Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis

Mammalian target of rapamycin, mTOR, is a major sensor of nutrient and energy availability in the cell and regulates a variety of cellular processes, including growth, proliferation, and metabolism. Loss of the tuberous sclerosis complex genes (TSC1 or TSC2) leads to constitutive activation of mTOR and downstream signaling elements, resulting in the development of tumors, neurological disorders, and at the cellular level, severe insulin/IGF-1 resistance. Here, we show that loss of TSC1 or TSC2 in cell lines and mouse or human tumors causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). The resulting ER stress plays a significant role in the mTOR-mediated negative-feedback inhibition of insulin action and increases the vulnerability to apoptosis. These results demonstrate ER stress as a critical component of the pathologies associated with dysregulated mTOR activity and offer the possibility to exploit this mechanism for new therapeutic opportunities.     

Spacing patterns of an Acacia tree in the Kalahari over a 61-year period: How clumped becomes regular and vice versa

Nearest tree neighbour distances and the tree spatial formation on a large scale over time and space replicates were examined. The study was conducted in a natural savanna ecosystem in the Southern Kalahari, South Africa. Nearest tree neighbour and point pattern analysis methods were used to investigate changes in the spatial pattern of trees in two plots. Trees larger than 2 m canopy diameter were mapped. We used aerial photographs of the study area from 1940, 1964, 1984, 1993, and a satellite image from 2001 to follow two plots over time. Field work was carried out too for classification accuracy. We were able to identify and individually follow over 2400 individual trees from 1940 until 2001. Nearest neighbour analysis results indicate that dead trees were on average closer to their nearest neighbouring trees than living trees were to their neighbours. Most dead trees were on average 6 m from their nearest neighbours, while most living trees were about 20 m apart. Point pattern analysis results show a cyclical transition from clumped to random and sequentially to regular tree spacing. These transitions were not correlated across two plots. Generally, decreases in small-scale clumping coincided with periods of high mortality. Our findings show that regular, clumped, and random tree pattern can occur, pending on time, location, and scale within the location.     

CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific monoclonal antibody, inhibits the release of HIV-1 and canine distemper virus (CDV)- but not measles virus (MV)-induced cell–cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell–cell contact areas. High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins β₁-integrin and EWI-F were co-clustered with CD9 at cell–cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within CD9 clusters, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell–cell fusion by controlling the access of the fusion machinery to cell contact areas.     

Ligand binding induces Cbl-dependent EphB1 receptor degradation through the lysosomal pathway

Eph receptor tyrosine kinases play a critical role in embryonic patterning and angiogenesis. In the adult, they are involved in carcinogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1 and then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1 as kinase-dead EphB1-K652R was resistant to Cbl. Glutathione S-transferase binding experiments showed that Cbl bound to EphB1 through its tyrosine kinase-binding domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.     

A plastid protein crucial for Ca2+-regulated stomatal responses

* Guard cell movements are regulated by environmental cues including, for example, elevations in extracellular Ca(2+) concentration. Here, the subcellular localization and physiological function of the Ca(2+)-sensing receptor (CAS) protein was investigated. * CAS protein localization was ascertained by microscopic analyses of green fluorescent protein (GFP) fusion proteins and biochemical fractionation assays. Comparative guard cell movement investigations were performed in wild-type and cas loss-of-function mutant lines of Arabidopsis thaliana. Cytoplasmic Ca(2+) dynamics were addressed in plants expressing the yellow cameleon reporter protein YC3.6. * This study identified CAS as a chloroplast-localized protein that is crucial for proper stomatal regulation in response to elevations of external Ca(2+). CAS fulfils this role through modulation of the cytoplasmic Ca(2+) concentration. * This work reveals a novel role of the chloroplast in cellular Ca(2+) signal transduction.     

Elucidation of the final reactions of DIMBOA-glucoside biosynthesis in maize: characterization of Bx6 and Bx7

Benzoxazinoids were identified in the early 1960s as secondary metabolites of the grasses that function as natural pesticides and exhibit allelopathic properties. Benzoxazinoids are synthesized in seedlings and stored as glucosides (glcs); the main aglucone moieties are 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). The genes of DIBOA-glc biosynthesis have previously been isolated and the enzymatic functions characterized. Here, the enzymes for conversion of DIBOA-glc to DIMBOA-glc are identified. DIBOA-glc is the substrate of the dioxygenase BENZOXAZINLESS6 (BX6) and the produced 2,4,7-trihydroxy-2H-1,4-benzoxazin-3-(4H)-one-glc is metabolized by the methyltransferase BX7 to yield DIMBOA-glc. Both enzymes exhibit moderate K(m) values (below 0.4 mm) and k(cat) values of 2.10 s(-1) and 0.25 s(-1), respectively. Although BX6 uses a glucosylated substrate, our localization studies indicate a cytoplasmic localization of the dioxygenase. Bx6 and Bx7 are highest expressed in seedling tissue, a feature shared with the other Bx genes. At present, Bx6 and Bx7 have no close relatives among the members of their respective gene families. Bx6 and Bx7 map to the cluster of Bx genes on the short arm of chromosome 4.     

