Dueling Ca2+ Sensors in Neurotransmitter Release

Ca2+-triggered neurotransmitter release is characterized by two kinetically distinct components: a fast synchronous phase and a slow asynchronous phase. Yao et al. (2011) now report that double C2 domain (Doc2) proteins function as high-affinity Ca2+ sensors to specifically regulate the asynchronous component of neurotransmitter release

Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 Through Poly-Ubiquitination

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues

Study od bHLH orange proteins in drosophila melanogaster: a) mode of action of E (spl) proteins during neurogenesis, b) analysis of the pattern of expression of hey and its relationship with notch signalling pathway

A) Neurogenesis in all animals is triggered by the activity of a group of bHLH transcription factors, the proneural proteins [i.e. Achaete (Ac) and Scute (Sc)], whose expression endows ectodermal regions with neural potential. The eventual commitment to a neural precursor fate involves the interplay of these proneural transcriptional activators with a number of other transcription factors, which fine-tune transcriptional responses at target genes. Most prominent among the factors antagonizing proneural protein activity are the HES [E(spl)] bHLH proteins. We show here that E(spl) proteins m7 and mγ are potent inhibitors of neural fate, even in the presence of excess Sc activity and even when their DNA-binding basic domain has been inactivated. Furthermore, these E(spl) proteins can efficiently repress target genes that lack cognate DNA binding sites, as long as these genes are bound by Ac/Sc activators. This activity of E(spl)m7 and mg correlates with their ability to interact with proneural activators, through which they are probably tethered on target enhancers. The co-repressor Groucho is always needed for target gene regulation. In addition, by using in vivo and in vitro assays we have discovered that the N-teminal region (including the bHLH domain) of E(spl)m7 interacts with the C-teminal domain of Sc. An important dual role for the Sc C-terminal domain is shown: on one hand it acts as a transcription activation domain and on the other it is used to recruit E(spl) proteins. In vivo, the Sc C-terminal domain is required for E(spl) recruitment in an enhancer-context dependent fashion, suggesting that in some enhancers alternative interaction surfaces can be used to recruit E(spl) proteins. As far as E(spl)m7 is concerned, the integrity of the Orange domain is important for in its activity. Analysis of reporter genes and sensory organ (bristle) patterns reveals that, in addition to this indirect recruitment of E(spl) onto enhancers via protein-protein interaction with bound Ac/Sc factors, direct DNA binding of target genes by E(spl) also takes place. B) E(spl) proteins belong to a family of bHLH proteins, called bHLH-Orange proteins. All members of this family share homology with respect to a region called the Orange domain, which is considered to play an important role in the regulation of the proteins’ intra- and intermolecular interactions and possibly thereby of their activity. Computational analysis of the Drosophila genome has shown that there are 13 representative members of this family. We were particularly interested in Hey protein. Hey protein belongs to a subgroup of bHLH-Orange proteins with distinct, evolutionarily conserved structural characteristics. Hey genes have been studied extensively in vertebrates and they have been shown to participate in numerous developmental processes such as angiogenesis and neurogenesis. They have also been shown to be novel targets of Notch signalling. Hey proteins are heterodimeric partners of Hes proteins, with which they co-operate to achieve a more efficient transcriotional repression of target genes. However, nothing is known about the corresponding gene in Drosophila. We have studied its expression pattern in various tissues and developmental stages. It has been revealed that it is always expressed in a subset of postmitotic neurons. Its expression pattern persists even after Notch signalling is seriously reduced, suggesting (at least under the present experimental conditions) that it is possibly not a target of the Notch pathway.A) Η έναρξη της νευρογένεσης βασίζεται στην ενεργότητα ενός συνόλου bHLH μεταγραφικών παραγόντων, των προνευρικών πρωτεϊνών [όπως οι πρωτεΐνες Achaete (Ac) και Scute (Sc) στη Drosophila melanogaster], των οποίων η έκφραση προσδίδει σε εκτοδερμικές περιοχές τη δυνατότητα να ακολουθήσουν νευρική αναπτυξιακή πορεία. Η τελική δρομολόγηση προς την ταυτότητα του νευρικού προδρόμου κυττάρου εξαρτάται από τη λειτουργική σχέση μεταξύ των προνευρικών ενεργοποιητών με άλλους μεταγραφικούς παράγοντες, μεταξύ των οποίων οι, ανταγωνιστικές, HES [E(spl)] πρωτεΐνες. Οι Ε(spl)m7 και mγ είναι ισχυροί καταστολείς της νευρικής τύχης ακόμα και όταν υπάρχει περίσσεια ενεργής Sc και ακόμα και όταν η πρόσδεσή τους σε αλληλουχίες DNA έχει απενεργοποιηθεί. Επίσης, είναι ικανές να καταστείλουν γονίδια στόχους μέσω πρωτεϊνικής αλληλεπίδρασης με τις προνευρικές πρωτεΐνες ήδη προσδεδεμένες σε αντίστοιχες αλληλουχίες σε ρυθμιστικές περιοχές των γονιδίων αυτών. Για τη κατασταλτική τους δράση είναι πάντα απαραίτητο να αλληλεπιδρούν με το συγκαταστολέα Groucho. Περαιτέρω ανάλυση έδειξε ότι η αμινοτελική περιοχή της Ε(spl)m7, συμπεριλαμβανομένου της bHLH περιοχής, και η καρβοξυτελική περιοχή της Sc αληλεπιδρούν in vivo και in vitro. Η καρβοξυτελική περιοχή της πρωτεΐνης Sc ενεργοποιεί τη μεταγραφή και είναι υπεύθυνη για την στρατολόγηση της Ε(spl)m7. In vivo είναι επίσης υπεύθυνη για τη στρατολόγηση της E(spl)m7 κατά τρόπο εξαρτώμενο από τον ενισχυτή στο οποίο αυτή προσελκύεται, καθώς σε πιο σύνθετους ενισχυτές πρόσθετοι μεταγραφικοί παράγοντες παρέχουν εναλλακτικές επιφάνειες πρόσδεσης και αλληλεπίδρασης. Από την άλλη μεριά η ακεριότητα της περιοχής Orange είναι απαραίτητη για την ενεργότητα της Ε(spl) in vivo. Ανάλυση γονιδίων ανταποκριτών και του προτύπου έκφρασης των εξωτερικών αισθητηρίων οργάνων στο θώρακα ενήλικων ατόμων δείχνει ότι υπάρχουν δύο τρόποι δράσης των Ε(spl) πρωτεϊνών in vivo: α) άμεση πρόσδεση στο DNA και β) έμμεση πρόσδεση στους ενισχυτές μέσω προνευρικών πρωτεϊνών. B) Οι Ε(spl) πρωτεΐνες ανήκουν στην οικογένεια των bHLH-Orange πρωτεϊνών. Βασικό χαρακτηριστικό των πρωτεϊνών αυτών είναι η περιοχή Orange, η οποία παίζει σημαντικό ρυθμιστικό ρόλο ενδο- και διαμορικών αλληλεπιδράσεων και κατ’ επέκταση ίσως της ενεργότητάς τους. Υπάρχουν 13 αντιπρόσωποι αυτής της οικογένειας στη Drosophila melanogaster. Μεταξύ αυτών είναι η πρωτεΐνη Hey, η οποία ανήκει σε ένα διακριτό σύνολο με εξελικτικά συντηρημένα δομικά χαρακτηριστικά. Τα ομόλογα γονίδια Hey των σπονδυλωτών έχουν εξεταστεί εκτεταμένα. Φαίνεται να παίζουν ιδαίτερο ρόλο σε πληθώρα αναπτυξιακών διαδικασιών όπως η νευρογένεση και η αγγειογένεση. Επίσης, φαίνεται να είναι άμεσοι μεταγραφικοί στόχοι του σηματοδοτικού μονοπατιού Notch. Οι Hey πρωτεΐνες σχηματίζουν ετεροδιμερή με τις Hes πρωτεΐνες, με τις οποίες συνεργάζονται για την αποτελεσματικότερη μεταγραφική καταστολή γονιδίων στόχων. Επειδή τίποτα δεν είναι γνωστό για την πρωτεΐνη Hey της Drosophila melanogaster, εξετάστηκε το πρότυπο έκφρασης της και η σχέση της με το σηματοδοτικό μονοπάτι Notch. Tα αποτελέσματα δείχνουν ότι εκφράζεται σε υποσύνολο νευρώνων του ΚΝΣ και ΠΝΣ στο έμβρυο, την προνύμφη και τη νύμφη και ότι η έκφρασή της παραμένει ακόμα και όταν η σηματοδότηση Notch έχει σημαντικά μειωθεί. Επομένως, δε φαίνεται να είναι στόχος του Notch (υπό τις παρούσες πειραματικές συνθήκες)

