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W2987126329 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. https://doi.org/10.7554/eLife.12039.001 eLife digest Most plants and animals, including humans, have cells that contain two copies of every chromosome, with one set inherited from each parent. However, reproductive cells (such as eggs and sperm) contain just one copy of every chromosome so that when they fuse together at fertilization, the resulting cell will have the usual two copies of each chromosome. Embryos that have incorrect numbers of chromosome copies either fail to survive or develop disorders such as Down syndrome. Therefore, it is important that when cells divide to form new reproductive cells, their chromosomes are correctly segregated. To end up with one copy of each chromosome, reproductive cells undergo a form of cell division called meiosis. During meiosis, pairs of chromosomes are held together by a zipper-like structure called the synaptonemal complex. While held together like this, each chromosome in the pair exchanges DNA with the other by forming junctions called crossovers. Once DNA exchange is completed, the synaptonemal complex disappears from certain regions of the chromosome. Using a range of genetic, biochemical and cell biological approaches, Nadarajan et al. have now investigated how crossover formation and the disassembly of the synaptonemal complex are coordinated in the reproductive cells of a roundworm called Caenorhabditis elegans. This revealed that a signaling pathway called the MAP kinase pathway regulates the removal of synaptonemal complex proteins from particular sites between the paired chromosomes. Turning off this pathway’s activity is required for the timely disassembly of this complex, and depends on proteins that are involved in crossover formation. This regulatory mechanism likely ensures that the synaptonemal complex starts to disassemble only after the physical attachments between the paired chromosomes are “locked in”, thus ensuring that reproductive cells receive the correct number of chromosomes. Given that the MAP kinase pathway regulates cell processes in many different organisms, a future challenge is to determine whether this pathway regulates the synaptonemal complex in other species as well. https://doi.org/10.7554/eLife.12039.002 Introduction Accurate chromosome segregation during meiosis is critical for sexually reproducing organisms. Meiosis is the specialized cell division program by which diploid germ cells generate haploid gametes that during fertilization will form a diploid zygote. This is accomplished by following a single round of DNA replication with two consecutive rounds of cell division (meiosis I and II) in which, first pairs of homologous chromosomes (bivalents), and then sister chromatids, separate away from each other. At center stage during prophase I of meiosis from yeast to humans is a tripartite proteinaceous structure known as the synaptonemal complex (SC) (Colaiácovo, 2006). The SC is a ladder-like structure comprised of lateral element proteins running along the axes of the homologs and central region proteins connecting these axes (Colaiácovo, 2006; Page and Hawley, 2003). The SC is required to stabilize the interactions between pairs of homologous chromosomes and for interhomolog crossover (CO) formation in budding yeast, worms, flies, and mice (Nag et al., 1995; Storlazzi et al., 1996; Page and Hawley, 2001; MacQueen et al., 2002; Colaiácovo et al., 2003; de Vries et al., 2005; Smolikov et al., 2007a; 2007b; 2009), all of which are prerequisite steps for achieving accurate chromosome segregation at meiosis I. Importantly, the SC must be disassembled prior to the end of prophase I to ensure timely and proper chromosome segregation (Sourirajan and Lichten, 2008; Jordan et al., 2009; Dix et al., 1997). Several proteins have been recently implicated in regulating the disassembly of SC proteins, including polo-like kinase in mammals and budding yeast, Ipl1/Aurora B kinase in budding yeast, the condensin complex component dcap-g in flies and both AKIR-1 (a member of the akirin protein family) and ZHP-3 (ortholog of budding yeast Zip3) in worms (Sourirajan and Lichten, 2008; Jordan et al., 2009; Resnick et al., 2009; Jordan et al., 2012; Clemons et al., 2013; Bhalla et al., 2008). However, whether these factors directly regulate the SC in vivo and the mechanisms by which they promote the disassembly of SC proteins are not fully understood. We previously showed that the SC disassembles asymmetrically in C. elegans, progressing from being localized along the full length of the interface between homologous chromosomes in early prophase to persisting at discrete chromosome locations, termed the short arm