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Conceived and designed the experiments: WZ NM MSZ AJM AMV. Performed the experiments: WZ NM MSZ AJM AS KN EM-P. Analyzed the data: WZ NM MSZ AJM AS PMC AMV. Contributed reagents/materials/analysis tools: SM-E. Wrote the paper: WZ AMV.
During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification.
For successful segregation of homologous chromosomes during sexual reproduction, homologs must first identify and pair with their correct partners. Further, many organisms stabilize and maintain alignment between paired homologs through assembly of a highly ordered structure known as the synaptonemal complex (SC). Pairing and synapsis must be tightly coordinated to ensure that SC assembly only occurs in a productive manner, linking the axes of correctly aligned homologous chromosomes. In this work, we identify HAL-2, a protein that concentrates in the nucleoplasm of germ cells, as a key player in mediating this coordination. We find that precursors of the SC have the potential to inhibit homolog pairing, interfering with the very process that the SC normally serves to stabilize. Moreover, we show that HAL-2 promotes homolog pairing and associated chromosome movement primarily by counteracting these detrimental inhibitory effects of SC precursors. Our data suggest that HAL-2 serves to prevent inappropriate association of SC precursors with chromosomes prior to licensing of SC assembly, and we propose that HAL-2 may enable precursors to accumulate in a manner that allows rapid, cooperative SC assembly upon homology verification.
Generation of haploid gametes during sexual reproduction depends critically on the ability of homologous chromosomes to identify and form pairwise associations with their appropriate partners. Pairing is essential to enable homologs to orient and segregate away from each other at the meiosis I division, thereby achieving the reduction in ploidy that is necessary to ensure restoration of the diploid state upon fertilization.
Pairing between homologous chromosomes is reinforced by a highly ordered tripartite structure known as the synaptonemal complex (SC) that assembles at the interface between aligned homologs [1]. The lateral elements (LEs) of the SC are comprised of cohesin complexes and meiosis-specific axial components that coalesce along the length of each homolog during early meiotic prophase. SC central region (CR) proteins, which contain extended coiled-coil domains, then load to create transverse connections between the axes of the paired homologs, conferring the characteristic zipper-like appearance of mature SC. SC proteins collaborate with meiosis-specific recombination proteins to promote crossover recombination events between the homologs and to promote maturation of chromosome structure surrounding crossover sites into temporary connections known as chiasmata that enable homolog segregation at the meiosis I division [2].
Although the SC normally assembles only between paired homologous chromosomes, the SC structure itself is indifferent to homology. Moreover, SC assembly is highly cooperative and processive, such that once synapsis is initiated, it can proceed along nonhomologous chromosome segments [1]. Further, CR proteins have a propensity to self-assemble into aggregates known as polycomplexes even when they are not associated with chromosomes [3]–[6]. Thus, homolog pairing and synapsis must be tightly coordinated to ensure that SC assembly only occurs in a productive fashion, linking the axes of correctly aligned homologs.
During Caenorhabditis elegans meiosis, much of the coordination between pairing and synapsis is mediated by specialized chromosomal domains termed pairing centers (PCs) that are located near one end of each chromosome [7]–[9]. PCs function both in conferring SC-independent local stabilization of pairing and in promoting SC assembly [8], [10]. A family of zinc finger proteins (HIM-8, ZIM-1, ZIM-2 and ZIM-3) that bind to specific PCs mediates functional connections between chromosomes and cytoplasmic dynein and microtubules through a conserved nuclear envelope-spanning complex (which includes KASH-domain protein ZYG-12 and SUN-domain protein SUN-1) that promotes dramatic intranuclear chromosome movements [11]–[17]. In addition to facilitating timely pairing between homologs, these movements also appear to play a role in licensing SC assembly [14]. Dynein-dependent forces acting at PCs have been proposed to function in a checkpoint-like mechanism that makes SC assembly contingent upon homology verification [14], [18]. Both the ZYG-12/SUN-1 complex and the HORMA domain LE component HTP-1 have been implicated in the operation of this proposed coupling mechanism, serving to impose a barrier to SC assembly that can be overcome by interactions between homologous PCs, thereby preventing SC assembly between nonhomologous chromosomes [14], [16], [18]. Further, HTP-1 has also been implicated in a second coordination mechanism that couples termination of chromosome movement with SC installation [18].
Here we identify a nucleoplasmic component of the meiotic machinery, HAL-2 (homolog alignment-2), that provides a distinct contribution to the coordination of homolog pairing and synapsis. We find that HAL-2 promotes pairing between homologous chromosomes predominantly by counteracting detrimental inhibitory effects of CR precursors (SYP proteins). By preventing uncontrolled behavior of SYP proteins that interferes with PC function and antagonizes HTP-1/2 localization, HAL-2 enables chromosome movement and homology assessment. Additionally, we find that HAL-2 also has SYP-independent roles in regulating progression of meiotic prophase events, by contributing to the coupling mechanism that maintains chromosome mobilization in response to unsynapsed chromosomes and by promoting initiation of recombination.
An essential role for HAL-2 in promoting nuclear reorganization and homolog pairing was revealed through analysis of the hal-2(me79) mutant, which was isolated in a screen for mutants with defective meiotic chromosome segregation [19], [20]. Cytological analysis of the hal-2(me79) mutant revealed defects in chromosome organization during meiotic prophase. In wild-type germ cell nuclei entering meiotic prophase (in a region of the gonad called the transition zone, TZ), chromosomes are dramatically reorganized into a clustered configuration that reflects active chromosome movement (Figure 1A) [14], [15], [21], [22]. In the hal-2 mutant, germ cell nuclei entering meiotic prophase lack this characteristic TZ organization and chromosomes are instead dispersed around the nuclear periphery (Figure 1A). Further, whereas wild-type nuclei at the mid-pachytene stage exhibit parallel tracks of DAPI-stained DNA corresponding to aligned homolog pairs, hal-2 mutant nuclei at this stage exhibit disorganized single DAPI tracks (Figure 1B). This organization reflects a severe impairment of homolog pairing in the hal-2 mutant, as assessed at the 5S locus on chromosome V (by fluorescence in situ hybridization [FISH]) and at the X chromosome PC (X-PC) (by immunofluorescence [IF] for X-PC binding protein HIM-8) (Figure 1C). In both assays, the hal-2 mutant exhibited two widely separated foci in most nuclei (see quantitation below), indicating a failure to achieve homolog pairing.
As expected given the pairing defect, diakinesis oocytes in the hal-2 mutant contain 12 univalents, indicating a lack of chiasmata connecting homologous chromosomes (Figure 1B). Absence of chiasmata results in impaired meiotic chromosome segregation, with hal-2 mutant hermaphrodites producing both a high frequency of inviable embryos (87.2%, n=1147), indicative of autosomal aneuploidy, and a high incidence of males (Him) among the surviving progeny, indicative of X chromosome missegregation (32.7%, n=147) (Table S1) [23].
Nuclear reorganization and initiation of homologous pairing in the TZs of wild-type worms are accompanied by dramatic chromosomal movements that are mediated by association of PCs and their binding proteins with patches of nuclear envelope (NE) proteins that are in turn connected to cytoplasmic microtubules [14]–[16]. SUN-1, an inner NE protein, is phosphorylated on multiple residues upon meiotic entry in a CHK-2-dependent fashion, and together with the outer NE protein ZYG-12, is reorganized into NE patches in wild-type TZ nuclei [11], [14]–[17]. Further, polo-like kinase PLK-2, which promotes phosphorylation of Ser12 (S12-Pi) of SUN-1, colocalizes during early meiotic prophase with PCs, ZYG-12 and phosphorylated SUN-1 in these NE patches and promotes their formation [24], [25]. IF analysis revealed that PLK-2 is not localized to NE patches in early prophase nuclei in hal-2 mutants, and SUN-1 S12-Pi is not detected (Figure 2A). Further, even though ZYG-12::GFP was localized to the nuclear envelope in the hal-2 mutant, it was not reorganized into patches, and CHK-2-dependent phosphorylation of Ser8 (S8-Pi) of SUN-1 was severely reduced and/or delayed (Figures 2B and S1). Finally, while live imaging of TZ nuclei in wild-type control germ lines showed chromosome-associated ZYG-12::GFP patches moving rapidly along the nuclear envelope, no evidence of mobile ZYG-12::GFP NE patches was visible in hal-2 mutant nuclei (Videos S1 and S2). Thus, the modification and reorganization of the NE proteins required for PC-mediated chromosomal movement is severely abrogated in hal-2 mutants.
We also assessed recruitment of autosomal PC-binding proteins ZIM-2 (chromosome V) and ZIM-3 (chromosomes I and IV) in the hal-2 mutant. Whereas the X-PC binding protein HIM-8 is associated with the X-PCs and the nuclear envelope throughout most of the germ line, association of the ZIM proteins with their respective PCs and the nuclear envelope occurs most prominently in the TZ during wild-type meiosis, beginning just prior to the onset of homolog pairing [12], [13]. We found that while bright NE-associated HIM-8 foci were detected in hal-2 mutant nuclei (Figures 1C and S2), no bright ZIM-2 or ZIM-3 foci were detected (Figures 2C and S3A), indicating a failure to concentrate ZIM-2 and ZIM-3 to their respective PCs and the nuclear envelope. In summary, our data indicate that hal-2 mutants are defective in the assembly of the linkage between PCs and cytoplasmic microtubules (via the NE bridge) required for PC-mediated chromosome movement.
During wild-type meiosis, assembly of the SC between successfully paired homologs is initiated in the TZ. LE components such as HIM-3 and HTP-3 [26], [27] coalesce to form discrete chromosome axes, and these axes are then linked by loading of CR components such as SYP-1 and SYP-3 between them [10], [28], [29]. In standard high-resolution IF images, LE components and SYP proteins colocalize at the interface between aligned homologous chromosomes, beginning in the TZ and during the pachytene stage (Figures 3A and 3B). In meiotic prophase nuclei from similar regions of hal-2 mutant germ lines, we detected HTP-3 and HIM-3 localized in tracks along the individual chromosomes, indicating that LEs assemble along the unpaired chromosomes in the hal-2 mutant. Whereas SYP proteins normally load only between paired LEs, however, we found that SYP proteins colocalized with LE components along the lengths of individual unpaired chromosomes in the hal-2 mutant (Figures 3A and 3B). Colocalization of SYP-1 with HTP-3 in the hal-2 mutant was detected at about the same time or soon after HTP-3 began to coalesce into discrete axial structures (Figure 3A). These data indicate that in the absence of HAL-2, SYP proteins are able to load onto unpaired chromosome axes.
