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. 2020 Jan 7;10(1):255-266.
doi: 10.1534/g3.119.400726.

Checkpoint Regulation of Nuclear Tos4 Defines S Phase Arrest in Fission Yeast

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Free PMC article

Checkpoint Regulation of Nuclear Tos4 Defines S Phase Arrest in Fission Yeast

Seong M Kim et al. G3 (Bethesda). .
Free PMC article

Abstract

From yeast to humans, the cell cycle is tightly controlled by regulatory networks that regulate cell proliferation and can be monitored by dynamic visual markers in living cells. We have observed S phase progression by monitoring nuclear accumulation of the FHA-containing DNA binding protein Tos4, which is expressed in the G1/S phase transition. We use Tos4 localization to distinguish three classes of DNA replication mutants: those that arrest with an apparent 1C DNA content and accumulate Tos4 at the restrictive temperature; those that arrest with an apparent 2C DNA content, that do not accumulate Tos4; and those that proceed into mitosis despite a 1C DNA content, again without Tos4 accumulation. Our data indicate that Tos4 localization in these conditions is responsive to checkpoint kinases, with activation of the Cds1 checkpoint kinase promoting Tos4 retention in the nucleus, and activation of the Chk1 damage checkpoint promoting its turnover. Tos4 localization therefore allows us to monitor checkpoint-dependent activation that responds to replication failure in early vs. late S phase.

Keywords: Cell cycle; Checkpoint; S phase; Tos4; fission yeast.

Figures

Figure 1
Figure 1
Tos4-GFP is present in nuclei of dividing cells. (A) Live cell imaging of WT cells containing Tos4-GFP and Sad1-DsRed was followed at 25°C for 8 hr. Nuclear Tos4-GFP signal is observed after duplication of SPBs and it disappears from the nucleus before cells enter the next round of mitosis. Scale bar, 5 µm. (B) Duration of the presence of nuclear Tos4-GFP. The presence of nuclear Tos4-GFP was determined by measuring the nuclear Tos4-GFP signals using the ImageJ software (Schindelin et al. 2012). Nuclear Tos4-GFP signals was counted as a positive when nuclear Tos4-GFP signal is greater than 50 (scale 0-255) after the background subtraction using ImageJ. (C) Ratio of nuclear Tos4-GFP duration vs. the cell cycle duration is presented. Duration of the cell cycle was determined by measurement of the timing between the first and second separation of SPBs. Sad1-DsRed was used to follow the separation of SPBs.
Figure 2
Figure 2
Tos4-GFP accumulate in the nuclei of cells arrested in early S phase. (A) WT cells were imaged for Tos4-GFP in asynchronous culture, after treatment with 12 mM HU for 4 h, and 1 h after release from HU. (B) Temperature-sensitive cell cycle mutants, cdc22-M45 (S phase arrest), cdc10-V50 (G1 phase arrest), cdc25-22 (G2 phase arrest) were imaged for Tos4-GFP in asynchronous culture at 25°C, after 4 h at 36°C, or after 1 h-1.5 h after release to 25°C. (C) Cold-sensitive nda3-KM311 (M phase arrest) was imaged for Tos4-GFP in asynchronous culture at 32°C, after 4 h at 17°C, or after 0.5 h after release to 32°C.
Figure 3
Figure 3
Replication mutants lack nuclear Tos4-GFP. Temperature-sensitive Mcm4 helicase mutants (mcm4-M68, mcm4-c106, mcm4-dg) (A), MCM loader mutant (cdc18-K46), ligase mutants (cdc17-M45 and cdc17-K42) (B), and polymerase delta mutants (cdc6-23, cdc6-ts2, and cdc27-K3) (C) were imaged for Tos4-GFP in asynchronous culture at 25°C or after 4 h at 36°C.
Figure 4
Figure 4
Tos4-GFP is not targeted for APC-mediated degradation. (A) Temperature-sensitive APC mutants (cut9-665, cut4-533, and nuc2-663) were imaged for Tos4-GFP in asynchronous culture at 25°C or after 4 h at 36°C. (B) APC mutants were imaged for Tos4-GFP after treatment with 12 mM HU for 4 h and 1 h after release from HU.
Figure 5
Figure 5
Tos4 protein level is increased in S phase cells arrested by HU. (A) WT cells with Tos4-GFP in asynchronous culture, after treatment with 12 mM HU, or after release from HU were lysed and immunoblotted for GFP and cdc2 (loading control). (B) WT cells used in (A) were fixed in 70% ethanol, and FACS analyzed for GFP and propidium iodide (PI). Green in scatter plot represents population with high GFP and low PI while red represents population with low GFP and high PI.
Figure 6
Figure 6
Cds1 is required for nuclear Tos4 accumulation in cells arrested in early S phase by HU or cdc22-M45. (A) WT, cds1Δ, cds1-FHA* cells were imaged for Tos4-GFP after treatment with 12 mM HU. Right, quantification of % cells with nuclear Tos4-GFP. (B) cdc22-M45 and cds1Δ cdc22-M45 cells were imaged for Tos4-GFP after 4 h at 36°C. Right, quantification of % cells with nuclear Tos4-GFP. N> 300 cells analyzed for each strain. A two-tailed Student’s t-test was used to determine significance: * P < 0.05, *** P < 0.001, n.s. not significant. Error bars represent Standard Error (SE).
Figure 7
Figure 7
Continued Cds1 activation induces S phase arrest in mcm4-M68 but not mcm4-dg. (A) mcm4-M68, cds1Δ mcm4-M68, mcm4-dg, chk1Δ mcm4-dg were imaged for Tos4-GFP after treatment with 12 mM HU at 25°C, after release from HU to 36°C, or after pre-treatment with HU at 25°C then transfer to 36°C. (B) Quantification of % cells with nuclear Tos4-GFP in (A). N> 300 cells analyzed for each strain. A two-tailed Student’s t-test was used to determine significance: * P < 0.05, ** P < 0.01, *** P < 0.001, Error bars represent Standard Error (SE).
Figure 8
Figure 8
Replication mutants that bypass cell cycle arrest lack nuclear Tos4 despite continued Cds1 activation by HU. (A) Temperature-sensitive hsk1-1312 and rad4-116 cells were imaged for Tos4-GFP in asynchronous culture at 25°C, after 4h at 36°C, after treatment with 12 mM HU at 25°C, or after pre-treatment with HU at 25°C then transfer to 36°C. (B) Left, early replication stress (1C DNA) such as HU and cdc22-M45 activates Cds1 and Tos4 expression is upregulated. Middle, replication mutants that complete most of DNA synthesis (2C DNA) fail to fully activate Cds1 and Tos4 expression is turned off unless in the continued presence of HU. Right, early replication mutant mcm4-dg (1C DNA) that enters mitosis fail to activate Cds1 and turn on Tos4 expression even in the presence of HU, in a Chk1-dependent manner.

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