The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ(+) neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ(-) neurons in CEl. Electrical silencing of PKC-δ(+) neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called CEl(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.
During tissue morphogenesis, simple epithelial sheets undergo folding to form complex structures. The prevailing model underlying epithelial folding involves cell shape changes driven by myosin-dependent apical constriction. Here we describe an alternative mechanism that requires differential positioning of adherens junctions controlled by modulation of epithelial apical-basal polarity. Using live embryo imaging, we show that before the initiation of dorsal transverse folds during Drosophila gastrulation, adherens junctions shift basally in the initiating cells, but maintain their original subapical positioning in the neighbouring cells. Junctional positioning in the dorsal epithelium depends on the polarity proteins Bazooka and Par-1. In particular, the basal shift that occurs in the initiating cells is associated with a progressive decrease in Par-1 levels. We show that uniform reduction of the activity of Bazooka or Par-1 results in uniform apical or lateral positioning of junctions and in each case dorsal fold initiation is abolished. In addition, an increase in the Bazooka/Par-1 ratio causes formation of ectopic dorsal folds. The basal shift of junctions not only alters the apical shape of the initiating cells, but also forces the lateral membrane of the adjacent cells to bend towards the initiating cells, thereby facilitating tissue deformation. Our data thus establish a direct link between modification of epithelial polarity and initiation of epithelial folding.
Deregulation in lysine methylation signaling has emerged as a common etiologic factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics1. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumors2-4. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP Kinase signaling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LAC), we found that abrogating SMYD3 catalytic activity inhibits tumor development in response to oncogenic Ras. We employed protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signaling module. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP Kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signaling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signaling.
Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH-MMOB (hereafter termed H-B) complex. Here we remedy this deficiency by providing a crystal structure of H-B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α(2)β(2) interface of α(2)β(2)γ(2) MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.
V(D)J recombination and class switch recombination employ overlapping but distinct non-homologous end-joining (NHEJ) pathways to repair DNA double strand break (DSB) intermediates. 53BP1 is a DNA damage response protein that is rapidly recruited to sites of chromosomal DSBs, where it appears to function in a subset of ataxia-telangiectasia mutated (ATM) kinase, H2AX- and MDC1- dependent events1,2. A 53BP1 dependent end joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination CSR3, 4. Here, we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1 deficient lymphocytes distinct from that found in classical NHEJ-, H2AX-, MDC1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of un-repaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes bearing antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long range joining of DNA breaks.
AFTER a radiation accident in July 1969 we had the opportunity of comparing radiation dose estimates made by biological and physical methods. The biological method involved counting chromosome aberrations in cultured peripheral blood lymphocytes, and the physical method was based on a reconstruction of the accident. For the conditions of this accident a reasonable agreement was obtained between the two methods, which adds to the growing confidence in the use of chromosome aberration counting for dose assessment in radiation accidents.
THE degree of malignancy of tumour cells may be related to the magnitude of the cell surface charge presented by exposed macromolecular groups of the enlarged muco-polysaccharide layers around tumour cells1, and it has also been suggested2 that this surface charge inhibits the recognition of tumour antigens by lymphocytes. Removal of excess mucopolysaccharide with neuraminidase restores fibroblast cells which are transformed by polyoma virus to normal3. Ehrlich ascites tumour cells, which have a large negative surface charge, provide a sensitive system for electrophoretic studies of some surface properties.
EXPOSURE to a number of carcinogenic agents results in a depression of immune status1. In detailed studies of chemical carcinogenesis in mice, Stjernswärd2,3 has shown that methylcholanthrene, benzpyrene and dimethylbenzanthracene (DMBA), all potent carcinogens, depress the immune response to sheep red cells (SRBC) for a long period following a single small carcinogenic dose of the agent. Although it seemed likely from these studies that the carcinogens act on the cells involved in the immune response, no direct evidence was presented. Recent work4–10 in a number of independent laboratories clearly demonstrates that a variety of cell-mediated immune responses, including those to SRBC, involve the interaction of two distinct classes of lymphocyte—the thymusderived cell responsible for reacting with antigen and the bone-marrow derived cell that is the precursor of the antibody-forming cell. The present study was designed to show whether carcinogen-induced immunodepression was a phenomenon linked to cells involved in the immune response and, if so, which of the interacting classes of cell was the target for this effect.
THE stability and integrity of the human foetal cell strain WI-38, which has been well demonstrated in the past ten years1–3, and its susceptibility to viruses infective for man1,4, explain the value of such material for the isolation of viruses and in the development of vaccines5–8. We have developed another strain of cells, also derived from foetal lung tissue, taken from a 14-week male foetus removed for psychiatric reasons from a 27 year old woman with a genetically normal family history and no sign of neoplastic disease both at abortion and for at least three years afterwards. The criteria used for characterizing the cells are those recommended internationally2,9.
BRIEF treatment with very low concentrations of proteolytic enzymes can bring about a change in the cell surface similar to that occurring in the chemical or viral transformation of normal to malignant cells1. This suggests to us that proteolytic enzymes may not only convert the surface structure into the type seen in malignant cells but that the whole cell may respond to the proteolytic enzyme with initiation of new rounds of cell division and a concomitant escape from contact inhibition of growth, as is the case for malignant cells in tissue culture2.