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. 2010 Apr 19;189(2):247-59.
doi: 10.1083/jcb.200909144.

Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis

Olivier Gavet  1 , Jonathon Pines
Affiliations

Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis

Olivier Gavet et al. J Cell Biol. .

Abstract

The cyclin B-Cdk1 kinase triggers mitosis in most eukaryotes. In animal cells, cyclin B shuttles between the nucleus and cytoplasm in interphase before rapidly accumulating in the nucleus at prophase, which promotes disassembly of the nuclear lamina and nuclear envelope breakdown (NEBD). What triggers the nuclear accumulation of cyclin B1 is presently unclear, although the prevailing view is that the Plk1 kinase inhibits its nuclear export. In this study, we use a biosensor specific for cyclin B1-Cdk1 activity to show that activating cyclin B1-Cdk1 immediately triggers its rapid accumulation in the nucleus through a 40-fold increase in nuclear import that remains dependent on Cdk1 activity until NEBD. Nevertheless, a substantial proportion of cyclin B1-Cdk1 remains in the cytoplasm. The increase in nuclear import is driven by changes in the nuclear import machinery that require neither Plk1 nor inhibition of nuclear export. Thus, the intrinsic link between cyclin B1-Cdk1 activation and its rapid nuclear import inherently coordinates the reorganization of the nucleus and the cytoplasm at mitotic entry.

