Cellular asymmetry is critical for the development of multicellular organisms. Here we show that homologues of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C (aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. We conclude that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signalling proteins that are implicated in mammalian cell polarity.

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.

Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.

No abstract

The neurotrophin receptor TrkA plays critical roles in the nervous system by recruiting signaling molecules that activate pathways required for the growth and survival of neurons. Here, we report APPL1 as a TrkAassociated protein. APPL1 and TrkA coimmunoprecipitated in sympathetic neurons. We have identified two routes through which this association can occur. APPL1 was isolated as a binding partner for the TrkAinteracting protein GIPC1 from rat brain lysate by mass spectrometry. The PDZ domain of GIPC1 directly engaged the C-terminal sequence of APPL1. This interaction provides a means through which APPL1 may be recruited to TrkA. In addition, the APPL1 PTB domain bound to TrkA, indicating that APPL1 may associate with TrkA independently of GIPC1. Isolation of endosomal fractions by high-resolution centrifugation determined that APPL1, GIPC1, and phosphorylated TrkA are enriched in the same fractions. Reduction of APPL1 or GIPC1 protein levels suppressed nerve growth factor (NGF)-dependent MEK, extracellular signal-regulated kinase, and Akt activation and neurite outgrowth in PC12 cells. Together, these results indicate that GIPC1 and APPL1 play a role in TrkA function and suggest that a population of endosomes bearing a complex of APPL1, GIPC1, and activated TrkA may transmit NGF signals.

The mechanisms that regulate symmetric, proliferative divisions versus asymmetric, neurogenic divisions of mammalian neural precursors are still not well understood. We found that Lfc (Arhgef2), a Rho-specific guanine nucleotide exchange factor that interacts with spindle microtubules, and its negative regulator Tctex-1 (Dynlt1) determine the genesis of neurons from precursors in the embryonic murine cortex. Specifically, genetic knockdown of Arhgef2 in cortical precursors either in culture or in vivo inhibited neurogenesis and maintained cells as cycling radial precursors. Conversely, genetic knockdown of Dynlt1 in radial precursors promoted neurogenesis and depleted cycling cortical precursors. Coincident silencing of these two genes indicated that Tctex-1 normally inhibits the genesis of neurons from radial precursors by antagonizing the proneurogenic actions of Lfc. Moreover, Lfc and Tctex-1 were required to determine the orientation of mitotic precursor cell divisions in vivo. Thus, Lfc and Tctex-1 interact to regulate cortical neurogenesis, potentially by regulating mitotic spindle orientation.

Mutations in p73 are rare in cancer. Emerging evidence suggests that the relative expression of various p73 isoforms may contribute to tumorigenesis. Alternative promoters and N-terminal splicing result in the transcription and processing of either full-length (TA) or N-terminally truncated (⌬N) p73 isoforms. TAp73 possesses pro-apoptotic functions, while ⌬Np73 has anti-apoptotic properties via functional inhibition of TAp73 and p53. Here, we report that TAp73, but not ⌬Np73, is covalently modified by NEDD8 under physiologic conditions in an Mdm2-dependent manner. Co-expression of NEDP1, a cysteine protease that specifically cleaves NEDD8 conjugates, was shown to deneddylate TAp73. In addition, blockage of the endogenous NEDD8 pathway increased TAp73-mediated transactivation of p53-and p73-responsive promoter-driven reporter activity, and in conjunction, neddylated TAp73 species were found preferentially in the cytoplasm. These results suggest that Mdm2 attenuates TAp73 transactivation function, at least in part, by promoting NEDD8-dependent TAp73 cytoplasmic localization and provide the first evidence of a covalent posttranslational modification exclusively targeting the TA isoforms of p73.p73 is a member of the p53 family that binds p53 DNAbinding sites, transactivates p53-target genes, and induces apoptosis (1-3). p73 mutations are rarely observed in cancer (4). However, p73 exists as multiple C-terminal (␣, ␤, ␥, ␦, ⑀, and ) and N-terminal (TA and ⌬N) isoforms, and accumulating evidence suggests that the relative expression and stability of the different N-terminal isoforms of p73 may play a role in tumorigenesis. Full-length TAp73 3 isoforms have pro-apoptotic properties, whereas the N-terminally truncated ⌬Np73 isoforms, which lack the transactivation domain due to the use of a promoter within intron 3 and alternative N-terminal mRNA splicing, have anti-apoptotic properties. ⌬Np73 acts as a negative inhibitor of both TAp73 and p53, either via hetero-oligomerization or through competition for DNA-binding sites (5-10). Furthermore, ⌬Np73 expression has been shown to be elevated in a number of human cancers, including breast, ovarian, hepatocellular, prostate, colon, and neuroblastoma tumors (11-15). Increased ⌬Np73 expression has been associated with poor prognosis in patients, and this finding has been attributed to ⌬Np73 inhibition of p53 and TAp73 causing chemoresistance (10,12,16,17). Conversely, a recent study demonstrated that aged p73 heterozygous mice develop spontaneous tumors, and in comparison with p53 heterozygous mice, mice heterozygous for both p53 and p73 have different tumor spectrums and higher tumor burden and incidence of metastasis, presumably due to the loss of function of the pro-apoptotic TAp73 isoforms (18). Studies have also established a role for TAp73 in chemotherapy-induced apoptosis and the status of TAp73 may be an important determinant of chemotherapeutic efficacy in humans (17,19,20). Therefore, therapeutic modulation of the relative levels of TAp73 and ⌬Np73 isoforms has pote...

We describe the design and execution of the BORTAS (Quantifying the impact of BOReal forest fires on Tropospheric oxidants over the Atlantic using Aircraft and Satellites) experiment, which has the overarching objective of understanding the chemical aging of air masses that contain the emission products from seasonal boreal wildfires and how these air masses subsequently impact downwind atmospheric composition. The central focus of the experiment was a two-week deployment of the UK BAe-146-301 Atmospheric Research Aircraft (ARA) over eastern Canada, based out of Halifax, Nova Scotia. Atmospheric ground-based and sonde measurements over Canada and the Azores associated with the planned July 2010 deployment of the ARA, which was postponed by 12 months due to UK-based flights related to the dispersal of material emitted by the Eyjafjallajökull volcano, went ahead and constituted phase A of the experiment. Phase B of BORTAS in July 2011 involved the same atmospheric measurements, but included the ARA, special satellite observations and a more comprehensive ground-based measurement suite. The high-frequency aircraft data provided a comprehensive chemical snapshot of pyrogenic plumes from wildfires, corresponding to photochemical (and physical) ages ranging from < 1 day to ~<45 sr 10 days, largely by virtue of widespread fires over Northwestern Ontario. Airborne measurements reported a large number of emitted gases including semi-volatile species, some of which have not been been previously reported in pyrogenic plumes, with the corresponding emission ratios agreeing with previous work for common gases. Analysis of the NO_y data shows evidence of net ozone production in pyrogenic plumes, controlled by aerosol abundance, which increases as a function of photochemical age. The coordinated ground-based and sonde data provided detailed but spatially limited information that put the aircraft data into context of the longer burning season in the boundary layer. Ground-based measurements of particulate matter smaller than 2.5 μm (PM_2.5) over Halifax show that forest fires can on an episodic basis represent a substantial contribution to total surface PM_2.5