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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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Related in: MedlinePlus

Wee1 dynamically localizes to the SPB. (A) GFP–Wee1 localizes in the nucleus and temporally accumulates at the SPB before SPB separation. Live images of a cell expressing GFP–Wee1 and Sid4–mRFP are shown at 1-min intervals. Time 0 represents when the two SPBs are separated for spindle assembly. GFP–Wee1 signal is observed in the nucleus and at the SPB. The fibrous structures observed in the cytoplasm are likely to come from the autofluorescence of mitochondrial flavoproteins (Kunz et al., 1997). The cell shape is outlined in white on a merged image. (B) Quantification of the maximal intensity of GFP–Wee1 observed at the SPB. Data from three cells are shown. Cell 3 represents data from the cell shown in (A). (C) Levels of Wee1 localized at the SPB fluctuate during late G2. Live images of the SPB region of a cell expressing GFP–Wee1 and Sid4–mRFP. Images at 18 min before SPB separation through 1 min after SPB separation are shown. Maximal GFP signal intensity measured at the SPB is labeled at each time point on the merged image. (D) Quantification of GFP–Wee1 at the SPB in cells showing a fluctuation of Wee1 levels in late G2. Data from three cells are shown. Cell 3 represents the data from the cell shown in (C). (E) Wee1 localized at the SPB decreases to the minimum level before Wee1 is highly enriched at the SPB. Time 0 represents when the GFP signal intensity at the SPB reaches the minimum level. The ratio of GFP signal intensity to the minimum—(It − Ibg)/(I0 − Ibg), where Ibg is the background GFP intensity—is plotted at each time point until the signal intensity reaches the maximum level. Note that in 5, 6, and 1 out of the 16 cells observed, Wee1 levels at the SPB sharply increase at 1, 2, and 4 min after it reaches the minimum, respectively. In the other four cells, the GFP signal intensity at the SPB decreases to the minimum and then goes up and down once before it sharply increases. Bars, 10 μm.
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Figure 1: Wee1 dynamically localizes to the SPB. (A) GFP–Wee1 localizes in the nucleus and temporally accumulates at the SPB before SPB separation. Live images of a cell expressing GFP–Wee1 and Sid4–mRFP are shown at 1-min intervals. Time 0 represents when the two SPBs are separated for spindle assembly. GFP–Wee1 signal is observed in the nucleus and at the SPB. The fibrous structures observed in the cytoplasm are likely to come from the autofluorescence of mitochondrial flavoproteins (Kunz et al., 1997). The cell shape is outlined in white on a merged image. (B) Quantification of the maximal intensity of GFP–Wee1 observed at the SPB. Data from three cells are shown. Cell 3 represents data from the cell shown in (A). (C) Levels of Wee1 localized at the SPB fluctuate during late G2. Live images of the SPB region of a cell expressing GFP–Wee1 and Sid4–mRFP. Images at 18 min before SPB separation through 1 min after SPB separation are shown. Maximal GFP signal intensity measured at the SPB is labeled at each time point on the merged image. (D) Quantification of GFP–Wee1 at the SPB in cells showing a fluctuation of Wee1 levels in late G2. Data from three cells are shown. Cell 3 represents the data from the cell shown in (C). (E) Wee1 localized at the SPB decreases to the minimum level before Wee1 is highly enriched at the SPB. Time 0 represents when the GFP signal intensity at the SPB reaches the minimum level. The ratio of GFP signal intensity to the minimum—(It − Ibg)/(I0 − Ibg), where Ibg is the background GFP intensity—is plotted at each time point until the signal intensity reaches the maximum level. Note that in 5, 6, and 1 out of the 16 cells observed, Wee1 levels at the SPB sharply increase at 1, 2, and 4 min after it reaches the minimum, respectively. In the other four cells, the GFP signal intensity at the SPB decreases to the minimum and then goes up and down once before it sharply increases. Bars, 10 μm.

Mentions: We studied the dynamic changes of the subcellular localization of Wee1 around the G2/M transition by live cell imaging using Wee1 tagged with N-terminal green fluorescent protein (GFP). We replaced wild-type wee1+ with GFP–wee1, the expression of which was under the control of the endogenous promoter. To observe mitotic progression of the GFP–Wee1 cells, the SPB was labeled using Sid4–monomeric red fluorescent protein (mRFP), or microtubules were labeled using mCherry–Atb2. We found that GFP–Wee1 was localized mostly in the nucleus at mid-late G2 phase, was enriched at the SPB at 2–3 min before SPB separation, and had disappeared from the SPB after SPB separation (Figure 1, A and B). Nuclear Wee1 lasted through M phase but then gradually decreased during M phase, ultimately disappearing in late anaphase. In most cells, the SPB localization of Wee1 was limited to a short period around the G2/M transition as described above; however, in some cells, it was also observed at late G2 phase at lower and fluctuating levels (Figure 1, C and D). In these cells, Wee1 decreased at the SPB to the minimum levels ∼1–2 min before its levels sharply increased before SPB separation (Figure 1E), suggesting that the removal of Wee1 from the SPB is strictly regulated at this point.