domains, corresponding to one end of every pair of homologs at late prophase I (Figure 1A) (Nabeshima et al., 2005). Importantly, factors required for CO formation are necessary for the asymmetric disassembly of the SC and localized retention of SC central region proteins. Since in C. elegans, a single CO occurs at the terminal third of every pair of homologous chromosomes, we proposed that chromosomes remodel around the single off-centered CO event at the pachytene-diplotene transition. This results in bivalents with a cruciform configuration comprised of two perpendicular chromosomal axes (namely the long and short arm domains) intersecting at the chiasma, where the long arms face the poles and the short arms occupy an equatorial position on the metaphase plate (Figure 1A) (Nabeshima et al., 2005; Riddle et al., 1997; Maddox et al., 2004). This remodeling includes changes in chromosome compaction as well as changes in both the localization and the types of proteins associated with the long and short arm domains (Figure 1A) (Nabeshima et al., 2005; Chan et al., 2004; de Carvalho et al., 2008; Martinez-Perez and Villeneuve, 2005). Subsequent studies in yeast, flies and mice (Newnham et al., 2010; Qiao et al., 2012; Bisig et al., 2012; Takeo et al., 2011; Gladstone et al., 2009) also observed an asymmetric disassembly of the SC, with a residual localized retention of the SC at centromeres. However, the mechanism linking CO recombination sites to asymmetric SC disassembly remained unknown. Figure 1 with 4 supplements see all Download asset Open asset ECT-2 regulates AIR-2 localization and SC dynamics in meiotic prophase I. (A) Schematic representation of SC dynamics and chromosome remodeling during prophase I of meiosis. A single pair of homologous chromosomes (bivalent) is shown for simplicity. Upon entrance into pachytene, the SC is present along the full length of the pairs of homologous chromosomes. CO formation is completed within the context of fully synapsed chromosomes, and in worms, a single CO is formed per homolog pair usually at an off-centered position. Chromosome remodeling has been proposed to take place around the off-centered CO (or CO precursor) resulting in a cruciform configuration comprised of two intersecting perpendicular chromosomal axes of different lengths (long and short arms of the bivalent, corresponding to the longest and shortest distances from the off-centered CO/CO precursor site to opposite ends of the chromosomes). This remodeling involves disassembly of central region components of the SC (SYP-1/2/3/4) along the long arms of the bivalents starting during late pachytene and diplotene resulting in the restricted localization of these proteins to the short arms. During diplotene and diakinesis, chromosomes undergo condensation as evidenced by a coiling of the arms and increased bivalent compaction. In late diakinesis, the SC proteins located on the short arms are replaced by AIR-2, which promotes the separation of the homologs at the end of meiosis I. CO – crossover, S – short arm and L – long arm. (B) Immunolocalization of HTP-3 and AIR-2 on -1 oocytes at diakinesis in wild type, ect-2(gf) and ect-2(rf) gonads. AIR-2 is localized to the short arm of the bivalents in wild type and ect-2(gf) mutants, but fails to localize onto chromosomes in ect-2(rf) mutants. Diagrams on the right illustrate the cruciform structure of the bivalents at this stage consisting of long (L) and short (S) arms and the localization of AIR-2 (red) and HTP-3 (green) in wild type and ect-2(rf) mutant. White box indicates the bivalent shown at a higher magnification on the right. Bivalents with both long and short arms clearly displayed were chosen for higher magnification. (C) Immunolocalization of HTP-3 and SYP-1 on leptotene/zygotene nuclei from gonads of the indicated genotypes. SYP-1 aggregates (polycomplexes) are detected in ect-2(gf) mutants. (D) SYP-1 and HTP-3 localize throughout the full length of the synapsed homologous chromosomes during pachytene in wild type and in most pachytene nuclei in ect-2(rf) mutants. Arrowhead indicates a nucleus where chromosomes persist in the DAPI-bright and tighter clustered configuration characteristic of the leptotene/zygotene stage in ect-2(rf). In ect-2(gf) mutants, meiotic nuclei that progressed through the leptotene/zygotene stage before the shift to the non-permissive temperature show wild type-like SYP-1 localization. Worms from all the indicated genotypes, including wild type, were grown at 15°C, shifted to 25°C at the L4 stage, and analyzed 18–24 hr post-L4. n>26 gonads were examined for each genotype in (B) and n>15 in (C) and (D). Bars, 2 μm. https://doi.org/10.7554/eLife.12039.003 Recently, a two-step CO specification process has been