Images obtained using 3D-structured illumination microscopy (3D-SIM) [30] support this interpretation. In 3D-SIM images of wild-type pachytene nuclei, the two LEs of the aligned homologs (visualized using an α-HTP-3 antibody) can be spatially resolved as two separate entities along much of their lengths, with SYP-1 localized between the two LEs and the associated chromatin of the paired homologs (Figure 3C and Video S3). In 3D-SIM images of hal-2 mutant pachytene nuclei, SYP-1 was detected mainly in association with single LEs (rather than between resolvable pairs of LEs) along the lengths of the conjoined sister chromatids of unaligned individual chromosomes (Figure 3C and Video S4). Further, in nuclei where short stretches of parallel LE tracks were observed, these corresponded to regions of fold-back near chromosome ends or regions where LEs of two different chromosomes were in close proximity (Figure S4). Thus, the 3D-SIM images are most consistent with SYP proteins loading along individual LEs in the hal-2 mutant, rather than with formation of SCs between sister chromatid pairs that each had assembled their own LE. Intersister SC formation has been reported to occur in Rec8 null mutant mice [31] and in the budding yeast pds5 meiotic-null mutant [32]; in both cases, the widths of the intersister SCs in the mutants were similar to those of interhomolog SCs in wild-type controls [31], [32]. While the lack of extensive resolvable pairs of LEs in our 3D-SIM images of hal-2 mutant nuclei rules out the formation of intersister SCs of normal width, however, we cannot exclude the possibility that intersister SC-like structures of smaller width might form in hal-2 mutants.
The loading of SYP proteins onto the axes of unaligned individual chromosomes in hal-2 mutants contrasts with a distinct class of C. elegans pairing mutants (including htp-1) in which chromosomes engage in extensive nonhomologous synapsis, with SYP proteins linking the axes of nonhomologous chromosomes [16], [18], [26], [33]. This aspect of the hal-2 phenotype also contrasts with chk-2 mutants, in which pairing is abolished and SYP loading is very delayed [18], [20]. We found that hal-2; htp-1 double mutants load HTP-3 and SYP-1 extensively onto unpaired chromosome axes (as in the hal-2 single mutant; Figure S5), indicating that nonhomologous synapsis in the htp-1 mutant is dependent on HAL-2 and suggesting that HAL-2 may be required for SYP proteins to connect chromosome axes together. Likewise, the hal-2; chk-2 double mutant also resembles the hal-2 single mutant with respect to loading of HTP-3 and SYP-1 (Figure S5), implying that the ability to inhibit SYP loading on unpaired axes requires HAL-2.
The inappropriate loading of SYP proteins onto unpaired homologs in hal-2 mutants raised the possibility that the defects related to homologous pairing might be a consequence of the improper association of SYP proteins with chromosomes. To test this hypothesis, we evaluated several features associated with homologous pairing in hal-2 mutants lacking the SYP proteins. First, we found that removal of SYP proteins (in hal-2; syp double mutants) substantially restored multiple features associated with PC-mediated chromosome movement that were eliminated in hal-2 single mutants. Nuclei with clustered chromosomes and NE patches of PLK-2, ZYG-12::GFP and phosphorylated SUN-1 (S12-Pi and S8-Pi) were detected in hal-2; syp-2 and/or syp-3; hal-2 double mutants (Figures 4A, S3B and S6). Further, live imaging of ZYG-12::GFP in hal-2; syp-2 germ lines indicated that these restored NE patches are competent for movement (Video S5). After the TZ region, hal-2; syp-2 nuclei also resembled wild-type nuclei in that most nuclei retained a single bright NE focus that was closely associated with HIM-8, indicating that the ZYG-12/SUN-1 NE patch attached to the X chromosomes persists longer than those associated with the autosomes (Figure S7) [14], [15]. In addition, localization of the autosomal PC-binding proteins ZIM-2 and ZIM-3 to bright foci at the nuclear periphery in nuclei with clustered chromosomes was also observed in hal-2; syp-2 double mutants (Figures 4B and S3A). Finally, restoration of chromosome clustering and SUN-1 S8-Pi in the syp-3; hal-2 mutants is dependent on CHK-2, as these features were absent in syp-3; hal-2; chk-2 triple mutants (Figure S6). Taken together, these findings indicate that CHK-2-/PLK-2-dependent PC-mediated chromosome mobilization can be inhibited by SYP proteins, and that HAL-2 antagonizes this inhibitory effect of SYP proteins on PC function.
Second, we found evidence that significant homolog pairing occurred in hal-2 mutants that lacked the SYP proteins. The syp mutants are proficient for establishment of pairing at PCs and can achieve a significant degree of lengthwise homolog alignment, but are defective in stabilization and maintenance of homologous associations [10], [28], [34]–[36]; this made it possible to test whether significant homolog pairing occurred in hal-2; syp double mutants. We quantified levels of pairing at the X chromosome PCs (using IF for HIM-8), at chromosome V PCs (V-PCs) (using IF for ZIM-2) and at an internal position of chromosome V (using FISH for the 5S rDNA locus) (Figures 4D–F). For all of these analyses, gonads were divided into 5 zones of equal lengths, as indicated in Figure 4C.
Pairing at the X-PC was quantified from Zones 2–5 (Figure 4D), as HIM-8 localization at the X-PCs is most robust from the TZ through late pachytene [13]. Whereas pairing at the X-PCs was effectively abolished in the hal-2 single mutant, high levels of pairing were restored in the hal-2; syp-2 double mutant, with X-PC pairing levels peaking in Zones 3 and 4 at levels approaching those of syp-2 and wild-type controls (Figure 4D). Although X-PC pairing was substantially restored in the hal-2; syp-2 double mutant, however, pairing in all zones was significantly lower than in the syp-2 single mutant, suggesting that restoration of synapsis–independent maintenance of PC pairing may be incomplete.
ZIM-2 pairing levels were analyzed specifically for Zone 2, the zone where ZIM-2 foci are most prominently detected during normal meiosis (Figure 4E). In wild-type gonads, Zone 2 includes some premeiotic nuclei, the TZ, and some early pachytene nuclei; hence, the wild-type data set included nuclei with two bright ZIM-2 foci, nuclei with a single focus (indicative of paired PCs), and some nuclei without ZIM-2 foci. As described above, hal-2 mutants lack nuclei with clear bright ZIM-2 foci, and these foci are restored in the hal-2; syp-2 double mutants (Figure 4B). Among the nuclei with ZIM-2 foci in Zone 2, the proportion of nuclei in which foci were paired in the hal-2; syp-2 double mutant was similar to the proportions observed for both wild-type and syp-2 mutant gonads (Figure 4E), indicating highly successful pairing at the V-PCs in early meiotic prophase nuclei with ZIM-2 foci in the hal-2; syp-2 double mutants. Whereas ZIM-2 foci (and other markers of PC-mediated movement) persisted into Zones 3 and 4 in syp-2 mutant gonads, ZIM-2 foci were not detected beyond Zone 2 in the hal-2; syp-2 double mutants (data not shown), thus precluding assessment of pairing at V-PCs throughout a larger portion of the hal-2; syp-2 mutant germ lines.
Pairing at the 5S locus on chromosome V was assessed for all five zones of the gonad (Figure 4F). While pairing at this locus was abolished throughout the gonad in the hal-2 single mutant, the syp-3; hal-2 double mutant exhibited a highly significant increase in pairing over the hal-2 single mutant specifically in Zone 2 (p<0.0001), the same zone in which chromosome V PCs were shown to be active in the hal-2; syp-2 double mutant. However, increases in pairing were not detected in other zones and pairing levels for syp-3; hal-2 for Zones 2–5 were consistently significantly lower than in syp-3 controls. As markers of active PCs persist beyond Zone 2 at the X-PCs but not at the autosomal PCs (in both hal-2; syp-2 and wild-type), it is likely that the lower level of pairing restoration observed for chromosome V upon removal of SYP proteins reflects a difference between the X chromosome and autosomes in the duration of PC activity.
Taken together, our analyses show that significant homologous pairing at the PCs can occur in hal-2 mutants when SYP proteins are absent, indicating that the SYP proteins can interfere with pairing, likely through inappropriate association with unaligned chromosomes. These findings imply that HAL-2 promotes PC function by counteracting inhibitory effects of the SYP proteins. However, incomplete restoration of pairing also indicates that HAL-2 plays additional role(s) in homolog alignment beyond antagonizing inhibitory effects of SYP proteins on PC function (see below).
hal-2 mutants show several phenotypic similarities with worms lacking both HTP-1 and HTP-2 (LE components that are paralogs of HIM-3 and HTP-3), including association of SYP proteins with the axes of unpaired chromosomes in pachytene nuclei [33], [37]. Thus, we assessed localization of HTP-1/2 in hal-2 mutant gonads by IF. (Since HTP-1 and HTP-2 are recognized by the same antibody, they are referred to as HTP-1/2 [37].) In wild-type germ lines, HTP-1/2 colocalize with other SC components (including the SYP proteins) along the full lengths of paired homologs until late pachytene, when HTP-1/2 and SYP proteins begin to become redistributed in a crossover-dependent fashion to reciprocal chromosomal domains such that their localization is almost mutually exclusive by diakinesis, with HTP-1/2 localizing on the long arms of the bivalent and SYP proteins localizing on the short arms (Figure 5) [37], [38]. The localization of HTP-1/2 on diakinesis chromosomes is markedly different from the other axis components, such as HTP-3, which localize to all four arms of the bivalent (Figure 5B) [27], [37].
We found that HTP-1/2 localization was impaired in the hal-2 mutant. During the pachytene stage, faint HTP-1/2 staining was detected along the axes of the unpaired chromosomes, but the level of HTP-1/2 relative to HTP-3 was much lower than in wild-type controls (Figure 5A). Further, while HTP-3 was present on univalent chromosomes of diakinesis-stage oocytes in the hal-2 mutant, HTP-1/2 staining was undetectable (Figure 5B).
The fact that SYP proteins load improperly onto unaligned chromosome axes in hal-2 mutants raised the possibility that the SYP proteins might be preventing the normal localization of HTP-1/2. Consistent with this hypothesis, HTP-1/2 localization was substantially restored in hal-2; syp-2 double mutant gonads, both at the pachytene and diakinesis stages (Figure 5). These results provide additional support for a previously proposed idea that SYP proteins and HTP-1/2 are incompatible, each antagonizing the chromosomal localization of the other [37]. Further, the data suggest that HAL-2 plays a role in promoting a temporary compatibility between SYP proteins and HTP-1/2 that enables them to coexist along the lengths of chromosomes from zygotene through mid-pachytene stages during wild-type meiosis.
Mapping of the hal-2(me79) mutation, phenocopy by RNAi, and identification of a nonsense mutation in the me79 allele by sequence analysis identified T16H12.11 as the hal-2 gene (see Materials and Methods) (Figure 6A). An independently generated deletion allele, tm4960, is missing 596 bp of the 927-bp coding region (Figure 6A) and fails to complement me79, confirming the molecular identity of hal-2 as T16H12.11. The two alleles cause similar phenotypic defects and both are likely null alleles; all of the analyses reported here were performed using the me79 mutant unless otherwise specified. Although the T16H12.11 gene model in WormBase (release WS227) predicts a 295 aa protein, we propose a modified gene model (that uses an upstream ATG) based on upstream conservation of amino acid sequence in other Caenorhabditis orthologs (Figure S8). The revised gene model encodes a predicted 308 aa protein with MW of 36.1 kDa. An antibody raised against the C-terminal 100 amino acids of HAL-2 detects a band of slightly less than 37 kDa in Western blot analysis of wild-type whole worm lysates, consistent with the protein size predicted by our revised gene model. This band was absent in lysates from both hal-2(me79) and hal-2(tm4960) mutant worms (Figure 6B), indicating that no full-length protein is produced in these mutants.
