Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint

Chromosoma - The meiotic recombination checkpoint blocks meiotic cell cycle progression in response to synapsis and/or recombination defects to prevent aberrant chromosome segregation. The...     

The perinuclear microtubule-organizing center and the synaptonemal complex of higher plants share a common antigen: its putative transfer and role in meiotic chromosomal ordering

Recognition of homologous chromosomes during meiotic prophase is associated in most cases with the formation of the synaptonemal complex along the length of the chromosome. Telomeres, located at the nuclear periphery, are preferential initiation sites for the assembly of the synaptonemal complex. In most eukaryotic cells, telomeres cluster in a restricted area, leading to the bouquet configuration in leptotene-zygotene, while this typical organization progressively disappears in late zygotene-pachytene. We wondered whether such striking changes in the intranuclear ordering and pairing of meiotic chromosomes during the progression of prophase I could be correlated with activity of the centrosome and/or microtubule-organizing center (MTOC). Plant cells may be used as a model of special interest for this study as the whole nuclear surface acts as an MTOC, unlike other cell types where MTOCs are restricted to centrosomes or spindle pole bodies. Using a monoclonal antibody (mAb 6C6) raised against isolated calf centrosomes we found that the 6C6 antigen is present over the entire surface of the plant meiotic nucleus, in early prophase I, before chromosomal pairing. At zygotene, short fragments of chromosomes become stained near the nuclear envelope and within the nucleus. At pachytene, after complete synapsis, the labeling specifically concentrates within the synaptonemal complexes, although the nuclear surface is no longer reactive. Ultrastructural localization using immunogold labeling indicates that the 6C6 antigen is colocalized with the synaptonemal complex structures. Later in metaphase I, the antigen is found at the kinetochores. Our data favor the idea that the 6C6 antigen may function as a particular chromosomal passenger-like protein. These observations shed new light on the molecular organization of the plant synaptonemal complex and on the redistribution of cytoskeleton-related antigens during initiation of meiosis. They suggest that antigens of MTOCs are relocated to chromosomes during the synapsis process starting at telomeres and contribute to the spatial arrangement of meiotic chromosomes. Such cytoskeleton-related antigens may acquire different functions depending on their localization, which is cell-cycle regulated.     

Reexamination of the role of ubiquitin-like modifier ISG15 in the phenotype of UBP43-deficient mice

UBP43/USP18 was described as a specific protease that removes conjugated ubiquitin-like modifier ISG15 from target proteins. The severe phenotype of UBP43(-/-) mice characterized by premature death, brain cell injury, and deregulated STAT1 signaling was ascribed to an enhanced conjugation of ISG15. In contrast, no phenotypic changes were detected in ISG15(-/-) mice. To verify the role of ISG15 in the phenotype of UBP43(-/-) mice, we employed mice deficient for both ISG15 and UBP43. Here, we show that the phenotype of UBP43(-/-) mice was not rescued by the absence of ISG15, as evident from unchanged mortality, neurological symptoms, and occurrence of hydrocephalus. Also, the reported hypersensitivity of UBP43(-/-) mice to an interferon inducer, poly(I . C), was ISG15 independent. Furthermore, no evidence for a role of ISG15 in the modulation of STAT1 signaling or in the resistance against lymphocytic choriomeningitis virus and vesicular stomatitis virus was found. Presented results clearly demonstrate that the phenotypic alterations of UBP43(-/-) mice are not caused by the lack of ISG15 deconjugation and must be due to another, non-ISG15-mediated molecular mechanism.     

Brca2/Xrcc2 dependent HR, but not NHEJ, is required for protection against O(6)-methylguanine triggered apoptosis, DSBs and chromosomal aberrations by a process leading to SCEs

O(6)-methylguanine (O(6)MeG) is a highly critical DNA adduct induced by methylating carcinogens and anticancer drugs such as temozolomide, streptozotocine, procarbazine and dacarbazine. Induction of cell death by O(6)MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O(6)MeG. Here, we compared the response of cells mutated in HR and NHEJ proteins to temozolomide and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (DNA-PK(cs) mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of O(6)-methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific hypersensitivity was due to O(6)MeG lesions. MNNG provoked H2AX phosphorylation 24-48h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The gammaH2AX foci formed in response to O(6)MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O(6)MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O(6)MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving cancer therapy with methylating agents.     