described to take place following SC assembly as prospective CO sites progressively differentiate during C. elegans and mouse meiosis (Yokoo et al., 2012; Holloway et al., 2014). This consists of CO licensing during mid-pachytene followed by CO designation at or just prior to the mid-to-late pachytene transition in a manner dependent of the pro-CO factor COSA-1/CNTD1. Since DSBs outnumber COs in most species (Martinez-Perez and Colaiácovo, 2009) this has been proposed as a strategy to pare down the number of early recombination sites that will become CO sites thus both ensuring and limiting the number of COs. These and other features of meiosis in C. elegans, including the existence of various markers that distinguish the short and long arm subdomains and the CO precursor sites, therefore provide an ideal scenario to understand the regulation of the disassembly of the SC proteins from distinct chromosome subdomains and late prophase I chromosome remodeling. Here, we report that regulation of the disassembly of the SC proteins from the long arms of the bivalents in C. elegans requires the mammalian Rho GEF homolog, ECT-2. We show that ECT-2 functions through the conserved MAP kinase pathway to regulate the asymmetric disassembly of SC proteins during prophase I of meiosis. We show that MPK-1 potentially directly phosphorylates SYP-2, a central region component of the SC, and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from the long arms. Moreover, inactivation of MPK-1 takes place in late pachytene in a manner dependent on pro-CO factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1 and concomitant with the initiation of SC disassembly. Therefore, we propose a model in which MPK-1 is inactivated in response to CO designation resulting in either de novo loading of unphosphorylated SYP-2 or dephosphorylation of chromatin-associated SYP-2, which triggers disassembly of SC proteins from along the long arms. Thus, coordination between CO designation and the disassembly of SC proteins executed via a conserved MAP kinase pathway is critical for ensuring accurate chromosome segregation during meiosis. Results ECT-2 regulates synaptonemal complex dynamics We identified ect-2, the homolog of mammalian Rho GEF, in a targeted RNAi screen for novel components regulating chromosome remodeling and short/long arm identity by using the mislocalization of Aurora B kinase, AIR-2, which localizes to the short arms of diakinesis bivalents in wild type oocytes as a readout (see Materials and methods). ECT-2 (Epithelial Cell Transforming sequence 2) is a highly conserved protein, initially identified as a proto-oncogene in cell culture (Miki et al., 1993). It encodes a Guanine nucleotide Exchange Factor (GEF) that belongs to the Dbl family and functions as a key activator of Rho GTPase mediated signaling with roles during cytokinesis, DNA damage-induced cell death, cell polarity establishment during embryogenesis, vulval development, and epidermal P cell migration (Prokopenko et al., 1999; Saito et al., 2003; Morita et al., 2005; Srougi and Burridge, 2011; Canevascini et al., 2005; Motegi and Sugimoto, 2006). Although ECT2 is expressed in testis and ovaries in humans (Hirata et al., 2009; Fields and Justilien, 2010), understanding its role during mammalian meiosis is challenging due to its earlier roles in development. The availability of conditional mutants, such as temperature-sensitive mutants in C. elegans, therefore provided a unique opportunity to discover a novel role for ECT-2 in meiosis. Analysis of ect-2(ax751rf) temperature-sensitive reduction-of-function mutants and of worms depleted of ect-2 by RNAi revealed a failure of AIR-2 to load on the chromosomes in late diakinesis even though AIR-2 is present inside the nucleus (Figure 1B and Figure 1—figure supplement 1). Importantly, we shifted ect-2(ax751rf) worms to the non-permissive temperature at the L4 larval stage to bypass the requirements for ECT-2 during somatic development and germ cell mitotic proliferation such as seen in ect-2(e1778) null mutants, which are sterile and exhibit fewer germ cells with abnormal nuclei (Figure 1—figure supplement 2). ect-2(ax751rf) mutants also exhibited a reduced brood size, increased embryonic lethality and a High Incidence of Males (Him) at the non-permissive temperature, all phenotypes indicative of increased meiotic chromosome nondisjunction (Table 1). Table 1 Worms were maintained at 15°C and then shifted to 25°C at the L4 larval stage. All the analyses were conducted at 25°C for all the genotypes indicated above. The 'Eggs Laid' column indicates the average number of eggs laid (including both hatched and non-hatched embryos) per P0 hermaphrodite ± standard deviation. % Embryonic lethality was calculated by dividing the number of non-hatched embryos by the total number of hatched and non-hatched embryos laid. % Males was calculated by dividing the total number of males observed by the total number of hatched (viable) progeny scored. N = total number of P0 worms for which entire broods were scored. N.A.