Perturbation of invadolysin disrupts cell migration in zebrafish (Danio rerio)

Invadolysin is an essential, conserved metalloprotease which links cell division with cell migration and is intriguingly associated with lipid droplets. In this work we examine the expression pattern, protein localisation and gross anatomical consequences of depleting invadolysin in the teleost Danio rerio. We observe that invadolysin plays a significant role in cell migration during development. When invadolysin is depleted by targeted morpholino injection, the appropriate deposition of neuromast clusters and distribution of melanophores are both disrupted. We also observe that blood vessels generated via angiogenesis are affected in invadolysin morphant fish while those formed by vasculogenesis appear normal, demonstrating an unanticipated role for invadolysin in vessel formation. Our results thus highlight a common feature shared by, and a requirement for invadolysin in, these distinct morphological events dependent on cell migration

Invadolysin, a conserved lipid-droplet-associated metalloproteinase, is required for mitochondrial function in Drosophila

Mitochondria are the main producers of ATP, the principal energy source of the cell, and reactive oxygen species (ROS), important signaling molecules. Mitochondrial morphogenesis and function depend on a hierarchical network of mechanisms in which proteases appear to be center stage. The invadolysin gene encodes an essential conserved metalloproteinase of the M8 family that is necessary for mitosis and cell migration during Drosophila development. We previously demonstrated that invadolysin is found associated with lipid droplets in cells. Here, we present data demonstrating that invadolysin interacts physically with three mitochondrial ATP synthase subunits. Our studies have focused on the genetic phenotypes of invadolysin and bellwether, the Drosophila homolog of ATP synthase a, mutants. The invadolysin mutation presents defects in mitochondrial physiology similar to those observed in bellwether mutants. The invadolysin and bellwether mutants have parallel phenotypes that affect lipid storage and mitochondrial electron transport chain activity, which result in a reduction in ATP production and an accumulation of ROS. As a consequence, invadolysin mutant larvae show lower energetic status and higher oxidative stress. Our data demonstrate an essential role for invadolysin in mitochondrial function that is crucial for normal development and survival.peer-reviewe

Glucocorticoid-Mediated Inhibition of Angiogenic Changes in Human Endothelial Cells Is Not Caused by Reductions in Cell Proliferation or Migration

Glucocorticoid-mediated inhibition of angiogenesis is important in physiology, pathophysiology and therapy. However, the mechanisms through which glucocorticoids inhibit growth of new blood vessels have not been established. This study addresses the hypothesis that physiological levels of glucocorticoids inhibit angiogenesis by directly preventing tube formation by endothelial cells.Cultured human umbilical vein (HUVEC) and aortic (HAoEC) endothelial cells were used to determine the influence of glucocorticoids on tube-like structure (TLS) formation, and on cellular proliferation (5-bromo-2'-deoxyuridine (BrdU) incorporation), viability (ATP production) and migration (Boyden chambers). Dexamethasone or cortisol (at physiological concentrations) inhibited both basal and prostaglandin F(2α) (PGF(2α))-induced and vascular endothelial growth factor (VEGF) stimulated TLS formation in endothelial cells (ECs) cultured on Matrigel, effects which were blocked with the glucocorticoid receptor antagonist RU38486. Glucocorticoids had no effect on EC viability, migration or proliferation. Time-lapse imaging showed that cortisol blocked VEGF-stimulated cytoskeletal reorganisation and initialisation of tube formation. Real time PCR suggested that increased expression of thrombospodin-1 contributed to glucocorticoid-mediated inhibition of TLS formation.We conclude that glucocorticoids interact directly with glucocorticoid receptors on vascular ECs to inhibit TLS formation. This action, which was conserved in ECs from two distinct vascular territories, was due to alterations in cell morphology rather than inhibition of EC viability, migration or proliferation and may be mediated in part by induction of thrombospodin-1. These findings provide important insights into the anti-angiogenic action of endogenous glucocorticoids in health and disease

Sequential Loading of Cohesin Subunits during the First Meiotic Prophase of Grasshoppers

A previous version of this article appeared as an Early Online Release on January 2, 2007 (doi:10.1371/journal.pgen.0030028.eor).The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.This work was supported by grants BFU2005–05668-C03–01, BFU2006–06655, BFU2005–01266, BFU2005–02431, and BFU2006–04406 from Ministerio de Educación y Ciencia, España, and grants 1001160016 and 11/BCB/013 from Universidad Autónoma de Madrid and Comunidad de Madrid. The Department of Immunology and Oncology was founded and is supported by the Spanish Council for Scientific Research (CSIC).Peer reviewe

Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling

Multi-cellular organisms need to successfully link cell growth and metabolism to environmental cues during development. Insulin receptor–target of rapamycin (InR–TOR) signalling is a highly conserved pathway that mediates this link. Herein, we describe poly, an essential gene in Drosophila that mediates InR–TOR signalling. Loss of poly results in lethality at the third instar larval stage, but only after a stage of extreme larval longevity. Analysis in Drosophila demonstrates that Poly and InR interact and that poly mutants show an overall decrease in InR–TOR signalling, as evidenced by decreased phosphorylation of Akt, S6K and 4E-BP. Metabolism is altered in poly mutants, as revealed by microarray expression analysis and a decreased triglyceride : protein ratio in mutant animals. Intriguingly, the cellular distribution of Poly is dependent on insulin stimulation in both Drosophila and human cells, moving to the nucleus with insulin treatment, consistent with a role in InR–TOR signalling. Together, these data reveal that Poly is a novel, conserved (from flies to humans) mediator of InR signalling that promotes an increase in cell growth and metabolism. Furthermore, homology to small subunits of Elongator demonstrates a novel, unexpected role for this complex in insulin signalling

Sequential Loading of Cohesin Subunits during the First Meiotic Prophase of Grasshoppers

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I

Dietary Modulation of Drosophila Sleep-Wake Behaviour

Background A complex relationship exists between diet and sleep but despite its impact on human health, this relationship remains uncharacterized and poorly understood. Drosophila melanogaster is an important model for the study of metabolism and behaviour, however the effect of diet upon Drosophila sleep remains largely unaddressed. Methodology/Principal Findings Using automated behavioural monitoring, a capillary feeding assay and pharmacological treatments, we examined the effect of dietary yeast and sucrose upon Drosophila sleep-wake behaviour for three consecutive days. We found that dietary yeast deconsolidated the sleep-wake behaviour of flies by promoting arousal from sleep in males and shortening periods of locomotor activity in females. We also demonstrate that arousal from nocturnal sleep exhibits a significant ultradian rhythmicity with a periodicity of 85 minutes. Increasing the dietary sucrose concentration from 5% to 35% had no effect on total sucrose ingestion per day nor any affect on arousal, however it did lengthen the time that males and females remained active. Higher dietary sucrose led to reduced total sleep by male but not female flies. Locomotor activity was reduced by feeding flies Metformin, a drug that inhibits oxidative phosphorylation, however Metformin did not affect any aspects of sleep. Conclusions We conclude that arousal from sleep is under ultradian control and regulated in a sex-dependent manner by dietary yeast and that dietary sucrose regulates the length of time that flies sustain periods of wakefulness. These findings highlight Drosophila as an important model with which to understand how diet impacts upon sleep and wakefulness in mammals and humans

A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity

Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes

Fermitins, the orthologs of mammalian Kindlins, regulate the development of a functional cardiac syncytium in Drosophila melanogaster.

The vertebrate Kindlins are an evolutionarily conserved family of proteins critical for integrin signalling and cell adhesion. Kindlin-2 (KIND2) is associated with intercalated discs in mice, suggesting a role in cardiac syncytium development; however, deficiency of Kind2 leads to embryonic lethality. Morpholino knock-down of Kind2 in zebrafish has a pleiotropic effect on development that includes the heart. It therefore remains unclear whether cardiomyocyte Kind2 expression is required for cardiomyocyte junction formation and the development of normal cardiac function. To address this question, the expression of Fermitin 1 and Fermitin 2 (Fit1, Fit2), the two Drosophila orthologs of Kind2, was silenced in Drosophila cardiomyocytes. Heart development was assessed in adult flies by immunological methods and videomicroscopy. Silencing both Fit1 and Fit2 led to a severe cardiomyopathy characterised by the failure of cardiomyocytes to develop as a functional syncytium and loss of synchrony between cardiomyocytes. A null allele of Fit1 was generated but this had no impact on the heart. Similarly, the silencing of Fit2 failed to affect heart function. In contrast, the silencing of Fit2 in the cardiomyocytes of Fit1 null flies disrupted syncytium development, leading to severe cardiomyopathy. The data definitively demonstrate a role for Fermitins in the development of a functional cardiac syncytium in Drosophila. The findings also show that the Fermitins can functionally compensate for each other in order to control syncytium development. These findings support the concept that abnormalities in cardiomyocyte KIND2 expression or function may contribute to cardiomyopathies in humans