5 Papers found (0.008 seconds)

Rog, Ofer et al. (2013) International Worm Meeting "In vivo visualization of chromosome synapsis in C. elegans."

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International Worm Meeting,

2013]

Meiosis is the special cell division process that enables the production of haploid gametes. During meiotic prophase, chromosomes form linkages with their homologous partners to enable reductional segregation during the first meiotic division. Essential to this process is the pairwise alignment of homologous chromosomes along their entire lengths. In most eukaryotes this alignment is reinforced through synapsis, the assembly of a structurally conserved polymer called the synaptonemal complex (SC), which links homologous chromosomes. The SC promotes genetic exchanges (crossovers) between homologs, and likely regulates their number and location. Synapsis is a dynamic process, the details of which are difficult to infer from images of fixed cells or tissues. Our goal is to illuminate this process through analysis in living nematodes, using fluorescently tagged SC components and high-resolution time-lapse microscopy. Our observations have revealed that initiation of synapsis is a relatively infrequent event that is rate-limiting for completion of synapsis, consistent with evidence that initiation is subject to strict regulation. Initiation occurs at the "Pairing Centers" - regions near one end of each chromosome where initial pairing of homologous chromosomes is achieved. Once initiated, synapsis extends along the chromosome at a rate of ~160nm per minute. Individual chromosomes thus complete synapsis within 20-30 minutes of initiation. In C. elegans, synapsis is accompanied by rapid chromosome motions driven by dynein, which is coupled through the nuclear envelope to the Pairing Centers. Under conditions where this motion is severely abrogated, synapsis remains processive but is ~5-fold slower. This suggests that chromosome motion promotes homolog alignment, enabling synapsis to proceed more rapidly. Moreover, it reveals a novel function for the rapid chromosome motions that have been observed in diverse organisms during meiotic prophase.

Daniel C. Bennett et al. (2007) International Worm Meeting "Identifying Interactors of ROG-1, a C. elegans FRS2 Homolog."

Michael J. Stern, Isaac E. Sasson, Daniel C. Bennett

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International Worm Meeting,

2007]

Signal transduction by mammalian receptor tyrosine kinases (RTKs) often requires intracellular signaling adaptors. One such adaptor is FRS2/snt-1, which mediates signaling through the neurotrophin (Trk) and FGF receptors to the Ras/MAPK cascade (Kouhara, et al. (1997)). FRS2 interacts with these RTKs through a PTB protein-protein interaction domain, originally named for its affinity for phosphorylated tyrosine residues (Forman-Kay and Pawson (1999)). The C. elegans genome contains one FRS2-like gene, rog-1. ROG-1 is required in the germ line for meiotic progression and interacts genetically with let-60 Ras, suggesting that it transduces signals to the Ras-MAP Kinase cascade in a manner similar to its mammalian counterpart FRS2 (Matsubara, et al. (2007)). Thus, ROG-1 may represent the link between an unidentified transmembrane RTK and the established Ras-MAP Kinase signaling core required for germ line meiotic progression (Church, et al.(1995)). However work in our lab suggests that, unlike mammalian FRS2, ROG-1 does not interact with the C. elegans FGFR pathway. Homozygotes for a putative null mutant, rog-1(tm1031), are sterile and display embryonic lethality (Matsubara, et al. (2007)) but do not show any sex myoblast migration defects, and tm1031 fails to suppress hyperactivation of the FGFR pathway in the hypodermis. Consistent with this, we have not detected a yeast two-hybrid interaction between ROG-1 and the C. elegans FGFR, EGL-15. Thus, to identify the pathway in which ROG-1 functions, we are pursuing two complementary yeast two-hybrid approaches. The first of these approaches is a traditional yeast two-hybrid screen to identify proteins which bind to the PTB domain of ROG-1. To this end, we have screened 1.65 x 107 cDNA clones from the RB2 cDNA library and have identified 29 candidate interactors. We are identifying and confirming these candidate interactors. The second of these approaches is a directed two-hybrid test with the intracellular domains of the entire set of 38 C. elegans RTKs. To facilitate this, we are forcing the auto-phosphorylation of these 38 C. elegans RTKs in yeast by creating fusions to both the DNA binding domain of LexA and the N-terminal coiled-coil domain of human TPR, which has been shown to constitutively activate receptor tyrosine kinases. We hope that these studies will provide a more complete model for ROG-1 function and help elucidate signal transduction through MAP Kinase in the C. elegans germ line, as well as provide insights into the mechanism of signal transduction through receptor tyrosine kinases in C. elegans.

Kim, Yumi et al. (2015) International Worm Meeting "Dynamic phosphoregulation of axis proteins underlies chromosome remodeling during meiosis."

Kostow, Nora, Rog, Ofer, Kim, Yumi, Corbett, Kevin D., Dernburg, Abby F., Rosenberg, Scott C.