= not applicable. https://doi.org/10.7554/eLife.12039.008 GENOTYPEEGGS LAID% EMBRYONIC LETHALITY% MALESNWild type192 ± 32.30020ect-2(ax751)109 ± 38.676.43.2530ect-2(zh8)4.5 ± 6.793.4032let-60(ga89)3.1 ± 5.683.8020mpk-1(ga111)5.8 ± 7.656020ect-2(ax751); let-60(ga89)0.7 ± 0.7100020ect-2(zh8); mpk-1(ga111)0N.A.N.A.30 To investigate the localization of ECT-2 in the germline, we utilized a ECT-2::GFP transgene driven by the ect-2 promoter (Chan and Nance, 2013). ECT-2::GFP is able to rescue the reduced brood size, embryonic lethality and Him phenotypes observed in the ect-2(ax751rf) mutants (Figure 1—figure supplement 3), confirming that mutation of ect-2 impairs fertility. We found that ECT-2::GFP localizes throughout the germline from the mitotically dividing nuclei at the premeiotic tip, where it is enriched at the germ cell membrane, to the end of diakinesis, where it exhibits a stronger nuclear signal (Figure 1—figure supplement 4). Importantly, we discovered a novel role for ECT-2 in regulating SC dynamics, a term we will use herein to refer to SC assembly and disassembly. In ect-2(ax751rf) mutants, SC formation is indistinguishable from wild type in most of the meiotic nuclei except for a few pachytene nuclei (11.1%, n=120/1080 pachytene nuclei) which display reduced SC assembly (Figures 1C and D). SC disassembly occurred as in wild type, initiating in late pachytene and resulting in the restricted localization of the central region proteins of the SC, as exemplified by SYP-1, to the short arm of the bivalents (Figure 2 and Figure 2—figure supplement 1). In contrast, ect-2(zh8gf) gain-of-function mutants, in which a mutation in an auto-inhibitory BRCT domain retains ECT-2 in its constitutively active configuration (Canevascini et al., 2005), exhibited defects in both SC assembly and disassembly at the non-permissive temperature. Specifically, the SC failed to assemble on the chromosomes and instead SYP-1 formed aggregates referred to as polycomplexes (Figure 1C). Those nuclei that had already gone through the leptotene/zygotene stage before the temperature shift showed normal synapsis during early and mid pachytene stages, but the central region components of the SC remained localized along the long arms of the bivalents and failed to become restricted to the short arms during late prophase (a defect herein referred to as impaired disassembly of the SC proteins) (Figure 2). Importantly, the HORMA domain containing lateral element protein HTP-3, exhibits a wild type pattern of localization in ect-2 mutants (Figures 1C,D and 2). This suggests that ECT-2 acts in a specific manner to regulate SC dynamics, which involves regulating central region components of the SC. Figure 2 with 6 supplements see all Download asset Open asset ECT-2 regulates the disassembly of SC proteins from the long arms of the bivalents through the MPK-1 pathway. Co-staining with HTP-3 (green), SYP-1 (red) and DAPI (blue) of diplotene and early diakinesis nuclei from the indicated genotypes. At diplotene and early diakinesis, SYP-1 localization is restricted to the short arm in both wild type and ect-2(rf) mutants. In contrast, SYP-1 fails to disassemble from the long arm and become restricted to the short arm of the bivalents in ect-2(gf), let-60(gf) and lip-1(rf) mutants. mpk-1(lf) mutants suppress the defect in disassembly of SC proteins from the long arms observed in ect-2(gf) mutants whereas ect-2(rf); let-60(gf) double mutants exhibit the phenotype of let-60(gf) mutants. Illustrations depict the bivalent configuration at this stage. White box indicates the bivalent shown at a higher magnification on the right. Bivalents with both long and short arms clearly displayed were chosen for higher magnification. Worms from all the indicated genotypes, including wild type, were grown at 15°C, shifted to 25°C at the L4 stage, and analyzed 18–24 hr post-L4. Histograms on the right indicate the percentage of diplotene and diakinesis stage nuclei with SYP-1 either only on the short arm (S, blue) or on both long and short arms (red, L&S) of the bivalents. All the bivalents were examined in every nucleus and the bivalents in the same nucleus either all exhibited SYP-1 staining on both the long and short arms or all exhibited staining only on the short arms. Numbers of nuclei scored are shown. (B) Schematic representation shows the crosstalk between the conserved ECT-2 and RAS/MAPK pathways and restriction of the SC to the short (S) arm