Tobacco mosaic virus movement protein interacts with green fluorescent protein-tagged microtubule end-binding protein 1

The targeting of the movement protein (MP) of Tobacco mosaic virus to plasmodesmata involves the actin/endoplasmic reticulum network and does not require an intact microtubule cytoskeleton. Nevertheless, the ability of MP to facilitate the cell-to-cell spread of infection is tightly correlated with interactions of the protein with microtubules, indicating that the microtubule system is involved in the transport of viral RNA. While the MP acts like a microtubule-associated protein able to stabilize microtubules during late infection stages, the protein was also shown to cause the inactivation of the centrosome upon expression in mammalian cells, thus suggesting that MP may interact with factors involved in microtubule attachment, nucleation, or polymerization. To further investigate the interactions of MP with the microtubule system in planta, we expressed the MP in the presence of green fluorescent protein (GFP)-fused microtubule end-binding protein 1a (EB1a) of Arabidopsis (Arabidopsis thaliana; AtEB1a:GFP). The two proteins colocalize and interact in vivo as well as in vitro and exhibit mutual functional interference. These findings suggest that MP interacts with EB1 and that this interaction may play a role in the associations of MP with the microtubule system during infection.     

The allene oxide cyclase family of Arabidopsis thaliana: localization and cyclization

Jasmonates are derived from oxygenated fatty acids (oxylipins) via the octadecanoid pathway and are characterized by a pentacyclic ring structure. They have regulatory functions as signaling molecules in plant development and adaptation to environmental stress. Recently, we solved the structure of allene oxide cyclase 2 (AOC2) of Arabidopsis thaliana, which is, together with the other three AOCs, a key enzyme in the biosynthesis of jasmonates, in that it releases the first cyclic and biologically active metabolite -- 12-oxo-phytodienoic acid (OPDA). On the basis of models for the bound substrate, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, and the product, OPDA, we proposed that a conserved Glu promotes the reaction by anchimeric assistance. According to this hypothesis, the transition state with a pentadienyl carbocation and an oxyanion is stabilized by a strongly bound water molecule and favorable pi-pi interactions with aromatic residues in the cavity. Stereoselectivity results from steric restrictions to the necessary substrate isomerizations imposed by the protein environment. Here, site-directed mutagenesis was used to explore and verify the proposed reaction mechanism. In a comparative analysis of the AOC family from A. thaliana involving enzymatic characterization, in vitro import, and transient expression of AOC-enhanced green fluorescent protein fusion proteins for analysis of subcellular targeting, we demonstrate that all four AOC isoenzymes may contribute to jasmonate biosynthesis, as they are all located in chloroplasts and, in concert with the allene oxide synthase, they are all able to convert 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid into enantiomerically pure cis(+)-OPDA.     

Small molecule, non-peptide p75 ligands inhibit Abeta-induced neurodegeneration and synaptic impairment

The p75 neurotrophin receptor (p75(NTR)) is expressed by neurons particularly vulnerable in Alzheimer's disease (AD). We tested the hypothesis that non-peptide, small molecule p75(NTR) ligands found to promote survival signaling might prevent Abeta-induced degeneration and synaptic dysfunction. These ligands inhibited Abeta-induced neuritic dystrophy, death of cultured neurons and Abeta-induced death of pyramidal neurons in hippocampal slice cultures. Moreover, ligands inhibited Abeta-induced activation of molecules involved in AD pathology including calpain/cdk5, GSK3beta and c-Jun, and tau phosphorylation, and prevented Abeta-induced inactivation of AKT and CREB. Finally, a p75(NTR) ligand blocked Abeta-induced hippocampal LTP impairment. These studies support an extensive intersection between p75(NTR) signaling and Abeta pathogenic mechanisms, and introduce a class of specific small molecule ligands with the unique ability to block multiple fundamental AD-related signaling pathways, reverse synaptic impairment and inhibit Abeta-induced neuronal dystrophy and death.     

The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation

The mouse placenta was unveiled as an important reservoir for hematopoietic stem cells (HSCs), yet the origin of placental HSCs was unknown. By tracking developing HSCs by expression of Runx1-lacZ and CD41, we have found that HSCs emerge in large vessels in the placenta. Analysis of Ncx1(-/-) embryos, which lack a heartbeat, verified that HSC development is initiated in the placental vasculature independent of blood flow. However, fewer CD41+ hematopoietic cells were found in Ncx1(-/-) placentas than in controls, implying that some HSCs/progenitors colonize the placenta via circulation and/or HSC emergence is compromised without blood flow. Importantly, placentas from Ncx1(-/-) embryos possessed equal potential to generate myelo-erythroid and B and T lymphoid cells upon explant culture, verifying intact multilineage hematopoietic potential, characteristic of developing HSCs. These data suggest that, in addition to providing a niche for a large pool of HSCs prior to liver colonization, the placenta is a true site of HSC generation.