Localisation of RAD50 and MRE11 in spermatocyte nuclei of mouse and rat

Synaptonemal complexes (SCs) are zipperlike structures that are assembled between homologous chromosomes during meiotic prophase. They consist of two axial elements (AEs) (one along each of the two homologous chromosomes), which, in mature SCs, are connected by numerous transverse filaments along their length. Several proteins involved in the later steps of meiotic recombination most probably function in close association with the AEs of SCs, because the proteins involved in these steps have all been localised along AEs or SCs by immunocytochemical methods. It is not known at which step in meiotic recombination this association with the AEs is established. In order to shed some light on this issue, we analysed the localisation of two proteins that are involved in early steps of meiotic recombination, RAD50 and MRE11, relative to AEs and SCs by immunofluorescence labelling of paraffin sections of the mouse testis, using affinity-purified polyclonal antibodies against RAD50 and MRE11, and monoclonal and polyclonal antibodies against SC components. The localisation patterns of MRE11 and RAD50 within spermatocytes were very similar. MRE11 and RAD50 appeared in high abundance in preleptotene spermatocytes, just before SC components could be detected. From preleptotene until early zygotene they were present throughout the nucleus. In mid and late zygotene, MRE11 and RAD50 concentrated in distinct areas; in early pachytene the two proteins had almost disappeared from the nucleus, except from the sex vesicle (the chromatin of the XY bivalent), where they persisted in high abundance until diplotene. We propose that MRE11 and RAD50, together with other proteins, prepare chromatin throughout the early meiotic prophase nucleus for the initiation of meiotic recombination. Possibly, only a small fraction of the RAD50- and MRE11-containing (pre)recombination complexes associates transiently with AEs, where further steps in meiotic recombination can take place. Received: 16 November 1999; in revised form: 29 December 1999 / Accepted: 3 January 2000     

In vivo analysis of synaptonemal complex formation during yeast meiosis

During meiotic prophase a synaptonemal complex (SC) forms between each pair of homologous chromosomes and is believed to be involved in regulating recombination. Studies on SCs usually destroy nuclear architecture, making it impossible to examine the relationship of these structures to the rest of the nucleus. In Saccharomyces cerevisiae the meiosis-specific Zip1 protein is found throughout the entire length of each SC. To analyze the formation and structure of SCs in living cells, a functional ZIP1::GFP fusion was constructed and introduced into yeast. The ZIP1::GFP fusion produced fluorescent SCs and rescued the spore lethality phenotype of zip1 mutants. Optical sectioning and fluorescence deconvolution light microscopy revealed that, at zygotene, SC assembly was initiated at foci that appeared uniformly distributed throughout the nuclear volume. At early pachytene, the full-length SCs were more likely to be localized to the nuclear periphery while at later stages the SCs appeared to redistribute throughout the nuclear volume. These results suggest that SCs undergo dramatic rearrangements during meiotic prophase and that pachytene can be divided into two morphologically distinct substages: pachytene A, when SCs are perinuclear, and pachytene B, when SCs are uniformly distributed throughout the nucleus. ZIP1::GFP also facilitated the enrichment of fluorescent SC and the identification of meiosis-specific proteins by MALDI-TOF mass spectroscopy.     