of the bivalent at diakinesis in wild type. Remaining schematic shows epistasis analysis in ect-2(gf); mpk-1(ga111lf) and ect-2(rf); let-60(gf) double mutants. S indicates short arm and L indicates long arm. n>15 gonads arms were analyzed for each genotype. Bar, 2 μm. https://doi.org/10.7554/eLife.12039.009 ECT-2 specifically regulates the disassembly of SC proteins and is not required for other aspects of late prophase chromosome remodeling Since the SC is no longer restricted to the short arms of the bivalents in ect-2(zh8gf) mutants, we tested whether late prophase chromosome remodeling is altered in this mutant. First, we observed that AIR-2 localization is still successfully restricted to the short arms of the bivalents at diakinesis in ect-2(zh8gf) mutants (Figure 1B). Second, LAB-1, a proposed functional ortholog of Shugoshin, is still lost from the short arms and restricted to the long arms of the bivalents as in wild type (de Carvalho et al., 2008; Schvarzstein et al., 2010; Tzur et al., 2012) (Figure 3). These data indicate that key aspects of late prophase chromosome remodeling, namely LAB-1 and AIR-2 restricted localizations, are not altered in ect-2(zh8gf) mutants and that ECT-2 specifically regulates the disassembly of the SC proteins during prophase I of meiosis. Figure 3 Download asset Open asset ECT-2 does not alter LAB-1 localization. Immunolocalization of LAB-1 and HTP-3 in -1 oocytes at diakinesis indicates that LAB-1 is restricted to the long arm of the bivalents in ect-2(rf) and ect-2(gf) mutants similar to wild type. Illustration depicts the cruciform structure of the bivalents at this stage and the localization of LAB-1 (red) to the long arm (L) and HTP-3 (green) to both long and short (S) arms in wild type. White box indicates the bivalent shown at a higher magnification on the right. Bivalents with both long and short arms clearly displayed were chosen for higher magnification. Worms from all the indicated genotypes, including wild type, were grown at 15°C, shifted to 25°C at the L4 stage, and analyzed 18–24 hr post-L4. n>15 gonads were analyzed for each genotype. Bar, 2 μm. https://doi.org/10.7554/eLife.12039.016 The ERK MAP kinase pathway is involved in regulating SC dynamics ECT-2 has been shown to activate the RAS/MAP kinase signaling cascade to promote primary vulval cell fate specification during vulval development in C. elegans (Canevascini et al., 2005). The RAS/ERK (Extracellular signal-regulated kinase) MAP Kinase signaling regulates various aspects of the cell cycle and components of the signaling cascade are highly conserved between C. elegans and mammals (Sundaram, 2013). In C. elegans, the ERK MAP kinase, MPK-1, is expressed in both somatic tissues as well as in the germline where it controls several aspects of germ line development including pachytene progression and germ cell survival (Church et al., 1995; Kritikou et al., 2006; Lee et al., 2007). However, the role of MPK-1 in SC dynamics has never been explored. We therefore tested whether the ERK MAP kinase pathway regulates SC dynamics in the germline. Analysis of mpk-1(ga117) null, mpk-1(ga111lf) and mpk-1(ku1lf) temperature-sensitive loss-of-function mutants revealed defects in SC assembly as indicated by the formation of polycomplexes, similar to ect-2(zh8gf) mutants at the non-permissive temperature (Figure 1C and Figure 2—figure supplement 3). However, germline nuclei that had already passed through the leptotene/zygotene stage before the temperature shift exhibited normal SC tracts along chromosomes at the pachytene stage (Figure 2—figure supplement 3). Since nuclei fail to progress from early/mid-pachytene to late-pachytene in mpk-1(ga117) null mutants (Lee et al., 2007), we analyzed the disassembly of SC proteins in mpk-1(ga111lf) mutants. The mpk-1(ga111) temperature sensitive loss-of-function mutant has been shown to still have a low level of dpMPK-1 activity and an incompletely penetrant pachytene arrest phenotype (Lee et al., 2007). Nuclei that were able to progress from pachytene to diplotene exhibited normal SC disassembly similar to the ect-2(ax751rf) mutant (Figure 2). To further examine the role of the ERK MAP kinase pathway in the disassembly of SC proteins, we analyzed let-60, which encodes for the RAS protein that functions upstream of the MAP kinase cascade to activate the MAP kinase pathway. A let-60(ga89gf) temperature-sensitive gain-of-function mutant leads to constitutive activation of MAP kinase both in somatic tissues and the germline (Lee et al., 2007). Similar to the ect-2(zh8gf) mutant, let-60(ga89gf) mutants exhibit defects in both SC assembly, as evidenced by the presence of polycomplexes, and the disassembly of the SC proteins from the long arms of the bivalents, where