Kang Q and Pomerening JR (2012), Punctuated cyclin synthesis drives early embryo...

XB-ART-44912

Mol Biol Cell 2012 Jan 01;232:284-96. doi: 10.1091/mbc.E11-09-0768.

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Punctuated cyclin synthesis drives early embryonic cell cycle oscillations.

Kang Q , Pomerening JR .

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Cyclin B activates cyclin-dependent kinase 1 (CDK1) at mitosis, but conflicting views have emerged on the dynamics of its synthesis during embryonic cycles, ranging from continuous translation to rapid synthesis during mitosis. Here we show that a CDK1-mediated negative-feedback loop attenuates cyclin production before mitosis. Cyclin B plateaus before peak CDK1 activation, and proteasome inhibition caused minimal accumulation during mitosis. Inhibiting CDK1 permitted continual cyclin B synthesis, whereas adding nondegradable cyclin stalled it. Cycloheximide treatment before mitosis affected neither cyclin levels nor mitotic entry, corroborating this repression. Attenuated cyclin production collaborates with its destruction, since excess cyclin B1 mRNA accelerated cyclin synthesis and caused incomplete proteolysis and mitotic arrest. This repression involved neither adenylation nor the 3' untranslated region, but it corresponded with a shift in cyclin B1 mRNA from polysome to nonpolysome fractions. A pulse-driven CDK1-anaphase-promoting complex (APC) model corroborated these results, revealing reduced cyclin levels during an oscillation and permitting more effective removal. This design also increased the robustness of the oscillator, with lessened sensitivity to changes in cyclin synthesis rate. Taken together, the results of this study underscore that attenuating cyclin synthesis late in interphase improves both the efficiency and robustness of the CDK1-APC oscillator.

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Species referenced: Xenopus laevis
Genes referenced: ccnb1 ccnb1.2 cdk1 neb