Bioinformatics analyses using PSI-BLAST and several protein structure prediction servers yielded little information regarding HAL-2 function. No homologs were detected outside of the Caenorhabditis genus, and no conserved motifs were detected by searches of the Conserved Domain Database. Further, while HAL-2 orthologs were found in other Caenorhabditis species, they exhibited relatively low sequence conservation (52% amino acid identity between ortholog pairs). This differs from conservation levels observed within Caenorhabditis for proteins implicated directly in meiotic recombination (which range from 74–98% amino acid identity) and is more in line with conservation levels for SYP proteins ortholog pairs (which range from 41–61% amino acid identity). Analysis using the COILS and Paircoil2 programs [39], [40] suggests that a short domain (33–46 amino acids) in HAL-2 orthologs may have the capacity to adopt a coiled-coil conformation, although the probability of this conformation differed widely among HAL-2 orthologs and reached significance threshold only for a subset of them.
IF analysis using our α-HAL-2 antibody demonstrated that the HAL-2 protein concentrates in the nuclei of germ cells. HAL-2 staining was first detected in germ cell nuclei before they entered the TZ and gradually weakened during diplotene until no signal was observed by the end of diakinesis (Figures 6C–E). As the majority of the IF signal did not colocalize with DAPI-stained chromatin, nucleoli (data not shown) or SC (see below), we infer that HAL-2 is predominantly nucleoplasmic. This nucleoplasmic staining was detected in wild-type gonads but was absent in hal-2 mutant gonads (Figure 6E), further verifying the specificity of the antibody. We also generated a functional GFP::HAL-2 transgene that rescues the Him and progeny lethality phenotypes of hal-2 mutants (Table S1). GFP::HAL-2 expressed from this transgene exhibited a similar nucleoplasmic localization in germ cells, although the protein persisted beyond diplotene (Figure 6F and data not shown), likely because of heterologous promoter and 3′UTR sequences.
Since our phenotypic analyses indicated that HAL-2 plays an important role in restraining the behavior of the SYP proteins, we used IF to investigate their spatial relationship in situ. In wild-type germ cell nuclei, little or no overlap was detected between the localization of HAL-2, which concentrates in the nucleoplasm, and SYP-1, which concentrates at the interface between aligned chromosomes (Figure 7).
However, when we analyzed the localization of HAL-2 in the scc-3 cohesin mutant, in which chromosome axes and SC assembly are severely disrupted and LE precursors (including the other cohesin subunits) and SYP proteins form aggregates (presumably polycomplexes) in the nuclei instead of loading extensively onto chromosomes, we detected HAL-2 both in the nucleoplasm and concentrated together with SYP-1 in the nuclear aggregates (Figure 7) [27], [41]. Localization of HAL-2 in nuclear aggregates is dependent on SYP-1, as HAL-2 nuclear aggregates are not observed in scc-3; syp-1 germ cell nuclei (Figure 7). Further, we found that HAL-2 colocalized with SYP-1 in nuclear aggregates in an htp-3 mutant, in which LEs do not assemble along the chromosomes but cohesin components are also not present in the nuclear aggregates (Figure 7) [42]. Finally, we examined HAL-2 localization in a him-3 null mutant, which does assemble LEs that contain cohesin complexes, HTP-1/2 and HTP-3, but is severely defective for SC assembly [26], [27], [37], [42]. In the him-3 mutant, SYP-1 appears as small nuclear aggregates at early pachytene that elongate into short stretches by late pachytene, and HAL-2 was found both in the nucleoplasm and colocalized together with SYP-1 in these structures (Figure 7). HAL-2 is not required for formation of these SYP-1 nuclear aggregates, however, as SYP-1 aggregates were still formed in hal-2; him-3 double mutants (Figure S9). Together, these analyses of SYP-dependent localization of HAL-2 to nuclear aggregates in various mutants with defective LE and SC assembly raise the possibility that HAL-2 might interact with SYP proteins or other SC-associated proteins; however these IF analyses do not address whether such potential interactions are direct or indirect.
Consistent with our finding that homolog pairing in the hal-2; syp double mutants is not restored to the levels observed in syp single mutants, closer examination of DAPI-stained chromosome morphology, SUN-1 phosphorylation, ZYG-12::GFP patches and PLK-2 localization in hal-2; syp-2 gonads provided further evidence that HAL-2 has additional roles in meiosis beyond regulating SYP loading (Figures 8A, S10 and data not shown). In contrast to the dramatically extended TZ observed in syp-2 mutant gonads, in which nuclei with clustered chromosomes and multiple bright NE patches reflecting ongoing chromosome mobilization persist until near the very end of the pachytene region (Figure 8A) [14], [15], [21], [34], hal-2; syp-2 mutant gonads lack persistent chromosome clustering, and only the single NE focus associated with HIM-8 persists beyond the TZ, as in wild-type gonads (Figures 8A and S7). The fact that hal-2; syp-2 nuclei can exit from the clustered chromosome organization despite lack of synapsis suggests that HAL-2 may be required for normal functioning of a checkpoint-like mechanism that operates to make redispersal of clustered chromosomes contingent upon SC assembly [18].
We also found that hal-2 mutants exhibit severely reduced levels of immunostaining for DNA strand exchange protein RAD-51 [34], [43]. Whereas multiple RAD-51 foci were detected in most early/mid-pachytene nuclei in wild-type germ lines, most nuclei in hal-2 mutant germ lines lacked RAD-51 foci (Figure 8B), suggesting that initiation of recombination may be impaired. Further, this deficit of RAD-51 foci was not suppressed in the syp-3; hal-2 double mutant, which also lacked RAD-51 foci in most nuclei, indicating that this phenotype is not a secondary consequence of inappropriate SYP loading. The reduced levels of RAD-51 foci in the syp-3; hal-2 double mutant contrasts with the increased levels of foci observed in the syp-3 single mutant (Figure 8B) [44], indicating that absence of HAL-2 prevents accumulation of RAD-51 foci. Reduction in RAD-51 foci in hal-2 mutants suggests either that hal-2 mutants have a reduced number of double-strand DNA breaks (DSBs) or that HAL-2 is needed for efficient loading of RAD-51 onto DSBs. To test whether HAL-2 is required for accumulation of RAD-51 on DSBs, we induced DSBs with ionizing irradiation in hal-2 animals and dissected them 1 hour post irradiation for RAD-51 immunostaining. Abundant RAD-51 foci were detected in nuclei throughout the irradiated hal-2 mutant germ lines (Figure 8C), indicating that HAL-2 is not required for accumulation of RAD-51 on DSBs. Taken together, these data suggest that HAL-2 has a role in promoting initiation of recombination that is distinct from its role in regulating SYP loading.
Variable defects in PC-mediated chromosome clustering in combination with impaired DSB formation had been reported previously for the C. elegans him-19 [45] mutant. Moreover, some of the defects in PC function in him-19 mutants were rescued by the introduction of DSBs by irradiation [45], raising the possibility that defects in PC function in hal-2 mutants might be similarly rescued by artificial induction of DSBs. We tested this by inducing DSBs in hal-2 mutants by ionizing irradiation and dissecting the animals 2 hours post irradiation for IF analysis. No chromosome clustering, recruitment of bright ZIM-3 foci to the PCs and nuclear envelope, or NE patches of phosphorylated SUN-1 S12-Pi were observed in the irradiated hal-2 gonads (Figure S11), indicating that these defects in the hal-2 mutant are not secondary consequences of its defect in recombination initiation.
The work reported here highlights the potential of SC central region (CR) precursors to exert detrimental effects that interfere with the process of homolog pairing. This potential emphasizes the need for SC precursors to be carefully shepherded during early meiotic prophase to avoid antagonizing the very process that they normally serve to stabilize. One way that CR precursors can cause trouble is by inappropriately linking the axes of nonhomologous chromosomes. If transient nonhomologous interactions that occur during early prophase are stabilized by synapsis, chromosomes will then have fewer chances to associate and pair with their correct partners. This is illustrated by the improved pairing observed when SYP proteins (and nonhomologous synapsis) were eliminated in a mutant with impaired SUN-1 function [16]. Nonhomologous synapsis may also prevent and/or restrain the active prophase chromosomal movements that promote pairing, as illustrated by the amelioration of restrained SUN-1::GFP NE patch movement in an htp-1 mutant by the removal of SYP proteins [21]. Our comparative analyses of hal-2; syp double mutants with hal-2 single mutants have revealed several additional ways in which CR precursors can be detrimental.
Firstly, SYP proteins can interfere with pairing by inhibiting CHK-2-dependent PC activities at the onset of meiotic prophase. In hal-2 mutants, NE protein SUN-1 is not phosphorylated on Ser12 (and S8-Pi is severely reduced and/or delayed), PCs fail to recruit PLK-2, autosomal PC-binding proteins fail to concentrate at their respective PCs or the NE, and no ZYG-12::GFP NE patches or PC-mediated chromosome movements are observed. Coordinate loss of these features suggests that lack of pairing in hal-2 mutants likely results primarily from the abrogation of vital CHK-2-dependent PC functions. Moreover, the fact that all of these problems are alleviated by removal of SYP proteins implies that the unconstrained behavior of SYP proteins in the absence of HAL-2 is responsible for preventing PC function in this context.
Secondly, SYP proteins can interfere with proper chromosomal association of HORMA domain proteins HTP-1/2. An incompatibility between CR proteins and (some) meiosis-specific HORMA domain proteins is evident from observations of apparently opposing spatial relationships of these classes of proteins along chromosomes in several different organisms. In S. cerevisiae, HORMA protein Hop1 and CR protein Zip1 exhibit largely complementary patterns of high and low abundance along pachytene chromosomes [46]. Further, mouse HORMAD1 and HORMAD2 lose their chromosomal localization upon installation of CR protein SYCP1 [47]. In C. elegans, all four HORMA proteins (HTP-3, HIM-3 and HTP-1/2) coexist with the SYP proteins along the lengths of chromosomes for most of the pachytene stage, but at late pachytene HTP-1/2 and SYP proteins are triggered by (nascent) COs to relocalize to reciprocal chromosomal domains [27], [37], [38]. These patterns all suggest a mutually antagonistic relationship between CR proteins and (some) HORMA proteins. In C. elegans, the coexistence of HTP-1/2 and SYP proteins from zygotene until late pachytene further implies a mechanism that temporarily counteracts this antagonism. The temporary compatibility between these proteins during early prophase requires HAL-2, as our data show that the unconstrained behavior of SYP proteins in hal-2 mutants inhibits axis association of HTP-1/2 during early prophase. Since HTP-1/2 is essential for homolog pairing [18], [33], reduced chromosomal HTP-1/2 likely contributes to the pairing defect in hal-2 mutants.