Pushing the (nuclear) envelope into meiosis

A recent study shows that a short isoform of a mammalian nuclear lamin is important for homologous chromosome interactions during meiotic prophase in mice.Meiosis is the specialized cell division process required for sexual reproduction. As cells enter meiotic prophase, a relatively long period preceding the two chromosome divisions, nuclei and chromosomes undergo remodeling to promote interactions between homologous chromosomes. Each chromosome must find and identify its unique partner within the volume of the nucleus, a process that obviously involves large-scale chromosome movements.Over 100 years ago, cytological analysis of meiotic cells revealed a unique chromosome configuration termed the meiotic ''bouquet'', in which chromosome ends seem to be attached to the nuclear periphery, frequently in a tight cluster. The presence of the bouquet was found to coincide with the stage during which homologous chromosomes undergo pairing and synapsis. This was the first indication that interactions between the chromosomes and the nuclear envelope might be important for meiotic pairing. More recent analysis in diverse model systems has revealed that the bouquet is a consequence of interactions between chromosomes and cytoskeletal elements - microtubules or actin cables - via a protein bridge that spans the nuclear envelope. A study recently published in PLOS Genetics [1] has shed further light on the role of the nuclear lamina in meiotic progression by studying the role of a meiosis-specific isoform of a nuclear lamin protein.In metazoans the nuclear envelope is fortified by the nuclear lamina, a meshwork of intermediate filament proteins (lamins) and associated proteins that underlies the inner nuclear membrane. The lamina confers structural rigidity to nuclei and also interacts with a wide variety of nucleoplasmic, transmembrane and chromosome-associated proteins. The composition of the lamina in metazoans shows tissue-specific variability and developmental regulation. Most differentiated mammalian cells express both A-type lamins (lamins A and C, which are generated by alternative splicing of the LMNA gene) and B-type lamins (encoded by two different genes), whereas some invertebrates express only a single lamin protein. Stem cells typically lack A-type lamins, which are also dispensable for early development in mice.Among the nuclear envelope components that interact with lamins are LINC (linker of nucleoskeleton and cytoskeleton) complexes. These versatile networks involve a pair of SUN/KASH proteins that bridge both membranes of the nuclear envelope. SUN domain proteins traverse the inner membrane, with their amino termini projecting into the nucleus and their SUN domains in the lumen between the two membranes. Their partners have membrane-spanning regions adjacent to their carboxy-terminal KASH domains, short peptides that bind to the SUN domains. Using a variety of interaction modules, LINC complexes create connections between nuclear structures such as the lamina or chromosomes and cytoskeletal elements such as actin filaments or microtubules. Throughout the eukaryotes, they have essential roles in diverse processes, including the positioning and migration of nuclei within cells and anchorage of centrosomes to the nuclear envelope. During meiosis, specific LINC complexes are recruited to interact with chromosomes through the expression of meiosis-specific proteins that bind to telomeres or, less frequently, to other specialized loci [2]. These connections, probably in conjunction with meiosis-specific modifications to the cytoskeleton and motor proteins, lead to large-scale chromosome motions that facilitate homologous chromosome pairing. These movements involve dramatic motion of the LINC proteins within the nuclear membrane, sometimes involving movements of up to several micrometers that occur within a few seconds [3]. This stands in sharp contrast to the behavior of some of the same protein complexes in somatic or premeiotic cells, in which they show highly constrained motion and minimal turnover [3].In the new PLOS Genetics study [1], groups led by Manfred Alsheimer and Ricardo Benavente, both of the University of Würzburg, have now engineered a disruption of an exon in the mouse LMNA gene that is specific to the meiotic isoform lamin C2 to generate C2-deficient mice (C2^-/- mice). These collaborators have previously provided important insights into the regulation and functions of cell-type specific lamin isoforms, particularly during meiosis. Using antibodies, they characterized the lamin isoforms present in rat spermatocytes [4]. Immunolocalization revealed that a truncated isoform of lamin C (lamin C2) was localized in a patchy pattern along the nuclear envelope, along with a short B-type lamin (lamin B3) [4]. Because these short isoforms lack domains implicated in interactions between lamin subunits, they and others