Organization of the synaptonemal complex during meiosis in Caenorhabditis elegans

Four different SYP proteins (SYP-1, SYP-2, SYP-3, and SYP-4) have been proposed to form the central region of the synaptonemal complex (SC) thereby bridging the axes of paired meiotic chromosomes in Caenorhabditis elegans. Their interdependent localization suggests that they may interact within the SC. Our studies reveal for the first time how these SYP proteins are organized in the central region of the SC. Yeast two-hybrid and co-immunoprecipitation studies show that SYP-1 is the only SYP protein that is capable of homotypic interactions, and is able to interact with both SYP-2 and SYP-3 directly, whereas SYP-2 and SYP-3 do not seem to interact with each other. Specifically, the coiled-coil domain of SYP-1 is required both for its homotypic interactions and its interaction with the C-terminal domain of SYP-2. Meanwhile, SYP-3 interacts with the C-terminal end of SYP-1 via its N-terminal domain. Immunoelectron microscopy analysis provides insight into the orientation of these proteins within the SC. While the C-terminal domain of SYP-3 localizes in close proximity to the chromosome axes, the N-terminal domains of both SYP-1 and SYP-4, as well as the C-terminal domain of SYP-2, are located in the middle of the SC. Taking into account the different sizes of these proteins, their interaction abilities, and their orientation within the SC, we propose a model of how the SYP proteins link the homologous axes to provide the conserved structure and width of the SC in C. elegans.     

Corona is required for higher-order assembly of transverse filaments into full-length synaptonemal complex in Drosophila oocytes

The synaptonemal complex (SC) is an intricate structure that forms between homologous chromosomes early during the meiotic prophase, where it mediates homolog pairing interactions and promotes the formation of genetic exchanges. In Drosophila melanogaster, C(3)G protein forms the transverse filaments (TFs) of the SC. The N termini of C(3)G homodimers localize to the Central Element (CE) of the SC, while the C-termini of C(3)G connect the TFs to the chromosomes via associations with the axial elements/lateral elements (AEs/LEs) of the SC. Here, we show that the Drosophila protein Corona (CONA) co-localizes with C(3)G in a mutually dependent fashion and is required for the polymerization of C(3)G into mature thread-like structures, in the context both of paired homologous chromosomes and of C(3)G polycomplexes that lack AEs/LEs. Although AEs assemble in cona oocytes, they exhibit defects that are characteristic of c(3)G mutant oocytes, including failure of AE alignment and synapsis. These results demonstrate that CONA, which does not contain a coiled coil domain, is required for the stable ‘zippering’ of TFs to form the central region of the Drosophila SC. We speculate that CONA's role in SC formation may be similar to that of the mammalian CE proteins SYCE2 and TEX12. However, the observation that AE alignment and pairing occurs in Tex12 and Syce2 mutant meiocytes but not in cona oocytes suggests that the SC plays a more critical role in the stable association of homologs in Drosophila than it does in mammalian cells.     

SYP-3 restricts synaptonemal complex assembly to bridge paired chromosome axes during meiosis in Caenorhabditis elegans

Synaptonemal complex (SC) formation must be regulated to occur only between aligned pairs of homologous chromosomes, ultimately ensuring proper chromosome segregation in meiosis. Here we identify SYP-3, a coiled-coil protein that is required for assembly of the central region of the SC and for restricting its loading to occur only in an appropriate context, forming structures that bridge the axes of paired meiotic chromosomes in Caenorhabditis elegans. We find that inappropriate loading of central region proteins interferes with homolog pairing, likely by triggering a premature change in chromosome configuration during early prophase that terminates the search for homologs. As a result, syp-3 mutants lack chiasmata and exhibit increased chromosome mis-segregation. Altogether, our studies lead us to propose that SYP-3 regulates synapsis along chromosomes, contributing to meiotic progression in early prophase.     

