Kazunori Tachibana

Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes

M phase promoting factor (MPF) is a major element controlling entry into the M phase of the eukaryotic cell cycle. MPF is composed of two subunits, p34cdc2 and cyclin B. Using indirect immunofluorescence staining with specific antibody against starfish cyclin B, we monitored the dynamics of the subcellular distribution of MPF during meiosis reinitiation in starfish oocytes. We found that all of the cyclin B is already associated with p34cdc2 in immature oocytes arrested at the G2/M border and that this inactive complex is present exclusively in the cytoplasm. After its activation, part of the p34cdc2‐cyclin B complex moves into the germinal vesicle before nuclear envelope breakdown, independently of either microtubules or actin filaments. Thereafter, some part of the complex accumulates in the nucleolus and condensed chromosomes. Another portion of the complex accumulates on meiotic asters and spindles, while the rest is still present throughout the cytoplasm. As these patterns of localization are detected in the detergent‐extracted oocytes, we propose at least four distinct subcellular states of the p34cdc2‐cyclin B complex: freely soluble, microtubule‐associated, detergent‐resistant cytoskeleton‐associated and chromosome‐associated. Thus, in addition to the intramolecular modification of p34cdc2‐cyclin B complex, its intracellular relocation plays a key role in promoting the M phase.

Akt inhibits Myt1 in the signalling pathway that leads to meiotic G2/M-phase transition

In eukaryotes, entry into M-phase of the cell cycle is induced by activation of cyclin B–Cdc2 kinase. At G2-phase, the activity of its inactivator, a member of the Wee1 family of protein kinases, exceeds that of its activator, Cdc25C phosphatase. However, at M-phase entry the situation is reversed, such that the activity of Cdc25C exceeds that of the Wee1 family. The mechanism of this reversal is unclear. Here we show that in oocytes from the starfish Asterina pectinifera, the kinase Akt (or protein kinase B (PKB)) phosphorylates and downregulates Myt1, a member of the Wee1 family. This switches the balance of regulator activities and causes the initial activation of cyclin B–Cdc2 at the meiotic G2/M-phase transition. These findings identify Myt1 as a new target of Akt, and demonstrate that Akt functions as an M-phase initiator.

MAP kinase links the fertilization signal transduction pathway to the G1/S-phase transition in starfish eggs.

The mechanism by which fertilization initiates S‐phase in the zygote is examined by manipulating the activity of MAP kinase in mature starfish eggs. These unfertilized eggs, which are arrested at G1‐phase after the completion of meiosis, have high MAP kinase activity but undetectable cdc2 kinase activity. Either fertilization or inhibition of protein synthesis causes a decrease in MAP kinase activity, which is followed by DNA synthesis. Inactivation of MAP kinase with its specific phosphatase, CL100, initiates DNA synthesis in the absence of fertilization, while constitutive activation of MAP kinase with MEK represses the initiation of DNA synthesis following fertilization. Thus, in unfertilized mature starfish eggs, a capacity for DNA replication is already acquired, but entry into S‐phase is negatively regulated by MAP kinase activity that is supported by a continuously synthesized protein(s) but not by cdc2 kinase. Upon fertilization, downregulation of MAP kinase activity is necessary and sufficient for triggering the G1/S‐phase transition.

Distinct regulators for Plk1 activation in starfish meiotic and early embryonic cycles

The Polo‐like kinase, Plk, has multiple roles in regulating mitosis. In particular, Plk1 has been postulated to function as a trigger kinase that phosphorylates and activates Cdc25C prior to the activation of cyclin B–Cdc2 and thereby initiates its activation. However, the upstream regulation of Plk1 activation remains unclear. Here we have studied the interplay between Plk1 and Cdc2 through meiotic and early embryonic cycles in starfish. Distinct kinases, cyclin B–Cdc2, MAPK along with cyclin B– and/or cyclin A–Cdc2 and cyclin A–Cdc2, were unique upstream regulators for Plk1 activation at meiosis I, meiosis II and embryonic M‐phase, respectively, indicating that Plk1 is not the trigger kinase at meiotic reinitiation. When Plk1 was required for cyclin B–Cdc2 activation, the action of Plk1 was mediated primarily through suppression of Myt1 rather than through activation of Cdc25. We propose that Plk1 can be activated by either cyclin A– or cyclin B–Cdc2, and its primary target is Myt1.

Heterochronic activation of VEGF signaling and the evolution of the skeleton in echinoderm pluteus larvae.

