Livia Pérez-Hidalgo

A Large-Scale Screen in S. pombe Identifies Seven Novel Genes Required for Critical Meiotic Events

Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.

Regulation of meiotic progression by the meiosis-specific checkpoint kinase Mek1 in fission yeast

During the eukaryotic cell cycle, accurate transmission of genetic information to progeny is ensured by the operation of cell cycle checkpoints. Checkpoints are regulatory mechanisms that block cell cycle progression when key cellular processes are defective or chromosomes are damaged. During meiosis, genetic recombination between homologous chromosomes is essential for proper chromosome segregation at the first meiotic division. In response to incomplete recombination, the pachytene checkpoint (also known as the meiotic recombination checkpoint) arrests or delays meiotic cell cycle progression, thus preventing the formation of defective gametes. Here, we describe a role for a meiosis-specific kinase, Mek1, in the meiotic recombination checkpoint in fission yeast. Mek1 belongs to the Cds1/Rad53/Chk2 family of kinases containing forkhead-associated domains, which participate in a number of checkpoint responses from yeast to mammals. We show that defects in meiotic recombination generated by the lack of the fission yeast Meu13 protein lead to a delay in entry into meiosis I owing to inhibitory phosphorylation of the cyclin-dependent kinase Cdc2 on tyrosine 15. Mutation of mek1+ alleviates this chekpoint-induced delay, resulting in the formation of largely inviable meiotic products. Experiments involving ectopic overexpression of the mek1+ gene indicate that Mek1 inhibits the Cdc25 phosphatase, which is responsible for dephosphorylation of Cdc2 on tyrosine 15. Furthermore, the meiotic recombination checkpoint is impaired in a cdc25 phosphorylation site mutant. Thus, we provide the first evidence of a connection between an effector kinase of the meiotic recombination checkpoint and a crucial cell cycle regulator and present a model for the operation of this meiotic checkpoint in fission yeast.

Nutritional Control of Cell Size by the Greatwall-Endosulfine-PP2A·B55 Pathway

Proliferating cells adjust their cell size depending on the nutritional environment. Cells are large in rich media and small in poor media. This physiological response has been demonstrated in both unicellular and multicellular organisms. Here we show that the greatwall-endosulfine (Ppk18-Igo1 in fission yeast) pathway couples the nutritional environment to the cell-cycle machinery by regulating the activity of PP2A·B55. In the presence of nutrients, greatwall (Ppk18) protein kinase is inhibited by TORC1 and PP2A·B55 is active. High levels of PP2A·B55 prevent the activation of mitotic Cdk1·Cyclin B, and cells increase in size in G2 before they undergo mitosis. When nutrients are limiting, TORC1 activity falls off, and the activation of greatwall (Ppk18) leads to the phosphorylation of endosulfine (Igo1) and inhibition of PP2A·B55, which in turn allows full activation of Cdk1·CyclinB and entry into mitosis with a smaller cell size. Given the conservation of this pathway, it is reasonable to assume that this mechanism operates in higher eukaryotes, as well.

Slk1 is a meiosis-specific Sid2-related kinase that coordinates meiotic nuclear division with growth of the forespore membrane

Septation and spore formation in fission yeast are compartmentalization processes that occur during the mitotic and meiotic cycles, and that are regulated by the septation initiation network (SIN). In mitosis, activation of Sid2 protein kinase transduces the signal from the spindle pole body (SPB) to the middle of the cell in order to promote the constriction of the actomyosin ring. Concomitant with ring contraction, membrane vesicles are added at the cleavage site to enable the necessary expansion of the cell membrane. In meiosis, the forespore membrane is synthesized from the outer layers of the SPB by vesicle fusion. This membrane grows and eventually engulfs each of the four haploid nuclei. The molecular mechanism that connects the SIN pathway with synthesis of the forespore membrane is poorly understood. Here, we describe a meiosis-specific Sid2-like kinase (Slk1), which is important for the coordination of the growth of the forespore membrane with the meiotic nuclear divisions. Slk1 and Sid2 are required for forespore membrane biosynthesis and seem to be the final output of the SIN pathway in meiosis.