proposed that these proteins might form a more flexible network. This idea was supported by experiments in which meiosis-specific lamin C2 was ectopically expressed in fibroblasts and found to be more mobile within the nuclear envelope than full-length lamin C [5]. Expression of lamin C2 also resulted in aberrant localization of Sun1 in these cells. The collaborators also demonstrated that spermatogenesis was disrupted in Lmna^-/- mice, although oocyte meiosis was not obviously perturbed [6]. Although defects in meiosis-specific processes were observed in the knockout mice, it was not possible to rule out an indirect effect of lamin depletion in somatic cells on meiosis in spermatocytes, prior to the new study.An important feature of the new research [1] is that the C2^-/- mice show normal expression of all other A-type lamins. The C2^-/- males recapitulate the meiotic failure seen in Lmna^-/- mice. Nevertheless, their chromosomes frequently fail to synapse and they engage in heterologous associations or show aberrant telomere-telomere interactions; all of these defects are rare in wild-type spermatocytes. As a result of extensive apoptosis and failure of sperm maturation, the males are completely infertile. However, females are fertile, despite some evidence for pairing defects in C2^-/- oocytes.These sex-specific differences in the effects of lamin C2 loss are somewhat surprising. They could in part reflect differential implementation of meiotic checkpoints, which cull defective spermatocytes more ruthlessly than oocytes [7]. However, analysis of homologous pairing and synapsis in the C2^-/- mutant mice also revealed more severe defects in males. Both male and female mice lacking Sun1 protein are completely sterile and show synaptic failure during meiotic prophase [8]. This suggests that LINC-mediated chromosome dynamics are essential for homolog interactions during meiosis in both sexes. The milder defects caused by loss of lamin C2 in both male and female meiosis suggest that it has a less direct role in mediating chromosome movement than Sun1. This is consistent with the idea that expression of short lamin isoforms during meiosis acts primarily to increase the mobility of proteins within the nuclear envelope, relative to somatic cells. It seems likely that the dynamics of pairing, synapsis and recombination differ dramatically between spermatocytes, which are produced continually during the adult life of the male, and oocytes, which undergo meiotic prophase during fetal development. Such differences might render male meiosis more sensitive to changes in nuclear envelope organization or dynamics.The modifications made to the mouse nuclear envelope during meiosis are likely to be conserved in concept, if not in detail, in other taxa. As mentioned above, the isoforms and expression patterns of lamin proteins have diverged rapidly among the metazoa, as have the structures and functions of LINC complexes. For example, amphibians lack lamin C (and lamin C2), suggesting that its meiotic role in mammals is a recent innovation. Furthermore, the mouse Sun1 protein has a C2H2 zinc finger lacking in primate orthologs, which might suggest that it has evolved a distinct way to connect with meiotic chromosomes. It is thus not currently clear which aspects of meiotic lamina remodeling in mice can be extrapolated to other species.In Caenorhabditis elegans, meiotic chromosome dynamics are probably mediated by post-translational modification of the amino-terminal (nucleoplasmic) domain of sun-1 [9]. It is not yet known how this modification contributes to the function of the meiotic LINC complex. Direct observation has indicated that the motion of LINC complexes within the nuclear envelope becomes much less constrained as cells enter meiosis [3]. Phosphorylation of sun-1 may weaken interactions between the LINC complexes and the lamina to increase their mobility within the nuclear envelope, and/or promote interactions between LINC complexes to create high load-bearing aggregates of these proteins necessary to drive chromosome movement. It is not currently known whether the lamina itself is modified in C. elegans meiotic nuclei, but it is easy to imagine that phosphorylation could also be used to tweak protein-protein interactions within the lamina to optimize its properties during meiosis and other specialized cellular processes. It is likely that metazoans have evolved a wide range of mechanisms to modify their nuclear envelopes to meet the special demands of meiotic prophase.Homologous chromosome pairing remains one of the most mysterious aspects of meiosis. This new work in mice [1] adds an important piece of the puzzle by illuminating how the nuclear lamina can be modified to facilitate meiotic chromosome dynamics. To understand this process will clearly require looking beyond the chromosomes, and even beyond the nucleus, to the cellular networks connected by LINC complexes.     