The evolution of the echinoderm larval skeleton was examined from the aspect of interactions between skeletogenic mesenchyme cells and surrounding epithelium. We focused on vascular endothelial growth factor (VEGF) signaling, which was reported to be essential for skeletogenesis in sea urchin larvae. Here, we examined the expression patterns of vegf and vegfr in starfish and brittle stars. During starfish embryogenesis, no expression of either vegfr or vegf was detected, which contrast with previous reports on the expression of starfish homologs of sea urchin skeletogenic genes, including Ets, Tbr, and Dri. In later stages, when adult skeletogenesis commenced, vegfr and vegf expression were upregulated in skeletogenic cells and in the adjacent epidermis, respectively. These expression patterns suggest that heterochronic activation of VEGF signaling is one of the key molecular evolutionary steps in the evolution of the larval skeleton. The absence of vegf or vegfr expression during early embryogenesis in starfish suggests that the evolution of the larval skeleton requires distinct evolutionary changes, both in mesoderm cells (activation of vegfr expression) and in epidermal cells (activation of vegf expression). In brittle stars, which have well‐organized skeletons like the sea urchin, vegfr and vegf were expressed in the skeletogenic mesenchyme and the overlying epidermis, respectively, in the same manner as in sea urchins. Therefore, the distinct activation of vegfr and vegf may have occurred in two lineages, sea urchins and brittle stars.

The starfish egg mRNA responsible for meiosis reinitiation encodes cyclin

During meiotic maturation and early embryonic cycles, the activity of maturation-promoting factor (MPF) cycles in exact correspondence with the mitotic cycles. For the appearance of MPF activity in starfish, protein synthesis is required except in the first meiotic cycle. In order to identify newly synthesized proteins involved in the regulation of MPF activity, we extracted poly(A)+ RNA from starfish eggs, and found that the egg poly(A)+ RNA induced germinal vesicle breakdown (GVBD) upon injection into immature oocytes of starfish and Xenopus. The molecular size of the poly(A)+ RNA responsible for GVBD was estimated to be approximately 22S by sucrose density gradient centrifugation. Since these characteristics of the starfish egg poly(A)+ RNA are similar to those of cyclin mRNAs from sea urchin and surf clam eggs, we synthesized a 50-mer antisense-cyclin oligonucleotide probe coding for a part of the sea urchin cyclin cDNA and used this to screen starfish RNA. The Northern blot analysis showed that the starfish egg RNA contained cyclin homologous transcripts. Incubation of the starfish egg poly(A)+ RNA and the antisense-cyclin oligonucleotide with RNase H completely destroyed its GVBD-inducing activity. These results indicated that starfish cyclin mRNA was the only poly(A)+ RNA responsible for GVBD. We constructed a starfish egg cDNA library to clone starfish cyclin cDNA. The longest cDNA clone containing 2190 base pairs was sequenced. The longest open reading frame consisted of 395 amino acid residues, and the predicted molecular size was 48 kDa. Comparison of the deduced amino acid sequences of starfish cyclin with known cyclins indicated that the starfish cyclin belongs to the B-type. Injection of synthetic mRNA of starfish cyclin caused GVBD in immature oocytes of starfish and Xenopus, while injection of synthetic mRNA of human CDC2 had no effect. The Northern blot analysis of starfish RNA extracted at various stages of the meiotic cycles suggested that the starfish cyclin transcript was stored in its polyadenylated form even in immature oocytes and was further polyadenylated at maturation.

Involvement of Mos–MEK–MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae

Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.

Cyclin B-Cdk1 Controls Pronuclear Union in Interphase

In sexual reproduction, the union of the male and female pronuclei occurs in fertilized eggs to mix genetic materials derived from both parents, thereby creating a new genome for the next generation [1-4]. The process leading to pronuclear union consists of pronuclear congression, which depends on astral microtubules derived from sperm centrosome [5-8], and the subsequent pronuclear fusion or karyogamy. The union process progresses in parallel with the first embryonic cell cycle, but the molecular mechanisms involved are poorly understood. Here, we devise a labeling method with Dendra2 to track both pronuclei individually in living starfish eggs. Although pronuclear union naturally proceeds while G1 arrest is released by fertilization and S phase progresses [9], we show that the cell-cycle resumption and progression are not prerequisites for pronuclear union. However, low levels of cyclin B- (but not cyclin A-) Cdk1 activity are detectable even in interphase, and are indispensable for pronuclear union, by contributing at least to pronuclear congression through formation of sperm aster. Pronuclear congression thus requires the activity of M-phase cell-cycle regulator in interphase, independently of the cell-cycle regulation. These findings not only provide a clue to the regulatory aspect of creation of new genome with fertilization, but also reveal a novel role for the M-phase Cdk1 during interphase.

