A chromatin remodelling SWI/SNF subunit, Snr1, regulates neural stem cell determination and differentiation.

Coordinated spatio-temporal regulation of the determination and differentiation of neural stem cells is essential for brain development. Failure to integrate multiple factors leads to defective brain structures or tumour formation. Previous studies suggest changes of chromatin state are needed to direct neural stem cell differentiation, but the mechanisms are unclear. Analysis of Snr1, the Drosophila orthologue of SMARCB1, an ATP-dependent chromatin remodelling protein, identified a key role in regulating the transition of neuroepithelial cells into neural stem cells and subsequent differentiation of neural stem cells into the cells needed to build the brain. Loss of Snr1 in neuroepithelial cells leads to premature neural stem cell formation. Additionally, loss of Snr1 in neural stem cells results in inappropriate perdurance of neural stem cells into adulthood. Snr1 reduction in neuroepithelial or neural stem cells leads to the differential expression of target genes. We find that Snr1 is associated with the actively transcribed chromatin region of these target genes. Thus, Snr1 likely regulates the chromatin state in neuroepithelial cells and maintains chromatin state in neural stem cells for proper brain development.

Recruitment of Peroxin 14 to lipid droplets affects lipid storage in Drosophila.

Both peroxisomes and lipid droplets regulate cellular lipid homeostasis. Direct inter-organellar contacts as well as novel roles for proteins associated with peroxisome or lipid droplets occur when cells are induced to liberate fatty acids from lipid droplets. We have shown a non-canonical role for a subset of peroxisome-assembly [Peroxin (Pex)] proteins in this process in Drosophila. Transmembrane proteins Pex3, Pex13 and Pex14 were observed to surround newly formed lipid droplets. Trafficking of Pex14 to lipid droplets was enhanced by loss of Pex19, which directs insertion of transmembrane proteins like Pex14 into the peroxisome bilayer membrane. Accumulation of Pex14 around lipid droplets did not induce changes to peroxisome size or number, and co-recruitment of the remaining Peroxins was not needed to assemble peroxisomes observed. Increasing the relative level of Pex14 surrounding lipid droplets affected the recruitment of Hsl lipase. Fat body-specific reduction of these lipid droplet-associated Peroxins caused a unique effect on larval fat body development and affected their survival on lipid-enriched or minimal diets. This revealed a heretofore unknown function for a subset of Pex proteins in regulating lipid storage. This article has an associated First Person interview with Kazuki Ueda, joint first author of the paper.

Role of nuclear-cytoplasmic protein localization during Drosophila neuroblast development.

Nuclear-cytoplasmic localization is an efficient way to regulate transcription factors and chromatin remodelers. Altering the location of existing protein pools also facilitates a more rapid response to changes in cell activity or extracellular signals. There are several examples of proteins that are regulated by nucleo-cytoplasmic shuttling, which are required for Drosophila neuroblast development. Disruption of the localization of homologs of these proteins has also been linked to several neurodegenerative disorders in humans. Drosophila has been used extensively to model the neurodegenerative disorders caused by aberrant nucleo-cytoplasmic localization. Here, we focus on the role of alternative nucleo-cytoplasmic protein localization in regulating proliferation and cell fate decisions in the Drosophila neuroblast and in neurodegenerative disorders. We also explore the analogous role of RNA binding proteins and mRNA localization in the context of regulation of nucleo-cytoplasmic localization during neural development and a role in neurodegenerative disorders.

Loss of the Drosophila melanogaster DEAD box protein Ddx1 leads to reduced size and aberrant gametogenesis.

Mammalian DDX1 has been implicated in RNA trafficking, DNA double-strand break repair and RNA processing; however, little is known about its role during animal development. Here, we report phenotypes associated with a null Ddx1 (Ddx1(AX)) mutation generated in Drosophila melanogaster. Ddx1 null flies are viable but significantly smaller than control and Ddx1 heterozygous flies. Female Ddx1 null flies have reduced fertility with egg chambers undergoing autophagy, whereas males are sterile due to disrupted spermatogenesis. Comparative RNA sequencing of control and Ddx1 null third instars identified several transcripts affected by Ddx1 inactivation. One of these, Sirup mRNA, was previously shown to be overexpressed under starvation conditions and implicated in mitochondrial function. We demonstrate that Sirup is a direct binding target of Ddx1 and that Sirup mRNA is differentially spliced in the presence or absence of Ddx1. Combining Ddx1 null mutation with Sirup dsRNA-mediated knock-down causes epistatic lethality not observed in either single mutant. Our data suggest a role for Drosophila Ddx1 in stress-induced regulation of splicing.

