Dicer interacts with the P-granule component GLH-1 in C. elegans

Abstracts Increasing female age also affected the rates and locations of sperm depletion. This study provides direct evidence that females influence sperm fate by regulating sperm depletion and that this influence is affected by both somatic condition and environment. doi:10.1016/j.ydbio.2010.05.360 513 localized, or at least not at significant levels, at ring channels. Using RNAi to knockdown actin-related proteins I will evaluate oocyte size, ring channel structure, actin and NMY-2 localization in the gonad. Furthermore, live-imaging will elucidate the progress of ring channel closure in manipulated animals. Data from these studies will provide a preliminary model for a cytoskeletal mechanism of ring channel closure. doi:10.1016/j.ydbio.2010.05.362 Program/Abstract # 350 Testes specific neurotransmitter transporter essential for male fertility in Drosophila melanogaster Nabanita Chatterjeea, Janet Rollinab, Christopher Bazineta a Dept Biological Sci, St John's Univ, USA b Col Mt Saint Vincent, Riverdale, NY, USA The predicted protein sequence for Neurotransmitter transporter like (Ntl) or CG7075 in Drosophila, shares a very strong homology with members of the sodium dependent neurotransmitter transporter family. Ntl expression is restricted to the male germline. Mobilization of a P element inserted in the 3′ end of the gene yields male sterile mutants defining a single complementation group. The mutant phenotype is completely rescued by germline transformation with Ntl cDNA under a testis specific promoter, also Ntl genomic DNA under it's native promoter. EM cross-sections show mutant cysts undergo normal individualization. Mutants counterstained using TRITC-phalloidin in don-juan and β-tubulin GFP background demonstrates perfect formation of actin cones around the axoneme and movement of the individualization complex along the length of the cyst. Individualized sperms in the mutants undergo coiling however they are not transferred into seminal vesicles and are immotile. To localize Ntl, an EGFP–Ntl fusion under a testis specific promoter was constructed. Germline transformation of the construct completely rescued the mutant phenotype. Initial studies suggest localization in intra cellular organelles, which is very unusual for a member of the SLC6a family. Sequence alignments of ntl with other SLC6a transporters suggest glycine as a likely substrate. One model currently being explored is that Ntl functions as a glycine transporter in the testes to provide the large amount of glycine required for polyglycylation of axonemal tubulins required in sperm maturation and motility (Rogowski et al., 2008). doi:10.1016/j.ydbio.2010.05.361 Program/Abstract # 351 Closing ring channels in the C. elegans gonad Erin Jezuita,b, Uta Wolkeb, Jim R. Priessb a Dept. of Biology, Knox College, Galesburg, IL, USA b Basic Sciences Dept., Fred Hutchinson Cancer Research Center, Seattle, WA, USA A fully-grown C. elegans oocyte is a large cell, the size of the 558 cells it will generate during embryogenesis. Most of the growth of the oocyte occurs during stages when its nucleus is transcriptionally quiescent. The distal region of the C. elegans gonad is a syncytium, with germ cells connected by ring channels. We determined that there is bulk streaming of the cytoplasmic materials in the core of the C. elegans gonad, and this streaming transfers material from a region of transcriptionally active pachytene-stage nuclei in the distal gonad into the proximal, enlarging, oocytes via ring channels. Since oocytes are uniform in size, and are ovulated every 23 min, there must be a cell size control mechanism. “Large oocyte mutants” have proximal oocytes that retain open ring channels for a prolonged period of time (Nadarajan et al., 2009). My aim is to better understand the mechanism that regulates ring channel closure. Actin and non-muscle myosin II (NMY-2) localize to ring channels. The closure of the ring channel is somewhat reminiscent of the actomyosin ring during cell division. However, midbody proteins are not Program/Abstract # 352 Trans-generational epigenetic regulation in C. elegans primordial germ cells Hirofumi Furuhashia, Teruaki Takasakib, Andreas Rechtsteinerb, Tengguo Lic, Hiroshi Kimurad, Susan Stromeb, William G. Kellyc a Grad. Sch. of Pharm. Sci., Tohoku Univ., Sendai, Miyagi, Japan b Dept. of MCD Biol., UCSC., Santa Cruz, CA, USA c Biol. Dept., Emory Univ., Atlanta, GA, USA d Grad. Sch. of Frontier Biosci., Osaka Univ., Suita, Osaka, Japan Epigenetic