Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.

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In cells of Old World and some New World monkeys, dominant factors restrict human immunodeficiency virus type 1 (HIV-1) infections after virus entry. The simian immunodeficiency virus SIV mac is less susceptible to these restrictions, a property that is determined largely by the viral capsid protein. For this study, we altered exposed amino acid residues on the surface of the HIV-1 capsid, changing them to the corresponding residues found on the SIV mac capsid. We identified two distinct pathways of escape from early, postentry restriction in monkey cells. One set of mutants that were altered near the base of the cyclophilin A-binding loop of the N-terminal capsid domain or in the interdomain linker exhibited a decreased ability to bind the restricting factor(s). Consistent with the location of this putative factor-binding site, cyclophilin A and the restricting factor(s) cooperated to achieve the postentry block. A second set of mutants that were altered in the ridge formed by helices 3 and 6 of the N-terminal capsid domain efficiently bound the restricting factor(s) but were resistant to the consequences of factor binding. These results imply that binding of the simian restricting factor(s) is not sufficient to mediate the postentry block to HIV-1 and that SIV mac capsids escape the block by decreases in both factor binding and susceptibility to the effects of the factor(s).

MIB1 ubiquitin ligase–mediated regulation of internalization of the Wnt receptor RYK is necessary for response to Wnt3a ligand in cell culture and C. elegans.

The cells of most Old World monkey species exhibit early, postentry restrictions on infection by human immunodeficiency virus type 1 (HIV-1) but not by simian immunodeficiency virus of macaques (SIVmac). Conversely, SIVmac, but not HIV-1, infection is blocked in most New World monkey cells. By using chimeric HIV-1/SIVmac viruses capable of a single round of infection, we demonstrated that a major viral determinant of this restriction is the capsid (CA) protein. The efficiency of early events following HIV-1 and SIVmac entry is apparently determined by the interaction of the incoming viral CA and species-specific host factors

Developmental and cancer models show Wnt/β-catenin-dependent signaling mediates diverse phenotypic outcomes in the pancreas that are dictated by context, duration and strength of activation. While generally assumed to be pro-tumorigenic, it is unclear to what extent dysregulation of Wnt/β-catenin signaling impacts tumor progression in pancreatic adenocarcinoma (PDAC). In the present study, Wnt/β-catenin activity was characterized across a spectrum of PDAC cell lines and primary tumors. Reporter and gene expression based assays revealed wide heterogeneity in Wnt/β-catenin transcriptional activity across PDAC cell lines and patient tumors, as well as variable responsiveness to exogenous Wnt ligand stimulation. An experimentally-generated, pancreas-specific gene expression signature of Wnt/β-catenin transcriptional activation was used to stratify pathway activation across a cohort of resected, early stage PDAC tumors (N=41). In this cohort, higher Wnt/β-catenin activation was found to significantly correlate with lymphvascular invasion and worse disease specific survival (median survival time 20.3 versus 43.9 months, log rank P=0.03). Supporting the importance of Wnt ligand in mediating autocrine Wnt signaling, Wnt/β-catenin activity was significantly inhibited in PDAC cell lines by WLS gene silencing and the small molecule inhibitor IWP-2, both of which functionally block Wnt ligand processing and secretion. Transcriptional profiling revealed elevated expression of WNT7B occurred in PDAC cell lines with high levels of cell autonomous Wnt/β-catenin activity. Gene knockdown studies in AsPC-1 and HPAF-2 cell lines confirmed WNT7B mediated cell autonomous Wnt/β-catenin activation, as well as an anchorage-independent growth phenotype. Our findings indicate WNT7B can serve as a primary determinant of differential Wnt/β-catenin activation in PDAC. Disrupting the interaction between Wnt ligands and their receptors may be a particularly suitable approach for therapeutic modulation of Wnt/β-catenin signaling in PDAC and other cancer contexts where Wnt activation is mediated by ligand expression rather than mutations in canonical pathway members.

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. Although several Wnt signal transduction mechanisms have been described in detail, it is still largely unknown how cells are specified to adopt such different Wnt signaling responses. Here, we have used the stereotypic migration of the C. elegans Q neuroblasts as a model to study how two initially equivalent cells are instructed to activate either β-catenin dependent or independent Wnt signaling pathways to control the migration of their descendants along the anteroposterior axis. We find that the specification of this difference in Wnt signaling response is dependent on the thrombospondin repeat containing protein MIG-21, which acts together with the netrin receptor UNC-40/DCC to control an initial left-right asymmetric polarization of the Q neuroblasts. Furthermore, we show that the direction of this polarization determines the threshold for Wnt/β-catenin signaling, with posterior polarization sensitizing for activation of this pathway. We conclude that MIG-21 and UNC-40 control the asymmetry in Wnt signaling response by restricting posterior polarization to one of the two Q neuroblasts.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.genome engineering | conditional-null