Twenty-year follow-up of newborn screening for patients with muscular dystrophy.

INTRODUCTION: An opt-out newborn screening (NBS) program for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) was implemented at 2 hospitals in Pittsburgh, Pennsylvania, between 1987 and 1995. METHODS: For patients and their parents in families who received a diagnosis of DMD or BMD, either by NBS or by traditional diagnostics after symptom onset, attitudes toward NBS for DMD and BMD were assessed. RESULTS: All patients and most parents supported NBS for DMD and BMD. In contrast to the NBS parent cohort, the non-NBS cohort felt that diagnosis by NBS would cause anxiety. CONCLUSIONS: There was strong support of NBS for DMD and BMD in both patients and their parents in families who received a diagnosis through NBS or through traditional diagnostics. No negative psychosocial impacts of NBS were identified among those families who received a diagnosis through NBS.

Regulation of cell proliferation and adhesion by means of a novel region of drosophila merlin interacting with Sip1.

BACKGROUND: The tumor suppressor protein merlin is thought to regulate cell proliferation and cell adhesion through interaction with protein partners. Loss of merlin is associated with Neurofibromatosis Type 2 (NF2) tumors. NHERF1 or EBP50 is a scaffolding protein that functions in apical organization of polarized cells. Merlin and NHERF1 have been shown to interact in vitro in vertebrates. We investigate how the Drosophila NHERF1 orthologue, Sip1, and Merlin function to regulate cell proliferation and adhesion. RESULTS: We identify two conserved arginine residues (R325 and R335) in Merlin which, in addition to the FERM domain, are required for interaction with Sip1. Mutation of the arginine residues result in reduced Sip1 binding to Merlin and loss of Merlin growth suppressor function. Over-expression of Merlin(R325A) and/or Merlin(R335L) in Drosophila wings result in increased proliferation in the adult wing (increase in size), which is rescued by co-over-expression of constitutively active Merlin protein. Reduced Sip1 binding to Merlin also produces defects in adhesion in follicle epithelial cells. CONCLUSIONS: Sip1 facilitates the activation of Merlin as a tumor suppressor protein. Thus, our work provides insight into how Merlin functions as a tumor suppressor and in adhesion and this provides insight into the mechanism of NF2 pathogenesis.

Understanding ERM proteins--the awesome power of genetics finally brought to bear.

In epithelial cells, the Ezrin, Radixin and Moesin (ERM) proteins are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. However, discerning the specific cellular roles of ERMs has been complicated by redundancy between these proteins. Recent genetic studies in model organisms have identified unique roles for ERM proteins. These include the regulation of morphogenesis and maintenance of integrity of epithelial cells, stabilization of intercellular junctions, and regulation of the Rho small GTPase. These studies also suggest that ERMs have roles in actomyosin contractility and vesicular trafficking in the apical domain of epithelial cells. Thus, genetic analysis has enhanced our understanding of these widely expressed membrane-associated proteins.

Parental attitudes toward newborn screening for Duchenne/Becker muscular dystrophy and spinal muscular atrophy.

INTRODUCTION: Disease inclusion in the newborn screening (NBS) panel should consider the opinions of those most affected by the outcome of screening. We assessed the level and factors that affect parent attitudes regarding NBS panel inclusion of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and spinal muscular atrophy (SMA). METHODS: The attitudes toward NBS for DMD, BMD, and SMA were surveyed and compared for 2 categories of parents, those with children affected with DMD, BMD, or SMA and expectant parents unselected for known family medical history. RESULTS: The level of support for NBS for DMD, BMD, and SMA was 95.9% among parents of children with DMD, BMD, or SMA and 92.6% among expectant parents. CONCLUSIONS: There was strong support for NBS for DMD, BMD, and SMA in both groups of parents. Given advances in diagnostics and promising therapeutic approaches, discussion of inclusion in NBS should continue.

The PP1 phosphatase flapwing regulates the activity of Merlin and Moesin in Drosophila.