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Figures

Figure 1.
Figure 1.
Activation and nuclear accumulation of cyclin B1–Cdk1 are coincident. (A) HeLa cells coexpressing the cyclin B1–Cdk1 activity FRET sensor and cyclin B1–mCherry were recorded at one image every 1 min 40 s by time-lapse fluorescence and differential interference contrast (DIC) microscopy as cells entered mitosis. Two cells entering mitosis are displayed for DIC (top), FRET efficiency determined by emission ratio (middle), and cyclin B1–mCherry (bottom). Bars, 10 µm. (B) Quantification of the nuclear accumulation of cyclin B1 and emission ratio in three different cells. The vertical dashed line indicates when signals started to increase.
Figure 2.
Figure 2.
Nuclear accumulation of cyclin B1–Cdk1 depends on Cdk activity. (A) HeLa cells coexpressing the cyclin B1–Cdk1 activity FRET sensor and cyclin B1–mCherry were recorded at one image every 2 min as cells entered mitosis. After cyclin B1 began to move into the nucleus, 300 nM Cdk1/2 inhibitor III was added. The quantification curves correspond to the cell displayed. Note the immediate decrease in the nuclear accumulation rate of cyclin B1 just after addition of the Cdk inhibitor before its reexport ∼3 min later. (B) Cells analyzed as in A were treated with 5 µM aurora B inhibitor ZM447439 in late G2 phase (n = 6). Bars, 10 µm.
Figure 3.
Figure 3.
Active cyclin B1–Cdk1 kinase is located both in the cytoplasm and nucleus during prophase. HeLa cells coexpressing cytoplasmic and nuclear targeted cyclin B1–Cdk1 biosensors (fused to an NES and histone H2B, respectively) were recorded as cells entered mitosis (one image every 1 min 40 s). DIC (top), YFP emission (inverted grayscale; middle), and emission ratio (bottom) images are shown. Quantifications of the emission ratio in the whole cell, the nucleus, and the cytoplasm correspond to the cell displayed. Bar, 10 µm.
Figure 4.
Figure 4.
Nuclear accumulation of cyclin B1 in prophase is independent of its phosphorylation and of Plk1 activity. (A) Cells coexpressing wild-type (WT) cyclin B1–mCherry and 5xA (Ser116, 126,128,133, and 147A)–cyclin B1–GFP were assayed, and the nuclear accumulation of the proteins was quantified. Mean curves of quantifications in different cells are displayed (n = 5). (B) Cells expressing wild-type cyclin B1–mCherry were assayed, and100 nM Plk1 inhibitor (BI 2536) was added during the nuclear import of cyclin B1 in prophase. Two examples are displayed (one image/minute). Error bars show SEM.
Figure 5.
Figure 5.
The nuclear/cytoplasmic distribution of cyclin B1 is only affected by phosphorylation in its N terminus domain in interphase. (A) The nuclear/cytoplasmic ratio of wild-type (WT) cyclin B1–GFP, Ser126– and 128A–cyclin B1–GFP, Ser126–,128A–, and 133A–cyclin B1–GFP, 5xA (Ser116,126,128,133, and 147A)–cyclin B1–GFP, Ser126– and 128E–cyclin B1–GFP, Ser126–,128A–, and 133E–cyclin B1–GFP, and 5xE (Ser116,126,128,133, and 147E) cyclin B1–GFP was quantified on optical sections of asynchronous interphase HeLa cells. Mean values ± SEM and numbers of cells assayed are displayed. (B) Cells expressing either wild-type or 5xE cyclin B1–GFP were recorded. Real time changes in the nuclear/cytoplasmic ratio were quantified on optical sections as cells entered mitosis. Five examples are displayed for each experiment.
Figure 6.
Figure 6.
The nuclear accumulation of wild-type and 5xE mutant of cyclin B1 in prophase is concurrent. (A) The nuclear accumulation of cyclin B1 in prophase was assayed in cells coexpressing wild-type (WT) cyclin B1–mCherry and 5xE cyclin B1–GFP. DIC (top), mCherry (middle), and GFP (bottom) images are shown. The region of interest used for quantification of the nuclear signal is displayed. Mean curves of quantifications in different cells are displayed (n = 6). Bar, 10 µm. (B) Cells were assayed as in A, and 300 nM Cdk inhibitor (Cdk1/2 inhibitor III) was added during prophase. Two examples are displayed. Error bars show SEM.
Figure 7.
Figure 7.
Cyclin B1 nuclear import rate increases significantly at mitotic entry. Cells expressing either wild-type (WT) or 5xE cyclin B1–GFP were recorded, and the nuclear import of wild-type and 5xE cyclin B1 were quantified after treating G2 phase cells with 20 nM LMB to inhibit nuclear export. Note the sudden and strong increase of the wild-type cyclin B1 nuclear import rate when the cell enters mitosis.
Figure 8.
Figure 8.
The nuclear accumulation of cyclin B1–Cdk1 in prophase correlates with a change in nuclear transport machinery. (A and B) Cells coexpressing cyclin B1–mCherry and IBB-GFP (A) or GFP-M9 (B) were recorded as they entered mitosis (one image/30 s), and the import of cyclin B1 and export of IBB or M9 quantified on optical sections. (A) The mCherry (top) and GFP (bottom) images and mean curves of quantification of nuclear cyclin B1 and cytoplasmic IBB are shown. (B) mCherry (top), GFP (middle), and pseudo-colored GFP (rainbow look up table [LUT]; bottom) images and mean curves of quantification of the nuclear cyclin B1 and cytoplasmic M9 are displayed. Boxed images show the time point when cyclin B1 begins to enter the nucleus and GFP-M9 begins to exit. Error bars show SEM. Bars, 10 µm.
Figure 9.
Figure 9.
Cyclin B1 but not Cdc25C moves into the nucleus before the nuclear pores become permeable to tubulin. (A and B) Cells coexpressing cyclin B1–GFP and mCherry–α-tubulin (A) or cyclin B1–mCherry and GFP-Cdc25C (B) were recorded as they entered mitosis (one image/40 s and one image/30 s, respectively), and the import of cyclin B1, α-tubulin, and Cdc25C was quantified on optical sections. (A) The GFP (top) and mCherry (bottom) images and mean curves of quantification of nuclear cyclin B1 and α-tubulin are shown. (B) mCherry (top) and GFP (bottom) images and mean curves of quantification of the nuclear cyclin B1 and Cdc25C are displayed. Error bars show SEM. Bars, 10 µm.
Figure 10.
Figure 10.
Model showing how activating cyclin B1–Cdk1 synchronizes the cytoplasm with the nucleus. The activation (1) of cyclin B1–Cdk1 kinase synchronizes cytoplasmic (left) and nuclear (right) events through triggering its own nuclear import (2) by modifying nuclear import adapters, the functional properties of nuclear pores, or both.
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