glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans

In the wild-type C. elegans germ line there are both mitotic and meiotic germ cells. Mutations in glp-1 cause germ cells that would normally divide mitotically to enter meiosis. This mutant phenotype mimics the effect of killing the distal tip cell, a somatic cell that interacts with the germ line to regulate the mitotic/meiotic decision. In addition, wild-type glp-1 product is required maternally for embryogenesis. Temperature-shift experiments indicate that the temporal requirement for glp-1 activity in the germ line is the same as that for distal tip cell regulation. Mosaic analyses suggest that glp-1 is produced in the germ line. We propose that glp-1 acts as part of the receiving mechanism in the interaction between the distal tip cell and germ line.     

The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells

A homolog of the fission yeast cdc2-encoded protein kinase (p34) is a component of M phase promoting factor in Xenopus oocytes. The homologous kinase in human HeLa cells is maximally active during mitosis, suggesting a mitotic role in mammalian somatic cells. This has been directly investigated by microinjection of anti-p34 antibodies into serum-stimulated rat fibroblasts. DNA synthesis was unaffected but cell division was quantitatively blocked in injected cells. Injection of antibodies against p13suc1, a component of the p34 kinase complex, did not block mitosis but caused mitotic abnormalities resulting in cells containing multiple micronuclei in the subsequent interphase. p34 localized in the nucleus during interphase. During mitosis, a fraction tightly associated with centrosomes. p13 was more evenly distributed between the nucleus and cytoplasm. These observations demonstrate that cdc2 is a nuclear and centrosomal protein that is required for mitosis in mammalian cells.     

Osmotin is a homolog of mammalian adiponectin and controls apoptosis in yeast through a homolog of mammalian adiponectin receptor

The antifungal activity of the PR-5 family of plant defense proteins has been suspected to involve specific plasma membrane component(s) of the fungal target. Osmotin is a tobacco PR-5 family protein that induces apoptosis in the yeast Saccharomyces cerevisiae. We show here that the protein encoded by ORE20/PHO36 (YOL002c), a seven transmembrane domain receptor-like polypeptide that regulates lipid and phosphate metabolism, is an osmotin binding plasma membrane protein that is required for full sensitivity to osmotin. PHO36 functions upstream of RAS2 in the osmotin-induced apoptotic pathway. The mammalian homolog of PHO36 is a receptor for the hormone adiponectin and regulates cellular lipid and sugar metabolism. Osmotin and adiponectin, the corresponding "receptor" binding proteins, do not share sequence similarity. However, the beta barrel domain of both proteins can be overlapped, and osmotin, like adiponectin, activates AMP kinase in C2C12 myocytes via adiponectin receptors.     

Forskolin and the meiosis inducing substance synergistically initiate meiosis in fetal male germ cells

We have shown that Meiosis Inducing Substance (MIS) and forskolin synergistically and dose dependently induce meiosis in germ cells of cultured fetal mouse testes. We used a bioassay which consists of fetal mouse testes and ovaries cultured for 6 days. In this study MIS media are spent culture media from 24 hour cultures of minced adult mouse testes. In the bioassay one gonad of each fetus is cultured either in MIS medium, in control medium with forskolin, or in MIS medium with forskolin. The other gonad serves as the control and is cultured in control medium. After culture the gonads are fixed, squashed, and DNA-stained. In these preparations germ cells and somatic cells can be distinguished, and the number of germ cells in the different stages of meiosis is counted as is the number of somatic cells in mitosis. MIS activity is defined to be present in a medium when meiosis is induced in male germ cells during culture. We found that MIS media as well as forskolin induced meiosis in fetal male germ cells in a dose-dependent manner. In addition, MIS media and forskolin acted synergistically by inducing meiosis. Female germ cells seem to be unaffected by the various culture media. These findings indicate that receptors for stimuli of meiotic initiation may exist in germ cells or neighbouring somatic cells. In addition to induction of meiosis, MIS media and forskolin also dose dependently increase the number of male germ cells compared to controls. This increase is correlated with induction of advanced stages of meiosis: Male germ cells seem to survive better if they are triggered to enter meiosis. Neither MIS media nor forskolin affected the growth of somatic cells. We therefore propose that MIS media has a growth factor activity with a specific effect on meiotic initiation. © 1993 Wiley-Liss, Inc.     