The signalling activities of Merlin and Moesin, two closely related members of the protein 4.1 Ezrin/Radixin/Moesin family, are regulated by conformational changes. These changes are regulated in turn by phosphorylation. The same sterile 20 kinase-Slik co-regulates Merlin or Moesin activity whereby phosphorylation inactivates Merlin, but activates Moesin. Thus, the corresponding coordinate activation of Merlin and inactivation of Moesin would require coordinated phosphatase activity. We find that Drosophila melanogaster protein phosphatase type 1 ß (flapwing) fulfils this role, co-regulating dephosphorylation and altered activity of both Merlin and Moesin. Merlin or Moesin are detected in a complex with Flapwing both in-vitro and in-vivo. Directed changes in flapwing expression result in altered phosphorylation of both Merlin and Moesin. These changes in the levels of Merlin and Moesin phosphorylation following reduction of flapwing expression are associated with concomitant defects in epithelial integrity and increase in apoptosis in developing tissues such as wing imaginal discs. Functionally, the defects can be partially recapitulated by over expression of proteins that mimic constitutively phosphorylated or unphosphorylated Merlin or Moesin. Our results suggest that changes in the phosphorylation levels of Merlin and Moesin lead to changes in epithelial organization.

Sip1, the Drosophila orthologue of EBP50/NHERF1, functions with the sterile 20 family kinase Slik to regulate Moesin activity.

Organization of the plasma membrane in polarized epithelial cells is accomplished by the specific localization of transmembrane or membrane-associated proteins, which are often linked to cytoplasmic protein complexes, including the actin cytoskeleton. In this study, we identified Sip1 as a Drosophila orthologue of the ezrin-radixin-moesin (ERM) binding protein 50 (EBP50; also known as the Na(+)/H(+) exchanger regulatory factor NHERF1). In mammals, EBP50/NHERF1 is a scaffold protein required for the regulation of several transmembrane receptors and downstream signal transduction activity. In Drosophila, loss of Sip1 leads to a reduction in Slik kinase protein abundance, loss of Moesin phosphorylation and changes in epithelial structure, including mislocalization of E-cadherin and F-actin. Consistent with these findings, Moesin and Sip1 act synergistically in genetic-interaction experiments, and Sip1 protein abundance is dependent on Moesin. Co-immunoprecipitation experiments indicate that Sip1 forms a complex with both Moesin and Slik. Taken together, these data suggest that Sip1 promotes Slik-dependent phosphorylation of Moesin, and suggests a mechanism for the regulation of Moesin activity within the cell to maintain epithelial integrity.

Phosphorylation and activity of the tumor suppressor Merlin and the ERM protein Moesin are coordinately regulated by the Slik kinase.

Merlin and Moesin are closely related members of the 4.1 Ezrin/Radixin/Moesin domain superfamily implicated in regulating proliferation and epithelial integrity, respectively. The activity of both proteins is regulated by head to tail folding that is controlled, in part, by phosphorylation. Few upstream regulators of these phosphorylation events are known. In this study, we demonstrate that in Drosophila melanogaster, Slik, a Ste20 kinase, controls subcellular localization and phosphorylation of Merlin, resulting in the coordinate but opposite regulation of Merlin and Moesin. These results suggest the existence of a novel mechanism for coordinate regulation of cell proliferation and epithelial integrity in developing tissues.

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development.

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.

Drosophila mRNA Localization During Later Development: Past, Present, and Future.

Multiple mechanisms tightly regulate mRNAs during their transcription, translation, and degradation. Of these, the physical localization of mRNAs to specific cytoplasmic regions is relatively easy to detect; however, linking localization to functional regulatory roles has been more difficult to establish. Historically, Drosophila melanogaster is a highly effective model to identify localized mRNAs and has helped identify roles for this process by regulating various cell activities. The majority of the well-characterized functional roles for localizing mRNAs to sub-regions of the cytoplasm have come from the Drosophila oocyte and early syncytial embryo. At present, relatively few functional roles have been established for mRNA localization within the relatively smaller, differentiated somatic cell lineages characteristic of later development, beginning with the cellular blastoderm, and the multiple cell lineages that make up the gastrulating embryo, larva, and adult. This review is divided into three parts-the first outlines past evidence for cytoplasmic mRNA localization affecting aspects of cellular activity post-blastoderm development in Drosophila. The majority of these known examples come from highly polarized cell lineages such as differentiating neurons. The second part considers the present state of affairs where we now know that many, if not most mRNAs are localized to discrete cytoplasmic regions in one or more somatic cell lineages of cellularized embryos, larvae or adults. Assuming that the phenomenon of cytoplasmic mRNA localization represents an underlying functional activity, and correlation with the encoded proteins suggests that mRNA localization is involved in far more than neuronal differentiation. Thus, it seems highly likely that past-identified examples represent only a small fraction of localization-based mRNA regulation in somatic cells. The last part highlights recent technological advances that now provide an opportunity for probing the role of mRNA localization in Drosophila, moving beyond cataloging the diversity of localized mRNAs to a similar understanding of how localization affects mRNA activity.

