Defects in Saccharomyces cerevisiae protein phosphatase type I activate the spindle/kinetochore checkpoint

Abstract

A conditional allele of type 1 protein phosphatase (glc7-129) in Saccharomyces cerevisiae causes first cycle arrest in G2/M, characterized by cells with a short spindle and high H1 kinase activity. Point-of-execution experiments indicate Glc7p function is required in G2/M just before anaphase for the completion of mitosis. Loss of the spindle/kinetochore checkpoint in glc7-129 cells abolishes the G2/M cell cycle arrest with a concomitant increase in chromosome loss and reduced viability. These results support a role for Glc7p in regulating kinetochore attachment to the spindle, an event monitored by the spindle/kinetochore checkpoint.

Figure 1

glc7-129 point of execution is in G₂/M. Images of glc7-129 NUF2-GFP (YAB128) cells logarithmically growing at 30°C were captured using DIC (left) and fluorescence (right) microscopy. The fluorescence images represent localization of the spindle pole body(s) (as detected by a Nuf2p–GFP fusion). After shift to 12°C for ∼20 hr, images were captured of the same cells. The numbers below each panel indicate the number of cells that arrest in the first cell cycle as large-budded cells with a short spindle.

Figure 2

glc7-129 cells delay with a short spindle. (A) A montage of GLC7 NUF2–GFP (YAB 131) (large arrow) and glc7-129 NUF2–GFP (YAB 128) cells growing logarithmically at 30°C, using combined fluorescence and DIC time-lapse microscopy. The Nuf2p–GFP fluorescence at each spindle pole body was pseudocolored black. (Small black arrow) A glc7-129 cell that goes through anaphase; (white arrow) a glc7-129 cell arrested with a short spindle oscillating between the mother and bud. (B) The distance (μm) between spindle pole bodies was calculated for three wild-type cells (left) and three glc7-129 cells (right) at 2-min time points.

Figure 3

DNA content, cell morphology, and H1 kinase activity indicate that the glc7-129 induced G₂/M arrest requires a functional spindle checkpoint. (A) Flow cytometry for DNA content of wild-type (KT1112), mad1 (YAB127), glc7-129 (YAB27), mad1 glc7-129 (YAB123), bub3 glc7-129 (YAB92), and rad9 glc7-129 (YAB82) strains at 30°C and 12°C for 12 hr. (B) The morphological types of cells used in A were quantified microscopically, n > 200. (C) Kinase activity towards histone H1 was assayed for cells incubated at the nonpermissive temperature (12°C) for 12 hr. Labeled histone H1 was monitored using SDS-PAGE and autoradiography. (D) Labeled histone H1 was quantified using a PhosphorImager in three independent kinase assays and the average activity and standard error for each of the strains are represented.

Figure 4

MAD1 is required for the G₂/M delay of cells containing glc7-129. (A) mad1 glc7-129 NUF2–GFP (YAB152) cells were treated and analyzed by DIC and fluorescence time-lapse microscopy as in Fig. 2A. In the first panel, an arrow marks a mad1 glc7-129 cell in approximately the same stage of the cell cycle (G₁/S) as the wild-type (Fig. 2A; large black arrow) and glc7-129 (Fig. 2A; small black arrow) cells. Note that by 64 min, the double mutant has proceeded into anaphase. (B) The distance (μm) between spindle pole bodies in three cells was plotted vs. time.