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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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Wee1 temporally accumulates at the SPB during the G2/M transition. (A) Wee1 accumulates at the SPB when cytoplasmic microtubules are reorganized into a mitotic spindle and disappears from the SPB after spindle assembly. Live images of a cell expressing GFP–Wee1 and mCherry–Atb2 are shown. Time 0 represents when the bipolar spindle forms. (B) Timing of Wee1 accumulation at the SPB compared with mitotic proteins accumulated at the SPB. Live images of nuclear regions in cells expressing GFP–Wee1 and Plo1–mCherry, Cut7–mCherry, or Msd1–mCherry are shown. Time 0 represents when the SPBs are separated. (C) Cdc13 accumulates at the SPB in late G2 and M phase. Live cell images of cells expressing Cdc13–GFP and Sid4–mRFP are shown at 12 min before SPB separation through 11 min after SPB separation. Maximal GFP intensity at the SPB is labeled at each time point. From time 0–11 min, the average of intensities at the two SPBs is shown. (D) Quantification of Cdc13–GFP and GFP–Wee1 localized at the SPB. Average intensities of GFP signals at the SPB from cells expressing either Cdc13–GFP (N = 10) or GFP–Wee1 (N = 7) are shown as a function of time after SPB separation. Error bars show SD. (E) Cdc13 levels decrease in cdc13.117 cells. Left, live cell images of cells expressing Cdc13.117–GFP and Cdc13–GFP. Right, quantification of Cdc13.117–GFP and Cdc13–GFP in mid-late G2 cells. Average of the maximum GFP signal intensities is shown. (F) Levels of GFP–Wee1 accumulated at the SPB during the G2/M transition are reduced in cdc13.117 cells. Left, live cell images of a GFP–wee1 cdc13.117 cell and a GFP–wee1 cdc13+ cell; and right, quantification of GFP–Wee1 at the SPB. Bars, 10 μm.
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Figure 2: Wee1 temporally accumulates at the SPB during the G2/M transition. (A) Wee1 accumulates at the SPB when cytoplasmic microtubules are reorganized into a mitotic spindle and disappears from the SPB after spindle assembly. Live images of a cell expressing GFP–Wee1 and mCherry–Atb2 are shown. Time 0 represents when the bipolar spindle forms. (B) Timing of Wee1 accumulation at the SPB compared with mitotic proteins accumulated at the SPB. Live images of nuclear regions in cells expressing GFP–Wee1 and Plo1–mCherry, Cut7–mCherry, or Msd1–mCherry are shown. Time 0 represents when the SPBs are separated. (C) Cdc13 accumulates at the SPB in late G2 and M phase. Live cell images of cells expressing Cdc13–GFP and Sid4–mRFP are shown at 12 min before SPB separation through 11 min after SPB separation. Maximal GFP intensity at the SPB is labeled at each time point. From time 0–11 min, the average of intensities at the two SPBs is shown. (D) Quantification of Cdc13–GFP and GFP–Wee1 localized at the SPB. Average intensities of GFP signals at the SPB from cells expressing either Cdc13–GFP (N = 10) or GFP–Wee1 (N = 7) are shown as a function of time after SPB separation. Error bars show SD. (E) Cdc13 levels decrease in cdc13.117 cells. Left, live cell images of cells expressing Cdc13.117–GFP and Cdc13–GFP. Right, quantification of Cdc13.117–GFP and Cdc13–GFP in mid-late G2 cells. Average of the maximum GFP signal intensities is shown. (F) Levels of GFP–Wee1 accumulated at the SPB during the G2/M transition are reduced in cdc13.117 cells. Left, live cell images of a GFP–wee1 cdc13.117 cell and a GFP–wee1 cdc13+ cell; and right, quantification of GFP–Wee1 at the SPB. Bars, 10 μm.

