[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) is an endogenous secreted peptide and, in preclinical studies, preferentially induces apoptosis in tumor cells rather than in normal cells. The acquisition of resistance in cells exposed to TRAIL or its mimics limits their clinical efficacy. Because ki-nases are intimately involved in the regulation of apoptosis, we systematically characterized kinases involved in TRAIL signaling. Using RNA interference (RNAi) loss-of-function and cDNA overexpression screens, we identified 169 protein kinases that influenced the dynamics of TRAIL-induced apoptosis in the colon adenocarcinoma cell line DLD-1. We classified the kinases as sensitizers or resistors or mod-ulators, depending on the effect that knockdown and overexpression had on TRAIL-induced apoptosis. Two of these kinases that were classified as resistors were PX domain–containing serine/threonine kinase (PXK) and AP2-associated kinase 1 (AAK1), which promote receptor endocytosis and may enable cells to resist TRAIL-induced apoptosis by enhancing endocytosis of the TRAIL receptors. We assembled protein interaction maps using mass spectrometry–based protein interaction analysis and quantitative phospho-proteomics. With these protein interaction maps, we modeled information flow through the networks and identified apoptosis-modifying kinases that are highly connected to regulated substrates downstream of TRAIL. The results of this analysis provide a resource of potential targets for the development of TRAIL combination therapies to selectively kill cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Development requires fertilization by a single sperm. In Caenorhabditis elegans, fertilization occurs in a sperm-filled spermatheca, implying the barrier to polyspermy is generated in this compartment. Eggshell chitin synthesis is initiated at fertilization, and chitin is deposited before the zygote exits the spermatheca. Whereas polyspermy is very rare in wild-type, here we report an incidence of 14%-51% in zygotes made chitin deficient by loss of chitin synthase-1 (CHS-1), the CHS-1 substrate UDP-N-acetylglucosamine, the CHS-1-interacting protein EGG-3, or the sperm-provided protein SPE-11. The spe-11(hc90) mutant deposits chitin at the male end but fails to complete a continuous layer. The polyspermy barrier is also compromised by loss of the chitin-binding protein CBD-1 or the GLD-1-regulated LDL receptor-like EGG-1, together with its homolog, EGG-2. Loss of CBD-1 or EGG-1/2 disrupts oocyte cortical distribution of CHS-1, as well as MBK-2 and EGG-3. In CBD-1 or EGG-1/2 deficiency, chitin is synthesized but the eggshell is fractured, suggesting aberrantly clustered CHS-1/MBK-2/EGG-3 may fail to support construction of a continuous eggshell. Together, our results show that eggshell chitin is required to prevent polyspermy in C. elegans, in addition to its previously reported requirement in polar body extrusion and polarization of the zygote.
Current biology: CB 10/2010; 20(21):1932-7. DOI:10.1016/j.cub.2010.09.059 · 9.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fertilization restores the diploid state and begins the process by which the single-cell oocyte is converted into a polarized, multicellular organism. In the nematode, Caenorhabditis elegans, two of the earliest events following fertilization are secretion of the chitinous eggshell and completion of meiosis, and in this report we demonstrate that the eggshell is essential for multiple developmental events at the one-cell stage.
We show that the GLD (Germline differentiation abnormal)-1-regulated hexosamine pathway enzyme, glucosamine-6-phosphate N-acetyltransferase (GNA)-2, is required for synthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the substrate for eggshell chitin synthesis by chitin synthase-1 (CHS-1). Furthermore, while chs-1(RNAi) or combined RNAi with the chitin-binding proteins, CEJ-1 and B0280.5, does not interfere with normal meiotic timing, lagging chromosomes are observed at meiosis, and polar-body extrusion fails. We also demonstrate that chitin, and either CEJ-1 or B0280.5, are essential for the osmotic/permeability barrier and for movement of the sperm pronucleus/centrosome complex to the cortex, which is associated with the initiation of polarization.