they persisted at the non-permissive temperature (Figure 2 and Figure 2—figure supplement 3). Similar defects in the disassembly of SC proteins are also observed in the lip-1(zh15rf) reduction-of-function mutant (Figure 2), where the LIP-1 phosphatase fails to inactivate, MPK-1 in late pachytene, which therefore persists throughout pachytene, diplotene and diakinesis (Hajnal and Berset, 2002; Rutkowski et al., 2011), suggesting that the constitutive presence of active MPK-1 impairs the disassembly of SC proteins from the long arms of the bivalents. In contrast, we did not observe any defects in SC assembly in lip-1(zh15rf) (Figure 2—figure supplement 3). Further, we found that LAB-1 localization was not altered in either let-60(ga89gf) or lip-1(zh15rf) mutants suggesting that MAP kinase specifically regulates the disassembly of SC proteins from the long arms of the bivalents and not other aspects of late prophase chromosome remodeling (Figure 2—figure supplement 4). Altogether these data suggest that the MAP kinase pathway plays an essential role in regulating the disassembly of SC proteins whereas the defects we see in SC assembly might be a secondary consequence of the role of MAP kinase in mitosis or due to several other germline functions of MPK-1. ECT-2 functions through the ERK MAP kinase pathway to regulate the disassembly of SC proteins from the long arms of the bivalents To examine the connection between ECT-2 and the ERK MAP kinase pathway, we took advantage of the tightly regulated windows where the activated diphosphorylated form of MPK-1 (dpMPK-1) can be detected in the germline. In the ect-2(ax751rf) mutant, we see reduced dpMPK-1 signal in the mid-late pachytene region compared to wild type (Figure 2—figure supplement 5A and B). We next tested whether the Rho GTPase, RHO-1, activates ERK MAP kinase signaling in the germline, similar to its role in the regulation of vulval development (Canevascini et al., 2005). Given the highly abnormal gonads observed following strong loss of rho-1 function, partial RNAi knockdown of rho-1 was performed to obtain germlines with essentially wildtype morphology/organization (see Materials and Methods). Similarly to the ect-2(ax751rf) mutant, partial depletion of rho-1 also results in reduced dpMPK-1 signal in the mid-late pachytene region (Figure 2—figure supplement 6). In addition, the expression pattern of RHO-1 is similar to that observed for ECT-2 in the germline (Figure 1—figure supplement 4). In contrast to ect-2(ax751rf) mutants and to rho-1 partial knock down, dpMPK-1 expression is not turned off at late pachytene and during diplotene in the ect-2(zh8gf) mutant. Instead, dpMPK-1 persists from pachytene to diakinesis similar to let-60/RAS gain-of-function mutants (Figure 2—figure supplements 5A and B; [Lee et al., 2007]). Taken together, these data indicate that ECT-2 and RHO-1 either promote MPK-1 activation or block MPK-1 inactivation in the germline. Next, we performed epistasis analysis to test whether ECT-2 functions through the MAP kinase pathway to regulate the disassembly of SC proteins. First, we analyzed the brood size in ect-2(ax751rf); let-60(ga89gf) double mutants (Table 1). let-60(ga89gf) mutants exhibit a severely reduced brood size compared to ect-2(ax751rf). Interestingly, ect-2(ax751rf); let-60(ga89gf) double mutants exhibit a severely reduced brood size similar to let-60(ga89gf). Further, ect-2(ax751rf); let-60(ga89gf) double mutants exhibit defects in the disassembly of SC proteins similar to let-60(ga89gf) mutants (Figure 2A and B). These data show that LET-60 functions downstream of ECT-2 in the germline. We also analyzed ect-2(zh8gf); mpk-1(ga11lf1) double mutants for defects in the disassembly of SC proteins. We found that mpk-1(ga111lf) is able to suppress the SC disassembly defect of ect-2(zh8gf) mutants (Figure 2A and B). Thus, our data demonstrates that ECT-2 functions through the MAP Kinase pathway to regulate the disassembly of the SC proteins on the long arms of the bivalents uncovering a novel mode of regulation for this structure. Phosphorylation of SYP-2 is dependent on the ERK MAP kinase pathway To determine how the disassembly of the SC proteins is regulated by MPK-1, we tested whether SC components might be a direct phosphorylation target of MPK-1. First we examined the central region components of the SC, SYP-1, SYP-2, SYP-3 and SYP-4, for the presence of potential MAP kinase phosphorylation sites using the phosphorylation site predictor programs GPS 2.1 and KinasePhos 2.0 (Wong et a" @default.
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W2987126329 title "Author response: The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis" @default.
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