What's hot today
Wednesday, December 31st, 2014
Galindo, K. A., Endicott, T. R., Avirneni-Vadlamudi, U. and Galindo, R. L. (2014). A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25491943
Rhabdomyosarcoma (RMS) is an aggressive childhood malignancy of neoplastic muscle-lineage precursors that fail to terminally differentiate into syncytial muscle. The most aggressive form of RMS, Alveolar-RMS (A-RMS), is driven by misexpression of the PAX-FOXO1 oncoprotein, which is generated by recurrent chromosomal translocations that fuse either the PAX3 or PAX7 gene (homologs of Drosophila Paired) to FOXO1 (homolog of Drosophila Foxo). The molecular underpinnings of PAX-FOXO1-mediated RMS pathogenesis remain unclear, however, and clinical outcomes poor. This study reports a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation. With this system, a comprehensive deletion screen was performed against the Drosophila autosomes, and mutation of Mef2, a myogenesis lynchpin in both flies and mammals, was demonstrated to dominantly suppresses PAX7-FOXO1 pathogenicity and act as a PAX7-FOXO1 gene target. Additionally, mutation of mastermind, a gene encoding a MEF2 transcriptional co-activator, was shown to similarly suppress PAX7-FOXO1, further pointing towards MEF2 transcriptional activity as a PAX-FOXO1 underpinning. These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease.
Saitoh, Y., Fujikake, N., Okamoto, Y., Popiel, H. A., Hatanaka, Y., Ueyama, M., Suzuki, M., Gaumer, S., Murata, M., Wada, K. and Nagai, Y. (2014). P62 plays a protective role in the autophagic degradation of polyglutamine protein oligomers in polyglutamine disease model flies. J Biol Chem [Epub ahead of print]. PubMed ID: 25480790
Oligomer formation and accumulation of pathogenic proteins are key events in the pathomechanisms of many neurodegenerative diseases, such as Alzheimer's disease, ALS, and the polyglutamine (polyQ) diseases. The autophagy-lysosome degradation system may have therapeutic potential against these diseases since it can degrade even large oligomers. Although p62/sequestosome1 plays a physiological role in selective autophagy of ubiquitinated proteins, whether p62 recognizes and degrades pathogenic proteins in neurodegenerative diseases has remained unclear. This study elucidates the role of p62 in such pathogenic conditions in vivo using Drosophila models of neurodegenerative diseases. p62 was shown to predominantly co-localize with cytoplasmic polyQ protein aggregates in the MJDtr-Q78 polyQ disease model flies. Loss of p62 function resulted in significant exacerbation of eye degeneration in these flies. Immunohistochemical analyses revealed enhanced accumulation of cytoplasmic aggregates by p62 knockdown in the MJDtr-Q78 flies, similarly to knockdown of Autophagy-related genes (Atgs). Knockdown of both p62 and Atgs did not show any additive effects in the MJDtr-Q78 flies, implying that p62 function is mediated by autophagy. Biochemical analyses showed that loss of p62 function delays the degradation of the MJDtr-Q78 protein, especially its oligomeric species. It was also found that loss of p62 function exacerbates eye degeneration in another polyQ disease fly model, as well as ALS model flies. It is therefore concluded that p62 plays a protective role against polyQ-induced neurodegeneration, by the autophagic degradation of polyQ protein oligomers in vivo, indicating its therapeutic potential for the polyQ diseases, and possibly for other neurodegenerative diseases.
Babcock, D. T., Shen, W. and Ganetzky, B. (2014). A neuroprotective function of NSF1 sustains autophagy and lysosomal trafficking in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 25519897
A common feature of many neurodegenerative diseases is the accumulation of toxic proteins that disrupt vital cellular functions. Degradative pathways such as autophagy play an important protective role in breaking down misfolded and long-lived proteins. Neurons are particularly vulnerable to defects in these pathways, but many of the details regarding the link between autophagy and neurodegeneration remain unclear. Previous studies found that temperature-sensitive (ts) paralytic mutants in Drosophila are enriched for those exhibiting age-dependent neurodegeneration. This study shows that one of these mutants, comatose (comt), in addition to locomotor defects, displays shortened lifespan and progressive neurodegeneration, including loss of dopaminerigic (DA) neurons. comt encodes N-ethyl-maleimide sensitive fusion protein (NSF1), which has a well-documented role in synaptic transmission. However, the neurodegenerative phenotypes observed in comt mutants do not appear to depend on defects in synaptic transmission, but rather from their inability to sustain autophagy under stress, due at least in part to a defect in trafficking of lysosomal proteases such as Cathepsin-L. Conversely, over-expression of NSF1 rescues alpha-synuclein-induced toxicity of DA neurons in a model of Parkinson's Disease. These results demonstrate a neuroprotective role for NSF1 that involves mediation of fusion events crucial for degradative pathways such as autophagy, providing greater understanding of cellular dysfunctions common to several neurodegenerative diseases (PubMed).