Taken together, our data indicate that HAL-2 plays a major role in homolog pairing by restricting the detrimental behavior of SYP proteins and constraining their loading to the appropriate context. Moreover, the contributions of HAL-2 to the coordination of pairing and synapsis are distinct from and parallel to the contribution of a previously identified checkpoint-like mechanism that ensures that SC central region assembly is contingent upon successful homology verification [14], [18]. LE component HTP-1, NE protein SUN-1, and dynein-mediated chromosome movements were shown to be involved in this mechanism [14], [16], [18]. Inhibition of dynein causes a significant delay between pairing and SYP loading (implying that motion and/or exertion of tension is required to license SYP loading), while HTP-1 loss or impaired SUN-1 function bypasses the dynein requirement and leads to nonhomologous synapsis (suggesting that these proteins are required to generate or respond to a "wait synapsis" signal). In contrast, neither pairing nor nonhomologous synapsis occurs in hal-2 mutants, and instead, SYP proteins load along the axes of individual unpaired chromosomes. This suggests the possibility that HAL-2 might function in promoting maturation of CR precursors, such that upon licensing of SC assembly, SYP installation invariably occurs in a manner that links LEs together. Thus, HAL-2 may have two potentially inter-related roles in regulating the behavior of SYP proteins: i) shepherding/restraining CR subunits to prevent them from wreaking havoc in the nucleus and ii) enabling their timely maturation into a form that can be assembled cooperatively into mature SCs.
These proposed roles suggest that HAL-2 might serve in the capacity of a chaperone to help ensure timely and appropriate SC assembly. Although the HAL-2 sequence does not identify it as a member of previously known chaperone families, there are numerous striking parallels between HAL-2 and Fpr3, a prolyl isomerase that regulates SC assembly during budding yeast meiosis [48]. Like HAL-2, Fpr3 localizes in the nucleoplasm of meiotic cells and colocalizes with CR precursors in polycomplexes that form when SC assembly is prevented [48]. Further, like HAL-2, Fpr3 functions in parallel with other mechanisms to prevent assembly of CR precursors onto chromosomes until licensing conditions have been met [48]. Thus, both HAL-2 and Fpr3 can be seen as guardians of CR precursors, inhibiting their inappropriate behavior.
The cooperative and processive nature of SC assembly and the inherent tendency for self-assembly of its precursors [1] present a challenge for meiotic cells: they must accumulate large pools of precursors needed to accomplish rapid SC assembly, while at the same time preventing these precursors from aggregating into nonfunctional structures and/or interfering with prerequisite events. In C. elegans, the SC central region consists of four interdependent coiled-coil SYP proteins that interact to span the distance between the LEs [10], [28], [29], [34], [35]. Thus, involvement of regulatory proteins not included in the mature SC (such as HAL-2) represents a practical solution for controlling maturation of these CR precursors and constraining their loading to occur only in the appropriate context.
In addition to HAL-2, assembly of the SC central region also requires CRA-1 [49], the C. elegans ortholog of the non-catalytic subunit of the NatB N-terminal acetyltransferase complex, which acetylates the N-terminal methionine of proteins containing Met-Asp-, Met-Glu- or Met-Asn- [50], [51]. As in hal-2 mutants, SYP proteins associate with unpaired LEs in cra-1 mutants [49], raising the possibility that maturation of SC precursors may require N-terminal acetylation of one or more subunits. While we have also suggested a role for HAL-2 in promoting CR subunits maturation, it is clear that HAL-2 and CRA-1 make different contributions to regulating SYP behavior. Loading of SYP proteins onto chromosome axes in cra-1 mutants occurs later than in hal-2 mutants and does not interfere with homolog pairing [49]. Further, whereas association of SYP proteins with unpaired chromosomes in cra-1 mutants is dependent on DSB formation and progression of meiotic DSB repair processes [49], SYP proteins load onto unpaired chromosomes in hal-2 mutants in the absence of DSBs (as in hal-2; spo-11 double mutants; Figure S5), indicating that the chromosomal association of SYP proteins in hal-2 mutants is not dependent on DSBs or recombination intermediates.
In addition to the checkpoint-like mechanism that couples SC assembly to homology verification, a related but distinct mechanism operates during C. elegans meiosis to render redispersal of clustered chromosomes and termination of active chromosome motion contingent upon SC assembly [18], [28]. HAL-2 may also play a role in this coupling mechanism that is distinct from its role in preventing SYP proteins from interfering with pairing. This is suggested by the fact that chromosome clustering and PC-mediated chromosome mobilization do not persist in hal-2; syp double mutants, in which synapsis cannot occur. It has been postulated that chromosome redispersal is regulated by a signaling mechanism that monitors synapsis progression, and HTP-1 has been suggested to be involved in generating and/or responding to an inhibitory "wait dispersal" signal that maintains chromosome clustering [18]. Since HTP-1/2 loading onto chromosomes is largely restored by removal of SYP proteins (in hal-2; syp double mutants), the inability to maintain chromosome clustering is not a consequence of lack of HTP-1 in the hal-2 mutant, but it may reflect a cooperation of HAL-2 with HTP-1 in regulating or responding to the "wait dispersal" inhibitory signal.
Implicit in such a coupling mechanism is that progression of synapsis must generate "start dispersal" activation signals to counteract the existing inhibitory signals. The SYP proteins themselves likely contribute, as suggested by analysis of the syp-3(me42) mutant, which expresses C-terminally truncated SYP-3 [28]. In contrast to the persistent chromosome clustering observed in syp null mutants, a premature exit from chromosome clustering is observed in the syp-3(me42) mutant; it was proposed that association of SYP proteins with unpaired chromosomes in this mutant may act as the trigger for chromosome redispersal, as removal of other SYP proteins in the syp-3(me42) mutant restored persistent chromosome clustering [28]. This contrasts with the hal-2 mutant, in which elimination of SYP proteins did not lead to persistent clustering, further supporting the notion that HAL-2 may play a SYP-independent role in the coupling mechanism and/or the maintenance of chromosome movement.
C. elegans strains were cultivated at 20°C under standard conditions [52], unless otherwise specified. For experiments involving meiotic mutants, homozygous mutants were picked from the progeny of heterozygous balanced parents by the absence of dominant markers associated with the balancers and/or by the presence of a recessive marker cis-linked with the mutant allele. For Figure S5, the spo-11 single mutant controls imaged were heterozygous for the hal-2 mutation as they were siblings of hal-2; spo-11 double mutants, both of which were derived from doubly balanced heterozygous parents. All experiments involving transgenic strains were performed in the presence of the wild-type gene unless otherwise noted. A list of strains used in this study is provided in Text S1.
hal-2(me79) was generated by EMS mutagenesis and isolated by screening for "Green eggs and Him" [19] and defects in prophase chromosome morphology and organization [20]. hal-2(me79) was mapped to a ~126 kb region between positions 10057573 and 10183653 on chromosome III using standard crosses and SNP mapping [53]. The interval contained 28 known or predicted genes; complementation tests and RNAi led to the identification of T16H12.11 as the likely hal-2 gene. RNAi targeted to the conserved region of T16H12.11 produced a partial hal-2 phenocopy: no apparent TZ, unaligned chromosomes at pachytene and 8–10 DAPI-stained bodies at diakinesis. Sequencing of T16H12.11 in the hal-2(me79) mutant revealed a C-to-T transition resulting in a premature stop (instead of glutamine) encoded at codon 96 of the 308 amino acid coding sequence.
hal-2(tm4960) was provided by Dr. S. Mitani at the C. elegans National BioResource Project, NIG, Japan; the 761 bp deletion was confirmed by PCR and sequencing.
Except for Figures 3C, S4, Video S3 and S4, immunostaining, DAPI-staining, image acquisition and processing using the DeltaVision deconvolution microscopy system were performed as in [18], except that slides were washed thrice in phosphate-buffered saline containing 0.1% Tween-20 (PBST) before blocking in 0.7% BSA. Dissections were performed on 20–24 hours post-L4 adults. A list of antibodies used is provided in Text S1. For experiments involving transgenic strains carrying GFP::SYP-3, GFP::HAL-2 or ZYG-12::GFP, L4 worms were placed at 25°C for 20–24 hrs before dissection.
For Figures 3C, S4, Video S3 and S4, gonads were dissected at 18 hours post L4 and fixed and processed for immunofluorescence as in [8]. Samples were stained with DAPI and mounted in glycerol containing 1% n-propyl gallate. Glass coverslips measured to 170±5 µm were placed over the samples and sealed with nail polish. 3D-SIM [30] was performed using a DeltaVision OMX microscope (Applied Precision), using an Olympus 1.4NA 100× UPlanSApo objective, with immersion oil (Cargille LaserLiquid) of refractive index 1.513, at 23–24°C. Image stacks of 8 µm thickness, 0.125 µm step size were acquired and processed to reconstruct high-resolution information. The videos each show a rotating series of 3D projections from a 3D-SIM data stack and were made with the IVE software package [54].
5S rDNA probes were amplified as in [36], [55] and labeled with Alexa Fluor 594 using the ULYSIS DNA labeling kit (Invitrogen). FISH was performed similarly to that in [20] with modifications described in Text S1.
Details of image acquisition and quantitative analysis of pairing are provided in Text S1.
L4 animals were selected and placed at 25°C for 20 hours before dissection. Worms were dissected in 10 µl of M9 containing tricaine (0.1%), tetramisole (0.01%) and Hoechst 33342 (Invitrogen; 11000) on a coverslip. The coverslip with the dissected worms was flipped onto a 2% agarose pad and imaged immediately using the DeltaVision deconvolution microscopy system with a 60× oil objective with 1.5× optivar. Images were acquired as stacks of 3 optical sections at 1.95 µm intervals every 15 s over a 5 min time period. Exposure times were kept constant for all images. Videos show maximum intensity projections of the 3D stacks displayed at 2 frames/s and were made with the Volocity (PerkinElmer) software.
For RAD-51 immunostaining, 20 hours post-L4 adults were exposed to 1 krad of γ irradiation from a Cs-137 source and dissected and fixed one hour post irradiation (21 hours post-L4). For ZIM-3 and SUN-1 S12-Pi immunostaining, 20 hours post-L4 adults were exposed to 5 krad of γ irradiation and dissected two hours post irradiation (22 hours post-L4). Unirradiated age-matched adults were dissected for immunostaining as controls for all irradiation experiments.
SUN-1 S8-Pi is severely reduced and/or delayed in hal-2 mutants. Immunolocalization of SUN-1 S8-Pi in DAPI-stained gonads of the indicated genotypes, with meiosis progressing from left to right. In the meiotic prophase region of the wild-type germ line, SUN-1 S8-Pi immunostaining is most robust from the TZ to mid-pachytene stage and concentrates as bright NE patches and also localizes weakly throughout the NE (as shown in zoomed-in image). In contrast, no SUN-1 S8-Pi is detected in the corresponding region of the chk-2 mutant germ line. Two representative examples of hal-2 mutant germ lines are shown; SUN-1 S8-Pi is not detected in the regions corresponding to the transition zone of wild-type germ lines, and exhibits only diffuse NE localization (as shown in zoomed-in images) in a subset of nuclei in the early and mid-pachytene regions. Bar, 50 µm. Bar in zoomed-in images, 10 µm.
