Matthew D. Rand

Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors

ABSTRACT Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (NEC) and a single-pass transmembrane signaling domain (NTM). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of NTM(NICD). In the current work, we show that the NEC and NTM subunits of DrosophilaNotch and human Notch1 (hN1) interact noncovalently. NEC-NTM interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca2+. Deletion of the Ca2+-binding Lin12-Notch (LN) repeats from the NEC subunit resulted in spontaneous shedding of NEC into conditioned medium, implying that the LN repeats are important in maintaining the interaction of NEC and NTM. The functional consequences of EDTA-induced NEC dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of NEC, the transient appearance of a polypeptide of the expected size of NICD, increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of NEC dissociation, which relieves inhibition of the intrinsically active NTMsubunit.

Delta-Notch signaling controls the generation of neurons/glia from neural stem cells in a stepwise process.

We examined the role of Notch signaling on the generation of neurons and glia from neural stem cells by using neurospheres that are clonally derived from neural stem cells. Neurospheres prepared from Dll1lacZ/lacZ mutant embryos segregate more neurons at the expense of both oligodendrocytes and astrocytes. This mutant phenotype could be rescued when Dll1lacZ/lacZ spheres were grown and/or differentiated in the presence of conditioned medium from wild-type neurospheres. Temporal modulation of Notch by soluble forms of ligands indicates that Notch signaling acts in two steps. Initially, it inhibits the neuronal fate while promoting the glial cell fate. In a second step, Notch promotes the differentiation of astrocytes, while inhibiting the differentiation of both neurons and oligodendrocytes.

Down-regulation of Delta by proteolytic processing

Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, we examined the biological activity of purified soluble Delta forms and conclude that Delta cleavage is an important down-regulating event in Notch signaling. The data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner.

Drosophotoxicology: The growing potential for Drosophila in neurotoxicology

Understanding neurotoxic mechanisms is a challenge of deciphering which genes and gene products in the developing or mature nervous system are targeted for disruption by chemicals we encounter in our environment. Our understanding of nervous system development and physiology is highly advanced due in large part to studies conducted in simple non-mammalian models. The paucity of toxicological data for the more than 80,000 chemicals in commercial use today, and the approximately 2000 new chemicals introduced each year, makes development of sensitive and rapid assays to screen for neurotoxicity paramount. In this article I advocate the use of Drosophila in the modern regimen of toxicological testing, emphasizing its unique attributes for assaying neurodevelopment and behavior. Features of the Drosophila model are reviewed and a generalized overall scheme for its use in toxicology is presented. Examples of where the fly has proven fruitful in evaluating common toxicants in our environment are also highlighted. Attention is drawn to three areas where development and application of the fly model might benefit toxicology the most: 1) optimizing sensitive endpoints for pathway-specific screening, 2) accommodating high throughput demands for analysis of chemical toxicant libraries, 3) optimizing genetic and molecular protocols for more rapid identification toxicant-by-gene interactions. While there are shortcomings in the Drosophila model, which exclude it from effective toxicological testing in certain arenas, conservation of fundamental cellular and developmental mechanisms between flies and man is extensive enough to warrant a central role for the Drosophila model in toxicological testing of today.

Methylmercury disruption of embryonic neural development in Drosophila

Methylmercury (MeHg) is a potent environmental neurotoxin that preferentially targets the developing embryonic nervous system. While a number of cytotoxic mechanisms of MeHg have been characterized in differentiated cells its mode of action in the developing nervous system in vivo is less clear. Studies in primate and rodent models demonstrate aberrant cell migration and disorganized patterning of cortical layers in the brain following MeHg exposure. However, defining the molecular and cellular pathways targeted by MeHg will require more genetically accessible animal models. In this study, we instigate a method of in vitro MeHg exposure using Drosophila embryos. We demonstrate dose-dependent inhibition of embryonic development with MeHg revealed by a failure of embryos to hatch to the larval stage. In addition, we document definitive phenotypes in neural development showing abnormalities in neuronal and glial cell patterning consistent with disrupted migration. We observe pronounced defects in neurite outgrowth in both central and peripheral neurons. Ectopic expression of the Nrf2 transcription factor in embryos, a core factor in the antioxidant response element (ARE) pathway, enhances embryonic development and hatching in the presence of MeHg, illustrating the power of this model for investigation of candidate MeHg tolerance genes. Our data establish a utility for the Drosophila embryo model as a platform for elucidating MeHg sensitive pathways in neural development.

Kuz and TACE can activate Notch independent of ligand

Abstract.A central mechanism in activation of the Notch signaling pathway is cleavage of the Notch receptor by ADAM metalloproteases. ADAMs also cleave Delta, the ligand for Notch, thereby downregulating Notch signals. Two ADAMs, Kuzbanian (Kuz) and TNF-α converting enzyme (TACE), are known to process both Delta and Notch, yet the role of these cleavages in signal propagation has remained controversial. Using an in vitro model, we show that Kuz regulates Notch signaling primarily by activating the receptor and has little overall effect on signaling via disabling Delta. We confirm that Kuz-dependent activation of Notch requires stimulation of Notch by Delta. However, over-expression of Kuz gives ligand-independent Notch activation. In contrast, TACE, which is elevated in expression in the developing Drosophila nervous system, can efficiently activate Notch in a ligand-independent manner. Altogether, these data demonstrate the potential for Kuz and TACE to participate in context- and mechanism-specific modes of Notch activation.