The mammalian synaptonemal complex: protein components, assembly and role in meiotic recombination

The synaptonemal complex (SC) is a proteinaceous structure of chromosome bivalents whose assembly is indispensable for the successful progression of the first meiotic division of sexually reproducing organisms. In this mini-review we will focus on recent progress dealing with the composition and assembly of the mammalian SC. These advances mainly resulted from the systematic use of knockout mice for all known mammalian SC proteins as well as from protein polymerization studies performed in heterologous systems.     

Sterile mutants in Caenorhabditis elegans: the synaptonemal complex as an indicator of the stage-specific effect of the mutation

Two sterile mutants of Caenorhabditis elegans hermaphrodites have been examined using the electron microscope and serial section analysis. The F4 and F80 mutants were described previously ( Mounier and Brun , 1980), and they were shown to be blocked at the start of oogenesis. In the F4 mutant, normal sperm are produced and the pachytene nuclei contain tripartite synaptonemal complexes (SC) between the homologously paired chromosomes. In the F80 asynaptic mutant, only a few sperm are produced and they have abnormal morphology. Whereas SCs and SC associated structures (termed 'SC knobs') are present in the F4, these structures are absent from the F80 . The mutation in F80 affects gametogenesis prior to the pachytene stage of meiosis and pairing of homologous chromosomes apparently does not occur. The SC knobs may influence the regulation of the disjunction of the chromosomes. For that reason, these structures are now termed 'disjunction regulator regions'.     

Shaping meiotic prophase chromosomes: cohesins and synaptonemal complex proteins

Recent progress in elucidating the function of synaptonemal complex (SC) proteins and of cohesins in meiocytes made possible, in particular, through the analysis of mice deficient in SC or cohesin proteins has significantly enriched our understanding of how meiotic chromosome architecture is determined. Cohesins and the SC proteins act together in generating the characteristic axis-loop structure of meiotic chromosomes, their pairing into bivalents, their ability to recombine, and to be properly segregated. This minireview attempts to summarize the current knowledge with a focus on higher eukaryotic systems and to ask questions that ought to be addressed in the future.The synaptonemal complex—50 years     

Components of the spindle assembly checkpoint regulate the anaphase-promoting complex during meiosis in Caenorhabditis elegans

Temperature-sensitive mutations in subunits of the Caenorhabditis elegans anaphase-promoting complex (APC) arrest at metaphase of meiosis I at the restrictive temperature. Embryos depleted of the APC co-activator FZY-1 by RNAi also arrest at this stage. To identify regulators and potential substrates of the APC, we performed a genetic suppressor screen with a weak allele of the APC subunit MAT-3/CDC23/APC8, whose defects are specific to meiosis. Twenty-seven suppressors that resulted in embryonic viability and larval development at the restrictive temperature were isolated. We have identified the molecular lesions in 18 of these suppressors, which correspond to five genes. In addition to a single intragenic suppressor, we found mutations in the APC co-activator fzy-1 and in three spindle assembly checkpoint genes, mdf-1, mdf-2, and mdf-3/san-1, orthologs of Mad1, Mad2, and Mad3, respectively. Reduction-of-function alleles of mdf-2 and mdf-3 suppress APC mutants and exhibit pleiotropic phenotypes in an otherwise wild-type background. Analysis of a single separation-of-function allele of mdf-1 suggests that MDF-1 has a dual role during development. These studies provide evidence that components of the spindle assembly checkpoint may regulate the metaphase-to-anaphase transition in the absence of spindle damage during C. elegans meiosis.     

Functional analysis of kinetochore assembly in Caenorhabditis elegans

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical "kinetochore null" phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A-containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.     

KLP-18, a Klp2 kinesin,is required for assembly of acentrosomal meiotic spindles in Caenorhabditis elegans

The proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.     

Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1

In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions—ASPM-1 recruitment to the spindle and microtubule severing—both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity.     