HIM-8 localizes close to the nuclear envelope in a hal-2 mutant. IF images of hal-2 early prophase nuclei co-stained with antibodies against HIM-8 and nuclear lamin LMN-1, which localizes immediately adjacent to the inner surface of the NE. Images shown are 3D projections of partial data stacks in which sections from the tops and bottoms of the nuclei were omitted in order to illustrate the close proximity of the HIM-8 foci to the NE; not all HIM-8 foci were contained within the projections shown. Bar, 2 µm.
Bright ZIM-3 foci and NE patches of ZYG-12::GFP and SUN-1 S8-Pi are restored in hal-2; syp-2 double mutants. (A) IF images of early prophase nuclei stained with antibodies against ZIM-3, the PC-binding protein specific for chromosomes I and IV. Bright ZIM-3 foci are detected near the NE of wild-type TZ nuclei (top panel), whereas the hal-2 mutant nuclei lack clear ZIM-3 foci (middle panel). ZIM-3 foci are present in TZ nuclei of the hal-2; syp-2 double mutant (bottom panel), indicating that the concentration of autosomal PC-binding proteins to bright NE-associated foci was restored. Bar, 5 µm. Animals used for these IF images contained the zyg-12::gfp transgene. (B) IF images of early prophase nuclei stained for ZYG-12::GFP and SUN-1 S8-Pi in hal-2 and hal-2; syp-2 germ lines. Chromosome clustering, SUN-1 Ser8 phosphorylation and NE patches containing ZYG-12::GFP and SUN-1 S8-Pi (features that are missing in the hal-2 nuclei) are restored in the hal-2; syp-2 nuclei. Bar, 2 µm. Animals used for these IF images contained the zyg-12::gfp transgene.
Regions of fold-back near chromosome ends observed in hal-2 mutants. 3D-SIM images of a hal-2 mutant pachytene nucleus stained for the LE component HTP-3 and CR protein SYP-1. A short stretch of parallel LEs flanking a single track of SYP-1 (as shown by right panel of zoomed-in images of region in white box) is detected in the hal-2 mutant nucleus and corresponds to a region of fold-back near the end of the chromosome. Images shown are 3D projections of partial data stacks in which the bottom half of the nucleus was excluded in order to highlight the region of fold-back. Bars, 1 µm.
SYP-1 localization in hal-2; chk-2 , hal-2; htp-1 and hal-2; spo-11 double mutants resembles that in hal-2 mutants. IF images of HTP-3 and SYP-1 in late pachytene nuclei of the indicated genotypes. Images show: 1) many asynapsed chromosomes (HTP-3 stretches lacking SYP-1) and limited SYP-1 localized at the interface between nonhomologous chromosome segments in the chk-2 mutant; 2) extensive synapsis (between paired X chromosomes and between nonhomologous chromosomes) in the htp-1 mutant; 3) full homologous synapsis in the spo-11 mutant; 4) SYP-1 localization in hal-2; chk-2, hal-2; htp-1 and hal-2; spo-11 double mutants, which appears similar to that observed in the hal-2 single mutant, in that extensive SYP-1 is observed to be colocalized with HTP-3 along the axes of unpaired homologs. Bar, 5 µm.
Markers of PC-mediated chromosomal movement in syp-3; hal-2 mutants are dependent on chk-2 . IF images of early prophase nuclei stained with antibodies against SUN-1 S8-Pi reveal the presence of TZ nuclei with clustered chromosomes and SUN-1 S8-Pi NE patches in a syp-3; hal-2 mutant (left panel) and the lack of these features in syp-3; hal-2; chk-2 triple mutant nuclei (right panel), indicating that these markers of PC-mediated chromosomal movement are dependent on chk-2. Bar, 5 µm.
A single ZYG-12::GFP focus associated with HIM-8 persists into pachytene of hal-2; syp-2 mutants. IF images of wild-type and hal-2; syp-2 mutant nuclei of the indicated meiotic stages, co-stained for ZYG-12::GFP and HIM-8. In wild-type germ lines, a single ZYG-12::GFP NE focus associated with HIM-8 persists in nuclei that have exited the clustered chromosome configuration (right panels). Likewise, hal-2; syp-2 double mutants exhibit TZ nuclei with multiple ZYG-12::GFP patches (circled nuclei in left panels), and after release from the clustered chromosome configuration, a single ZYG-12::GFP NE focus colocalizing with HIM-8 persists in pachytene nuclei (left and middle panels). Bar, 5 µm. Animals used for these IF images contained the zyg-12::gfp transgene.
Modified gene prediction for hal-2/T16H12.11. (A) Modified predicted coding sequence for the hal-2/T16H12.11 gene is shown in capital letters, with the additional coding sequence highlighted in yellow; upstream untranslated sequence is shown in lower case. (B) Alignment of the N-terminal portions of HAL-2 orthologs from five different Caenorhabditis species, showing strong conservation of the first N-terminal 13 amino acids. Red indicates identity among all five orthologs; blue indicates conservative substitutions; green indicates semi-conservative substitutions.
SYP-1 forms aggregates and does not localize on chromosomes in hal-2; him-3 double mutants. IF images of pachytene nuclei from a hal-2; him-3 double mutant, showing that SYP-1 is not loaded on chromosomes but does form nuclear aggregates, indicating that HAL-2 is not required for aggregate formation. Bar, 4 µm.
Diffuse SUN-1 S8-Pi NE localization in hal-2 mutant gonad shown in Figure 8A . Immunolocalization of SUN-1 S8-Pi in gonads of the indicated genotypes, with meiosis progressing from left to right. Images shown are the same gonads as those shown in Figure 8A. In the hal-2 mutant gonad shown, early meiotic prophase nuclei lack SUN-1 S8-Pi, and only a small subset of nuclei at a later stage of meiotic prophase exhibit diffuse SUN-1 S8-Pi NE signal, as demonstrated in the zoomed-in image (blue box). The SUN-1 S8-Pi signal was boosted in the zoomed-in image to illustrate the weak diffuse NE localization. Bars, 10 µm.
Irradiation-induced DSBs do not restore chromosome clustering, bright NE-associated ZIM-3 foci or NE patches of SUN-1 S12-Pi in hal-2 mutant gonads. IF images of wild-type and hal-2 mutant early prophase nuclei stained for SUN-1 S12-Pi and ZIM-3. As in unirradiated hal-2 controls, chromosome clustering, ZIM-3 recruitment to PCs and the nuclear envelope, and formation of SUN-1 S12-Pi NE patches are not observed in hal-2 gonads exposed to 5 krad of γ irradiation. Bar, 2 µm.
High embryonic lethality and Him phenotype in hal-2 mutants are rescued by GFP::HAL-2.
Supplemental materials and methods.
ZYG-12::GFP patches move rapidly along the NE in wild-type TZ nuclei. Live imaging of ZYG-12::GFP in Hoechst 33342-stained dissected wild-type worms, showing chromosome-associated ZYG-12::GFP patches moving rapidly along the NE of TZ nuclei. Videos are 3D projections of images captured at 15 sec intervals, displayed at 2 frames/sec. ZYG::GFP is in pink and DNA is in blue. Bar, 4 µm.
ZYG-12::GFP does not reorganize into moving NE patches in hal-2 nuclei. Live imaging of ZYG-12::GFP in Hoechst 33342-stained dissected hal-2 worms reveals ZYG-12::GFP localization to the NE. However ZYG-12::GFP did not concentrate into the movement-competent NE patches observed in wild-type TZ nuclei. Videos are 3D projections of images captured at 15 sec intervals, displayed at 2 frames/sec. ZYG::GFP is in pink and DNA is in blue. Bar, 4 µm.
High-resolution 3D-SIM image of a wild-type pachytene nucleus. A rotating series of 3D projections of the wild-type pachytene nucleus shown in Figure 3C, highlighting the two spatially resolved LEs of the paired homologs. HTP-3 is in green and HIM-8 is in red.
High-resolution 3D-SIM image of a hal-2 mutant pachytene nucleus. A rotating series of 3D projections of the hal-2 mutant pachytene nucleus shown in Figure 3C, which exhibits single LEs rather than resolvable pairs of LEs as were observed in wild-type nuclei. HTP-3 is in green and HIM-8 is in red.
Movement-competent ZYG-12::GFP NE patches are restored in hal-2; syp-2 nuclei. Live imaging of ZYG-12::GFP in Hoechst 33342-stained dissected hal-2; syp-2 worms showing the restoration of movement-competent ZYG-12::GFP NE patches in hal-2; syp-2 TZ nuclei. Videos are 3D projections of images captured at 15 sec intervals, displayed at 2 frames/sec. ZYG::GFP is in pink and DNA is in blue. Bar, 4 µm.
We thank S. Mitani for the hal-2(tm4960) allele, A. Dernburg, V. Jantsch, C. Malone, A. la Volpe, M. Zetka, and the Caenorhabditis Genetics Center for reagents and M. Hayashi for technical assistance. We thank members of the Villeneuve lab for helpful discussions.
This work was supported by an A*STAR National Science Scholarship to WZ, National Institutes of Health grant RO1GM53804 to AMV, and WPI (The World Premier International Research Center Initiative) program for both AS and PMC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Question: A company with 200 000 in receivables could sell them
A company with $ 200,000 in receivables could sell them for $ 169,000 without recourse or for $ 195,000 with recourse. What do the terms "with recourse" and "without recourse" mean? Why is the "without recourse" alternative more expensive? How could the transfer be recorded?
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Answer to relevant QuestionsUnder what conditions will a transfer of receivables be recognized as a sale/derecognition? As a borrowing? What financial statement ratios are particularly affected by these alternatives?Vish Limited reported a cash balance of $ 36,000 in the general ledger, but the bank statement reported a balance of $ 27,900 at the end of September 20X9. You have ascertained that the bank cashed a cheque on this account, ...The net accounts receivable on the books of GJY Corporation as of 1 January 20X3 are as follows:Accounts receivable .......... $ 562,000 Less: Allowance for sales discounts.... 12,000 Allowance for doubtful accounts.... ...Belanger Ld. reports a current ratio of 2- to- 1 in its 20X2 financial statements. The statement of Financial position shows current assets of $ 2,540,500 and current liabilities of $ 1,284,000. Accounts receivable are $ ...On 1 January 20X5, Spencer Inc. sold merchandise ( cost, $ 8,000; sales value, $ 14,000) to Bryden, Inc. and received a non- interest- bearing note in return. The note requires $ 15,730 to be paid in a lump sum on 31 ...