Putative homeodomain transcription factor 1 interacts with the feminization factor homolog fem1b in male germ cells

The Phtf1 gene encodes a membrane protein abundantly expressed in male germinal cells. Using a two-hybrid screening procedure we have identified FEM1B, an ortholog of the C. elegans feminization factor 1 (FEM-1), as a binding partner for PHTF1. We studied FEM1B expression in the rodent testis and found that Fem1b mRNA is present at high levels during meiosis and after, during spermiogenesis, in a similar manner to Phtf1 mRNA. Accordingly, Western blot and immunofluorescence revealed the presence of PHTF1 and FEM1B in the same cell types, and by coimmunoprecipitation we demonstrated the association between these proteins. We characterized some aspects of this interaction and showed that the ANK domain of FEM1B is necessary for the interaction with the amino extremity of PHTF1. Next, we found that FEM1B can bind several intracellular organelles and demonstrated that PHTF1 would recruit FEM1B to the endoplasmic reticulum membrane. Previous in vitro experiments had suggested that the human FEM1B was involved in apoptosis. After comparing expression profiles of FEM1B and PHTF1 with apoptotic events occurring in the normal seminiferous tubules, we suggest that neither FEM1B nor PHTF1 are directly implicated in apoptosis in this tissue.     

Leucine carboxyl methyltransferase-1 is necessary for normal progression through mitosis in mammalian cells

Protein phosphatase 2A (PP2A) is a multifunctional phosphatase that plays important roles in many cellular processes including regulation of cell cycle and apoptosis. Because PP2A is involved in so many diverse processes, it is highly regulated by both non-covalent and covalent mechanisms that are still being defined. In this study we have investigated the importance of leucine carboxyl methyltransferase-1 (LCMT-1) for PP2A methylation and cell function. We show that reduction of LCMT-1 protein levels by small hairpin RNAs causes up to a 70% reduction in PP2A methylation in HeLa cells, indicating that LCMT-1 is the major mammalian PP2A methyltransferase. In addition, LCMT-1 knockdown reduced the formation of PP2A heterotrimers containing the Balpha regulatory subunit and, in a subset of the cells, induced apoptosis, characterized by caspase activation, nuclear condensation/fragmentation, and membrane blebbing. Knockdown of the PP2A Balpha regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A(BalphaAC) heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or Balpha knockdown. LCMT-1 knockdown cells and Balpha knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and Balpha are important for spindle checkpoint. Treatment of LCMT-1 and Balpha knockdown cells with thymidine dramatically reduced cell death, presumably by blocking progression through mitosis. Consistent with these results, homozygous gene trap knock-out of LCMT-1 in mice resulted in embryonic lethality. Collectively, our results indicate that LCMT-1 is important for normal progression through mitosis and cell survival and is essential for embryonic development in mice.     

The teflon gene is required for maintenance of autosomal homolog pairing at meiosis I in male Drosophila melanogaster

In recombination-proficient organisms, chiasmata appear to mediate associations between homologs at metaphase of meiosis I. It is less clear how homolog associations are maintained in organisms that lack recombination, such as male Drosophila. In lieu of chiasmata and synaptonemal complexes, there must be molecules that balance poleward forces exerted across homologous centromeres. Here we describe the genetic and cytological characterization of four EMS-induced mutations in teflon (tef), a gene involved in this process in Drosophila melanogaster. All four alleles are male specific and cause meiosis I-specific nondisjunction of the autosomes. They do not measurably perturb sex chromosome segregation, suggesting that there are differences in the genetic control of autosome and sex chromosome segregation in males. Meiotic transmission of univalent chromosomes is unaffected in tef mutants, implicating the tef product in a pairing-dependent process. The segregation of translocations between sex chromosomes and autosomes is altered in tef mutants in a manner that supports this hypothesis. Consistent with these genetic observations, cytological examination of meiotic chromosomes suggests a role of tef in regulating or mediating pairing of autosomal bivalents at meiosis I. We discuss implications of this finding in regard to the evolution of heteromorphic sex chromosomes and the mechanisms that ensure chromosome disjunction in the absence of recombination.     