Mentions: We also found that Wee1 started to accumulate at the SPB after the interphase arrays of cytoplasmic microtubules started to disassemble at 3–4 min before spindle formation and disappeared after a bipolar spindle formed (Figure 2A). To confirm that Wee1 accumulated at the SPB during the G2/M transition but not in the late G2 phase, the timing of Wee1 accumulation was compared with the timing when the mitotic proteins Plo1, Cut7, and Msd1 were enriched at the SPBs (Figure 2B). Polo-like kinase Plo1 is a marker of mitotic entry, and a major accumulation of Plo1 at the SPB is observed after Cdc2 kinase activation (Mulvihill et al., 1999). Cut7 belongs to a family of kinesin-5, which is required for bipolar spindle assembly. Cut7 localizes in the nucleus during interphase and accumulates at the SPB and on spindles during mitosis (Hagan and Yanagida, 1992). Msd1 is involved in anchoring spindle microtubules to the SPB and localizes to the SPB in early M phase (Toya et al., 2007). We observed that, in comparison to these proteins, Wee1 accumulation at the SPB was preceded by the recruitment of Plo1 to the SPB, almost concomitant with Cut7, and was consequently followed by Msd1 (Figure 2B). In addition, we also examined the timing of accumulation of the B-type cyclin Cdc13 at the SPB. Cdc13 localizes predominantly in the nucleus and at the SPB in the G2 phase of the cell cycle (Decottignies et al., 2001). The accumulation of Cdc13 at the SPB started to increase sharply ∼6 min before SPB separation, peaked at 3 min before separation, and decreased to the levels of late G2 after separation (Figure 2, C and D). The beginning of increase in the levels of Cdc13 accumulated at the SPB seemed to correspond to the start of the G2/M transition, due to always being followed by SPB separation. The levels of Wee1 at the SPB stayed low when Cdc13 was accumulating at the SPB and then temporally increased around the time when levels of Cdc13 at the SPB reached their peak (Figure 2D). Thus the peak of SPB accumulation of Cdc13 is followed by the accumulation of Wee1 at the SPB. On the basis of these observations, we define here that the G2/M transition starts when Cdc13 starts to increase in levels at the SPB (at ∼6 min before SPB separation) and ends when a bipolar spindle forms (at the time of SPB separation) (Figure 2D). To study whether Cdc13–Cdc2 has a role in localization of Wee1 to the SPB, we observed Wee1 localization in a temperature-sensitive cdc13-117 mutant. The cdc13-117 cells have low levels of Cdc13 at the permissive temperatures but still enter mitosis (Booher et al., 1989) (Figure 2E). We found that levels of GFP–Wee1 accumulated at the SPB at the G2/M transition were significantly reduced in the cdc13-117 cells (Figure 2F). These results suggest that Wee1 localization to the SPB depends on Cdc13–Cdc2.


Spatiotemporal regulations of Wee1 at the G2/M transition.

