Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development

SummaryNuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation

Central chemoreception in the neonatal rat transverse medullary slice preparation

grantor: University of TorontoThis study investigates several aspects of central chemoreception in the neonatal rat, transverse medullary slice preparation. I found that the frequency of bursting recorded from hypoglossal nerves changed significantly with step changes in pH produced by varying the CO2 of the bathing solution of the slice. I also found that application of 1 mM acetazolamide dissolved in DMSO to the slice preparation produced no significant alterations in these responses to CO2. However, acetazolamide application did provoke a significant and prolonged decrease in hypoglossal nerve burst duration. Although the pH sensitive fluorescent dye, BCECF-AM, performed satisfactorily in bench tests, when it was used to label transverse medullary slice preparations it exhibited a faster rate of decay in fluorescence emission. Furthermore, I observed no change in fluorescence emission when pH was varied in the slice, either globally or locally using a CO2 diffusion pipette. I concluded that (1) the transverse medullary slice does contain central chemoreceptors, which elicit a respiratory response; (2) the central chemoreceptor response to pH/CO2 variations is not dependent upon carbonic anhydrase, and (3) the pH sensitive fluorescent dye BCECF-AM is not suitable for assessing regional changes of pH in the transverse medullary slice preparation.M.Sc

The in vivo Function of Nuclear Receptors During Drosophila Development

Nuclear receptors (NR’s) comprise a large, ancient, superfamily of eukaryotic transcription factors that govern a wide range of metabolic, homeostatic, and developmental pathways, and which have been implicated in disease states including cancer, inflammation, and diabetes. The ability of NRs to activate or repress gene transcription is modulated through direct binding of small lipophilic ligands which induce conformational changes in their cognate receptor. These changes are structural in nature and lead to the recruitment of coactivator or corepressor complexes, ultimately regulating the expression of target genes to whose response elements NRs are bound. In Drosophila 18 NRs have been identified which have representative members belonging to each of the six major NR subfamilies, and which show a high degree of homology to their vertebrate counterparts. This fact, in addition to the power and ease of genetic manipulation, make Drosophila an excellent model system in which to study NR function. When I began my project, 17 of the 18 NRs in Drosophila were ‘orphan’ receptors for which no cognate ligand had been identified. As a first step in an effort to identify potential ligands for these 17 receptors I first set out to determine how, where and when nuclear receptors are regulated by small chemical ligands and/or their protein partners. In order to do so I contributed to developing a ‘ligand sensor’ system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live, developing animals. This system is based upon transgenic lines that express the ligand binding domain of each Drosophila NR fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, these fusion proteins show tissue- and stage-specific patterns of activation. Analysis using this system has revealed the stage and tissue specificity of NR activation for each of the fly NRs. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production are also a prominent feature that has been revealed using this approach. In addition, I went on to characterize the ligand regulated function of a single Drosophila nuclear receptor, Ecdysone inducible protein 75 (E75). Previous work from our lab has demonstrated that E75 binds to heme, and that its function as a transcriptional repressor is regulated in vitro by binding of the small diatomic gases nitric oxide (NO) and carbon monoxide (CO) to its heme moiety. In an effort to validate and to further understand the in vivo relevance of E75 regulation by NO I used gain and loss of function transgenes, as well as tissues manipulated in culture to show that NO acts directly on the Drosophila nuclear receptor E75, reversing its ability to block the activity of its heterodimer partner Drosophila Hormone Receptor 3 (DHR3). By specifically focusing on the Drosophila larval ring gland, the principal endocrine organ responsible for the production of the metamorphosis-inducing hormone, ecdysone, I have shown that failure to produce NO and to inactivate E75 results in failure to recognize the signals that normally trigger metamorphosis.Ph

Human Diseases Associated with Notch Signalling: Lessons from Drosophila melanogaster

Drosophila melanogaster has been used as a model system to identify and characterize genetic contributions to development, homeostasis, and to investigate the molecular determinants of numerous human diseases. While there exist many differences at the genetic, structural, and molecular level, many signalling components and cellular machineries are conserved between Drosophila and humans. For this reason, Drosophila can and has been used extensively to model, and study human pathologies. The extensive genetic resources available make this model system a powerful one. Over the years, the sophisticated and rapidly expanding Drosophila genetic toolkit has provided valuable novel insights into the contribution of genetic components to human diseases. The activity of Notch signalling is crucial during development and conserved across the Metazoa and has been associated with many human diseases. Here we highlight examples of mechanisms involving Notch signalling that have been elucidated from modelling human diseases in Drosophila melanogaster that include neurodegenerative diseases, congenital diseases, several cancers, and cardiac disorders

Chronic AMPK Activation Reduces the Expression and Alters Distribution of Synaptic Proteins in Neuronal SH-SY5Y Cells

Neuronal growth and synaptic function are dependent on precise protein production and turnover at the synapse. AMPK-activated protein kinase (AMPK) represents a metabolic node involved in energy sensing and in regulating synaptic protein homeostasis. However, there is ambiguity surrounding the role of AMPK in regulating neuronal growth and health. This study examined the effect of chronic AMPK activation on markers of synaptic function and growth. Retinoic-acid-differentiated SH-SY5Y human neuroblastoma cells were treated with A-769662 (100 nM) or Compound C (30 nM) for 1, 3, or 5 days before AMPK, mTORC1, and markers for synapse function were examined. Cell morphology, neuronal marker content, and location were quantified after 5 days of treatment. AMPK phosphorylation was maintained throughout all 5 days of treatment with A-769662 and resulted in chronic mTORC1 inhibition. Lower total, soma, and neuritic neuronal marker contents were observed following 5 d of AMPK activation. Neurite protein abundance and distribution was lower following 5 days of A-769662 treatment. Our data suggest that chronic AMPK activation impacts synaptic protein content and reduces neurite protein abundance and distribution. These results highlight a distinct role that metabolism plays on markers of synapse health and function

Supplementary Table S3 - Restriction enzymes used for cloning: PlayBack cloning: simple, reversible, cost-effective cloning for the combinatorial assembly of complex expression constructs

Supplementary tables</p

Supplementary Table S4 - Plasmids made and deposited to Addgene: PlayBack cloning: simple, reversible, cost-effective cloning for the combinatorial assembly of complex expression constructs

Supplementary tables</p

Supplementary Table S1 - Plasmids ordered or synthesized for experiments: PlayBack cloning: simple, reversible, cost-effective cloning for the combinatorial assembly of complex expression constructs

Supplementary tables</p