The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility

During meiosis, the homologous chromosomes pair and recombine. An evolutionarily conserved protein structure, the synaptonemal complex (SC), is located along the paired meiotic chromosomes. We have studied the function of a structural component in the axial/lateral element of the SC, the synaptonemal complex protein 3 (SCP3). A null mutation in the SCP3 gene was generated, and we noted that homozygous mutant males were sterile due to massive apoptotic cell death during meiotic prophase. The SCP3-deficient male mice failed to form axial/lateral elements and SCs, and the chromosomes in the mutant spermatocytes did not synapse. While the absence of SCP3 affected the nuclear distribution of DNA repair and recombination proteins (Rad51 and RPA), as well as synaptonemal complex protein 1 (SCP1), a residual chromatin organization remained in the mutant meiotic cells.     

Unusual nuclear structures in meiotic prophase of fission yeast: a cytological analysis

Earlier results from sectioned nuclei indicating that Schizosaccharomyces pombe does not develop a classical tripartite synaptonemal complex (SC) during meiotic prophase are confirmed by spreading of whole nuclei. The linear elements appearing during prophase I resemble the axial cores (SC precursors) of other organisms. The number of linear elements in haploid, diploid, and tetraploid strains is always higher than the chromosome number, implying that they are not formed continuously along the chromosomes. Time course experiments reveal that the elements appear after DNA replication and form networks and bundles. Later they separate and approximately 24 individual elements with a total length of 34 microns are observed before degradation and meiotic divisions. Parallel staining of DNA reveals changes in nuclear shape during meiotic prophase. Strains with a mei4 mutation are blocked at a late prophase stage. In serial sections we additionally observed a constant arrangement of the spindle pole body, the nucleolus, and the presumptive centromere cluster. Thus, S. pombe manages to recombine and segregate its chromosomes without SC. This might correlate with the absence of crossover interference. We propose a mechanism for chromosome pairing with initial recognition of the homologs at the centromeres and suggest functions of the linear elements in preparation of the chromosomes for meiosis I disjunction. With the spreading technique combined genetic, molecular, and cytological approaches become feasible in S. pombe. This provides an opportunity to study essential meiotic functions in the absence of SCs which may help to clarify the significance of the SC and its components for meiotic chromosome structure and function.     

Identification of a structural protein component of rat synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved nuclear structures of meiotic cells which form during the zygotene stage of the first meiotic prophase and are responsible for the pairing of homologous chromosomes. Their formation appears to be a prerequisite for crossing-over events and proper chromosome segregation during the first meiotic division. Despite knowledge of their central role in genetic recombination processes very little is known about the molecular composition and the mechanisms governing the assembly of the SCs. In the present study we report on the characterization of a monoclonal antibody (SC14f10) which enabled us to identify a novel SC protein termed SC48. Protein SC48 has a Mr of 48,000 and migrates in two-dimensional gels with a pH value of 6.9. By means of immunogold EM we localized this protein to the central region of the SC. In cell fractionation experiments we recovered protein SC48 together with SC-residual structures in a karyoskeletal fraction of pachytene spermatocytes. Our results indicate that SC48 is a meiosis-specific structural protein component of the SC probably involved in the pairing of homologous chromosomes.     

Synaptonemal complex analysis in spermatocytes of tilapia, Oreochromis niloticus (Pisces, Cichlidae).

Some adaptations of the synaptonemal complex (SC) whole-mounting technique first used in plants permitted its application to meiotic studies in tilapia, Oreochromis niloticus. Direct observation of the chromosome pairing process and bivalent structure during the meiotic prophase of this fish species by light and electron microscopy permitted the analysis of SCs in autosomes and the possible identification of sex chromosomes. The analysis of SCs in spermatocytes of O. niloticus revealed that all 22 bivalent chromosomes completely paired, except for the occurrence of a size heteromorphism in the terminal region of the largest bivalent associated with the presence of an incompletely paired segment during the synapsis process, which may be the cytological visualization of an XX/XY sex chromosome system in this species.     

Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes

Background

The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition.

Results

In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents.

Conclusions

The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.     

Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.     

Atm Inactivation Results in Aberrant Telomere Clustering during Meiotic Prophase

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.     

Peculiarities of chromosome organization in meiosis

Meiotic and mitotic chromosomes have a complex of differences. (1) At the early prophase I of meiosis, chromosomes acquire protein axial elements (AEs) that were absent in mitosis; in addition to somatic cohesins, AEs contain the meiosis-specific cohesins REC8, SMC1β, and STAG3. (2) At the middle prophase I, protein lateral elements (LEs) of synaptonemal complexes (SCs) are formed on the basis of AEs. The LE proteins are not conserved, but in Saccharomyces cerevisiae and Arabidopsis thaliana they contain functional domains with conserved secondary structures. Among the almost 679 thousand proteins of primitive eukaryotes that we studied by bioinformatics methods, in green and brown algae, some lower fungi, and Coelenterata, we revealed proteins or functional domains similar to SC proteins. (3) During the pachytene and diplotene stages of meiosis, chromosomes of spermatocytes and mother pollen cells acquire a general structure resembling the structure of amphibian and avian lampbrush chromosomes in miniature. Lateral chromatin loops with sizes of 90, 160, and even over 480 Kb were observed in human spermatocytes during the diplotene stage. In combination, all these observations confirm the considerable conservation of the scheme of molecular and ultrastructural organization of meiotic chromosomes in a large variety of eukaryotic organisms.     

