases in nud1-A308T cells. Wild-type (AS513) and nud1-A308T (AS517) cells harboring Cdc15-eGFP and Tub1-mCherry fusions were grown to log phase in SC + glucose medium and living cells were imaged. (A) Brighfield (BF), Cdc15, and Cdc15 and Tub1 composite representative wild-type and nud1-A308T cells from S phase, metaphase, and anaphase are shown. Arrows indicate Cdc15 localization to SPBs. (B) The percentage of G1/S, metaphase, and anaphase cells containing Cdc15 on one or more SPBs in wild-type (black) or nud1-A308T (striped) cells is plotted (n ≥ 80 cells for each stage).

Figure 7

nud1-A308T recruits Dbf2 asymmetrically to the mSPB in metaphase. (A) and (B): wild-type (AS476) and nud1-A308T (AS477) cells harboring Dbf2-eGFP and Tub1-mCherry fusions were grown to log phase in SC + glucose media. Live cells were imaged. Representative metaphase and anaphase cells for each genotype are shown. The percentage of cells in each phase with Dbf2-eGFP localized to one or two SPBs is indicated in the column to the right of the images (n = 100). (C) nud1-A308T (AS477) cells harboring Dbf2-eGFP and Tub1-mCherry fusions were grown on an SC + glucose agar pad and imaged every 4 min for a total of 4 h. The start of metaphase was determined by identifying spindles that were 2 microns in length and this time was marked as 0 (n = 20). A representative cell is shown. The bud compartment is labeled “B” and the mother compartment is labeled “M”.

Figure 8

Nud1-A308T function requires CDC15 and MOB1 but not CDC5. (A) Ten-fold dilutions of cells containing NUD1 (AS3), nud1-A308T (AS387), cdc5-1 (AS564), and nud1-A308T, cdc5-1 (AS570) were spotted onto YEPD plates that were incubated at 25 °C or 37 °C for 2 days before imaging. (B) Ten-fold dilutions of cells containing NUD1 (AS3), cdc15-2 (AS179), nud1-A308T (AS387), or nud1-A308T, cdc15-2 (AS447) were spotted onto YEPD plates and incubated at 25 °C or 37 °C for 2–3 days before imaging. (C) Ten-fold dilutions of cells containing GAL-mob1-77 (AS402), nud1-A308T (AS387), or both GAL-mob1-77 and nud1A308T (AS478) were spotted onto YEPRG (top row) or YEPD (bottom row) plates to activate or to repress the expression of mob1-77. The plates were incubated at 25 °C or 37 °C for 2 days before imaging.

Figure 9

The mutant Nud1-A308T protein recruits Mob1 in both metaphase and anaphase cells. MOB1-6HA (AS407), MOB1-6HA NUD1-3V5 (AS228), and MOB1-6HA nud1-A308T-V5 (AS439) cells harboring the cdc14-3 mutation were grown to log phase. Cell cultures were split, and half of each culture was incubated at 37 °C for two hours to inactivate Cdc14 and produce an anaphase arrest (samples 1–3). The other half of each culture was arrested with 15 μg/mL nocodazole at 25 °C to produce a metaphase arrest (samples 4–6). Protein extracts were produced from each sample and Mob1-6HA was immunoprecipitated using anti-HA agarose beads. The samples were analyzed by SDS-PAGE and Western blotting using anti-HA antibody to detect the amount of Mob1-6HA immunoprecipitated in each sample and anti-V5 antibodies to detect the amount of Nud1-3V5 or Nud1-A308T-3V5 co-immunoprecipitated. Mob1, Nud1 (samples 2 and 5), and Nud1-A308T (samples 3 and 6) are labeled on the blot. Higher migrating phosphorylated species of Nud1 and Nud1-A308T are labeled (pNud1).

Figure 10

nud1-A308T suppresses the dominant GAL-nud1-T78A. NUD1 (AS326), nud1-A308T (AS387), NUD1 GAL-nud1-T78A (AS384), nud1-A308T GAL-nud1-T78A (AS395), NUD1 GAL-nud1-S53A, S63A, T78A (AS388), and nud1-A308T GAL-nud1-S53A, S63A, T78A (AS390) cells were grown to log phase in YEPR and subsequently induced with 2% galactose addition. Samples were taken at the indicated times and processed for tubulin immunofluorescence. The percentage of cells with anaphase spindles at each time point in each strain was determined and plotted (n = 100–200).