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There are a number of factors that can affect a person's ability to lose weight, and it's different for everybody. Some people struggle thanks to genetics or lower resting metabolisms, whereas others simply can't – or feel like they can't – fit exercise and healthy eating into their schedules. It's true that now more than ever it's become hard to exercise and easy to overindulge – and the statistics are there to back it up. According to government figures, around one in four people in the UK is obese. So what makes weight loss so difficult? Well, as I mentioned, that depends – but here are a few of the most common reasons.
I don't have the time – If you work long hours and commute to work then this is likely to be a factor in your struggle. But that doesn't mean that you can't do it. Making time for fitness – even if it's two or three hours at your local gym a week – can make all the difference in the world. And once you get into the routine of exercising it'll likely become a welcome part of your day.
Temptation – Whether it's at work or home, there are few things that make weight loss more difficult than temptation. Try to limit your contact with the things that you know you struggle to resist, but also don't be too hard on yourself! Sometimes the temptation is too much and you will break, but this is okay so long as you eat well generally. Eating less bad stuff than you did before is always a positive change to make.
Your support system – Linking with temptation in some ways, having a support system is one of the best ways to ensure that you lose as much weight as you possibly can. If your partner or friends aren't dieting, it might be worth letting them know that you are. That way they can avoid tempting you with bad foods that might force you to crack.
Eating what you lose – A lot of people fall into the trap of thinking that because they've spent an hour or two in the gym, they're fine to eat whatever they want. It isn't true. Exercising is always a benefit, but you're far more likely to lose weight if you combine it with a healthy diet. So next time you pick up a pizza because you've just left the gym and feel good, remember that the pizza makes it so you may as well not have gone to the gym at all.
Not cooking – One of the easiest ways to fail in a diet is to stop cooking. Ready meals and easy options are almost always worse for you then freshly cooked food and you can't always guarantee the content of what you're eating - have you checked how many calories there are in that microwave chicken korma? The best way to ensure that you receive the nutrients that you need is to make sure that you prepare the food yourself.
It's a well-known fact that smoking is harmful not only for smokers, but... Continue reading
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We have had posts in these forums where members have used such 'driver update' utilities and they have ended up with unworkable systems.
What you are doing voluntarily is great, and I am sure your family and friends appreciate the help, but I just wonder if it is so necessary so often.
There's an old maxim, "if it ain't broke, don't fix it", (sorry, ain't is quaint old English for isn't). It applies to driver updates as well. If a system is working well, why does it need driver updates? If it is not working well then if there is a specific need for an updated driver, (because the old driver is faulty), then we download and install just the one specific driver file and install that, otherwise we diagnose the problem and fix it in other ways.
One thing I can safely say is that we never use Windows own driver update checker to check for updates. Windows doesn't do that very well. But as to the others, RadarSync was known to cause problems some time ago and I have heard nothing that suggests it is any better now.
The hard way is usually best. If the problem is driver based, find the updated driver yourself and install it.
Sorry, I don't mean to be dismissive, I just think that you are creating a lot of unnecessary work for yourself.
Hello all,
I am looking for a driver update utility that I could recommend to some poeple I am helping. The thing is, I am giving support free to many friends, family and such but, with that many, I would have to spend most of my time doing a round to update everyone has none of them can hold on and not fall in the abyss when I explain them how, where and why to keep their drivers updated.
I have given Driver Genius Pro a go and found it to do a good job so far but, I have searched a bit and found there are a lot of those out there (radarSync, DriverDoctor, DriverMax, Drivercure etc...) so, I would like to have recommendation from you peeps.


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Sunday 10 March 1918
It was Sunday, under the sign of Pisces. The US president was Woodrow Wilson (Democratic).
Famous people born on this day include Heywood Hale and Gunther Rall.
Hearts of the World, directed by D.W. Griffith, was one of the most viewed movies released in 1918
But much more happened that day: find out below..
You can also have a look at the whole 1918 or at March 10 across the years.
Old Newspapers
Have a look at the old newspapers from 10 March 1918 and get them!
Which were the most popular movies released in the last months ?
Find out your future
Get a FREE Numerology report based on the digits of 10 March 1918!
Wish him/her..
Historical Events
Which were the important events of 10 March 1918 ?
- Saint Anastasia
- Saint Macharius (d.335)
- Saint John Ogilvie (died 1615)
- Saint Himelin
- Saint Blankaard
- Tibetian national Uprising day
- Fans of video game character Mario celebrate Mar10 Day, by singing songs about him, watching the movie and show, and playing Mario games.
- Doctors Day - (Venezuela).
Famous Birthdays:
- Heywood Hale: Broun journalist.
- Gunther Rall: German ace fighter pilot (d. 2009).
- Victor Blanco: Puerto Rican astronomer who in 1959 discovered "Blanco 1," a galactic cluster.
- Isaac Rosenfeld: American writer, a critic, essayist, novelist and short story writer.
- Günther Rall: German general and pilot (d. 2009).
Music Charts
Which were the top hits in that special week of March 1918 ?
Top #3 songs in the USA
- Gems from Leave It To Jane - Victor Light Opera Co.
- For You a Rose - Prince's Orchestra
- I Don't Want to Get Well - Van & Schenck
Search For Your Ancestors
Subscribe to our Newsletter
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...and if 10 March 1918 was your Birth Date then Join our Birthday Club!
Make this date unforgettable
Genealogy: find out your family heritage
Which were the most popular books released in the last weeks ?
Remember when
What videogames would you be playing back in the days ?


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obit: Carolyn H. Sharp Spacek
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Carolyn H. Spacek, 78, went to be with her Lord December 8, 2012.
She is preceded in death by her parents Herman and Mary Sharp.
Survived by her loving children: Marvin Ellisor, Karen Smith, Jeff Ellisor, and Deidra Tolleson. Also survived by 5 grandchildren, and 7 great grandchildren.
Visitation will be at Pasadena Funeral Chapel Tuesday, December 11, 2012 from 5:00 PM till 8:00 PM. A funeral service will be held at Pasadena Funeral Chapel 2203 Pasadena Blvd. Pasadena, TX Wednesday, December 12, 2012 at 2:00 PM with interment to follow at Forest Park Lawndale Cemetery.
Please visit our website: pasadenafuneralchapel.com
Published in Houston Chronicle on December 10, 2012
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From Vietnam comes a video that suggests we will see a Type-C USB port on the Samsung Galaxy Note 7. We had an idea that this was going to be the case when a listing on India's import-export website Zauba hinted that a new version of Sammy's Gear VR headset is on the way. Since the current Gear VR works with a micro USB port only, one reason to prep a new variant would be to make it compatible with the Type-C USB port found on a new Samsung device. And that would point to the Galaxy Note 7.
The video that we alluded to earlier shows three different charging cables. One is a Type-C USB cable for the Microsoft Lumia 950 (seen in black). The other two white cables are made by Samsung in Vietnam; while one features a micro USB connector, the other carries the Type-C plug. You might notice that the white Type-C USB cord has a plastic wrap around it that contains the N930 model number belonging to Samsung's eagerly awaited phablet.
With the Type-C port, users can transfer data faster, charge batteries quicker, and the plug is reversible. The Samsung Galaxy Note 7 will be introduced during the next Unpacked event, which will be held in New York City on August 2nd.
The Samsung Galaxy Note 7 will use a Type-C USB port
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The Samsung Galaxy Note 7 will use a Type-C USB port
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source: Tinhte.vn (translated)


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Have Honda Questions? Ask a Honda Repair Expert.
Hi, I can help you with this. Your car has a hydraulic clutch system. Either the system has developed a leak and there is now air in the lines or there was a mechanical failure with the pressure plate or release fork. Check the fluid reservoir and see if there is any fluid in it. Its the smaller one next to the brake master cylinder. If its empty look under the dash where the clutch pedal meets the firewall. You may see the fluid leak in that area. If so you need a new clutch master cylinder. If no leak there, the cylinder on the transmission or the line may have a leak.
It should come out of gear if you release the parking brake and push the car foreword slightly. If it wont there could be a cable or internal transmission problem.
All of the fluids are full. In the small one beside the master cylinder, I see fluid. I don't see a full level gauge on it. So I am presuming that seeing fluid means it is full.
My sons and I tried pushing the car foward. We did get it to move a little bit. The car still remains in reverse.
I did notice some fluid in my parking space at work the other day so perhaps there is a leak. I need to know how to find and repair the leak or something?
I suspect its not a hydraulic issue if you see fluid. The fact that is wont shift is bad too.
With the engine off, you should be able to operate the shifter normally even with a clutch problem. If you can, locate the 2 large shift cables on top of the tranmission and disconnect them and see if you can move the 2 levers manually.
If not there is an internal problem, I attached this link. There is a picture of the cables on the first page. CLICK
Have a good evening.


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Charlotte Crosby: My relationship's fine
Charlotte Crosby and Mitch Jenkins are still going strong, despite recent reports they'd called it quits.
The Geordie Shore star confirmed her split with Mitch in September, after he dumped her by text, but they reconciled following her slim down at the start of the year.
The pair have delayed buying a house together in London because of work commitments, but despite that all is well.
"There's no trouble, we're stronger than ever," Charlotte told Now magazine.
"We've put moving in together on hold at the minute because I'm so busy. I've got a two week book tour coming up – and then I have other projects I need to get working on. It's just not the right time to be juggling that with going to look at different houses as well."
Charlotte's autobiography Me Me Me hits shelves on July 16.
The 25-year-old isn't just busy with work though, she's also been planning a romantic surprise for Mitch's birthday – though she doesn't know what.
"I haven't got a clue what I'm going to get him yet – for my birthday he was really good and took him to a spa and bought us a lovely watch. So I need to make it up to him!" she smiled.
Since getting back together the pair has been going strong, with Charlotte even getting the letter M tattooed on the back of her arm.
However she is realistic when it comes to love, and recently admitted that they need to see each other more.
"We need to spend more time together to be able to say things are amazing. But it's fine," she told Star magazine.
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who we are
For three decades, The Five O'Clock Club has been applying a proven, targeted, strategic, research-driven approach to career development.
Ask around. Do your homework. You'll find we provide unmatched support, resources, coaching and insights for individuals, whether they're outbound or looking for new ground.
We power the success and growth of individuals and their careers.
Back in the Day
In the 1880s, while the rest of the world was discussing mustache fashion, businessmen in Philadelphia were establishing a reputable member's-only club that brought together prominent members of the community to share cognac and cigars while they traded ideas and supported one another's endeavors. As this original club charter says, it was all "in a spirit of fellowship and good humor." These original Five O'Clock Club members clearly intended to have a good time while navigating their business lives.
Today's Club
Fast forward to the age of the internet, smart phones and electric cars — it's more than a hundred years later and our modern day Five O'Clock Club has similar aspirations. We've been tirelessly researching and adapting a method that's proven to work with every twist and turn of the job market, every new advent in technology, every shift in business culture. Our method, thirty years in the making, is as solid and innovative as ever but it's all for one purpose, much like the original 19th century club: to move our member's careers forward and support them every step of the way.