Expression profiling of MAP kinase-mediated meiotic progression in Caenorhabditis elegans

The LET-60 (Ras)/LIN-45 (Raf)/MPK-1 (MAP kinase) signaling pathway plays a key role in the development of multiple tissues in Caenorhabditis elegans. For the most part, the identities of the downstream genes that act as the ultimate effectors of MPK-1 signaling have remained elusive. A unique allele of mpk-1, ga111, displays a reversible, temperature-sensitive, tissue-specific defect in progression through meiotic prophase I. We performed gene expression profiling on mpk-1(ga111) animals to identify candidate downstream effectors of MPK-1 signaling in the germ line. This analysis delineated a cohort of genes whose expression requires MPK-1 signaling in germ cells in the pachytene stage of meiosis I. RNA in situ hybridization analysis shows that these genes are expressed in the germ line in an MPK-1-dependent manner and have a spatial expression pattern consistent with the location of activated MPK-1. We found that one MPK-1 signaling-responsive gene encoding a C2H2 zinc finger protein plays a role in meiotic chromosome segregation downstream of MPK-1. Additionally, discovery of genes responsive to MPK-1 signaling permitted us to order MPK-1 signaling relative to several events occurring in pachytene, including EFL-1/DPL-1 gene regulation and X chromosome reactivation. This study highlights the utility of applying global gene expression methods to investigate genes downstream of commonly used signaling pathways in vivo.     

ZHP-3 acts at crossovers to couple meiotic recombination with synaptonemal complex disassembly and bivalent formation in C. elegans

Crossover recombination and the formation of chiasmata normally ensure the proper segregation of homologous chromosomes during the first meiotic division. zhp-3, the Caenorhabditis elegans ortholog of the budding yeast ZIP3 gene, is required for crossover recombination. We show that ZHP-3 protein localization is highly dynamic. At a key transition point in meiotic prophase, the protein shifts from along the length of the synaptonemal complex (SC) to an asymmetric localization on the SC and eventually becomes restricted to foci that mark crossover recombination events. A zhp-3::gfp transgene partially complements a null mutation and reveals a separation of function; although the fusion protein can promote nearly wild-type levels of recombination, aneuploidy among the progeny is high, indicating defects in meiotic chromosome segregation. The structure of bivalents is perturbed in this mutant, suggesting that the chromosome segregation defect results from an inability to properly remodel chromosomes in response to crossovers. smo-1 mutants exhibit phenotypes similar to zhp-3::gfp mutants at higher temperatures, and smo-1; zhp-3::gfp double mutants exhibit more severe meiotic defects than either single mutant, consistent with a role for SUMO in the process of SC disassembly and bivalent differentiation. We propose that coordination of crossover recombination with SC disassembly and bivalent formation reflects a conserved role of Zip3/ZHP-3 in coupling recombination with SC morphogenesis.     

The synaptonemal complex and meiotic recombination in humans: new approaches to old questions

Meiotic prophase serves as an arena for the interplay of two important cellular activities, meiotic recombination and synapsis of homologous chromosomes. Synapsis is mediated by the synaptonemal complex (SC), originally characterized as a structure linked to pairing of meiotic chromosomes (Moses (1958) J Biophys Biochem Cytol 4:633–638). In 1975, the first electron micrographs of human pachytene stage SCs were presented (Moses et al. (1975) Science 187:363–365) and over the next 15 years the importance of the SC to normal meiotic progression in human males and females was established (Jhanwar and Chaganti (1980) Hum Genet 54:405–408; Pathak and Elder (1980) Hum Genet 54:171–175; Solari (1980) Chromosoma 81:315–337; Speed (1984) Hum Genet 66:176–180; Wallace and Hulten (1985) Ann Hum Genet 49(Pt 3):215–226). Further, these studies made it clear that abnormalities in the assembly or maintenance of the SC were an important contributor to human infertility (Chaganti et al. (1980) Am J Hum Genet 32:833–848; Vidal et al. (1982) Hum Genet 60:301–304; Bojko (1983) Carlsberg Res Commun 48:285–305; Bojko (1985) Carlsberg Res Commun 50:43–72; Templado et al. (1984) Hum Genet 67:162–165; Navarro et al. (1986) Hum Reprod 1:523–527; Garcia et al. (1989) Hum Genet 2:147–53). However, the utility of these early studies was limited by lack of information on the structural composition of the SC and the identity of other SC-associated proteins. Fortunately, studies of the past 15 years have gone a long way toward remedying this problem. In this minireview, we highlight the most important of these advances as they pertain to human meiosis, focusing on temporal aspects of SC assembly, the relationship between the SC and meiotic recombination, and the contribution of SC abnormalities to human infertility.The synaptonemal complex–50 years     