Cell Adhesion, the Backbone of the Synapse: “Vertebrate” and “Invertebrate” Perspectives

Synapses are asymmetric intercellular junctions that mediate neuronal communication. The number, type, and connectivity patterns of synapses determine the formation, maintenance, and function of neural circuitries. The complexity and specificity of synaptogenesis relies upon modulation of adhesive properties, which regulate contact initiation, synapse formation, maturation, and functional plasticity. Disruption of adhesion may result in structural and functional imbalance that may lead to neurodevelopmental diseases, such as autism, or neurodegeneration, such as Alzheimer's disease. Therefore, understanding the roles of different adhesion protein families in synapse formation is crucial for unraveling the biology of neuronal circuit formation, as well as the pathogenesis of some brain disorders. The present review summarizes some of the knowledge that has been acquired in vertebrate and invertebrate genetic model organisms

Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila

The adult Drosophila external sensory organ (ESO), comprising the hair, socket, neuron, sheath and glia cells, arises through the asymmetric division of sensory organ precursor cells (SOPs). In a mosaic screen designed to identify new components in ESO development, we isolated mutations in sequoia, which encodes a putative zinc-finger transcription factor that has previously been shown to have a role in dendritogenesis. Here, we show that adult clones mutant for seq exhibit a loss of hair cells and a gain of socket cells. We propose that the seq mutant phenotype arises, in part, owing to the loss of several crucial transcription factors known to be important in peripheral nervous system development such as D-Pax2, Prospero and Hamlet. Thus, Sequoia is a new upstream regulator of genes that orchestrates cell fate specification during development of the adult ESO lineage

Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.status: publishe

Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse

<div><p>Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the <i>PEX</i> genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila <i>pex16</i> and <i>pex2</i> mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.</p></div

Ubr3 regulates the ubiquitination of Cos2.