The fission yeast meiotic checkpoint kinase Mek1 regulates nuclear localization of Cdc25 by phosphorylation.

In eukaryotic cells, fidelity in transmission of genetic information during cell division is ensured by the action of cell cycle checkpoints. Checkpoints are surveillance mechanisms that arrest or delay cell cycle progression when critical cellular processes are defective or when the genome is damaged. During meiosis, the so-called meiotic recombination checkpoint blocks entry into meiosis I until recombination has been completed, thus avoiding aberrant chromosome segregation and the formation of aneuploid gametes. One of the key components of the meiotic recombination checkpoint is the meiosis-specific Mek1 kinase, which belongs to the family of Rad53/Cds1/Chk2 checkpoint kinases containing forkhead-associated domains. In fission yeast, several lines of evidence suggest that Mek1 targets the critical cell cycle regulator Cdc25 to delay meiotic cell cycle progression. Here, we investigate in more detail the molecular mechanism of action of the fission yeast Mek1 protein. We demonstrate that Mek1 acts independently of Cds1 to phosphorylate Cdc25, and this phosphorylation is required to trigger cell cycle arrest. Using ectopic overexpression of mek1+ as a tool to induce in vivo activation of Mek1, we find that Mek1 promotes cytoplasmic accumulation of Cdc25 and results in prolonged phosphorylation of Cdc2 at tyrosine 15. We propose that at least one of the mechanisms contributing to the cell cycle delay when the meiotic recombination checkpoint is activated in fission yeast is the nuclear exclusion of the Cdc25 phosphatase by Mek1-dependent phosphorylation.

Modified Cell Cycle Regulation in Meiosis

The study of meiosis regulation has always been carried out in parallel with mitotic cell cycle discoveries. The basic cell cycle machinery that regulates mitosis, based on fluctuations in the activity of cyclin-dependent kinases (CDKs), is responsible for the main transitions that occur during meiosis. However, the special characteristics of meiosis (e.g., the absence of an S-phase between meiosis I and meiosis II, a long prophase in which homologous recombination events occur, etc.) require specific regulation, and cells respond to this challenging situation in different ways. In some cases, mitotic regulators carry out the new functions or change their relative importance in a particular process, while in other cases novel meiosis-specific regulators emerge.

Coupling TOR to the Cell Cycle by the Greatwall–Endosulfine–PP2A-B55 Pathway

Cell growth and division are two processes tightly coupled in proliferating cells. While Target of Rapamycin (TOR) is the master regulator of growth, the cell cycle is dictated by the activity of the cyclin-dependent kinases (CDKs). A long-standing question in cell biology is how these processes may be connected. Recent work has highlighted that regulating the phosphatases that revert CDK phosphorylations is as important as regulating the CDKs for cell cycle progression. At mitosis, maintaining a low level of protein phosphatase 2A (PP2A)-B55 activity is essential for CDK substrates to achieve the correct level of phosphorylation. The conserved Greatwall–Endosulfine pathway has been shown to be required for PP2A-B55 inhibition at mitosis in yeasts and multicellular organisms. Interestingly, in yeasts, the Greatwall–Endosulfine pathway is negatively regulated by TOR Complex 1 (TORC1). Moreover, Greatwall–Endosulfine activation upon TORC1 inhibition has been shown to regulate the progression of the cell cycle at different points: the G1 phase in budding yeast, the G2/M transition and the differentiation response in fission yeast, and the entry into quiescence in both budding and fission yeasts. In this review, we discuss the recent findings on how the Greatwall–Endosulfine pathway may provide a connection between cell growth and the cell cycle machinery.

Fission Yeast Cell Cycle Synchronization Methods

Fission yeast cells can be synchronized by cell cycle arrest and release or by size selection. Cell cycle arrest synchronization is based on the block and release of temperature-sensitive cell cycle mutants or treatment with drugs. The most widely used approaches are cdc10-129 for G1; hydroxyurea (HU) for early S-phase; cdc25-22 for G2, and nda3-KM311 for mitosis. Cells can also be synchronized by size selection using centrifugal elutriation or a lactose gradient. Here we describe the methods most commonly used to synchronize fission yeast cells.

Nutrients control cell size.