Further characterization of the phenotype of mCry1/mCry2-deficient mice

Mice lacking cryptochromes (mCry1^-/- mCry2^-/-) were kept in a 16h light, 8h dark, light-dark (16:8 LD) cycle and were given additional pulses of light of different brightness, starting 2h after dark onset and lasting for 1h. The suppression of wheel running during these light pulses (i.e., masking) was compared to that of wild types. No evidence of any decrement in the masking response to light was detected. As well as studying masking, minor bouts of activity occurring in the main light portion of a light-dark cycle were quantified. One possible explanation of such predark activity is that some damped endogenous process is spared in mCry1/mCry2 double-knockout mice. (Chronobiology International, 18(4), 613-625, 2001)     

Cytoskeleton abnormalities and meiotic restitution in the higher plants

Cytoskeleton abnormalities leading to the formation of restitution nuclei, and to the reunion of segregated daughter chromosome groups in the higher plant meiosis are reviewed.     

The role of DNA helicases and their interaction partners in genome stability and meiotic recombination in plants

DNA helicases are enzymes that are able to unwind DNA by the use of the energy-equivalent ATP. They play essential roles in DNA replication, DNA repair, and DNA recombination in all organisms. As homologous recombination occurs in somatic and meiotic cells, the same proteins may participate in both processes, albeit not necessarily with identical functions. DNA helicases involved in genome stability and meiotic recombination are the focus of this review. The role of these enzymes and their characterized interaction partners in plants will be summarized. Although most factors are conserved in eukaryotes, plant-specific features are becoming apparent. In the RecQ helicase family, Arabidopsis thaliana RECQ4A has been shown before to be the functional homologue of the well-researched baker's yeast Sgs1 and human BLM proteins. It was surprising to find that its interaction partners AtRMI1 and AtTOP3α are absolutely essential for meiotic recombination in plants, where they are central factors of a formerly underappreciated dissolution step of recombination intermediates. In the expanding group of anti-recombinases, future analysis of plant helicases is especially promising. While no FBH1 homologue is present, the Arabidopsis genome contains homologues of both SRS2 and RTEL1. Yeast and mammals, on the other hand. only possess homologues of either one or the other of these helicases. Plants also contain several other classes of helicases that are known from other organisms to be involved in the preservation of genome stability: FANCM is conserved with parts of the human Fanconi anaemia proteins, as are homologues of the Swi2/Snf2 family and of PIF1.     

Control of the meiotic cell division program in plants

While the question of why organisms reproduce sexually is still a matter of controversy, it is clear that the foundation of sexual reproduction is the formation of gametes with half the genomic DNA content of a somatic cell. This reduction in genomic content is accomplished through meiosis that, in contrast to mitosis, comprises two subsequent chromosome segregation steps without an intervening S phase. In addition, meiosis generates new allele combinations through the compilation of new sets of homologous chromosomes and the reciprocal exchange of chromatid segments between homologues. Progression through meiosis relies on many of the same, or at least homologous, cell cycle regulators that act in mitosis, e.g., cyclin-dependent kinases and the anaphase-promoting complex/cyclosome. However, these mitotic control factors are often differentially regulated in meiosis. In addition, several meiosis-specific cell cycle genes have been identified. We here review the increasing knowledge on meiotic cell cycle control in plants. Interestingly, plants appear to have relaxed cell cycle checkpoints in meiosis in comparison with animals and yeast and many cell cycle mutants are viable. This makes plants powerful models to study meiotic progression and allows unique modifications to their meiotic program to develop new plant-breeding strategies.     

Biochemical characterization of metnase's endonuclease activity and its role in NHEJ repair

Metnase (SETMAR) is a SET-transposase fusion protein that promotes nonhomologous end joining (NHEJ) repair in humans. Although both SET and the transposase domains were necessary for its function in DSB repair, it is not clear what specific role Metnase plays in the NHEJ. In this study, we show that Metnase possesses a unique endonuclease activity that preferentially acts on ssDNA and ssDNA-overhang of a partial duplex DNA. Cell extracts lacking Metnase poorly supported DNA end joining, and addition of wt-Metnase to cell extracts lacking Metnase markedly stimulated DNA end joining, while a mutant (D483A) lacking endonuclease activity did not. Given that Metnase overexpression enhanced DNA end processing in vitro, our finding suggests a role for Metnase's endonuclease activity in promoting the joining of noncompatible ends.     