Our results indicate that the eggshell is required in single-cell C. elegans development, playing an essential role in multiple actin-dependent early events. Furthermore, the earliest meiotic roles precede osmotic barrier formation, indicating that the role of the eggshell is not limited to generation of the osmotic barrier.
[Show abstract][Hide abstract] ABSTRACT: Members of the UNC-5 protein family are transmembrane receptors for UNC-6/netrin guidance cues. To analyze the functional roles of different UNC-5 domains, we sequenced mutations in seven severe and three weak alleles of unc-5 in Caenorhabditis elegans. Four severe alleles contain nonsense mutations. Two weak alleles are truncations of the cytodomain, but one is a missense mutation in an extracellular immunoglobulin domain. To survey the function of different regions of UNC-5, wild-type and mutant unc-5::HA transgenes were tested for their ability to rescue the unc-5(e53) null mutant. Our data reveal partial functional requirements for the extracellular domains and identify a portion of the cytoplasmic juxtamembrane (JM) region as essential for rescue of migrations. When nine cytodomain tyrosines, including seven in the JM region, are mutated to phenylalanine, UNC-5 function and tyrosine phosphorylation are largely compromised. When F482 in the JM region of the mutant protein is reverted to tyrosine, UNC-5 tyrosine phosphorylation and in vivo function are largely recovered, suggesting that Y482 phosphorylation is critical to UNC-5 function in vivo. Our data also show that part of the ZU-5 motif is required for UNC-40-independent signaling of UNC-5.
[Show abstract][Hide abstract] ABSTRACT: UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-TV) is a regulator of polylactosamine-containing N-glycans and is causally involved in T cell regulation and tumor metastasis. The Caenorhabditis elegans genome contains a single orthologous gene, gly-2, that is transcribed and encodes a 669-residue type II membrane protein that is 36.7% identical to mammalian GlcNAc-TV (Mgat-5). Recombinant GLY-2 possessed GlcNAc-TV activity when assayed in vitro, and protein truncations demonstrated that the N-terminal boundary of the catalytic domain is Ile-138. gly-2 complemented the Phaseolus vulgaris leucoagglutinin binding defect of Chinese hamster ovary Lec4 cells, whereas GLY-2(L116R), an equivalent mutation to that which causes the Lec4A phenotype, could not. We conclude that the worm gene is functionally interchangeable with the mammalian form. GlcNAc-TV activity was detected in wild-type animals but not those homozygous for a deletion allele of gly-2. Activity was restored in mutant animals by an extrachromosomal array that encompassed the gly-2 gene. Green fluorescent protein reporter transgenes driven by the gly-2 promoter were expressed by developing embryos from the late comma stage onward, present in a complex subset of neurons in larvae and, in addition, the spermathecal and pharyngeal-intestinal valves and certain vulval cells of adults. However, no overt phenotypes were observed in animals homozygous for deletion alleles of gly-2.
[Show abstract][Hide abstract] ABSTRACT: The Caenorhabditis elegans genome contains 18 sequences related to mammalian core 2/I N-acetylglucosaminyltransferases. The six most closely related genes (gly-1 and gly-15 to gly-19) likely encode active enzymes, because are all transcribed and do not appear to be pseudogenes. Polypeptide divergence and the gene structures are both concordant with a common ancestor at the time of radiation from mammals that underwent three rounds of duplication and, most recently, a tandem duplication. Polypeptide alignments with mammalian homologues do not indicate whether the enzyme specificities are core 2, 4, or I-like or novel, but do clearly demonstrate the secondary structure characteristics of glycosyltransferases. The six homologues have essentially nonoverlapping expression patterns, unrelated by tissue type or cell lineage. The extent varies widely; gly-15 is expressed only in two gland cells, whereas gly-18 is broadly expressed in diverse cell types. gly-1, -15, -18 and -19 are expressed during adulthood; gly-16 and gly-17 appear to be restricted to embryonic or early larval stages. The parsimonious interpretation of the expression pattern and sequence data is that the catalytic activities are similar but with diverged promoters. Null alleles of three of the genes were generated without causing gross abnormality in homozygous animals. RNA-mediated interference experiments also failed to induce defects in the four genes tested. Nevertheless, the nematode has evolved six diverged core 2 GlcNAc-T-like genes, and we postulate that these arose in response to selection pressures to which C. elegans is not ordinarily subjected in the laboratory.