Flavor preferences produced by backward pairing with wheel running.

In 3 experiments rats given 8 sessions of preexposure to wheel running acquired a preference for a flavor that was given immediately after each of 4 subsequent sessions of wheel running. Such flavor preference was less likely when rats were given the same conditioning procedure but without preexposure to wheels (Experiment 1) or when access to flavor was delayed by 30 min following a wheel session (Experiment 2). When rats were given a flavor before each wheel session, the resulting conditioned aversion was greater in rats that had no prior exposure to wheel running (Experiment 3). These results show that whether an aversion or preference for a flavor is produced by wheel running depends on an interaction between prior wheel experience and the sequence of events.

Moesin is involved in polarity maintenance and cortical remodeling during asymmetric cell division.

An intact actomyosin network is essential for anchoring polarity proteins to the cell cortex and maintaining cell size asymmetry during asymmetric cell division of Drosophila neuroblasts (NBs). However, the mechanisms that control changes in actomyosin dynamics during asymmetric cell division remain unclear. We find that the actin-binding protein, Moesin, is essential for NB proliferation and mitotic progression in the developing brain. During metaphase, phosphorylated Moesin (p-Moesin) is enriched at the apical cortex, and loss of Moesin leads to defects in apical polarity maintenance and cortical stability. This asymmetric distribution of p-Moesin is determined by components of the apical polarity complex and Slik kinase. During later stages of mitosis, p-Moesin localization shifts more basally, contributing to asymmetric cortical extension and myosin basal furrow positioning. Our findings reveal Moesin as a novel apical polarity protein that drives cortical remodeling of dividing NBs, which is essential for polarity maintenance and initial establishment of cell size asymmetry.

Evaluation of melanoma antigen (MAGE) gene expression in human cancers using The Cancer Genome Atlas.

Melanoma antigen (MAGE) cancer-testis (CT) (or cancer-germline) genes are frequently over-expressed in cancer. However, the types of cancer in which the MAGE genes are over-expressed and the effect of over-expression on outcomes have been difficult to ascertain, and their relevance to progression is unclear. We queried transcriptomic and survival data from 26 cancer studies through the cBio Cancer Genomics Portal (www.cbioportal.org) to investigate dysregulation of 34 MAGE genes in cancer. MAGE genes are dysregulated in many cancers. Up to 44% of samples had over-expression of 22 Type 1 MAGE, which are not expressed in most normal tissues, or dysregulation of 12 Type 2 MAGE genes, which are expressed in normal tissues. Dysregulation of specific MAGE genes correlated with reduced overall survival in four cancer types (brain low grade gliomas, renal clear cell carcinomas, head and neck squamous cell carcinomas, and uterine corpus endometrioid carcinomas), with weaker correlations in four additional cancer types. This study demonstrates (1) the value of cancer-germline antigen expression profiling as a complement to mutation analysis for prognosis in cancer; (2) that over-expression of MAGE genes in a subset of cancers is associated with reduced survival; and (3) that patterns of MAGE expression could inform individualized treatment in cancer.

Interaction with the effector dynamin-related protein 1 (Drp1) is an ancient function of Rab32 subfamily proteins.

The mitochondria-associated membrane (MAM) is an endoplasmic reticulum (ER) domain that forms contacts with mitochondria and accommodates Ca2+ transfer between the two organelles. The GTPase Rab32 regulates this function of the MAM via determining the localization of the Ca2+ regulatory transmembrane protein calnexin to the MAM. Another function of the MAM is the regulation of mitochondrial dynamics mediated by GTPases such as dynamin-related protein 1 (Drp1). Consistent with the importance of the MAM for mitochondrial dynamics and the role of Rab32 in MAM enrichment, the inactivation of Rab32 leads to mitochondrial collapse around the nucleus. However, Rab32 and related Rabs also perform intracellular functions at locations other than the MAM including melanosomal trafficking, autophagosome formation and maturation, and retrograde trafficking to the trans-Golgi network (TGN). This plethora of functions raises questions concerning the original cellular role of Rab32 in the last common ancestor of animals and its possible role in the last eukaryotic common ancestor (LECA). Our results now shed light on this conundrum and identify a role in Drp1-mediated mitochondrial dynamics as one common denominator of this group of Rabs, which includes the paralogues Rab32A and Rab32B, as well as the more recently derived Rab29 and Rab38 proteins. Moreover, we provide evidence that this mitochondrial function is dictated by the extent of ER-association of Rab32 family proteins.