Cyclin E and Cdk2 control GLD-1, the mitosis/meiosis decision, and germline stem cells in Caenorhabditis elegans

Coordination of the cell cycle with developmental events is crucial for generation of tissues during development and their maintenance in adults. Defects in that coordination can shift the balance of cell fates with devastating clinical effects. Yet our understanding of the molecular mechanisms integrating core cell cycle regulators with developmental regulators remains in its infancy. This work focuses on the interplay between cell cycle and developmental regulators in the Caenorhabditis elegans germline. Key developmental regulators control germline stem cells (GSCs) to self-renew or begin differentiation: FBF RNA-binding proteins promote self-renewal, while GLD RNA regulatory proteins promote meiotic entry. We first discovered that many but not all germ cells switch from the mitotic into the meiotic cell cycle after RNAi depletion of CYE-1 (C. elegans cyclin E) or CDK-2 (C. elegans Cdk2) in wild-type adults. Therefore, CYE-1/CDK-2 influences the mitosis/meiosis balance. We next found that GLD-1 is expressed ectopically in GSCs after CYE-1 or CDK-2 depletion and that GLD-1 removal can rescue cye-1/cdk-2 defects. Therefore, GLD-1 is crucial for the CYE-1/CDK-2 mitosis/meiosis control. Indeed, GLD-1 appears to be a direct substrate of CYE-1/CDK-2: GLD-1 is a phosphoprotein; CYE-1/CDK-2 regulates its phosphorylation in vivo; and human cyclin E/Cdk2 phosphorylates GLD-1 in vitro. Transgenic GLD-1(AAA) harbors alanine substitutions at three consensus CDK phosphorylation sites. GLD-1(AAA) is expressed ectopically in GSCs, and GLD-1(AAA) transgenic germlines have a smaller than normal mitotic zone. Together these findings forge a regulatory link between CYE-1/CDK-2 and GLD-1. Finally, we find that CYE-1/CDK-2 works with FBF-1 to maintain GSCs and prevent their meiotic entry, at least in part, by lowering GLD-1 abundance. Therefore, CYE-1/CDK-2 emerges as a critical regulator of stem cell maintenance. We suggest that cyclin E and Cdk-2 may be used broadly to control developmental regulators.     

The Arabidopsis ATK1 gene is required for spindle morphogenesis in male meiosis

The spindle plays a central role in chromosome segregation during mitosis and meiosis. In particular, various kinesins are thought to play crucial roles in spindle structure and function in both mitosis and meiosis of fungi and animals. A group of putative kinesins has been previously identified in Arabidopsis, called ATK1-ATK4 (previously known as KATA-KATD), but their in vivo functions have not been tested with genetic studies. We report here the isolation and characterization of a mutant, atk1-1, which has a defective ATK1 gene. The atk1-1 mutant was identified in a collection of Ds transposon insertion lines by its reduced fertility. Reciprocal crosses between the atk1-1 mutant and wild type showed that only male fertility was reduced, not female fertility. Molecular analyses, including revertant studies, indicated that the Ds insertion in the ATK1 gene was responsible for the fertility defect. Light microscopy revealed that, in the atk1-1 mutant, male meiosis was defective, producing an abnormal number of microspores of variable sizes. Further cytological studies indicated that meiotic chromosome segregation and spindle organization were both abnormal in the mutant. Specifically, the atk1-1 mutant male meiotic cells had spindles that were broad, unfocused and multi-axial at the poles at metaphase I, unlike the typical fusiform bipolar spindle found in the wild-type metaphase I cells. Therefore, the ATK1 gene plays a crucial role in spindle morphogenesis in male Arabidopsis meiosis.