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Spatiotemporal regulations of Wee1 at the G2/M transition.

Masuda H, Fong CS, Ohtsuki C, Haraguchi T, Hiraoka Y - Mol. Biol. Cell (2011)

Bottom Line: At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB).During the G2/M transition when cyclin B-Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B-Cdc2-dependent manner and locally suppresses both cyclin B-Cdc2 activity and spindle assembly to counteract a Polo kinase-dependent positive feedback loop.Then Wee1 disappears from the SPB during spindle assembly.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Regulation, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3LY, United Kingdom. hiro.masuda@cancer.org.uk

ABSTRACT
Wee1 is a protein kinase that negatively regulates mitotic entry in G2 phase by suppressing cyclin B-Cdc2 activity, but its spatiotemporal regulations remain to be elucidated. We observe the dynamic behavior of Wee1 in Schizosaccharomyces pombe cells and manipulate its localization and kinase activity to study its function. At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB). During the G2/M transition when cyclin B-Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B-Cdc2-dependent manner and locally suppresses both cyclin B-Cdc2 activity and spindle assembly to counteract a Polo kinase-dependent positive feedback loop. Then Wee1 disappears from the SPB during spindle assembly. We propose that regulation of Wee1 localization around the SPB during the G2/M transition is important for proper mitotic entry and progression.

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Regulation of Wee1 localization and activity by Cdr2–Cdr1 is not required for Wee1 accumulation at the SPB during the G2/M transition. (A) Levels of Wee1 localized to the cortical nodes decrease before Wee1 accumulation at the SPB. GFP–Wee1 was overproduced in the absence of thiamine using a thiamine-repressive promoter (P81nmt1) at 30ºC for 20 h. The first and second rows of the panels show live images projected from 14 Z-sections 10 min before SPB separation through 4 min after SPB separation. The maximum GFP intensity at the SPB is labeled at each time point. The third and fourth rows of the panels show images from the top and the middle Z-sections. Arrows indicate GFP–Wee1 localized to the cortical nodes. (B) Wee1 accumulates at the SPB during the G2/M transition in cdr2 deletion and cdr1 deletion cells. Live images of a cdr2 deletion and a cdr1 deletion cell expressing GFP–Wee1 and Sid4–mRFP are shown from 6 min before SPB separation through 2 min after SPB separation. Bars, 10 μm.
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Figure 3: Regulation of Wee1 localization and activity by Cdr2–Cdr1 is not required for Wee1 accumulation at the SPB during the G2/M transition. (A) Levels of Wee1 localized to the cortical nodes decrease before Wee1 accumulation at the SPB. GFP–Wee1 was overproduced in the absence of thiamine using a thiamine-repressive promoter (P81nmt1) at 30ºC for 20 h. The first and second rows of the panels show live images projected from 14 Z-sections 10 min before SPB separation through 4 min after SPB separation. The maximum GFP intensity at the SPB is labeled at each time point. The third and fourth rows of the panels show images from the top and the middle Z-sections. Arrows indicate GFP–Wee1 localized to the cortical nodes. (B) Wee1 accumulates at the SPB during the G2/M transition in cdr2 deletion and cdr1 deletion cells. Live images of a cdr2 deletion and a cdr1 deletion cell expressing GFP–Wee1 and Sid4–mRFP are shown from 6 min before SPB separation through 2 min after SPB separation. Bars, 10 μm.

Mentions: We were not able to observe Wee1 localization at the medial cortical nodes by expressing GFP–Wee1 under the control of the endogenous promoter. However, as previously reported (Moseley et al., 2009), we observed localization of Wee1 at the cortical nodes, at the SPB, and in the nucleus in cells overproducing GFP–Wee1. Although Wee1 overproduction arrested cells in G2 phase, we observed mitotic entry in some cells that showed localization of Wee1 at the cortical nodes in G2 phase (indicated by the arrows in Figure 3A). Under this overproduced condition, Wee1 showed localization at the SPB in G2 phase, decreased in SPB level at the G2/M transition, then temporally increased this level, and almost disappeared from the SPB after SPB separation (Figure 3A and Supplemental Figure S1). This was consistent with the observations made under the expression condition using the endogenous promoter (Figure 1, A and E). In these cells, the Wee1 localized to the cortical nodes in G2 phase disappeared from the nodes at the G2/M transition before accumulating at the SPB (Figure 3A). To examine whether Wee1 at the cortical nodes is involved in its accumulation at the SPB, we observed Wee1 localization in cdr2Δ cells that do not exhibit Wee1 localization at the nodes (Moseley et al., 2009). We found that Wee1 does accumulate at the SPB during the G2/M transition in cdr2Δ cells (Figure 3B). We also examined Wee1 localization in cdr1Δ cells, in which Wee1 localizes at the cortical nodes, but the Wee1 activity is not suppressed by Cdr1 (Moseley et al., 2009). Wee1 also accumulated at the SPB during the G2/M transition in cdr1Δ cells (Figure 3B). These observations suggest that regulation of Wee1 localization and activity by Cdr2–Cdr1 is not required for Wee1 accumulation at the SPB, although it is unclear how Wee1 localized at the cortical nodes behaves in the unperturbed cell cycle.