Protein SYCP2 provides a link between transverse filaments and lateral elements of mammalian synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved meiosis-specific nuclear structures critically involved in synapsis, recombination, and segregation of homologous chromosomes. SCs are proteinaceous structures composed of (a) two lateral elements (LEs), to which the chromatin of the homologs is attached, (b) numerous transverse filaments (TFs) that link the LEs, and (c) a central element (CE). Major protein components of mammalian SCs are the TF protein SYCP1 and the LE proteins SYCP2 and SCYP3. How SCs become assembled is presently poorly understood, in particular, it is not known how TFs assemble at the plane of LEs to interconnect the homologous chromosomes. Therefore, we have investigated possible interactions between SYCP1 and other SC proteins. In immunoprecipitation experiments we could find that SYCP1 and SYCP2 interact in extracts of meiotic cells. Using the yeast two-hybrid system, we were able to demonstrate that the C-terminus of SYCP1 directly interacts with SYCP2. These results were confirmed by different interaction traps. Furthermore, we could narrow down the interacting domain of the SYCP2 molecule to its C-terminal region. We propose that SYCP2 acts as a linker between SYCP1 and SYCP3 and therefore would be the missing connecting link between LEs and TFs essential for proper chromosome synapsis.     

Characterization of a Caenorhabditis elegans recA-like gene Ce-rdh-1 involved in meiotic recombination.

A recA-like gene was identified in the Caenorhabditis elegans genome project database. The putative product of the gene, termed Ce-rdh-1 (C. elegans RAD51 and DMC1/LIM15 homolog 1), consists of 357 amino acid residues. The predicted amino acid sequence of Ce-rdh-1 showed 46-60% identity to both RAD51 type and DMC1/LIM15 type genes in several eukaryote species. The results of RNAi (RNA-mediated interference) indicated that repression of Ce-rdh-1 blocked chromosome condensation of six bivalents and dissociation of chiasmata in oocytes of F1 progeny. Oogenesis did not proceed to the diakinesis stage. Accordingly, all the eggs produced (F2) died in early stages. These results suggest that Ce-rdh-1 participates in meiotic recombination.     

Zip1-induced changes in synaptonemal complex structure and polycomplex assembly

The yeast Zip1 protein is a component of the synaptonemal complex (SC), which is an elaborate macromolecular structure found along the lengths of chromosomes during meiosis. Mutations that increase the length of the predicted coiled coil region of the Zip1 protein show that Zip1 influences the width of the SC. Overexpression of the ZIP1 gene results in the formation of two distinct types of higher order structures that are found in the nucleus, but not associated with chromatin. One of these structures resembles the polycomplexes that have been observed in many organisms and are thought to be aggregates of SC components. The second type of structure, which we have termed "networks," does not resemble any previously identified SC-related structure. Assembly of both polycomplexes and networks can occur independently of the Hop1 or Red1 protein, which are thought to be SC components. Our results demonstrate that Zip1 is a structural component of the central region of the SC. More specifically, we speculate that Zip1 is a component of the transverse filaments that lie perpendicular to the long axis of the complex.     