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	FIGURE 1:. Cyclin B translation is attenuated before and during mitosis. (A) Time course of cyclin B levels and H1 kinase activities in cycling extracts. 35S-Met-labeled cyclin was precipitated using p13 agarose; H1 kinase assays were performed to measure CDK1 activity; both were subjected to SDS-PAGE separation and visualized by phosphorimaging. Sperm chromatin was added to extracts; aliquots were taken at various times and stained with DAPI. Sperm chromatin forms nuclei in interphase (60 min is shown as representative), and then nuclei undergo mitosis, as indicated by chromatin condensation and NEB (70 and 80 min). Data were shifted to align the peak H1 kinase activities of all three replicates, with two replicates shifted 2 and 10 min earlier, respectively, excluding the earliest and latest lone time points from the analysis. Quantitated data are plotted as means ± SEM (error bars; N = 3). Bars above micrographs and H1 kinase activities denote time points where NEB was observed. (B, C) Time course of cyclin B levels and H1 kinase activities for cycling extracts treated with DMSO (1%) or MG132 (1 mM; 1% final volume). Data were shifted to align the peak H1 kinase activities of all four replicates, with two replicates shifted 10 and 20 min earlier, respectively, excluding the earliest and latest lone time points from the analysis. Data were analyzed as described in A and plotted as means ± SEM (N = 4). Bars denote time points where NEB was observed. Also see Supplemental Figure S1.
	FIGURE 2:. Inhibiting or prematurely activating CDK1 alters the level of cyclin B synthesis. (A–C) Time course of cyclin B levels (left) and H1 kinase activities (right) for cycling extracts treated with (A) DMSO (1%) and ethanol (0.64%), MG132 (1 mM) and ethanol, or MG132 (1 mM) and roscovitine (0.18 mM); (B) DMSO (1%) and buffer, MG132 (1 mM) and buffer, or MG132 (1 mM) and Δ65XCycB1 (200 nM); (C) CHX (100 μg/ml) added at different time points prior to and during mitosis in cycling egg extract. Data were shifted to align the peak H1 kinase activities of all three replicates, with one replicate shifted 5 min earlier, excluding the earliest and latest lone time points from the analysis. Data were analyzed as described in Figure 1A and plotted as means ± SEM (A, N = 3; B, N = 2; C, N = 3). Bars denote time points where NEB was observed (A–C), and histograms correspond to cyclin levels and H1 kinase activities before and after CHX addition (C); control (no addition), dark gray bars; 30 min addition, light green bars; 40 min addition, light blue bars; 50 min, orange bars; 55 min, light gray bars.
	FIGURE 3:. Polyadenylation of the cyclin B1 mRNA 3′ UTR does not vary with changes in cyclin B1 synthesis during cycling. (A, B) Time course of cyclin B levels (left) and H1 kinase activities (right) for cycling extracts treated with (A) water or 6 or 12 ng/μl in vitro–transcribed cyclin B1 mRNA coding region; (B) DMSO (1%), MG132 (1 mM), 6 ng/μl cyclin B1 mRNA coding region and MG132 (1 mM). For A, data were shifted to align the H1 kinase activity peaks of all three replicates, with two replicates shifted 10 min earlier, excluding the earliest and latest lone time points from the analysis; for B, one replicate was shifted 30 min earlier to align peak H1 kinase activities. Data were analyzed as described in Figure 1A and plotted as means ± SEM; A, N = 3; B, N = 2. Bars denote time points where NEB was observed. (C) Pooled data of cyclin B levels (left) and H1 kinase activities (right) plotted as means ± SEM (N = 3) for results shown in D. Data were shifted to align the H1 kinase activity peaks of all three replicates, with one replicate shifted 15 min earlier, excluding the earliest and latest lone time points from the analysis. (D) 32P-Labeled, in vitro–transcribed cyclin B1 3′ UTR was incubated in cycling extracts treated with either DMSO (1%; left) or the polyadenylation inhibitor cordycepin (1 mM; right). Dot indicates addition of nonpolyadenylated UTR. Aliquots were taken at indicated time points, and total RNAs were purified, separated on denaturing urea PAGE, and visualized by phosphorimaging. H1 kinase activities and cyclin B levels were analyzed as described in Figure 1A. Representative of one of three independent experiments is shown. (E) Pooled data of cyclin B levels (left) and H1 kinase activities (right) plotted as means ± SEM (N = 3) for results shown in F. (F) 32P-Labeled, in vitro–transcribed and polyadenylated Xenopus cyclin B1 3′ UTR incubated in extracts treatment were performed as described in D. Dot indicates addition of in vitro–polyadenylated UTR. Bars denote time points where NEB was observed. Representative of one of three independent experiments is shown. Also see Supplemental Figure S2.