Spatiotemporal regulations of Wee1 at the G2/M transition.

Masuda H, Fong CS, Ohtsuki C, Haraguchi T, Hiraoka Y - Mol. Biol. Cell (2011)

Regulation of Wee1 localization and activity by Cdr2–Cdr1 is not required for Wee1 accumulation at the SPB during the G2/M transition. (A) Levels of Wee1 localized to the cortical nodes decrease before Wee1 accumulation at the SPB. GFP–Wee1 was overproduced in the absence of thiamine using a thiamine-repressive promoter (P81nmt1) at 30ºC for 20 h. The first and second rows of the panels show live images projected from 14 Z-sections 10 min before SPB separation through 4 min after SPB separation. The maximum GFP intensity at the SPB is labeled at each time point. The third and fourth rows of the panels show images from the top and the middle Z-sections. Arrows indicate GFP–Wee1 localized to the cortical nodes. (B) Wee1 accumulates at the SPB during the G2/M transition in cdr2 deletion and cdr1 deletion cells. Live images of a cdr2 deletion and a cdr1 deletion cell expressing GFP–Wee1 and Sid4–mRFP are shown from 6 min before SPB separation through 2 min after SPB separation. Bars, 10 μm.
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Figure 3: Regulation of Wee1 localization and activity by Cdr2–Cdr1 is not required for Wee1 accumulation at the SPB during the G2/M transition. (A) Levels of Wee1 localized to the cortical nodes decrease before Wee1 accumulation at the SPB. GFP–Wee1 was overproduced in the absence of thiamine using a thiamine-repressive promoter (P81nmt1) at 30ºC for 20 h. The first and second rows of the panels show live images projected from 14 Z-sections 10 min before SPB separation through 4 min after SPB separation. The maximum GFP intensity at the SPB is labeled at each time point. The third and fourth rows of the panels show images from the top and the middle Z-sections. Arrows indicate GFP–Wee1 localized to the cortical nodes. (B) Wee1 accumulates at the SPB during the G2/M transition in cdr2 deletion and cdr1 deletion cells. Live images of a cdr2 deletion and a cdr1 deletion cell expressing GFP–Wee1 and Sid4–mRFP are shown from 6 min before SPB separation through 2 min after SPB separation. Bars, 10 μm.
Mentions: We were not able to observe Wee1 localization at the medial cortical nodes by expressing GFP–Wee1 under the control of the endogenous promoter. However, as previously reported (Moseley et al., 2009), we observed localization of Wee1 at the cortical nodes, at the SPB, and in the nucleus in cells overproducing GFP–Wee1. Although Wee1 overproduction arrested cells in G2 phase, we observed mitotic entry in some cells that showed localization of Wee1 at the cortical nodes in G2 phase (indicated by the arrows in Figure 3A). Under this overproduced condition, Wee1 showed localization at the SPB in G2 phase, decreased in SPB level at the G2/M transition, then temporally increased this level, and almost disappeared from the SPB after SPB separation (Figure 3A and Supplemental Figure S1). This was consistent with the observations made under the expression condition using the endogenous promoter (Figure 1, A and E). In these cells, the Wee1 localized to the cortical nodes in G2 phase disappeared from the nodes at the G2/M transition before accumulating at the SPB (Figure 3A). To examine whether Wee1 at the cortical nodes is involved in its accumulation at the SPB, we observed Wee1 localization in cdr2Δ cells that do not exhibit Wee1 localization at the nodes (Moseley et al., 2009). We found that Wee1 does accumulate at the SPB during the G2/M transition in cdr2Δ cells (Figure 3B). We also examined Wee1 localization in cdr1Δ cells, in which Wee1 localizes at the cortical nodes, but the Wee1 activity is not suppressed by Cdr1 (Moseley et al., 2009). Wee1 also accumulated at the SPB during the G2/M transition in cdr1Δ cells (Figure 3B). These observations suggest that regulation of Wee1 localization and activity by Cdr2–Cdr1 is not required for Wee1 accumulation at the SPB, although it is unclear how Wee1 localized at the cortical nodes behaves in the unperturbed cell cycle.

Bottom Line: At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB).During the G2/M transition when cyclin B-Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B-Cdc2-dependent manner and locally suppresses both cyclin B-Cdc2 activity and spindle assembly to counteract a Polo kinase-dependent positive feedback loop.Then Wee1 disappears from the SPB during spindle assembly.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Regulation, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3LY, United Kingdom. hiro.masuda@cancer.org.uk

ABSTRACT
Wee1 is a protein kinase that negatively regulates mitotic entry in G2 phase by suppressing cyclin B-Cdc2 activity, but its spatiotemporal regulations remain to be elucidated. We observe the dynamic behavior of Wee1 in Schizosaccharomyces pombe cells and manipulate its localization and kinase activity to study its function. At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB). During the G2/M transition when cyclin B-Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B-Cdc2-dependent manner and locally suppresses both cyclin B-Cdc2 activity and spindle assembly to counteract a Polo kinase-dependent positive feedback loop. Then Wee1 disappears from the SPB during spindle assembly. We propose that regulation of Wee1 localization around the SPB during the G2/M transition is important for proper mitotic entry and progression.

Show MeSH
Related in: MedlinePlus