In VivoRegulation of Cyclin A/Cdc2 and Cyclin B/Cdc2 through Meiotic and Early Cleavage Cycles in Starfish

Okano-Uchida, Takayuki; Sekiai, Tohru; Lee, Kyon-su; Okumura, Eiichi; Tachibana, Kazunori; Kishimoto, Takeo

doi:10.1006/dbio.1998.8881

Cited by 56 publications

(66 citation statements)

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“…3A; see also ref. 15). Neither a single knockdown of Cdk2 nor a triple knockdown of cyclins A, B, and E with respective morpholino oligonucleotides prevented initiation of DNA replication after fertilization ( Fig.…”

Section: Resultsmentioning

confidence: 94%

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

Tachibana

Mori

Matsuhira

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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-MAPKp90Rsk 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-Cdk's 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.Cdc45 | G1 arrest | Mcm complex | Mos-MAPK pathway | oocyte-to-embryo transition D NA replication in eukaryotic cells is initiated through an ordered assembly of protein complexes at replication origins (1, 2). Replication origins are first recognized and bound by the origin recognition complex (ORC). During late M or early G1 phase, Cdc6 associates onto ORC-containing DNA. Then, MCM (minichromosome maintenance) proteins associate with the ORC-and Cdc6-containing replication origins, requiring Cdt1 to form a prereplicative complex (pre-RC). At the onset of S phase, Cdc45 associates with the pre-RC to form a preinitiation complex (pre-IC) that is capable of origin unwinding and of promoting assembly of replication forks at replication origin. Thus, Cdc45 plays a crucial role in activation of replication origins.Although the mechanism of initiation of DNA replication is well conserved, its control is diverse (3). In addition to evolutionary variation, DNA replication is regulated in response to developmental events in multicellular organisms. Fertilization is the first major event in development and is necessary for both releasing meiotic arrest and restarting the cell cycle with initiation of the first round of DNA replication. In some organisms, including Drosophila and echinoderms (4-6), fertilization is not a prerequisite for the completion of meiosis, but required to trigger entry into the first S phase and the subsequent cleavage cycles. In starfish Ast...

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Section: Resultsmentioning

confidence: 94%

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

Tachibana

Mori

Matsuhira

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Immediately downstream of MAPK, Rsk (p90 ribosomal S6 kinase, p90 pronuclei were inseminated. Oocyte extracts for immunoblots and histone H1 kinase assays were prepared as described (Okano-Uchida et al, 1998). Microinjection into oocytes was performed according to Kishimoto (Kishimoto, 1986).…”

Section: Oocytes and Eggsmentioning

confidence: 99%

“…Polyclonal anti-starfish Rsk antisera (1 and 2) were raised in rabbits against the His-RskNTD and were purified using a His-RskNTD-transferred membrane according to Okano-Uchida et al (Okano-Uchida et al, 1998). Antisera against Rsk were used for immunoblot and immunoprecipitation, while the purified antibodies were injected into oocytes at 3 ng to neutralize endogenous Rsk activity.…”

Section: Antibodiesmentioning

confidence: 99%

p90Rsk is required for G1 phase arrest in unfertilized starfish eggs

et al. 2006

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DEVELOPMENT 1823 RESEARCH ARTICLE INTRODUCTIONThe female gamete of most animals arrests in the meiotic cell cycle while awaiting fertilization, and thus parthenogenesis is prevented. Depending on the organism, this arrest occurs at the beginning of meiosis I, metaphase of meiosis I, metaphase of meiosis II (meta-II) or the pronucleus stage after the completion of meiosis II (Sagata, 1996). How each of these different meiotic cell cycle arrests is executed and whether there is a common molecular principle are key issues in cell cycle arrest.The meta-II arrest that is observed in most of unfertilized vertebrate eggs has been most extensively studied. The activity that keeps eggs arrested in meta-II was first identified in the amphibian Rana pipiens and was called cytostatic factor (CSF) (Masui and Markert, 1971). Later studies established that the Mos-MAPK (mitogen-activated protein kinase) pathway is an essential core component of CSF in Xenopus and mouse eggs (Sagata et al., 1989;Haccard et al., 1993;Shibuya and Ruderman, 1993;Colledge et al., 1994;Hashimoto et al., 1994;Verlhac et al., 1996) (for reviews, see Sagata, 1996;Masui, 2000;Kishimoto, 2003;. The Mos-MAPK pathway maintains the activity of cyclin B-Cdc2 kinase at an elevated level (Yamamoto et al., 2005), and thereby arrests the cell cycle at meta-II in mature eggs of frog and mouse, resulting in the prevention of parthenogenetic activation.By contrast, unfertilized mature eggs of echinoderms, including starfish and sea urchin, are arrested at the pronucleus stage. Nonetheless, the same Mos-MAPK pathway causes the G1 phase arrest at the pronucleus stage in starfish Asterina pectinifera eggs (Tachibana et al., 1997;Tachibana et al., 2000) (see Kishimoto, 2004). Mature starfish eggs lacking in Mos or MAPK activity are activated parthenogenetically in the absence of fertilization. In unfertilized sea urchin eggs, MAPK is also responsible for the G1 arrest (Carroll et al., 2000), and it is plausible that a sea urchin homolog of Mos might be present and function upstream of MAPK.Considering the different arrest phases in frog and mouse versus starfish and sea urchin eggs, it is likely that a downstream effector of the Mos-MAPK pathway should determine the arrest phase. In Xenopus eggs, Rsk (p90 ribosomal S6 kinase) generates the CSF activity, immediately downstream of MAPK (Bhatt and Ferrell, Jr, 1999;Gross et al., 1999). Constitutively active forms of Rsk induce metaphase arrest independently of the activation of the Mos-MAPK pathway, while the metaphase arrest fails to occur after depletion of Rsk. In mouse eggs, however, Dumont et al. (Dumont et al., 2005) reported recently that Rsk is not involved in meta-II arrest, even though Rsk is activated during mouse meiotic cycles (Kalab et al., 1996). Mouse eggs from the triple Rsk knockout normally arrest at meta-II, and constitutively active mutant forms of Rsk do not restore meta-II arrest in mos-deficient oocytes. Thus, it is intriguing whether the G1 arrest in unfertilized starfish eggs requires Rsk activit...