To better your career. Welcome to the Club.
Top Photo: The Five O'Clock Club at Tammany Pea Shore Fish-House by Mr. Edmund Stirling.
Inset Photo: William B. MacKellar, a member of The Five O'Clock Club in the 1880's from
"History of the Five O'Clock Club of Philidelphia" by J. Hampton Moore
Every great company has a dedicated team behind the curtain, powering the magic.
This is ours.
Darren Kimball
Chief Executive Officer
Darren Kimball
Chief Executive Officer
Wheezing his way through a high school summer job at a birdseed factory, Darren learned a lesson he's been applying ever since – choose your job with care. Some years later, following a highly successful career on Wall Street, he's now serving as CEO of The Five O'Clock Club, bringing thoughtful analysis and insight to others on their own career journeys.
After graduating with a Bachelor's degree from the Wharton School, Darren rose through the ranks to become one of the top stock analysts in the country, according to the Institutional Investor Magazine. At only 27, he was named a Managing Director at Merrill Lynch. Later on, he held senior roles at Lehman Brothers and the hedge fund George Weiss Associates.
Notwithstanding his success on Wall Street, Darren craved a hands-on entrepreneurial opportunity. He seized on that opportunity in 2013 with the acquisition of The Five O'Clock Club.
Darren was greatly inspired by the awesome work that The Five O'Clock Club was doing to help individuals with their careers. By making the jump from portfolio manager to entrepreneur, Darren hopes to inspire others to pursue their own stretch career goals.
Favorite Quote: Everybody falls, everybody fails, everybody, it is how you recover. Billy Joel
Anita Attridge
SVP, Executive Coaching & Leadership Development
Anita Attridge
SVP, Executive Coaching & Leadership Development
One of Anita's first jobs was teaching classes at a self-improvement school, and she hasn't wavered in her career path since. She's held senior leadership roles in HR and Training and Organization Development at Xerox, Merck and the Nature Conservancy.
As SVP of Executive Coaching at The Five O'Clock Club, Anita's focused experience allows her to quickly discern the bare bones of any situation and then support her clients through to the best solution. She believes in efficiency, dedication, and heart — a hard-to-balance combination that she uniquely champions with unique flair and grace. She recharges in her garden and long walks along the Jersey Shore.
Favorite Quote: It's never too late to be who you might have been. George Elliot
Jerry Iannaccone
SVP, Client Success
Jerry Iannaccone
SVP, Client Success
Born and raised in NYC, "Brooklyn Jerry" or "the Jobfather" has a wealth of experience hiding behind his wide smile. He's spent more than 30 years in Fortune 500 corporate operations, starting in data processing, computer programming, and client relations, and working up to facility operations, corporate budgeting and labor operations. He also knows first-hand what it's like to be unemployed, which gives him the ability to talk clients through the first days of a new job search with the perfect balance of heart and hope. Now a grandfather of three, he's instilling qualities of persistence and hard work in his grandkids, and teaching them how to scope out the best hot dog cart in the city. (Dirty Water Dogs, folks. He swears by 'em!)
When the road gets difficult, Jerry has learned that it's best to talk it out, which is why he's particularly gifted at matching clients to coaches: He listens and discerns, and usually comes up with a solution that's going to work best for the job searcher and the coach.
Favorite Quote: If everyone is thinking alike, then somebody isn't thinking. George S. Patton.
Nancy Karas
SVP, Business Development
Nancy Karas
SVP, Business Development
Nancy cut her coaching teeth as a teenager, working with other teens in Rockland Psychiatric Center. It scared her half to death, but taught her some pretty big life lessons like gratitude, graciousness, and self-confidence.
Her education and experience launched a career advocating for employee rights within the workplace. After getting her Master's degree in Human Resources Management, Nancy spent the next 20 years climbing the ladder in corporate America (and South America), including senior roles at Milbank Tweed, Coinbra Frutesp Agroindustrial, David Yurman, and Morgen-Walke. After many years at the helm, she moved back out West to the mountains and sunshine and dedicated herself to coaching.
As TFC's most charismatic brand ambassador, she is now a well-known speaker and executive coach and helps people remain positive and future-focused in their careers. She lives by the principle that you must know your own worth, follow your own heart, and fire the committee that lives in your head.
Favorite Quote: Go out on a limb. That's where the fruit is. Will Rogers
Linsey Odre
VP, Strategic Accounts & Communications
Linsey Odre
VP, Strategic Accounts & Communications
This pen-wielding former D-1 athlete, Instagramming foodie has a penchant for living life as fully as possible. Linsey's wit is as quick as the fast-paced culinary, fashion, and tradeshow industries she came from. Her confidence in who she is has allowed her to excel in every job she's held and to find creative solutions during stressful moments. A giver in every sense of the word, she'll show you the hidden gems around NYC she has collected over the years just as fast as she'll share a coveted family recipe.
At TFC, she manages key client relationships and strategic projects, as well as spearheading TFC's ad hoc culture committee.
Favorite Quote: What we fear most is usually what we most need to do. Ralph Waldo Emerson
Robert Hellmann
Social Media Black Belt
Robert Hellmann
Social Media Black Belt
Robert Hellmann has been a career and executive coach with the Five O'Clock Club since 2003. Rob has served as VP, Associate Director of the Five O'Clock Club Coach Network, and has made substantial contributions to the Club's coach training curriculum. He is an adjunct professor at New York University, teaching career development courses. Rob also brings 20 years of corporate management experience at organizations such as the Federal Reserve Bank of New York, JP Morgan Chase, and American Express.
Robert is the author of The Social Media Boost: Accessible Techniques To Accelerate Your Job Search And Career Growth With LinkedIn®, Twitter® And Other Social Media, published by The Five O'Clock Club in 2016.
Favorite Quote: "In 20 years you'll regret more the things you didn't do than the ones you did." – Mark Twain
Chip Conlin
Director, TFC Central
In a world of tough relationships, Chip is the master of smoothing the road for all involved. His experience in various HR, management and customer service roles in the corporate world and his experience serving as president of New York Technical Recruiters Association puts him squarely on both sides of the fence. He understands hiring from the corporate perspective as well as the job hunter's vantage point, which in turn makes him a masterful coach.
He's been leading Five O'Clock Club branch meetings, strategic small groups, and peer supervision groups for almost twenty years. Suffice it to say, he knows how to lead and motivate.
Favorite Quote: The most important thing to do if you find yourself in a hole is to stop digging. Warren Buffett


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COMMENT: GM faces a long road to its hydrogen future
GM chose the Detroit motor show this week to exhibit its latest hydrogen-fuelled prototype, a descendant of the Hy-wire - the company's first model unveiled in 2002.
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With Purchase of Both Skis and Bindings (Price Adjusted in Cart)
- Description
You just don't mess with this kind of success. The Spitfire LT is back unchanged this year featuring G3's proven semi-cap construction, lightweight Paulownia/Poplar wood core and classic dimensions. There's no other ski that offers this kind of versatility and solid, dependable performance in all snow conditions. The Spitfire LT embodies G3's standard for ski touring versatility.
Tip/Waist/Tail Width: 123 / 89 / 111 mm Available Lengths: 170, 177, 184 cm Weight: 3.2kg / 7.1lb (177cm) Skiing Style: Technical, Recreational Construction: CoreLight Ski Type: All Mountain Ability: Intermediate, Advanced, Expert Terrain: Piste, Touring
- Additional Information
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The refined 80's retro style of the Timex classic digital watch makes it easy to add a bold, modern finish to your wardrobe.
Timex T80 Classic T2N805
Wholesale Price
Do you want to know the wholesale price? Registered Users benefit from discounts up to 90% off, register now.
Retail price: €. 42
- Quartz digital with day, date and month
- Gender: Unisex
- Resina band and case, acrylic glass
- WR 30mt
- Measures: case 34x10mm, band 18cm x 18mm
Prices VAT excluded
Out of stock


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Most of these have been taking each other on in the qualifiers for this Sprint Series final, but David Evans won it last year and HAADEETH looks to have the best chance of his three runners this time around. Formerly with Richard Fahey, the latter scored over C&D at the first time of asking for the Monmouthshire trainer and is preferred to stablemates Nubar Boy and Captain Dimitrios, who are closely matched on earlier running here. Hatta Stream has a bit to find with the selection and Aldemoor, but Sulis Minerva has solid claims after going close over 5f last time having beaten The Strig over C&D last month. Speak The Truth, Picansort, Waterloo Dock and Waabel were all beaten in last year's renewal though the latter has tumbled down the weights since and may finish closer today.
Sulis Minerva (IRE) 5-1 (9-2) Held up towards rear, headway entering final furlong, ridden and ran on to go 2nd near finish, 2nd of 10, 1/2l behind Taajub (9-5) at Lingfield 5f hcp 0-85 (4) pol.
Haadeeth 8-1 (9-0) Tracked leaders, ridden to lead inside final furlong, stayed on, won at Lingfield 6f hcp 0-75 (5) pol beating Roman Strait (9-3) by nk, 11 ran.
Hatta Stream (IRE) 4-1fav (9-7) Tracked leaders, ridden 2f out, stayed on inside final furlong, not quicken close home, 3rd of 11, 3/4l behind Haadeeth (9-0) at Lingfield 6f hcp 0-75 (5) pol.
The Strig 14-1 (8-8) Went left start, mid-division, ridden over 1f out, one pace, 9th of 10, 7l behind Chilli Green (9-2) at Kempton 7f hcp 0-85 (4) pol.
Aldermoor (USA) 15-2 (9-1) Towards rear, weakened 2f out, last of 9, 30l behind Triple Dream (9-3) at Kempton 5f hcp 0-75 (5) pol.
Nubar Boy 11-2 (9-8) Held up towards rear, headway over 1f out, soon ridden, stayed on well, never nearer, 6th of 10, 2 1/4l behind Waterloo Dock (9-3) at Lingfield 6f hacp 0-70 (5) pol.
Diamond Vine (IRE) 9-4fav (9-6) Raced wide held up, improved to track leaders over 2f out, soon ridden, effort entering final furlong, ran on to lead towards finish, won at Lingfield 6f hcp 0-65 (6) pol beating Riflessione (8-12) by 3/4l, 7 ran.
Speak The Truth (IRE) 9-1 (8-11) Went right start, mid-division, headway over 1f out, never nearer, 4th of 12, 3/4l behind Aldermoor (9-1) at Lingfield 6f hcp 0-75 (5) pol in Jan.
Waterloo Dock 9-1 (9-3) Chased leaders, effort over 1f out, ran on to lead inside final furlong, held on gamely, won at Lingfield 6f hacp 0-70 (5) pol beating Cliffords Reprieve (8-11) by shd, 10 ran.
Picansort 9-2 (8-11) Dwelt, headway to chase leaders over 4f out, ridden to chase leader over 2f out until over 1f out, every chance when edged left inside final furlong, not quicken, went 2nd post, 2nd of 10, 1/2l behind Nubar Boy (9-0) at Lingfield 6f hcp 0-75 (5) pol.