mechanisms are thought to help regulate the unique transcription program that is established in germ cell development and ensures germline continuity across generations. However, the mechanisms remain poorly understood. We show that a histone H3K36 methyltransferse, MES-4, is an epigenetic modifier that prevents aberrant transcription activity in C. elegans primordial germ cells (PGCs). In mes-4 mutants, RNA Pol II activation is abnormally regulated and the PGCs degenerate. Genetic and genome-wide analyses of MES-4-mediated H3K36 methylation suggest that MES4 is predominantly responsible for maintenance, not de novo or transcription-coupled, methylation of H3K36. MES-4 appears to maintain an epigenetic memory of transcription occurring in germ cells of previous generations, and thus marks germline-expressed loci, probably to maintain their proper regulation as the genome is transferred across generations. doi:10.1016/j.ydbio.2010.05.363 Program/Abstract # 353 Dicer interacts with the P-granule component GLH-1 in C. elegans Karen L. Bennetta, T.J. McEwena, M.C. Judb, J.L. Marshalla, J.A. Schisab, E.L. Beshorea a MMI Dept, U Missouri, Columbia, MO, USA b Bio. Dept, Central Michigan University, Mt. Pleasant, MI, USA P granules, which are ribonuclear protein complexes specific to the outer, cytoplasmic side of the nuclear pores of C. elegans germ cells, are implicated in post-transcriptional control of maternally-transcribed mRNAs. Here we show a relationship in C. elegans between Dicer, the riboendonuclease processing enzyme of the RNA interference and microRNA pathways, and a constitutive component of P granules, the RNA helicase, GLH-1. Based on results from immuno-precipitations and GST-pull-downs, GLH-1 binds to DCR-1 and this binding does not require RNA. Both GLH-1 protein and mRNA levels are reduced in the dcr-1(ok247) null mutant background; conversely, a reduction of DCR-1 protein is observed in the glh-1(gk100) deletion strain. Thus, in the C. elegans germline, DCR-1 and GLH-1 demonstrate a germline-specific interdependence and both are necessary for the maintenance of the germline lineage. In addition, evidence indicates that levels of DCR-1 protein, like that of GLH-1, are regulated by proteosomal degradation, likely targeted by the Jun N-terminal kinase KGB-1. In adult germ cells DCR-1 is located in uniformly distributed small puncta throughout the cytoplasm, as well as being localized to the inner side of the nuclear pores, and to P granules. In arrested oocytes, GLH-1 and DCR-1 re- 514 Abstracts localize to cortically-distributed RNP granules, and are necessary to recruit other components to these complexes. We predict that the GLH1/DCR-1 complex may function in the transport, deposition, or regulation of maternally-transcribed mRNAs and their associated miRNAs. TRY-5 triggers activation by cleaving sperm surface proteins, thereby coupling rapid and irreversible acquisition of motility to transfer to a hermaphrodite. Supported by R01-GM087705 and T32-GM007464. doi:10.1016/j.ydbio.2010.05.366 doi:10.1016/j.ydbio.2010.05.364 Program/Abstract # 354 Regulation of motility in C. elegans sperm Gillian M. Stanfield Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Cellular motility is critical for many processes, from gastrulation to organogenesis to wound healing to fertility. However, cell movement must be tightly regulated to ensure that cells only migrate when and where they should. We are using C. elegans sperm, which move by crawling, as a model for studying signals that induce cells to become motile and guide their directional migration. Sperm motility is acquired during a process known as activation, in which a round spermatid undergoes subcellular morphogenesis, rapidly transforming into a polarized, fully-mature spermatozoon. In a genetic screen for regulators of activation, we have identified a likely extracellular trigger, the serine protease TRY-5, as well as a number of potential targets on the sperm cell, including the SLC6 family transporter SNF10. In other screens for sperm function, we have identified a mutant in which male sperm fail to migrate efficiently toward eggs, resulting in failure to outcompete hermaphrodite sperm; we are working to identify the affected gene. Analysis of these factors is yielding insight into how cellular motility can be modulated to achieve an important developmental event, the union of sperm and egg. doi:10.1016/j.ydbio.2010.05.365 Program/Abstract # 355 Regulation of C. elegans sperm motility by extracellular protease signaling Joseph R. Smith, Gillian