Spatiotemporal regulations of Wee1 at the G2/M transition.

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

Wee1 dynamically localizes to the SPB. (A) GFP–Wee1 localizes in the nucleus and temporally accumulates at the SPB before SPB separation. Live images of a cell expressing GFP–Wee1 and Sid4–mRFP are shown at 1-min intervals. Time 0 represents when the two SPBs are separated for spindle assembly. GFP–Wee1 signal is observed in the nucleus and at the SPB. The fibrous structures observed in the cytoplasm are likely to come from the autofluorescence of mitochondrial flavoproteins (Kunz et al., 1997). The cell shape is outlined in white on a merged image. (B) Quantification of the maximal intensity of GFP–Wee1 observed at the SPB. Data from three cells are shown. Cell 3 represents data from the cell shown in (A). (C) Levels of Wee1 localized at the SPB fluctuate during late G2. Live images of the SPB region of a cell expressing GFP–Wee1 and Sid4–mRFP. Images at 18 min before SPB separation through 1 min after SPB separation are shown. Maximal GFP signal intensity measured at the SPB is labeled at each time point on the merged image. (D) Quantification of GFP–Wee1 at the SPB in cells showing a fluctuation of Wee1 levels in late G2. Data from three cells are shown. Cell 3 represents the data from the cell shown in (C). (E) Wee1 localized at the SPB decreases to the minimum level before Wee1 is highly enriched at the SPB. Time 0 represents when the GFP signal intensity at the SPB reaches the minimum level. The ratio of GFP signal intensity to the minimum—(It − Ibg)/(I0 − Ibg), where Ibg is the background GFP intensity—is plotted at each time point until the signal intensity reaches the maximum level. Note that in 5, 6, and 1 out of the 16 cells observed, Wee1 levels at the SPB sharply increase at 1, 2, and 4 min after it reaches the minimum, respectively. In the other four cells, the GFP signal intensity at the SPB decreases to the minimum and then goes up and down once before it sharply increases. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 1: Wee1 dynamically localizes to the SPB. (A) GFP–Wee1 localizes in the nucleus and temporally accumulates at the SPB before SPB separation. Live images of a cell expressing GFP–Wee1 and Sid4–mRFP are shown at 1-min intervals. Time 0 represents when the two SPBs are separated for spindle assembly. GFP–Wee1 signal is observed in the nucleus and at the SPB. The fibrous structures observed in the cytoplasm are likely to come from the autofluorescence of mitochondrial flavoproteins (Kunz et al., 1997). The cell shape is outlined in white on a merged image. (B) Quantification of the maximal intensity of GFP–Wee1 observed at the SPB. Data from three cells are shown. Cell 3 represents data from the cell shown in (A). (C) Levels of Wee1 localized at the SPB fluctuate during late G2. Live images of the SPB region of a cell expressing GFP–Wee1 and Sid4–mRFP. Images at 18 min before SPB separation through 1 min after SPB separation are shown. Maximal GFP signal intensity measured at the SPB is labeled at each time point on the merged image. (D) Quantification of GFP–Wee1 at the SPB in cells showing a fluctuation of Wee1 levels in late G2. Data from three cells are shown. Cell 3 represents the data from the cell shown in (C). (E) Wee1 localized at the SPB decreases to the minimum level before Wee1 is highly enriched at the SPB. Time 0 represents when the GFP signal intensity at the SPB reaches the minimum level. The ratio of GFP signal intensity to the minimum—(It − Ibg)/(I0 − Ibg), where Ibg is the background GFP intensity—is plotted at each time point until the signal intensity reaches the maximum level. Note that in 5, 6, and 1 out of the 16 cells observed, Wee1 levels at the SPB sharply increase at 1, 2, and 4 min after it reaches the minimum, respectively. In the other four cells, the GFP signal intensity at the SPB decreases to the minimum and then goes up and down once before it sharply increases. Bars, 10 μm.
Mentions: We studied the dynamic changes of the subcellular localization of Wee1 around the G2/M transition by live cell imaging using Wee1 tagged with N-terminal green fluorescent protein (GFP). We replaced wild-type wee1+ with GFP–wee1, the expression of which was under the control of the endogenous promoter. To observe mitotic progression of the GFP–Wee1 cells, the SPB was labeled using Sid4–monomeric red fluorescent protein (mRFP), or microtubules were labeled using mCherry–Atb2. We found that GFP–Wee1 was localized mostly in the nucleus at mid-late G2 phase, was enriched at the SPB at 2–3 min before SPB separation, and had disappeared from the SPB after SPB separation (Figure 1, A and B). Nuclear Wee1 lasted through M phase but then gradually decreased during M phase, ultimately disappearing in late anaphase. In most cells, the SPB localization of Wee1 was limited to a short period around the G2/M transition as described above; however, in some cells, it was also observed at late G2 phase at lower and fluctuating levels (Figure 1, C and D). In these cells, Wee1 decreased at the SPB to the minimum levels ∼1–2 min before its levels sharply increased before SPB separation (Figure 1E), suggesting that the removal of Wee1 from the SPB is strictly regulated at this point.

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