Captain Dimitrios 5-1 (8-10) Tracked leader, driven to lead well over 1f out, soon strongly challenged, headed post, 2nd of 11, hd behind Gorgeous Goblin (8-7) at Lingfield 6f hacp 0-70 (5) pol.
Waabel 4-1fav (8-12) Always behind, 9th of 11, 5l behind Gorgeous Goblin (8-7) at Lingfield 6f hacp 0-70 (5) pol.


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Wedding Pictures: Siddharth-Neha Big Fat Wedding; Nakuul, Anil, Vahbbiz Present!
Indian Wedding are always big as they are star studded and the same happened when RJ Siddharth Kannan got married to Neha Agarwal. Pyar Ka Dard Hai's Nakuul Mehta, 24's Anil Kapoor, Yeh Rishta Kya Kehlata Hai's Karan Mehra and Vivian Dsena's wife Vahbbiz Dorabjee were among the guests. Also spotted were Vivek Oberoi, Poonam Dhillon, Tanaaz Irani, Delnaaz Irani, Suchitra Pillai and others.
Siddharth And Neha's Wedding A Starry Affair!
Siddharth and Neha's wedding reception was a grand affair and had lots of stars coming to grace the occassion and bless the couple.
Karan Mehra And Nakuul Mehta At The Wedding Reception!
Karan Mehra with wife and Nakuul Mehta with wife Jankee at the wedding.
Vahbbiz At The Wedding Reception!
Vivian Dsena's wife Vahbiz Dorabjee too was spotted at the wedding reception of Siddharth and Neha.
Anil Kapoor At The Wedding Reception!
Anil Kapoor was spotted at the reception with wife Shobha Kapoor.
Vivek Oberoi At The Wedding Reception!
Vivek happnes to be good friends with Siddharth so was present at the reception.
Neha Kakkar At The Wedding Reception!
Singer Neha Kakkar at the reception along with her sister Sonu Kakkar.
Poonam Dhillon At The Wedding Reception!
Yester year actress Poonam Dhillon snapped at the wedding reception.
Suchitra Pillai At The Wedding Reception!
Zain's mother in Beintehaa Suchitra Pillai graced the occassion and looked elegant.
The Married Couple Pose At Their Wedding!
Shama Sikander along with newly wedded couple Siddharth and Neha.
Aman And Gurdeep At The Wedding Reception!
Aman Punj arrives with wife Gurdeep Kohli at the wedding reception.
Claudia At The Wedding Reception!
Bigg Boss contestant Claudia too was present to wish the couple a happy married life.
Delnaaz Irani At The Wedding Reception!
Bigg Boss 6 contestant Delnaaz Irani arrives at the wedding reception.
Tanaaz Irani At The Wedding Reception!
Tanaaz Irani dressed in Indian attire for the wedding reception.
Shama Sikander At The Wedding Reception!
Shama Sikander along with beau Alex O' Neil at the wedding reception of Siddharth and Neha.
Rajesh Khatter At The Wedding Reception!
Actor Rajesh Khatter along with his wife at the party of Siddharth and Neha's wedding.


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Creativity, integrity and diligence are at the heart of all we do so that we can look back at every piece of work we have done with pride and then look forward to how we'll do it even better tomorrow.
From our offices in Hull (UK City of Culture 2017 no less!) we serve clients throughout the UK, though if an opportunity is exciting and we might be the right team for the job; we're always happy to travel!
eskimosoup works with a devoted internal team and highly skilled delivery partners. This approach enables us to have the right talent on the job with impressive turnaround time that often cannot be achieved by agencies that limit themselves by their salaried capability alone.


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- Annapolis Siding Roofing
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- Contact Us
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Bowie Siding and Roofing serves the following areas and surrounding cities: Aberdeen, Annapolis, Baltimore, Bel Air, Beltsville, Bethesda, Bowie, Brookeville, Catonsville, College Park, Columbia, Crofton, Cumberland, Ellicott City, Glenn Dale, Greenbelt, Laurel, Severna Park, Silver Spring, Upper Marlboro, Waldorf, and provides roofing, siding, and window services in more locations across Maryland and Washington, DC.
"We had this company replace our roof and install windows about 12 years ago. The workmanship was great and still looks great. They were on time, on budget, cleaned up and have been available since then whenever I had a question. Great people - very customer oriented. This is a company that has passed the test of time." JE from Bowie, MD


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I've been noticing a lot of guys bash on chubby girls and my best friend takes it to heart when she overhears rude comments even if there not directed to her and I was just wondering why its such a big deal for guys to NOT engage with a "fat" girl
Most Helpful Guy
Yes they can. If they have a pretty face and a pleasant and confident personality they can most definitely get a cute date. It also depends on what you define as a "çute date " too. Let's be real here most of us want a partner who aesthetically looks pleasing and who lives a health lifestyle. Very overweight people and very underweight people do get the short straws in terms of the pool of potential partners they can choose from. Most if not all of the bigger girls I know who are in relationships are incredibly comfortable in their skin. They are happy and confident and don't allow digs at their weight to get them down.00|00|0


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A solution to the problem you will run in to if you lose your emachines restore disk and try to use your ekb -5190(a) keyboard. Or if you just want to do a clean install of windows98.
|Discussion Thread
|emachines em250-1162 (Windows 7) [PCI / ISA]
|Jun 22, 2016
|Emachines ekb 5190 (Windows XP Professional)
|Aug 31, 2008
|Emachines T5048 9 in 1 reader (Windows XP Professional) [IDE]
|Dec 16, 2007
|Emachines Internet Keyboard (Windows XP Professional) [Parallel Port]
|May 22, 2007
|Emachines etower 466id (Windows 98SE)
|Jun 23, 2006
|HELP NEEDED: Emachines kb-0108 (Windows XP Home)
|Feb 23, 2006
|Emachines kb-0108 (Windows XP Home)
|Feb 15, 2006


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By SAM GOLDAPER, Special to the New York Times
Published: April 19, 1987
EAST RUTHERFORD, N.J., April 18— It has been this kind of season for the Nets: In the Detroit airport last week, Coach Dave Wohl leaned against a barrier and inadvertently set off the security-alarm system.
Then there was the cold December night in Boston when the Nets stood in a hallway of Boston Garden, locked out of their dressing room, as Fritz Massman, their trainer, searched for someone to open it.
And once, after Leon Wood ran the wrong play, Wohl asked out loud, ''How many years would I get for manslaughter?''
These are but a few incidents that illustrate the frustrations of the Nets' season, one that began with thoughts of the playoffs and ended tonight in Byrne Meadowlands Arena with the Indiana Pacers beating the Nets, 112-103. They finished their season with a 24-58 record, which qualifies them for the draft lottery.
Indiana, which is in the playoffs for the first time since the 1980-81 season, clinched seventh place in the Eastern Conference and will oppose the loser of Sunday's Boston Celtics-Atlanta Hawks game in the opening round. Worst Record in Years
How did things get so bad that the Nets, after a major off-season facelift, ended up with their worst record since the 1980-81 season? ''If this was the kind of team we had going into the season,'' said Wohl, ''it would have been simple to explain what happened because then your expectations are not high. But not once this season have we been able to put the team on the floor that we thought we had.''
Wohl was alluding to the total of 252 games his players missed with injuries in the 82 games this season. Darryl Dawkins missed 76 games, Otis Birdsong 69 and James Bailey 34. In addition, Albert King struggled all season with nagging knee and ankle injuries.
Wohl did receive some good news today, as the Nets announced that they are picking up their option to re-sign him as coach. Wohl originally signed a two-year contract with the team at the start of the 1985-86 season, with the 1987-88 season being the option year. The length of Wohl's new contract has not yet been determined.
After the Nets were eliminated in the opening round of last season's playoff, General Manager Lewis Schaffel promised, ''that the team that finished the season would not be the same team to take the floor next season.'' #7 New Players on Roster While Schaffel was to leave several months later to join the group seeking to put a National Basketball Association expansion franchise in Miami, the Nets did make the promised wholesale changes. There were 7 new players on the 12-man roster, players that were to have provided the lacking ingredients - a point guard to replace Micheal Ray Richardson; a high-scoring small forward, and additional height, speed and strength.
Wohl said the changes were forced on the Nets by the loss of Richardson, who was banned from the league in February 1986 for repeated drug use.
''When Micheal got into drugs, he was probably playing some of his best ball,'' Wohl said. ''Once he left, we became a different team. It was a team that lacked a leader and lost a certain amount of its competitiveness.''
The changes began with the drafting of the charismatic Dwayne (Pearl) Washington, who not only was supposed to run Wohl's controlled fast-break offense, but also fill a lot of empty seats. Unaware that the Bullets, who picked before the Nets, had no intention of drafting Washington, they gave up Darwin Cook, a solid six-season contributor, for the Bullets' promise that they would not draft Washington. Disarray in Backcourt
Washington, who has called his indoctrination into the pro ranks, ''tough,'' was less than the Pearl the Nets had hoped for, and the backcourt was in disarray from the start of the season.
Wohl, who is not ready to give up on Washington, insisted that ''Pearl had the typical rookie year.''
''We never felt he would be an instant star,'' said Wohl. ''Whether he eventually becomes a great player will depend on his work habits.''
Yet, if the Nets expected Washington to encounter early problems, they did not provide insurance in the backcourt. Not only did they give away Cook, but they failed to sign Kelvin Ransey and Ray Williams, both free-agent playmakers. The Nets finally signed Williams in late February and, though he has provided the lacking experience, the Nets were already virtually out the playoff picture. Birdsong Signs Late
Another thing that hurt the Nets was the late signing of Otis Birdsong, a free agent, who did not come to terms until seven games into the season, leaving the backcourt short and inexperienced. Then, after playing only in seven games, Birdsong, the most consistent outside shooter, was sidelined for the season with a sore knee and leg.
Perhaps the best off-season move was the trade that brought 6-foot-9-inch Orlando Woolridge from the Chicago Bulls. Although he has had some below-average games, he did provide the needed height up front and averaged 20.5 points a game, the best on the team.
But N.B.A. games are not won with small forwards but centers, and nothing hurt more than the loss of Dawkins, who slipped in the shower of his home Nov. 13. He needed back surgery and was lost for the season.
''Losing Darryl hurt the most,'' said Wohl. ''Without him, we lost the ability to defend the middle, to be more physical. Mike Gminski lost some of his effectiveness and our running game suffered.''
The Nets, who were without a general manager until March, recently hired Harry Weltman, and it is now his job to reconstruct the team round Buck Williams, the power forward who had his usual outstanding season.
Photo of coach Dave Wohl of the N.J. Nets (NYT/ Clifford Hausner)


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from me
Welcome to Vanessa Plana … handmade silver jewellery, created with love in the heart of Edinburgh, Scotland. Our pieces make perfect gifts for the people who matter to you the most.
The Newness of Spring
Inspired by a wonderful week in Turkey in April, I have been creating new designs with an arabesque feel. Trying…Read more