The Smc5/Smc6/MAGE complex confers resistance to caffeine and genotoxic stress in Drosophila melanogaster.

The SMC5/6 protein complex consists of the Smc5, Smc6 and Non-Smc-Element (Nse) proteins and is important for genome stability in many species. To identify novel components in the DNA repair pathway, we carried out a genetic screen to identify mutations that confer reduced resistance to the genotoxic effects of caffeine, which inhibits the ATM and ATR DNA damage response proteins. This approach identified inactivating mutations in CG5524 and MAGE, homologs of genes encoding Smc6 and Nse3 in yeasts. The fact that Smc5 mutants are also caffeine-sensitive and that Mage physically interacts with Drosophila homologs of Nse proteins suggests that the structure of the Smc5/6 complex is conserved in Drosophila. Although Smc5/6 proteins are required for viability in S. cerevisiae, they are not essential under normal circumstances in Drosophila. However, flies carrying mutations in Smc5, Smc6 and MAGE are hypersensitive to genotoxic agents such as ionizing radiation, camptothecin, hydroxyurea and MMS, consistent with the Smc5/6 complex serving a conserved role in genome stability. We also show that mutant flies are not compromised for pre-mitotic cell cycle checkpoint responses. Rather, caffeine-induced apoptosis in these mutants is exacerbated by inhibition of ATM or ATR checkpoint kinases but suppressed by Rad51 depletion, suggesting a functional interaction involving homologous DNA repair pathways that deserves further scrutiny. Our insights into the SMC5/6 complex provide new challenges for understanding the role of this enigmatic chromatin factor in multi-cellular organisms.

A Drosophila model for the Zellweger spectrum of peroxisome biogenesis disorders.

Human peroxisome biogenesis disorders are lethal genetic diseases in which abnormal peroxisome assembly compromises overall peroxisome and cellular function. Peroxisomes are ubiquitous membrane-bound organelles involved in several important biochemical processes, notably lipid metabolism and the use of reactive oxygen species for detoxification. Using cultured cells, we systematically characterized the peroxisome assembly phenotypes associated with dsRNA-mediated knockdown of 14 predicted Drosophila homologs of PEX genes (encoding peroxins; required for peroxisome assembly and linked to peroxisome biogenesis disorders), and confirmed that at least 13 of them are required for normal peroxisome assembly. We also demonstrate the relevance of Drosophila as a genetic model for the early developmental defects associated with the human peroxisome biogenesis disorders. Mutation of the PEX1 gene is the most common cause of peroxisome biogenesis disorders and is one of the causes of the most severe form of the disease, Zellweger syndrome. Inherited mutations in Drosophila Pex1 correlate with reproducible defects during early development. Notably, Pex1 mutant larvae exhibit abnormalities that are analogous to those exhibited by Zellweger syndrome patients, including developmental delay, poor feeding, severe structural abnormalities in the peripheral and central nervous systems, and early death. Finally, microarray analysis defined several clusters of genes whose expression varied significantly between wild-type and mutant larvae, implicating peroxisomal function in neuronal development, innate immunity, lipid and protein metabolism, gamete formation, and meiosis.

Alternative requirements for Vestigial, Scalloped, and Dmef2 during muscle differentiation in Drosophila melanogaster.

Vertebrate development requires the activity of the myocyte enhancer factor 2 (mef2) gene family for muscle cell specification and subsequent differentiation. Additionally, several muscle-specific functions of MEF2 family proteins require binding additional cofactors including members of the Transcription Enhancing Factor-1 (TEF-1) and Vestigial-like protein families. In Drosophila there is a single mef2 (Dmef2) gene as well single homologues of TEF-1 and vestigial-like, scalloped (sd), and vestigial (vg), respectively. To clarify the role(s) of these factors, we examined the requirements for Vg and Sd during Drosophila muscle specification. We found that both are required for muscle differentiation as loss of sd or vg leads to a reproducible loss of a subset of either cardiac or somatic muscle cells in developing embryos. This muscle requirement for Sd or Vg is cell specific, as ubiquitous overexpression of either or both of these proteins in muscle cells has a deleterious effect on muscle differentiation. Finally, using both in vitro and in vivo binding assays, we determined that Sd, Vg, and Dmef2 can interact directly. Thus, the muscle-specific phenotypes we have associated with Vg or Sd may be a consequence of alternative binding of Vg and/or Sd to Dmef2 forming alternative protein complexes that modify Dmef2 activity.