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“…Depending on the species and cell type, the regulators of cdc2 are compartmentalized differently and undergo translocations between nucleus and cytoplasm. In starfish, the inhibitory myt1 and the activating phosphatase cdc25 are both cytoplasmic, with cdc25 translocating into the nucleus before nuclear envelope breakdown (Okano-Uchida et al, 1998;Kishimoto, 1999), whereas in mammalian cells, wee1 is nuclear and cdc25C is cytoplasmic and undergoes translocation to the nucleus (Heald et al, 1993). In addition, cyclin B apparently must be phosphory- …”

Section: Controlled Damage By Multiphoton Excitationmentioning

confidence: 99%

Controlled Damage in Thick Specimens by Multiphoton Excitation

Galbraith

Terasaki

2003

MBoC

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Controlled damage by light energy has been a valuable tool in studies of cell function. Here, we show that the Ti:Sapphire laser in a multiphoton microscope can be used to cause localized damage within unlabeled cells or tissues at greater depths than previously possible. We show that the damage is due to a multiphoton process and made wounds as small as 1 m in diameter 20 m from the surface. A characteristic fluorescent scar allows monitoring of the damage and identifies the wound site in later observations. We were able to lesion a single axon within a bundle of nerves, locally interrupt organelle transport within one axon, cut dendrites in a zebrafish embryo, ablate a mitotic pole in a sea urchin egg, and wound the plasma membrane and nuclear envelope in starfish oocytes. The starfish nucleus collapsed ϳ1 h after wounding, indicating that loss of compartmentation barrier makes the structure unstable; surprisingly, the oocyte still completed meiotic divisions when exposed to maturation hormone, indicating that the compartmentalization and translocation of cdk1 and its regulators is not required for this process. Multiphoton excitation provides a new means for producing controlled damage deep within tissues or living organisms. INTRODUCTIONLight energy is an effective means for producing a localized cellular disruption (Berns et al., 1998). It is possible to photodynamically destroy individual structures or cells labeled with fluorescent dyes (e.g., Tamm, 1978), or specific proteins that have been targeted with fluorescent antibodies (CALI; Buchstaller and Jay, 2000). With the appropriate fluorescent label, the plasma membrane can be disrupted using a laser in a confocal microscope (Bi et al., 1995), and it has been shown that neurons loaded with specific dyes are susceptible to ablation (Liu and Fetcho, 1999). It is also possible to damage unlabeled structures. UV light from a mercury arc lamp has been used to cut microtubules (Walker et al., 1989), whereas individual Caenorhabditis elegans cells can be killed with energy from a dye laser (Bargmann and Avery, 1995), and centrosomes in cultured cells have been ablated with pulses from an Nd-YAG laser (Khodjakov et al., 1997).We investigated the use of multiphoton excitation as a potential new way of producing a highly localized disruption at greater depths than has been possible. Under conventional continuous illumination conditions, a single photon causes the transition of an electron to a higher energy state within a molecule. A mode-locked Ti-Sapphire laser can provide high-density photon pulses such that two or more photons combine to cause transitions, and because the probability of excitation depends on the square of photon density, excitation occurs only in the focal plane rather than all along the light path (Denk et al., 1990(Denk et al., , 1995. This small excitation volume has proven effective at reducing photodamage during live cell imaging (e.g., Jontes et al., 2000). Additional benefits of multiphoton microscopes are the longer wavelengths that pr...

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In VivoRegulation of Cyclin A/Cdc2 and Cyclin B/Cdc2 through Meiotic and Early Cleavage Cycles in Starfish

Cited by 56 publications

References 73 publications

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

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

p90Rsk is required for G1 phase arrest in unfertilized starfish eggs

Controlled Damage in Thick Specimens by Multiphoton Excitation

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