How are gametes formed through cell division

The regulation of the second meiotic division

Research Report 2016 - Max Planck Institute for Biochemistry

Chromosome Biology Research Group
Gametes such as egg cells, sperm or spores are created in a special cell division called meiosis. The chromosomes are divided into four nuclei in the course of two nuclear divisions, each of which contains only half the chromosome set. In contrast to the first division, little is known about the control of the second division. The chromosome biology research group has now been able to show in baker's yeast cells how the processes of the second division, namely the separation of the chromatids, the gamete differentiation and the completion of meiosis, are coordinated by the conserved Hrr25 kinase.

The essence of reduction

Most cells in animals, plants, and fungi are diploid, which means they contain two copies of each chromosome. These cells multiply through normal, mitotic cell division. The chromosomes are first duplicated so that they each consist of two identical sister chromatids that are held together by cohesins along their entire length. Cohesins are protein complexes made up of three subunits that form a ring that surrounds the sister chromatids. During cell division, the cohesins are cut open at their kleisin subunits by a protease called a separase, so that the spindle apparatus can distribute the chromatids between the two daughter cells. Ultimately, however, these cells are derived from a single cell, the zygote, which was created by the fusion of two gametes. Since both gametes contribute the same number of chromosomes to the zygote, they must be haploid and result from a special cell division called meiosis, in which the number of chromosomes is halved.

Meiosis also begins with a replication phase in which each chromosome is converted into a pair of sister chromatids (Fig. 1, S-phase). Then the corresponding paternal and maternal chromosomes attach to each other and are linked in the process of recombination, with paternal and maternal chromatids being spliced ​​together crosswise. "Double chromosomes" (tetrads) are created in which the cohesins hold all four homologous chromatids together (Fig. 1, prophase I). The double chromosomes are broken down into their chromatids again in the following two meiotic divisions: In the first meiotic division (meiosis I), Separase cuts the cohesins on the chromosome arms but not on the centromere. As a result, the meiosis I spindle distributes X-shaped chromosomes, the chromatids of which are only connected by cohesins at the centromere (Fig. 1, anaphase I). The centromere is that part of the chromosome to which the spindle fibers attach to the chromatin via a protein complex, the kinetochore. Since this division produces nuclei whose number of chromosomes is reduced by half, it is also called reduction division. In the second division (meiosis II or equation division), the cohesin at the centromeres is cut open by Separase so that the meiosis II spindle can now separate the individual chromatids from one another (Fig. 1, anaphase II). Four haploid cells are created, each containing a chromatid from each chromosome. As a result of the recombination, however, these chromatids now consist of different combinations of maternal and paternal chromatid segments, which guarantees the genetic diversity of the subsequent generation.

The conditions under which meiosis is induced and the development of gametes (egg cells, sperm or spores) are very different in different organisms. In contrast, the behavior of the chromosomes is regulated by evolutionarily conserved processes. Fundamental mechanisms of meiotic chromosome segregation can therefore be elucidated in organisms that are easy to manipulate experimentally, such as baker's yeast, a unicellular fungus.

Regulation of cohesin splitting during meiosis

The mechanism of the differential cleavage of cohesin in meiosis I could already be elucidated in earlier work with yeast cells (Fig. 2). In meiotic cells, the cleavable cohesin subunit is exchanged for a meiosis-specific variant, Rec8. Only this variant can be saved from splitting at the centromere. In order to be recognized by Separase, Rec8 must first be phosphorylated. In yeast this happens through the conserved protein kinases Cdc7-Dbf4 and Hrr25 [1]. At the centromere, however, this phosphorylation is prevented by protein phosphatase 2A (PP2A) and thus Rec8 is protected from cleavage; PP2A in turn binds to centromeres via the conserved Shugoshin protein (Sgo1). Ultimately, the cleavage of the cohesin is determined by the “fight” for the phosphorylation of Rec8. In meiosis I, Cdc7-Dbf4 and Hrr25 win this battle on the chromosome arms, but lose it on the centromeres, since PP2A is concentrated here by Sgo1. It was not previously known which kinase phosphorylates the centromere Rec8 in meiosis II and, like Sgo1 or PP2A, is either inactivated or removed from the centromere.

Meiosis II, a core division with special tasks

Meiosis I and meiosis II each begin with the activation of the cyclin-dependent kinase (Cdk1), which induces the development of the spindle apparatus and also for the activity of the ubiquitin ligase APCCdc20 is necessary (Fig. 1, below). APCCdc20 attaches ubiquitin to proteins, which are then degraded. This is how APC worksCdc20 the degradation of the cyclins and the separase inhibitor Pds1. This leads to the inactivation of CDK1 or the activation of Separase and consequently entry into the anaphase. The mutual regulation of CDK1 and APCCdc20 creates an oscillator that waves from Cdk1 and APCCdc20-Activity and thus triggers a series of core divisions. In order to prevent an additional meiotic division ("meiosis III"), meiosis II must contain a mechanism that switches off this oscillator after exactly two waves. Meiosis II also contains processes that are important for later fertilization. In male animals and fungi, the sperm or spore formation program is already started during meiosis II. In contrast, mammalian oocytes activate an APC in metaphase IICdc20-Inhibitor that does not allow entry into anaphase II until fertilization. How the cleavage of the centromeric cohesin, the differentiation of the gametes and the exit from meiosis are coordinated was unclear. One of the reasons for this is that meiosis II is difficult to manipulate experimentally, as it follows meiosis I briefly and has important regulators in common with meiosis I. However, a more detailed understanding of the control mechanisms of meiosis II is urgently needed, as there are increasing indications that a considerable part of the chromosome distribution errors in human oocytes originate in meiosis II.

The split of the centromeric cohesin

To study meiosis II, the research group has developed a new synchronization system for yeast. All cells pass through the two divisions at the same time and meiosis II can be manipulated without disturbing meiosis I. It was found that in meiosis II only one kinase, namely Hrr25, phosphorylates the cohesin subunit Rec8 so that it can be cleaved by Separase. However, this is only possible because the Hrr25 kinase simultaneously removes its opponent, the phosphatase PP2A, from the centromere (Fig. 3). Hrr25 causes Sgo1 and the kinase Mps1 of APCCdc20 recognized and marked for degradation [2]. The Mps1 kinase is required in meiosis II so that Sgo1 can bind to centromeres. APCCdc20 via two different mechanisms to remove PP2A from the centromere, making this process extremely robust. At the same time activates APCCdc20 the separase by causing the degradation of the inhibitor Pds1. It follows that in meiosis II the removal of PP2A and thus the phosphorylation of Rec8 are coupled with the activation of the separase. Thus, the centromer cohesin is protected from splitting by separase not only during meiosis I, but also until it enters anaphase II. This appears particularly useful in view of the fact that the amount of Pds1 in metaphase II is less than in metaphase I and therefore the separase may not be completely inhibited.

A robust mechanism to protect centromeric cohesin is particularly important in mammalian oocytes. After ovulation has triggered meiosis I, these oocytes arrest in metaphase II until fertilization causes entry into anaphase II. The centromer cohesin is thus exposed to bipolar spindle forces for a long time. How the cleavage of centromer cohesin in oocytes is regulated is unclear. It has been postulated that bipolar spindle forces spatially separate Sgo-PP2A from cohesin and thus expose Rec8 to phosphorylation and cleavage by separase. Since the amount of Pds1 in oocytes is considerably lower in metaphase II than in metaphase I, such a mechanism would make the centromer cohesin extremely susceptible to premature cleavage. In contrast, the "yeast mechanism" would protect the centromer cohesin from separase until APCCdc20 activated by fertilization.

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