G2/M arrest caused by actin disruption is a manifestation of the cell size checkpoint in fission yeast

Abstract

In budding yeast, actin disruption prevents nuclear division. This has been explained as activation of a morphogenesis checkpoint monitoring the integrity of the actin cytoskeleton. The checkpoint operates through inhibitory tyrosine phosphorylation of Cdc28, the budding yeast Cdc2 homolog. Wild-type Schizosaccharomyces pombe cells also arrest before mitosis after actin depolymerization. Oversized cells, however, enter mitosis uninhibited. We carried out a careful analysis of the kinetics of mitotic initiation after actin disruption in undersized and oversized cells. We show that an inability to reach the mitotic size threshold explains the arrest in smaller cells. Among the regulators that control the level of the inhibitory Cdc2-Tyr15 phosphorylation, the Cdc25 protein tyrosine phosphatase is required to link cell size monitoring to mitotic control. This represents a novel function of the Cdc25 phosphatase. Furthermore, we demonstrate that this cell size-monitoring system fulfills the formal criteria of a cell cycle checkpoint.

Figure 1

Cells delay mitosis and reduce cell growth in the presence of latrunculin A. (A) Filamentous actin displays a varying degree of derangement depending on the dose of latrunculin A. Exponentially growing wild-type cells were treated with the drug for 10 min, fixed, and stained with tetramethylrhodamine B isothiocyanate-phalloidin. (B) Percentage of cells that have initiated mitosis (top) and the average cell size in the population (bottom) in wild-type cells. Cells were synchronized in early G2 and released into the indicated concentrations of latrunculin A. In one culture, 10 μM latrunculin A was added at time = 40 min after the release. (C) Thickness of cells from experiment in B measured in the middle of the cell length (left) and the contribution to the total cell volume attributable to thickening of cells in the presence of 10 μM latrunculin A (right). (D) Percentage of cells that have initiated mitosis (top) and the average cell size in the population (bottom) in wild-type cells in which 1 μM latrunculin A (Lat A) was added at indicated times after the synchronization in early G2.

Figure 2

A morphogenesis checkpoint does not operate before mitosis in S. pombe. (A) Percentage of cdc25-22^ts cells that have initiated mitosis after the release from cdc arrest. Cells were incubated at 36°C for 4 h (3 h in the case of the t = −120 sample) and 10 μM latrunculin A was added at indicated times before the release to 25°C. (B) Percentage of cdc25-22^ts cells that have initiated the second mitosis after release from high-temperature arrest. Cells were synchronized in early G2 and arrested at 36°C for 1 (left) or 3 h (right) and then released to 25°C. Latrunculin A (Lat A; 10 μM) or 0.6 M KCl was added before the second mitosis at indicated times.

Figure 3

Cells arrest before mitosis or progress through mitosis unperturbed in the presence of latrunculin A. (A) Microtubules (green), Sad1 (red), and nuclear DNA (blue) in cdc25-22^ts cells. Cells were arrested at 36°C for 1 (left) and 3 (right) h, 10 μM latrunculin A was added, and cells were incubated at high temperature for another hour. Then they were released to 25°C (t = 0) and samples for immunostaining were taken at indicated times. Representative cells are presented. (B) Percentages of cdc25-22^ts cells from the previous experiment at different stages of mitosis. PAA, postanaphase array of microtubules. (C) Cellular localization of Plo1-GFP. Wild-type cells expressing Plo1-GFP were synchronized in early G2, 10 μM latrunculin A was added 20 min after the release, cells incubated for another 60 min, and examined under the microscope. (D) Histone H1 kinase activity in cells treated as described in A.

Figure 4

Size of cells in mitosis after latrunculin A pulse treatment. Cells were grown at 35°C for 4 h then pulsed for 2 min with 10 μM latrunculin A and the size of uninuclear cells with condensed chromosomes was measured at each time point.

Figure 5

Mitotic initiation correlates with cell size in wee1-50^ts mik1 cells. (A) Average cell sizes in an exponential culture of wee1-50^ts mik1Δ cells. The cells were shifted to 36°C at t = 0. (B) Percentage of wee1-50^ts mik1Δ cells synchronized in early G2 that have initiated mitosis (left) and average cell sizes at t = 0 and at mitosis (right). Cells were shifted to 36°C (t = 0) after indicated times spent at 25°C. The second mitosis is plotted for one culture (2nd) and 10 μM latrunculin A was added to another at time = 0 (Lat A) and entry into the first mitosis plotted. (C) Cdc2-Tyr15 phosphorylation in wee1-50^ts mik1Δ cells synchronized in early G2 and grown at 25°C for indicated times before the shift to 36°C at t = 0.

Figure 6

Cell size control over initiation of mitosis is preserved in cells lacking the Wee1 and Mik1 kinase activities. (A) Diagram summarizing the manipulations to obtain undersized and oversized cdc10-129^ts wee1-50^ts mik1Δ cells. Latrunculin A (Lat A; 10 μM) was added at time = 0. (B) Percentage of cells that have initiated mitosis after the treatment outlined in A. (C) Percentage of cells from the previous experiment undergoing mitosis (uninucleate or binucleate cells with condensed chromosomes) at each time point. (D) Cdc2-Tyr15 phosphorylation in cells treated as outlined in A. Asterisk indicates a dramatic drop in the Tyr15 phosphorylation level.

Figure 7

Cell size control over initiation of mitosis is not preserved in cells lacking cdc25. (A) Percentage of cdc10-129 cdc25Δ cdc2-3w cells that have initiated mitosis. Cells were synchronized in late G2, and incubated at 36°C for 2.5 h to induce one mitotic cycle and a brief G1 arrest. Cells were then released to 25°C and 10 μM latrunculin A (Lat A) or 12 mM hydroxyurea (HU) was added at indicated times after the release. (B) Percentage of the cdc25Δ cells expressing T-cell PTPase that have initiated mitosis (left) and average cell sizes in the population (right). A low-level T-cell PTPase expression was under the control of the inducible nmt1 promoter maintained in the “off” state. Cells were synchronized in early G2 and Lat A at indicated concentrations was added at t = 60.

Figure 8

Cells initiate mitosis after mitotic cells size threshold is lowered by a nutritional downshift. (A) Percentage of wild-type cells undergoing mitosis after a nutritional downshift. At time = 0, exponential culture of cells was shifted from the EMM to EMM-N medium containing or lacking 10 μM latrunculin A. (B) Percentage of cdc25Δ cells expressing the T-cell phosphatase that undergo mitosis after the same treatment as described in A.

Figure 9

Model summarizing the interaction between the Cdc2-Tyr15 phosphorylation and dephosphorylation pathways involved in cell size control.