[13] Factor V

Publisher Summary The proteolytic activation of prothrombin is catalyzed by an enzyme complex composed of the serine protease factor Xa reversibly associated with the cofactor factor Va on membranes containing negatively charged phospholipid in the presence of calcium ions. Studies of the structure–function relationships in factor Va have been aided by the availability of the sequence of the cDNA coding for factor V by the development of binding and kinetic measurements to examine individually the discrete interactions of the cofactor within prothrombinase and by the isolation and characterization of proteolytic fragments through protein chemistry techniques. This chapter discusses the methodology for the isolation of factor Va, its subunits, and proteolytic derivatives of the cofactor and describes the various steps of the purification and partial characterization of a small region of the Va LC of the cofactor, which possess a phospholipid binding domain. The chapter describes the enzymatic degradation of Va LC in presence of phospholipid vesicles.

Permeabilization of Drosophila embryos for introduction of small molecules

Pharmacological manipulations in the Drosophila embryo have been hindered by the impermeability of the eggshell. The ultimate barrier to delivery of small molecule solutes to the embryo is the waxy layer that lies beneath the external chorion layers and encases the underlying vitelline membrane of the eggshell. Conventional protocols call for heptane or octane to permeablize the dechorionated eggshell however, these solvents are toxic and can result in low viability. Furthermore, heptane and octane require transition of the embryo between aqueous and organic phase solvents making it challenging to avoid desiccation. Here we describe an embryo permeabilization solvent (EPS) composed of d-limonene and plant-derived surfactants that is water miscible and highly effective in rendering the dechorionated eggshell permeable. EPS permeabilization enables embryo uptake of several different dyes of various molecular mass up to 995Da. We find that the embryo undergoes an age-dependent decrease in the ability to be permeabilized in the first six to eight hours after egg laying. This apparent developmental change in the vitelline membrane contributes to the heterogeneity in permeabilization seen even among closely staged embryos. However, using fluorescent properties of Rhodamine B dye and various conditions of EPS treatment we demonstrate the ability to obtain optimally permeabilized viable embryos. We also demonstrate the ability to assess teratogenic activity of several compounds applied to embryos in vitro, using both early and late developmental endpoints. Application of the method to transgenic strains carrying GFP-reporter genes results in a robust readout of pharmacological alteration of embryogenesis. The straightforward and rapid nature of the manipulations needed to prepare batches of permeabilized embryos has the potential of establishing the Drosophila embryo as an alternative model in toxicology and for small molecule screening in a high-throughput format.

Methylmercury activates enhancer-of-split and bearded complex genes independent of the notch receptor.

Methylmercury (MeHg) is a persistent environmental toxin that has targeted effects on fetal neural development. Although a number of cytotoxic mechanisms of MeHg have been characterized in cultured cells, its mode of action in the developing nervous system in vivo is less clear. Studies of MeHg-affected rodent and human brains show disrupted cortical and cerebellar architecture suggestive of mechanisms that augment cell signaling pathways potentially affecting cell migration and proliferation. We previously identified the Notch receptor pathway, a highly conserved signaling mechanism fundamental for neural development, as a target for MeHg-induced signaling in Drosophila neural cell lines. Here we have expanded our use of the Drosophila model to resolve a broader spectrum of transcriptional changes resulting from MeHg exposure in vivo and in vitro. Several Notch target genes within the Enhancer-of-split (E(spl)C) and Bearded (BrdC) complexes are upregulated with MeHg exposure in the embryo and in cultured neural cells. However, the profile of MeHg-induced E(spl)C and BrdC gene expression differs significantly from that seen with activation of the Notch receptor. Targeted knockdown of Notch and of the downstream coactivator Suppressor of Hairless (Su(H)), shows no effect on MeHg-induced transcription, indicating a novel Notch-independent mechanism of action for MeHg. MeHg transcriptional activation is partially mimicked by iodoacetamide but not by N-ethylmaleimide, two thiol-specific electrophiles, revealing a degree of specificity of cellular thiol targets in MeHg-induced transcriptional events.

Thrombolytic Therapy and Proteolysis of Factor V

OBJECTIVES We sought to determine the extent of Factor V proteolysis during thrombolytic therapy. BACKGROUND Thrombin- or Factor Xa-activated Factor V is an essential cofactor in the prothrombinase complex. In purified systems, plasmin, the major product of thrombolytic therapy, is known to first activate then inactivate Factor V. METHODS We used quantitative gel electrophoresis and Western blotting to analyze the cleavages in plasma Factor V after thrombolytic therapy. RESULTS The addition of streptokinase to plasma resulted in the activation then inactivation of Factor V, confirming previous results using purified reagents. We also identified the Factor V fragments resulting from the action of thrombin and plasmin. After thrombolytic therapy, there was considerable Factor V cleavage. The cleavage patterns were consistent with the action of plasmin, with little evidence for the action of thrombin. In the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries trial (n = 17), we observed an average 58% loss of intact Factor V at 6 h (range 1% to 91%). Samples from the Thrombolysis in Myocardial Infarction trial, Phase II (n = 12), collected on a shorter time scale, showed a loss of up to 99% at 50 min, with the loss of intact Factor V associated with the plasma concentration of plasminogen activator. Samples from patients with bleeding (n = 12) showed extensive Factor V cleavage. CONCLUSIONS Factor V cleavage in thrombolytic therapy is primarily plasmin mediated, rapid and often extensive. It is likely that transient increases, as well as longer term losses, of Factor V cofactor activity play a role in both ischemic and hemorrhagic events subsequent to thrombolytic therapy. The extensive loss of Factor V in some patients may affect the estimation of heparinization.