[Show abstract][Hide abstract] ABSTRACT: Cell and growth cone migrations along the dorsoventral axis of Caenorhabditis elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, we have examined genetic interactions between unc-5 and unc-40 in the migrations of the hermaphrodite distal tip cells. We report that cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. We show that hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, we observed that unc-5 hypomorphs exhibit dominant negative effects. We propose that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes.
[Show abstract][Hide abstract] ABSTRACT: Cell and growth cone migrations along the dorsoventral axis of Caenorhabditis elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, we have examined genetic interactions between unc-5 and unc-40 in the migrations of the hermaphrodite distal tip cells. We report that cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. We show that hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, we observed that unc-5 hypomorphs exhibit dominant negative effects. We propose that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes. S EVERAL genes, including unc-6, unc-5, and unc-40, malformation (rcm) gene product (Ackerman et al. 1997), now renamed UNC5H3 (Przyborski et al. 1998), and are known to interact in guiding circumferential two rat homologues, UNC5H1 and UNC5H2 (Leo-cell and growth cone migrations in Caenorhabditis elegans nardo et al. 1997). unc5h3 mutants have defects in cell (Hedgecock et al. 1990; McIntire et al. 1992). unc-6 en-migrations in the developing cerebellum (Ackerman et codes a member of the secreted, laminin-related protein al. 1997; Przyborski et al. 1998), and all three homo-family called netrins (Ishii et al. 1992; Kennedy et al. logues have been shown to bind directly to netrins (Leo-1994; Serafini et al. 1994). unc-6 is expressed ventrally nardo et al. 1997). in C. elegans, and mutations cause defects in both dor-C. elegans unc-40 is required primarily for ventrally sally and ventrally directed cell and growth cone migra-oriented migrations, but also contributes to dorsally ori-tions (Hedgecock et al. 1990; Wadsworth et al. 1996). ented and longitudinal migrations (Hedgecock et al. Similar functions have been observed for insect and 1987, 1990). UNC-40 is related to the product of the vertebrate netrins (Colamarino and Tessier-Lavigne mammalian deleted-in-colorectal-cancer (Dcc) gene (Chan 1995; Harris et al. 1996; Mitchell et al. 1996; Serafini et al. 1996), which is involved in axon guidance in the et al. 1996). Although the mechanisms of function of mouse spinal cord (Fazeli et al. 1997), and to the prod-the UNC-6/netrins are poorly understood, migrating uct of the frazzled gene of Drosophila, also involved cells and growth cones are thought to transduce relative in axon guidance (Kolodziej et al. 1996). DCC binds differences in extracellular UNC-6/netrin concentra-directly to netrins (Keino-Masu et al. 1996) and medi-tion into local, intracellular changes in the actin cy-ates responses to Netrin-1 of Xenopus retinal ganglion toskeleton. cells in culture (de la Torre et al. 1997). Thus, the unc-5 and unc-40 encode transmembrane receptors of UNC-6/netrin ligands together with the UNC-5 and the immunoglobulin (Ig) superfamily (Leung-Hages-UNC-40/DCC receptors compose a highly conserved teijn et al. 1992; Chan et al. 1996). Expression of the system for the guidance of migrating cells and neuronal C. elegans UNC-5 protein is, in most cases, sufficient to growth cones. cause repulsion of migrating cells or growth cones away The functional relationships between the UNC-5 and from ventral concentrations of UNC-6 (Hamelin et al. UNC-40/DCC receptors are not entirely clear. Pre-1993, Wadsworth et al. 1996; Su et al. 2000). Vertebrate sumed null unc-40 mutations in C. elegans disrupt the homologues of UNC-5 include the murine rostrocerebellar same dorsally oriented cell and growth cone migrations as do unc-5 mutations, but with a lower penetrance (Hedgecock et al. 1990). These weak effects of