STEM Analysis of Caenorhabditis elegans muscle thick filaments: evidence for microdifferentiated substructures

In the thick filaments of body muscle in Caenorhabditis elegans, myosin A and myosin B isoforms and a subpopulation of paramyosin, a homologue of myosin heavy chain rods, are organized about a tubular core. As determined by scanning transmission electron microscopy, the thick filaments show a continuous decrease in mass-per-length (MPL) from their central zones to their polar regions. This is consistent with previously reported morphological studies and suggests that both their content and structural organization are microdifferentiated as a function of position. The cores are composed of a second distinct subpopulation of paramyosin in association with the alpha, beta, and gamma-filagenins. MPL measurements suggest that cores are formed from seven subfilaments containing four strands of paramyosin molecules, rather than the two originally proposed. The periodic locations of the filagenins within different regions and the presence of a central zone where myosin A is located, implies that the cores are also microdifferentiated with respect to molecular content and structure. This differentiation may result from a novel "induced strain" assembly mechanism based upon the interaction of the filagenins, paramyosin and myosin A. The cores may then serve as "differentiated templates" for the assembly of myosin B and paramyosin in the tapering, microdifferentiated polar regions of the thick filaments.     

Caenorhabditis elegans evolves a new architecture for the multi-aminoacyl-tRNA synthetase complex

MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains.     

Mutual antagonism between the anaphase promoting complex and the spindle assembly checkpoint contributes to mitotic timing in Caenorhabditis elegans

The anaphase promoting complex/cyclosome (APC/C) triggers the separation of sister chromatids and exit from mitosis across eukaryotic evolution. The APC/C is inhibited by the spindle assembly checkpoint (SAC) until all chromosomes have achieved bipolar attachment, but whether the APC/C reciprocally regulates the SAC is less understood. Here, we report the characterization of a novel allele of the APC5 component SUCH-1 in Caenorhabditis elegans. We find that some such-1(t1668) embryos lack paternally contributed DNA and centrioles and assemble a monopolar spindle in the one-cell stage. Importantly, we show that mitosis is drastically prolonged in these embryos, as well as in embryos that are otherwise compromised for APC/C function and assemble a monopolar spindle. This increased duration of mitosis is dependent on the SAC, since inactivation of the SAC components MDF-1/MAD1 or MDF-2/MAD2 rescues proper timing in these embryos. Moreover, partial depletion of the E1 enzyme uba-1 significantly increases mitosis duration upon monopolar spindle assembly. Taken together, our findings raise the possibility that the APC/C negatively regulates the SAC and, therefore, that the SAC and the APC/C have a mutual antagonistic relationship in C. elegans embryos.     

Multi-pathway control of the proliferation versus meiotic development decision in the Caenorhabditis elegans germline

An important event in the development of the germline is the initiation of meiotic development. In Caenorhabditis elegans, the conserved GLP-1/Notch signaling pathway regulates the proliferative versus meiotic entry decision, at least in part, by spatially inhibiting genes in the gld-1 and gld-2 parallel pathways, which are proposed to either inhibit proliferation and/or promote meiotic development. Mutations that cause constitutive activation of the GLP-1 pathway, or inactivation of both the gld-1 and gld-2 parallel pathways, result in a tumorous germline in which all cells are thought to be proliferative. Here, to analyze proliferation and meiotic entry in wild-type and mutant tumorous germlines, we use anti-REC-8 and anti-HIM-3 specific antibodies as markers, which under our fixation conditions, stain proliferative and meiotic cells, respectively. Using these makers in wild-type animals, we find that the border of the switch from proliferation to meiotic entry is staggered in late-larval and adult germlines. In wild-type adults, the switch occurs between 19 and 26 cell diameters from the distal end, on average. Our analysis of mutants reveals that tumorous germlines that form when GLP-1 is constitutively active are completely proliferative, while tumors due to inactivation of the gld-1 and gld-2 pathways show evidence of meiotic entry. Genetic and time course studies suggest that a third pathway may exist, parallel to the GLD-1 and GLD-2 pathways, that promotes meiotic development.