Radial spindle and the phenotype of the maize meiotic mutant, dv

The morphological phenotype of the maize meiotic mutant dv (divergent spindle) has been further analysed by visualization of the division spindle and examination of its fine structure in mother cells of pollen. Previous research showed that dv blocks convergence of spindle fibres at the poles. New observations reveal abnormalities caused by this mutation, with dv showing disturbances in nuclear envelope breakdown during vesiculation, preventing the spindle fibres from adopting a bipolar orientation (with convergence on the poles). The anomalies result in radial spindles which are similar to monoastral spindles in animal cells.     

Characterization of connexin30.3-deficient mice suggests a possible role of connexin30.3 in olfaction

We have generated connexin30.3-deficient mice in which the coding region of the connexin30.3 gene was replaced by the lacZ reporter gene. The expression pattern of this connexin was characterized using beta-galactosidase staining and immunoblot analyses. In skin, beta-galactosidase/connexin30.3 protein was expressed in the spinous and granulous layers of the epidermis. Specific beta-galactosidase/connexin30.3 expression was also detected in the thin ascending limb of Henle's loop in the kidney. In addition, we found beta-galactosidase/connexin30.3 in progenitor cells of the olfactory epithelium and in a subpopulation of cells in the apical layer of the vomeronasal organ. Connexin30.3-deficient mice were fertile and displayed no abnormalities in the skin or in the chemosensory systems. Furthermore, they showed normal auditory thresholds as measured by brain stem evoked potentials. These mice did, however, exhibit reduced behavioural responses to a vanilla scent.     

The dual role of HOP2 in mammalian meiotic homologous recombination

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1^−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.     

Seeds of Arabidopsis plants expressing dominant-negative AtSKD1 under control of the GL2 promoter show a transparent testa phenotype and a mucilage defect

We have recently shown that overexpression of dominant-negative AtSKD1 versions under control of the trichome and non-root-hair-cell specific GL2 promoter (GL2_pro) blocks trafficking of soluble cargo to the vacuole, resulting in its fragmentation and ultimately cell death. GL2_pro is also active in the Arabidopsis seeds. When we inspected seeds of the dominant-negative AtSKD1 variants we found two phenotypes. The seeds display a transparent testa phenotype caused by a lack of proanthocyanidin (PA) and do not possess seed coat mucilage. Both phenotypes could be connected by cell death induced by the overexpression of dominant-negative AtSKD1.Key words: VPS4, ESCRT, plant, Arabidopsis, SKD1, ATPase, MVB, proanthocyanidin, transparent testa, mucilage, tannin, seed coat, AtSKD1AAA ATPases are important regulators of a plethora of cellular functions such as peroxisome biogenesis, vesicle-mediated transport, control of cell divisions and gene expression. This variety is based on a common mechanism, the energy dependent unfolding, remodeling and disassembly of proteins and protein complexes.¹Mammalian SKD1 and its yeast homolog VPS4 are AAA ATPases involved in the sorting of monoubiquitylated trans-membrane cargo to the lysosome/vacuole by dismantling the members of the endosomal sorting complex required for transport (ESCRT) complexes from the endosomal membrane.² The Arabidopsis SKD1 homolog, AtSKD1, has been characterized recently and has been shown to be an ortholog of SKD1/Vps4.^3,4 Mutations for all three ATPases are known that alone or in combination render them dominant-negative.^3–6 Overexpression of dominant-negative AtSKD1 is, however, lethal for Arabidopsis plants. Therefore, we have used the trichome and non-root-hair-cell-specific GL2_pro promoter to address the function of AtSKD1 in planta. Cells that express the dominant-negative versions show multiple nuclei, fragmented vacuoles and ultimately die. These phenotypes are most likely due to a block in vacuolar trafficking of soluble cargo that is instead secreted.⁴GL2_pro is also active in the coat of developing Arabidopsis seeds. We therefore inspected seeds of lines overexpressing dominant-negative and wild-type AtSKD1.     

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.     

Extreme cytokinesis phenomenon in the phenotype of meiotic mutation pam of Zea mays

Our study of cytological phenotype of meiotic mutation pam resulted in detecting a failure of cytokinesis in mutant pollen mother cells in the form of a block of fusion of membrane vesicles of the cell plate, and an impossibility of formation of daughter cell membranes. The mutation does not disturb the division spindle structure and function. Asynchrony of meiosis in pam is the result of arrest of pollen mother cells at metaphase 1 and metaphase 2.     

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.