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

Wee1 temporally accumulates at the SPB during the G2/M transition. (A) Wee1 accumulates at the SPB when cytoplasmic microtubules are reorganized into a mitotic spindle and disappears from the SPB after spindle assembly. Live images of a cell expressing GFP–Wee1 and mCherry–Atb2 are shown. Time 0 represents when the bipolar spindle forms. (B) Timing of Wee1 accumulation at the SPB compared with mitotic proteins accumulated at the SPB. Live images of nuclear regions in cells expressing GFP–Wee1 and Plo1–mCherry, Cut7–mCherry, or Msd1–mCherry are shown. Time 0 represents when the SPBs are separated. (C) Cdc13 accumulates at the SPB in late G2 and M phase. Live cell images of cells expressing Cdc13–GFP and Sid4–mRFP are shown at 12 min before SPB separation through 11 min after SPB separation. Maximal GFP intensity at the SPB is labeled at each time point. From time 0–11 min, the average of intensities at the two SPBs is shown. (D) Quantification of Cdc13–GFP and GFP–Wee1 localized at the SPB. Average intensities of GFP signals at the SPB from cells expressing either Cdc13–GFP (N = 10) or GFP–Wee1 (N = 7) are shown as a function of time after SPB separation. Error bars show SD. (E) Cdc13 levels decrease in cdc13.117 cells. Left, live cell images of cells expressing Cdc13.117–GFP and Cdc13–GFP. Right, quantification of Cdc13.117–GFP and Cdc13–GFP in mid-late G2 cells. Average of the maximum GFP signal intensities is shown. (F) Levels of GFP–Wee1 accumulated at the SPB during the G2/M transition are reduced in cdc13.117 cells. Left, live cell images of a GFP–wee1 cdc13.117 cell and a GFP–wee1 cdc13+ cell; and right, quantification of GFP–Wee1 at the SPB. Bars, 10 μm.
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Figure 2: Wee1 temporally accumulates at the SPB during the G2/M transition. (A) Wee1 accumulates at the SPB when cytoplasmic microtubules are reorganized into a mitotic spindle and disappears from the SPB after spindle assembly. Live images of a cell expressing GFP–Wee1 and mCherry–Atb2 are shown. Time 0 represents when the bipolar spindle forms. (B) Timing of Wee1 accumulation at the SPB compared with mitotic proteins accumulated at the SPB. Live images of nuclear regions in cells expressing GFP–Wee1 and Plo1–mCherry, Cut7–mCherry, or Msd1–mCherry are shown. Time 0 represents when the SPBs are separated. (C) Cdc13 accumulates at the SPB in late G2 and M phase. Live cell images of cells expressing Cdc13–GFP and Sid4–mRFP are shown at 12 min before SPB separation through 11 min after SPB separation. Maximal GFP intensity at the SPB is labeled at each time point. From time 0–11 min, the average of intensities at the two SPBs is shown. (D) Quantification of Cdc13–GFP and GFP–Wee1 localized at the SPB. Average intensities of GFP signals at the SPB from cells expressing either Cdc13–GFP (N = 10) or GFP–Wee1 (N = 7) are shown as a function of time after SPB separation. Error bars show SD. (E) Cdc13 levels decrease in cdc13.117 cells. Left, live cell images of cells expressing Cdc13.117–GFP and Cdc13–GFP. Right, quantification of Cdc13.117–GFP and Cdc13–GFP in mid-late G2 cells. Average of the maximum GFP signal intensities is shown. (F) Levels of GFP–Wee1 accumulated at the SPB during the G2/M transition are reduced in cdc13.117 cells. Left, live cell images of a GFP–wee1 cdc13.117 cell and a GFP–wee1 cdc13+ cell; and right, quantification of GFP–Wee1 at the SPB. Bars, 10 μm.
Mentions: We also found that Wee1 started to accumulate at the SPB after the interphase arrays of cytoplasmic microtubules started to disassemble at 3–4 min before spindle formation and disappeared after a bipolar spindle formed (Figure 2A). To confirm that Wee1 accumulated at the SPB during the G2/M transition but not in the late G2 phase, the timing of Wee1 accumulation was compared with the timing when the mitotic proteins Plo1, Cut7, and Msd1 were enriched at the SPBs (Figure 2B). Polo-like kinase Plo1 is a marker of mitotic entry, and a major accumulation of Plo1 at the SPB is observed after Cdc2 kinase activation (Mulvihill et al., 1999). Cut7 belongs to a family of kinesin-5, which is required for bipolar spindle assembly. Cut7 localizes in the nucleus during interphase and accumulates at the SPB and on spindles during mitosis (Hagan and Yanagida, 1992). Msd1 is involved in anchoring spindle microtubules to the SPB and localizes to the SPB in early M phase (Toya et al., 2007). We observed that, in comparison to these proteins, Wee1 accumulation at the SPB was preceded by the recruitment of Plo1 to the SPB, almost concomitant with Cut7, and was consequently followed by Msd1 (Figure 2B). In addition, we also examined the timing of accumulation of the B-type cyclin Cdc13 at the SPB. Cdc13 localizes predominantly in the nucleus and at the SPB in the G2 phase of the cell cycle (Decottignies et al., 2001). The accumulation of Cdc13 at the SPB started to increase sharply ∼6 min before SPB separation, peaked at 3 min before separation, and decreased to the levels of late G2 after separation (Figure 2, C and D). The beginning of increase in the levels of Cdc13 accumulated at the SPB seemed to correspond to the start of the G2/M transition, due to always being followed by SPB separation. The levels of Wee1 at the SPB stayed low when Cdc13 was accumulating at the SPB and then temporally increased around the time when levels of Cdc13 at the SPB reached their peak (Figure 2D). Thus the peak of SPB accumulation of Cdc13 is followed by the accumulation of Wee1 at the SPB. On the basis of these observations, we define here that the G2/M transition starts when Cdc13 starts to increase in levels at the SPB (at ∼6 min before SPB separation) and ends when a bipolar spindle forms (at the time of SPB separation) (Figure 2D). To study whether Cdc13–Cdc2 has a role in localization of Wee1 to the SPB, we observed Wee1 localization in a temperature-sensitive cdc13-117 mutant. The cdc13-117 cells have low levels of Cdc13 at the permissive temperatures but still enter mitosis (Booher et al., 1989) (Figure 2E). We found that levels of GFP–Wee1 accumulated at the SPB at the G2/M transition were significantly reduced in the cdc13-117 cells (Figure 2F). These results suggest that Wee1 localization to the SPB depends on Cdc13–Cdc2.

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