<p>(A) S2 cells expressing HA::Cos2 fusion proteins were treated with CHX and MG132, or DMSO as a negative control, for the indicated time. The cell lysate was then examined by Western blot with antibodies. The HA::Cos2 signal intensity was normalized with α-tub and quantified in the lower panel. (B) S2 cells were transfected with Hh ligand and HA::Cos2 fusion proteins, and some cells were also co-transfected with Ubr3^RNAi or a construct to overexpress Ubr3. These cells were treated with Cyclohexamide for the indicated time. The cell lysate was then examined by Western blot (top panel). The levels of the Ubr3 protein were assessed with the anti-Ubr3 antibody. Anti-Actin served as loading control (bottom panel). (C) S2 cells were transfected with HA::Cos2 with GFP (lane 1) or UBR::GFP (lane 2) or Ubr3::GFP (lane 3) constructs. We performed co-immunoprecipitation assays with anti-GFP agarose followed by western blot assays on the cell lysates. Both UBR domain fragments (lane 2) and the Ubr3 full length protein (lane 3) co-precipitate with Cos2, whereas GFP (lane 1) does not. S2 cells co-transfected with HA::Cos2 and Ubr3::GFP were treated with Colchicine for 5 hours prior to harvest. Co-immunoprecipitation assay with anti-GFP agarose beads and western blots were performed from the cell lysate. (D) Schematic diagram of Cos2 deletion constructs. (E) Co-immunoprecipitation assays with lysates from S2 cells, transfected with UBR::GFP and the constructs indicated in C reveal that the UBR domain of Ubr3 interacts physically with full length Cos2 and Cos2ΔC1-ΔC3 (red bars in C, lanes 1, 4–6 in D), but not with Cos2ΔN1-ΔN2 (gray bars in B, lanes 2, 3 in D). (F) S2 cells were co-transfected with Myc::Cos2 and HA::Ub, in combination with Ubr3, Ubr3^RNAi or GFP^RNAi. Following immunoprecipitation and Western blot analysis with anti-HA (upper panel), we found that Cos2 is ubiquitinated, as indicated by the smeary shift of the protein (lane 1). Over-expression of Ubr3 increases ubiquitinated Cos2 (lane 2), whereas down-regulation of Ubr3 through Ubr3 RNAi (lane 3) decreases ubiquitinated Cos2. GFP^RNAi was used as a negative control. The asterisks indicate ubiquitinated Cos2 which exhibits a higher molecular weight.(G) S2 cells were co-transfected with Myc::Cos2 or Myc::Cos2ΔN1, and HA::Ub, in combination with wild type Ubr3 or E3 dead form of Ubr3 (Ubr3^D), following immunoprecipitation and Western blot analysis.</p

Up-regulation of Cos2 in <i>ubr3</i> mutant clones is responsible for the loss of Hh signaling.

<p>(A-A’) <i>ubr3</i>^<i>B</i> mutant cells (marked by GFP) up-regulate Cos2 (red; arrows). (B-B’) Over-expression of Cos2 by <i>eyg-Gal4</i> (indicated by expression of GFP) leads to loss of Ci¹⁵⁵ (arrow in B’) at the morphogenetic furrow. (C-C’) Over-expression of Cul1 by <i>eyg-Gal4</i> (indicated by expression of GFP) does not cause loss of Ci¹⁵⁵ at the morphogenetic furrow. (D-D’) Down-regulation of Cos2 by over-expression of <i>cos2</i>^<i>RNAi</i> in <i>ubr3</i>^<i>B</i> mutant clones (marked by the expression of GFP) suppresses loss of Ci¹⁵⁵ (red) in the morphogenetic furrow (arrows in D’). (E-E’) Down-regulation of Cul1 by over-expression of <i>cul1</i>^<i>RNAi</i> in <i>ubr3</i>^<i>B</i> mutant clones induces ectopic activation of Hh signaling anterior to the morphogenetic furrow (shown by expression of Ci¹⁵⁵, arrow) but does not rescue Ci¹⁵⁵ loss in the morphogenetic furrow (arrowheads). (F-F’) Over-expression of <i>ptc</i>^<i>DN</i> in <i>ubr3</i>^<i>B</i> mutant clones does not rescue Ci¹⁵⁵ loss in the morphogenetic furrow (arrowhead).</p