M. Stanfield Department of Human Genetics, University of Utah, Salt Lake City, UT, USA Sperm motility is a necessary aspect of sperm function and is thus generally required for sexual reproduction. In C. elegans, sperm develop motility during a process termed sperm activation (or spermiogenesis), which is regulated differentially in males and hermaphrodites to ensure optimal fertility. For male sperm, activation must be triggered rapidly after sperm is transferred to a hermaphrodite; if sperm become motile within the male or activate slowly upon transfer to the hermaphrodite, fertility is greatly reduced or eliminated. A variety of evidence suggests that a male signal, which is distinct from the hermaphrodite signal, likely instructs sperm to activate after mating. We are interested in how this rapid activation is accomplished and regulated via the presumptive signal. We have identified a pair of candidate regulators, a serine protease, TRY-5, and a trypsin inhibitor-like (TIL) domain protein, SWM-1. swm-1 mutant sperm activate within males, and TRY-5 activity is required for this premature activation. However, try-5 mutant males are fertile, likely due to redundancy with hermaphrodite sperm activation signals. We have found that try-5 mutant males are defective for transfer of a male activator, and preliminary evidence suggests that TRY-5 is expressed in and secreted by the vas deferens. Thus, TRY-5 is likely the seminal fluid sperm activator. We propose that during mating, Program/Abstract # 356 Feeding and mating are required for ovarian development and egg production in the predaceous minute pirate bug Orius pumilio Paul D. Shirk, Jeffrey P. Shapiro Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL 32608, USA Female minute pirate bugs, Orius pumilio (Champion) require food and mating as adults to achieve maximum egg production. Last instar nymphs, isolated individually in single wells of 96-well microtiter plates, yielded low mortalities and assured virginity. Using morphological characters of these nymphs, correct sex identification was achieved with 96% accuracy. The availability of food (eggs of Ephestia kuehniella Zeller) and mates for these isolated females was conveniently controlled. Unfed adult females, whether mated or not, did not produce detectible yolk protein when assayed by ELISA, nor did they show any follicle development when examined microscopically. Fed but unmated females produced a significant, detectible amount of yolk protein, and some oocyte development was observed, but they contained no fully mature eggs. Females that were both fed and mated fell into two categories: 44% produced mature eggs at a mean rate of 6.4 eggs/female, while 56% had ovaries similar to those of fed but unmated females. We conclude that there is a twostage process of egg development in adult female O. pumilio, in which early vitellogenesis depends on acquiring a nutritious adult diet, while completion of vitellogenesis and choriogenesis also requires mating. Unlike other Heteroptera, O. pumilio did not initiate vitellogenesis and yolk uptake under the influence of a juvenile hormone analog, indicating that juvenile hormone may not have a critical regulatory function in controlling egg production. doi:10.1016/j.ydbio.2010.05.367 Program/Abstract # 357 An ancient molecular circuit specifying multipotency S. Zachary Swartza, Celina E. Julianob, Tal Razc, Doron Lipsonc, Patrice Milosc, Amro Hamdound, Gary M. Wessela a MCB Dept., Brown University, Providence, RI, USA b Dept. of Cell Biology, Yale University School of Medicine, New Haven, CT, USA c Helicos Biosciences Corporation, Cambridge, MA, USA d Scripps Institution of Oceanography, UCSD, La Jolla, CA, USA Animal development requires a finely choreographed process of cell differentiation. Equally important, however, is the prevention of differentiation in stem cell lineages. Stem cells have various roles in the soma and also in the germ line, where they create sperm and eggs. In diverse species, many somatic and germ line functions are delegated to a single class of multipotent progenitor cells. The molecular control of these multipotent cells has remained largely unexplored. The sea urchin, an echinoderm, segregates multipotent cells in early embryogenesis that we hypothesize give rise to adult somatic tissues as well as the germ line. These cells possess gene expression homologous to multipotent progenitors and germ lines of multiple organisms, implying conservation of the molecular circuitry. We are determining the gene regulatory network (GRN) of sea urchin