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International Worm Meeting,

2015]

Segregation of chromosomes in meiosis requires their dramatic, stepwise reorganization during meiotic prophase. Each chromosome must pair, synapse, and recombine with its homolog to achieve the stable bivalent structures that biorient and then divide during Meiosis I. A fundamental mystery is how these events are coordinated during the meiotic cell cycle. Pairing, synapsis, and recombination depend on the formation of linear "axes" along each chromosome at meiotic entry, followed by assembly of the synaptomemal complex (SC) between paired axes. Chromosome axes in C. elegans are comprised of cohesins and four related HORMA domain proteins: HIM-3, HTP-1, HTP-2, and HTP-3. We recently reported that the largest of these proteins, HTP-3, recruits the other three paralogs through short peptide sequences (closure motifs) in its C-terminal tail, which are bound by the HORMA domains of HTP-1, HTP-2, and HIM-3 (Kim, Rosenberg, et al., 2014). Here we show that these interactions are dynamically regulated by phosphorylation of the closure motifs in HTP-3 by two meiotic kinases, CHK-2 and PLK-2. Phosphorylation of the four central motifs reduces their binding affinity for HIM-3, and occurs in two temporally distinct waves. In early meiosis, phosphorylation along the entire axis by CHK-2 is required for efficient synapsis. A second wave of phosophorylation by PLK-2 occurs after crossover formation, and specifies the "short arm" of the bivalent where cohesion will be released during the first meiotic division. This second wave of HTP-3 phosphorylation requires both crossover formation and recruitment of PLK-2 to the chromosomes through a binding site in a SC component, SYP-1. Thus, phosphorylation-dependent regulation of HORMA domain protein assembly promotes dynamic remodeling of chromosome axes during meiotic progression, and is essential for proper segregation of holocentric chromosomes in C. elegans meiosis.

Te-Wen Lo et al. (2007) International Worm Meeting "C. elegans FGF Receptor Signaling Can Occur via Direct Binding of the SEM-5/Grb2 Adaptor Protein."

Michael Stern, Te-Wen Lo

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International Worm Meeting,

2007]

The components of receptor tyrosine kinase(RTK)signaling complexes help to define the specificity of the effects of their activation. The C. elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2 binding motifs present in the CTD (Y1009 and Y1087), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. Although SEM-5 is required for the fluid homeostasis function of EGL-15, these sites are not required for that function, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multisubstrate adaptor protein FRS2 serves to link vertebrate FGFRs to GRB2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation1 but is not required for EGL-15 function2 . Thus, unlike the vertebrate FGFRs, which require the multisubstrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly. 1Matusbara et al., 2007. Genes to Cells; 12(3): 407-420. 2see D. Bennett abstract.

Susan J. Jun et al. (2007) International Worm Meeting "Identification of FGF Receptor Signaling Components Involved in Fluid Homeostasis in Caenorhabditis elegans."

Michael J. Stern, Judith S. Janette, Susan J. Jun

[

International Worm Meeting,

2007]

Fibroblast growth factor receptors (FGFRs) are a class of receptor tyrosine kinases (RTKs) known to play critical roles in the normal development of all higher metazoa. FGF signaling is crucial to a broad range of biological processes such as angiogenesis, mitogenesis, organogenesis, cell migration, proliferation, and differentiation. In humans, misregulation of FGFR signaling has been linked to skeletal disorders, cancer, and kidney disease. FGF signaling events in C. elegans are mediated by the EGL-15 FGFR and its two known ligands, EGL-17 and LET-756. EGL-15 signaling is essential for fluid homeostasis and is required for the proper guidance of sex myoblast (SM) migration. Hyperactivation of the EGL-15 pathway, such as by mutating clr-1, results in a Clr (Clear) phenotype characterized by fluid accumulation within the pseudocoelomic space. Mutations that compromise EGL-15 pathway activity confer the Soc (Suppressor of Clr) phenotype. A genetic screen to look for soc mutants in a clr-1(e1745ts) background identified a set of soc genes, including egl-15, sem-5, soc-1, and soc-2, that helped define the EGL-15 signaling pathway that mediates fluid homeostasis. EGL-15 RTK signaling triggers activation of RAS via SEM-5, the GRB2 adaptor protein in C. elegans. Protein sequence analysis identifies three putative SEM-5 binding sites within EGL-15. EGL-15 can directly recruit SEM-5 at two sites within the carboxy-terminal domain (CTD) to mediate SM chemoattraction (see abstract by T.-W. Lo). In the absence of these sites, however, such as in the egl-15(n1457) CTD-truncation mutant, EGL-15 can still signal in a SEM-5-dependent manner to mediate fluid homeostasis. The third remaining SEM-5 SH2 binding motif in EGL-15 does not directly recruit SEM-5, implying an indirect route to do this. Although FRS2 carries out this function in vertebrates, the FRS2-like homolog, ROG-1, is not involved in EGL-15 signaling (see abstract by D. Bennett), implicating another adaptor component or a different means of EGL-15-SEM-5 interaction. To identify components that can link EGL-15 to SEM-5, we carried out a genetic screen to identify mutations that can suppress the Clr phenotype of a clr-1(e1745ts); egl-15(n1457) strain that lacks the direct SEM-5 binding sites. We isolated 70 Soc mutants from approximately 75,000 mutagenized haploid genomes. Complementation analyses assigned 67/70 of these mutations to egl-15 and 1/70 to sem-5. The remaining 2/70 are still being analyzed. Based on sequence analysis, two egl-15 alleles identify key residues in the extracellular IG domains. The characterization of these extracellular mutations will be used to understand the ligand-binding parameters of FGF receptors.