Cells must grow to reach a critical size before cell division. In 1977 Fantes and Nurse established that this critical size depends on the nutritional environment. This seminal work was carried out in the fission yeast Schizosaccharomyces pombe, but further work revealed that the environmental control of cell size at division was also conserved in mammalian cells. S. pombe is an ideal model organism for the analysis of cell size. Fission yeast cells are rod-shaped, grow by tip elongation, and their cell length is proportional to their volume. Nutrients in the media determine cell length: cells growing in rich medium are larger than those growing in poor medium, and a rapid readjustment of the cell size is produced when fission yeast cells are shifted from a nitrogen-rich to a nitrogen-poor medium or vice versa. However, the molecular mechanisms connecting the nutritional control of the cell size with the cell cycle machinery are poorly understood. TORC1 (target of rapamycin 1), a central controller of cell growth, is highly conserved from yeast to mammals. In media with nutrients, TORC1 is active and promotes growth by inducing ribosome biogenesis and protein synthesis. In addition, TORC1 signalling inhibits catabolic processes such as autophagy. When nutrients are scarce, the TORC1 pathway is inhibited, and cells stop growing as a result of the inactivation of the anabolic processes induced by TORC1, while autophagy is active to allow survival under starvation conditions. Concomitant with the inactivation of TORC1, cells divide at a reduced size. Taking this observation into account, we wondered if there was a connection between the master regulator of cell growth, TORC1, and the basic cell cycle machinery to regulate cell size at division. Recent experiments in fission yeast suggest this may be the case. Phosphorylation of proteins by mitotic kinases such as Cdk1-CyclinB is essential for the G2/M transition and mitosis to proceed properly. But for the accurate progression of the cell cycle, the regulation of counteracting phosphatases is as essential as the regulation of kinases. As Cdk1 activity is reversed by PP2A¢B55, this phosphatase must be inhibited at mitosis to achieve a complete and timely phosphorylation of Cdk1 substrates. Among these substrates there are two Cdk1 regulators, a positive one, the Cdc25 phosphatase, and a negative one, the Wee1 kinase. The phosphorylation of these regulators by Cdk1, that leads to the activation and inhibition of Cdc25 and Wee1, respectively, contributes to the amplification of the Cdk1 signal. In recent years, work from several laboratories using different model systems has shown that the inhibition of PP2A¢B55 is carried out by the endosulfines, ENSA and Arpp19. These small proteins are phosphorylated by the Greatwall kinase, which is also activated by Cdk1, and become potent inhibitors of PP2A¢B55 at mitotic entry and during mitosis. Accordingly, depletion of Greatwall induces defects of entry into and progression through mitosis in different model systems. In S. pombe, the igo1C gene encodes for the only endosulfine present in the fission yeast genome, and Ppk18 and Cek1 kinases are orthologous to Greatwall, although Ppk18 seems to carry out most of Igo1 phosphorylation. However, under standard laboratory conditions, that is, in media with plenty of nutrients, cells deleted for either of these genes do not show apparent problems to enter into and proceed through mitosis. Surprisingly, when cells are transferred into media with a poornitrogen source, such as phenylalanine, they are unable to accelerate mitosis and show a delay in G2, in contrast to wildtype cells, which reduce cell size to adapt to starvation conditions. This result suggests that in fission yeast the GreatwallEndosulfine pathway is conserved and promotes the timely entry into mitosis, but its function is only required when cells are grown in media with low nutrients (Fig. 1). In this poor medium inhibition of the PP2A¢B55 complex is required for the complete phosphorylation of mitotic substrates to occur at an earlier point in G2, thus allowing cells to enter mitosis with a smaller cell size. In fission yeast a shorter G2 phase makes the extension of the G1 phase necessary, which is cryptic in rich medium, for the cells to grow and reach the minimal size required to enter into a new cell cycle. This modification of the cell cycle phases is essential for survival under starvation conditions, because cells from G1 can exit the cell cycle to enter the

Chemical inactivation of Pat1: A novel approach to synchronize meiosis

Comment on: Guerra-Moreno A, et al. Cell Cycle 2012; 11:1621-5 and Cipak L, et al. Cell Cycle 2012; 11:1626-33