	FIGURE 4:. Cyclin B translational repression is mitosis specific and collaborates with APC function. (A, B) Polyadenylation of cyclin B1 mRNA promotes translation. Water, 12 ng/μl in vitro–transcribed cyclin B1 mRNA coding region, or in vitro–polyadenylated cyclin B1 mRNA coding region were added to cycling extracts arrested in interphase (I) by roscovitine (0.18 mM) and MG132 (1 mM; A) and mitosis (M) by Δ65XCycB1 (67 nM) and MG132 (1 mM; B), respectively, in the presence of 35S-Met. Aliquots were collected at 15-min intervals and analyzed as described in Figure 1A. Cyclin B levels and H1 kinase activities are plotted as means ± SEM (N = 2). Bars denote time points where NEB was observed. (C) Cyclin B is translated in CSF-M extract. CSF-M extracts were treated with DMSO (1%) and buffer, DMSO and Δ65XCycB1 (65 nM), MG132 (1 mM) and buffer, or MG132 (1 mM) and Δ65XCycB1 (65 nM). Samples were collected at 15-min intervals and analyzed as described in Figure 1A. Quantitated data are plotted as means ± SEM (N = 2). Bars denote time points where NEB was observed. (D) Rapid production of cyclin B1 hampers its destruction and causes mitotic arrest. Time course of H1 kinase activities (right) and cyclin B levels (left) for cycling extracts treated with water, 24 or 48 ng/μl cyclin B1 mRNA coding region. Quantitated data are plotted as means ± SEM (N = 2). Bars denote time points where NEB was observed. Also see Supplemental Figures S3 and S4.
	FIGURE 5:. A reduction of cyclin B1 mRNA content in polysome fractions at M phase corresponds to its diminished translation. (A) Luciferase mRNA is poorly translated in mitosis-arrested extract. In vitro–transcribed luciferase mRNA (5 ng/μl) was added to cycling extracts treated with either buffer or Δ65XCycB1 (200 nM). Samples were collected at 15-min intervals in a 96-well plate cooled on ice, and luciferase assays were then performed. H1 kinase activities were analyzed as described in Figure 1A (bottom) and plotted with luciferase signals as means ± SEM (N = 2; top), in cycling egg extract (boxes), and Δ65XCycB1-arrested extract (dots). Data were shifted to align the H1 kinase activity peaks of both replicates, with one replicate shifted 15 min earlier, excluding the earliest and latest lone time points from the analysis. Bars above H1 kinase activities denote time points where NEB was observed. (B) Top, samples of interphase extract and mitotic extract (just prior to NEB) were separated by sucrose gradient centrifugation. Total RNA from 40S, 60S, 80S, and polysome fractions was purified, and the distribution of cyclin B1 mRNA was analyzed by qRT-PCR. Left of the dashed line is defined as nonpolysome fraction (40S, 60S, and 80S) and on its right is defined as the polysome fraction. The percentage cyclin B1 mRNA in each fraction was quantitated and normalized as a percentage of total across all fractions, in duplicate. (B) Bottom, differences in the percentage of cyclin B1 mRNA in nonpolysome and polysome fractions (n − p) between interphase and mitosis is presented as mean ± SEM (N = 3). Also see Supplemental Figure S5.
	FIGURE 6:. Attenuation of cyclin synthesis at M phase yields a CDK1 oscillator with some functional advantages. (A) Schematic of ODE model with constant cyclin synthesis (left); three oscillations of cyclin (gray) and CDK1 activity (black) from ODE model with constant cyclin synthesis (ksynthcycB = 0.2; ksynthcycBoff = 0; right). In this model, APC degrades all cyclin complexed with CDK1 and newly synthesized cyclin monomer. (B) Schematic of ODE model with pulses of cyclin synthesis caused by CDK1 activity repressing cyclin synthesis (left); three oscillations of cyclin (gray) and CDK1 activity (black) from ODE model with pulses of cyclin synthesis (ksynthcycB = 0.33; ksynthcycBoff = 0.2; right). In this model, CDK1 activity is inhibitory toward cyclin synthesis. Note that although the inhibitory leg of APC toward cyclin monomer shown in B is included in this computational model, it effectively disappears because no new cyclin synthesis occurs once APC is active. (C) Cyclin levels (top) and CDK1 activity (bottom) of ODE models with either constant cyclin synthesis (black) or cyclin pulses (gray) during a single oscillation (shown as the third oscillation in the simulation from 150 to 220 min to avoid minor variations in initial transients). (D) Peak cyclin levels (left) and trough cyclin levels (right) generated by ODE models with constant cyclin synthesis (black) or cyclin pulses (gray) as the parameter ksynthcycB (rate of cyclin synthesis) is varied. For consistency, peak and trough values of the limit cycle oscillations were measured. Arrows extending from the abscissa demarcate ksynthcycB values that produce a frequency of CDK1 oscillations representative of that observed in cycling egg extracts (shown in A and B, for constant synthesis and pulse, respectively); horizontal lines toward the ordinate indicate the median value required to produce the oscillations in the constant cyclin synthesis (black) and cyclin pulse (gray) models as shown in A and B, respectively. To generate oscillations in the cyclin pulse model, ksynthcycB values >1.8 were required. (E) Comparison of the sensitivity to changes in cyclin synthesis rate (ksynthcycB) between CDK1 oscillator models with constant or attenuated cyclin synthesis. Black arrow indicates changes in ksynthcycB required for an increase from three to four oscillations over a period of 250 min; gray arrow indicates changes in ksynthcycB required for an increase from four to five oscillations (left). Histogram (right) plotting differences in the average change in oscillation number over a period of 250 min per change in ksynthcycB, showing differences in sensitivity to increasing the oscillation number from three to four (left histogram pair; refer to black arrow in left plot) and from four to five (right histogram pair; refer to gray arrow in left plot).

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