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and redistribution of lamins during nuclear differentiation in mouse spermatogenesis

Chromatin may be attached to the nuclear envelope through interaction of the nuclear membrane lamins A, B, and C. Such a hypothesis requires that these proteins are present in all cells with chromatin attachment to the nuclear envelope. We have investigated the distribution of the lamins during spermatogenesis in mouse, which exhibits extremes in nuclear envelope structural changes. By immunohistochemical techniques using human auto-antibodies and monoclonal antibodies against these molecules, we found that the lamins persist through all stages of spermatogenesis, though in highly variable amounts. They are also present during meiotic prophase (pachytene) when chromosomes are only locally attached to the nuclear envelope, analogous to the early prophase of somatic cells. Restructuring of the early spermatid nuclear envelope is accompanied by the appearance of a new lamin at the acrosomal fossa. In the epididymal spermatozoon the distribution of different lamins varies markedly over the nucleus suggesting special structural functions. The presence of lamins throughout spermatogenesis supports the concept that they are a general feature of the nuclear envelope structure, even where a lamina is not recognizable ultrastructurally.     

DNA repeated sequences may be involved in synaptonemal complex formation

Synaptonemal complexes (SCs) are intranuclear structures that facilitate the reversible lateral synapsis of homologous chromosomes in the course of meiosis. It is still unclear which DNA nucleotide sequences are responsible for the attachment of chromatin to SC lateral elements. Considering the features of the dispersed repeated sequences (RSs), it is possible to assume that they participate in the structure and functional organization of the meiotic chromosomes. Using numerical analysis, we have investigated the relationship between the RS and the distribution of meiotic recombination events in mouse chromosome 1. Using in situ hybridization on spread mouse spermatocytes, we have examined the arrangement of different types of RSs relative to SCs. Hybridization signals of B1(Alu), B2, and minisatellite probes were localized predominantly in SCs regions. Based on the results, we proposed a model of meiotic chromosome organization. According to the model, RSs participate in the attachment of chromatin loops to SCs.     

Nuclear envelope breakdown requires overcoming the mechanical integrity of the nuclear lamina

In prophase cells, lamin B1 is the major component of the nuclear lamina, a filamentous network underlying the nucleoplasmic side of the nuclear membrane, whereas lamin A/C is dissociated from the scaffold. In vivo fluorescence microscopy studies have shown that, during the G2/M transition, the first gap in the nuclear envelope (NE) appears before lamin B1 disassembly and is caused by early spindle microtubules impinging on the NE. This result suggests that the mechanical tearing of the NE by microtubules plays a central role to the progression of mitosis. To investigate whether this microtubule-induced NE deformation is sufficient for NE breakdown, we assess the mechanical resilience of a reconstituted lamin B1 network. Quantitative rheological methods demonstrate that human lamin B1 filaments form stiff networks that can resist much greater deformations than those caused by microtubules impinging on the NE. Moreover, lamin B1 networks possess an elastic stiffness, which increases under tension, and an exceptional resilience against shear deformations. These results demonstrate that both mechanical tearing of the lamina and biochemical modification of lamin B1 filaments are required for NE breakdown.     

Meiotic sister chromatid cohesion in holocentric sex chromosomes of three heteropteran species is maintained in absence of axial elements

It has been suggested that in species with monocentric chromosomes axial element (AE) components may be responsible for sister chromatid cohesion during meiosis. To test this hypothesis in species with holocentric chromosomes we selected three heteropteran species with different sex-determining mechanisms. We observed in surface-spreads and sections using transmission electron microscopy that the univalent sex chromosomes form neither AEs nor synaptonemal complexes (SCs) during pachytene. We also found that a polyclonal antibody recognizing SCP3/Cor1, a protein present at AEs and SC lateral elements of rodents, labels the autosomal SCs but not AEs or SC stretches corresponding to the sex chromosomes. Cytological analysis of the segregational behaviour of the sex univalents demonstrates that although these chromosomes segregate equationally during anaphase I they never show precocious separation of sister chromatids during late prophase I or metaphase I. These results suggest that AEs are not responsible for sister cohesion in sex chromosomes. The segregational behaviour of these chromosomes during both meiotic divisions also indicates that different achiasmate modes of chromosome association exist in heteropteran species. Received: 22 September 1999; in revised form: 20 December 1999 / Accepted: 21 December 1999