Initiation of DNA replication after fertilization is regulated by p90Rsk at pre-RC/pre-IC transition in starfish eggs

Initiation of DNA replication in eukaryotic cells is controlled through an ordered assembly of protein complexes at replication origins. The molecules involved in this process are well conserved but diversely regulated. Typically, initiation of DNA replication is regulated in response to developmental events in multicellular organisms. Here, we elucidate the regulation of the first S phase of the embryonic cell cycle after fertilization. Unless fertilization occurs, the Mos-MAPK-p90Rsk pathway causes the G1-phase arrest after completion of meiosis in starfish eggs. Fertilization shuts down this pathway, leading to the first S phase with no requirement of new protein synthesis. However, how and in which stage the initiation complex for DNA replication is arrested by p90Rsk remains unclear. We find that in G1-arrested eggs, chromatin is loaded with the Mcm complex to form the prereplicative complex (pre-RC). Inactivation of p90Rsk is necessary and sufficient for further loading of Cdc45 onto chromatin to form the preinitiation complex (pre-IC) and the subsequent initiation of DNA replication. However, cyclin A-, B-, and E-Cdks activity and Cdc7 accumulation are dispensable for these processes. These observations define the stage of G1 arrest in unfertilized eggs at transition point from pre-RC to pre-IC, and reveal a unique role of p90Rsk for a negative regulator of this transition. Thus, initiation of DNA replication in the meiosis-to-mitosis transition is regulated at the pre-RC stage as like in the G1 checkpoint, but in a manner different from the checkpoint.

Role of Mos/MEK/ERK cascade and Cdk1 in Ca2+ oscillations in fertilized ascidian eggs

Intracellular calcium ion concentration ([Ca(2+)](i)) transients are observed in the fertilized eggs of all species investigated so far, and are critical for initiating several events related to egg activation and cell cycle control. Here, we investigated the role of the Mos/MEK/ERK cascade and Cdk1 on Ca(2+) oscillations in fertilized ascidian eggs. The egg of the ascidian Phallusia nigra shows [Ca(2+)](i) oscillations after fertilization: Ca(2+) waves immediately following fertilization (phase I), and [Ca(2+)](i) oscillations between the first and second polar body extrusions (phase II). Our results show that in P. nigra eggs, ERK activity peaked just before the extrusion of the first polar body, and decreased gradually, eventually disappearing at the extrusion of the second polar body. Cyclin-dependent protein kinase 1(Cdk1) activity decreased to undetectable levels immediately after fertilization, and then periodically increased according to the meiotic and mitotic cell cycle. When the unfertilized eggs were incubated with U0126, an inhibitor of MEK, before insemination, ERK was immediately inactivated, and the phase II [Ca(2+)](i) oscillations disappeared. Alternatively, when the constitutively active Mos protein (GST-Mos) was injected into the unfertilized eggs, ERK activity was preserved for at least 120 min after fertilization, and the phase II [Ca(2+)](i) oscillations lasted for more than 120 min after the second polar body extrusion. These results suggest that ERK activity is necessary for maintaining [Ca(2+)](i) oscillations. GST-ΔN85-cyclin, which maintains Cdk1 activity, caused ERK activity in the eggs to persist for over 120 min after fertilization, and prolonged [Ca(2+)](i) oscillations. Moreover, the effects of GST-ΔN85-cyclin on the egg were abrogated by the application of U0126. Thus, Cdk1-mediated [Ca(2+)](i) oscillations seem to require ERK activity. However, GST-Mos triggered [Ca(2+)](i) oscillations after the second polar body extrusion, whereas GST-ΔN85-cyclin did not, although it prolongs the duration of [Ca(2+)](i) oscillations. Interestingly, GST-ΔN85-cyclin increased the frequency of [Ca(2+)](i) transients in the Mos-induced [Ca(2+)](i) oscillations after the extrusion of the second polar body. Thus, Cdk1 could maintain, but not activate, ERK and [Ca(2+)](i) oscillations. ERK activity and [Ca(2+)](i) oscillations seem to form a negative feedback loop which may be responsible for maintaining the meiotic period.