Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

Abstract

The fertilising sperm triggers a transient Ca(2+) increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/C(cdc20) inhibitor, links release from metaphase II arrest with the Ca(2+) transient and its degradation is triggered by the Ca(2+)-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca(2+) transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca(2+)-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca(2+) increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates.

Keywords: CaMKII; Calcineurin; Egg activation; Meiotic arrest; PP2A.

Fig. 1.

CaMKII is not active after ascidian egg activation. (A) KN93 (150 μM) has no effect on Cyclin B Y170A destruction. Example traces of control eggs and eggs injected with KN93, both expressing cyclin B1 Y170A, were activated with ionomycin and the decrease in fluorescence as a result of cyclin destruction recorded. (A′) Analysis of destruction rates expressed as t_1/2 values ±s.e.m. for all the eggs recorded in A show there is no significant difference in the destruction rates in control and KN93-injected eggs (n in parentheses). (B) CaMKII assays on ascidian eggs sampled at the times shown after activation with ionomycin. CaMKII activity (expressed as a percentage of the value at time 0) shows no increase up to and including 16 minutes post-activation; the rise at 20 minutes is not significant (P=0.7) (n=3, 15 eggs used per time point). Error bars represent s.e.m. (C) The CaMKII assay is sufficiently sensitive. Varying numbers of eggs expressing CA CaMKII were harvested along with the same number of control eggs. Each sample was assayed for CaMKII activity and the results show activity is detectable at more than three times that in controls in four eggs. (D) Exogenously expressed CA CaMKII activity is inhibited by KN93. Three eggs expressing CA CaMKII were injected with KN93 (final concentration 150 μM) and assayed for CaMKII activity along with three control eggs and three eggs expressing CA CaMKII (n=3). Error bars represent s.e.m.

Fig. 2.

CN activity is necessary for full activation of the APC following activation with calcium. (A) Cyclin B Y170A destruction is impaired by CsA (1 μM) (post-activation time given on x-axis; n=10 from three animals). The spindle (green) remains intact near the egg cortex 92 minutes after activation, whereas by 44 minutes controls have extruded two polar bodies and the chromatin (red) has fully decondensed within a pronucleus. (A′) Analysis of Cyclin B Y170A destruction rates, expressed as mean t_1/2 values ±s.e.m. showing that destruction is approximately four times slower in CsA (n in parentheses). (B) Time course of MAPK T¹⁸³ dephosphorylation at the times shown (in minutes) after fertilisation, showing complete dephosphorylation by 40 minutes (n=3). (B′) T¹⁸³ remains phosphorylated 40 minutes after fertilisation in the presence of CsA (n=3). (B′) T¹⁸³ remains phosphorylated 40 minutes after fertilisation in the presence of Δ90 cyclin B (n=3). (B′′) Image shows the pronucleus (arrow) formed after fertilisation in the presence of CsA and roscovitine. Graph shows the percentage of eggs forming pronuclei in the treatments shown [n in parentheses; error bars represent counting error (√n) from one experiment]. (C) Fertilisation-triggered calcium oscillations persist in the presence of CsA but cease ∼15 minutes after U0126 (20 μM) addition (n=4). (C′) Pattern of calcium oscillations in a control egg, ceasing at ∼25 minutes. Time shown represents minutes post-fertilisation. (C′,C′′) Eggs fertilised in CsA and then treated with U0126 after 40 minutes destroy Cyclin B Y170A ∼30 minutes later (C′, red trace) and eventually undergo cytokinesis (n=15/17) as shown in the bright-field (C′′, left) and Map7::GFP (C′′, right) images. All graphs shown are example traces. Scale bars: in A, 20 μm in whole egg images,10 μm in enlarged images; in B′′, 10 μm.

Fig. 3.

ST protein is a specific inhibitor of PP2A. (A,A′) Inhibition of APC/C activity by ST protein, as measured by Cyclin B Y170A destruction, can be competed out by co-injection with a 1.5 to 2-fold excess of ascidian PP2A A subunit protein over ST protein (n in parentheses; error bars represent s.e.m.). (B,B′) APC/C activity, again measured by Cyclin B Y170A destruction, is unperturbed when STR7A is injected instead of ST (n in parentheses, error bars represent s.e.m.). All graphs shown are example traces.

Fig. 4.

Inhibition of PP2A with ST protein prevents activation both by U1026 and calcium. (A) U0126 triggers Cyclin B Y170A destruction, whereas injection of ST protein prevents U0126-mediated destruction of Cyclin B Y170A (n=7 from three animals). The spindle (green) remains intact 37 minutes after addition of U0126 (20 μM), at which time control eggs had exited meiosis with DNA decondensed (red) within a pronucleus. (A′) Cyclin B Y170A destruction rate, expressed as mean of t_1/2 values ±s.e.m., in U0126-treated eggs is approximately half that of control fertilised eggs (n in parentheses). (B) Schematic of our hypothesis for metaphase I (Meta I) arrest and how it is broken by a calcium signal. (C) Injection with ST protein fails to prevent dephosphorylation of T¹⁸³ in U0126-activated eggs after 40 minutes (n=3). (C′) T¹⁸³ remains phosphorylated after 40 minutes in eggs injected with ST and activated with ionomycin (n=3). (D) Cyclin B Y170A destruction is impaired (n=10 from three animals). DNA decondensation (red) and polar body emission inhibited in eggs injected with ST protein and activated with ionomycin (n=8 from three animals), whereas destruction is blocked in eggs injected with ST protein and treated with CsA prior to activation with ionomycin. (D′) Cyclin B Y170A destruction rate, expressed as mean t_1/2 values ±s.e.m., is five times slower in ST-injected eggs (n in parentheses). (D′) Comparison of APC/C inhibition by ST protein and CsA showing a similar level of inhibition. Error bars represent s.e.m. All graphs shown are example traces. Scale bars: in A and D, 20 μm in whole egg images, 10 μm in enlarged images.

Fig. 5.

Our current model. During metaphase I arrest, Cdk1 maintains the activity of Mos. This maintains the MEK/MAPK pathway active and suppresses the APC/C. After egg activation, calcineurin and PP2A activate the APC/C through dephosphorylation, initially in the continuing presence of MAPK activity. As the increase in APC/C activity causes inactivation of Cdk1, Mos is inactivated and so MEK/MAPK activity falls.