Lucey, B. P., Leahy, A., Rosas, R. and Shaw, P. J. (2014). A new model to study sleep deprivation-induced seizure. Sleep [Epub ahead of print]. PubMed ID: 25515102
A relationship between sleep and seizures is well-described in both humans and rodent animal models; however, the mechanism underlying this relationship is unknown. Using Drosophila melanogaster mutants with seizure phenotypes, this study demonstrated that seizure activity can be modified by sleep deprivation. Seizure activity was evaluated in an adult bang-sensitive seizure mutant, stress sensitive B (sesB9ed4), and in an adult temperature sensitive seizure mutant seizure (seits1) under baseline and following 12 h of sleep deprivation. The long-term effect of sleep deprivation on young, immature sesB9ed4 flies was also assessed. Sleep deprivation increased seizure susceptibility in adult sesB9ed4 and seits1 mutants. Sleep deprivation also increased seizure susceptibility when sesB was disrupted using RNAi. The effect of sleep deprivation on seizure activity was reduced when sesB9ed4/+ flies were given the anti-seizure drug, valproic acid. In contrast to adult flies, sleep deprivation during early fly development resulted in chronic seizure susceptibility when sesB9ed4/+ became adults. These findings show that Drosophila is a model organism for investigating the relationship between sleep and seizure activity.
Tuesday, December 30th
Papanikolopoulou, K. and Skoulakis, E. M. (2014). Temporally distinct phosphorylations differentiate Tau-dependent learning deficits and premature mortality in Drosophila. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25524708
Abnormally phosphorylated Tau protein, the major component of neurofibrillary tangles, is critical in the pathogenesis of Alzheimer's disease and related Tauopathies. This study used Drosophila to examine the role of key disease-associated phosphorylation sites on Tau-mediated neurotoxicity. Evidence is presented that the late-appearing phosphorylation on Ser238 rather than hyper-phosphorylation per se is essential for Tau toxicity underlying premature mortality in adult flies. This site is also occupied at the time of neurodegeneration onset in a mouse Tauopathy model and in damaged brain areas of confirmed Tauopathy patients suggesting a similar critical role on Tau toxicity in humans. In contrast, occupation of Ser262 is necessary for Tau-dependent learning deficits in adult Drosophila. Significantly, occupation of Ser262 precedes and is required for Ser238 phosphorylation and these temporally distinct phosphorylations likely reflect conformational changes. Because sequential occupation of Ser262 and Ser238 is required for the progression from Tau-mediated learning deficits to premature mortality in Drosophila, they may also play similar roles in the escalating symptom severity in Tauopathy patients, congruent with their presence in damaged regions of their brains.
Huang, Y., Wu, Z. and Zhou, B. (2014). hSOD1 promotes tau phosphorylation and toxicity in the Drosophila model. J Alzheimers Dis [Epub ahead of print]. PubMed ID: 25524953
Tau hyperphosphorylation has been found in several neurodegenerative diseases such as Alzheimer's disease (AD), Down syndrome, and amyotrophic lateral sclerosis (ALS). However, factors affecting tau hyperphosphorylation are not yet clearly understood. SOD1, a Cu/Zn superoxide dismutase whose mutations can cause adult-onset ALS, is believed to be involved in the pathology of Down syndrome. This work used Drosophila to study the possible link between hSOD1 and tau. The results show that hSOD1, and to a higher degree hSOD1(A4V), can increase tau toxicity in Drosophila and exacerbate the corresponding neurodegeneration phenotype. The increased tau toxicity appears to be explainable by elevated tau phosphorylation. Tau(S2A), a tau mutant with impaired phosphorylation capabilities, does not respond to expression of hSOD1 and hSOD1(A4V). It is suggested that increased SOD1 expression can lead to tau hyperphosphorylation, which might serve as an important contributing factor to the etiology of Down syndrome and SOD1-related ALS disease.
Buttner, S., Broeskamp, F., Sommer, C., Markaki, M., Habernig, L., Alavian-Ghavanini, A., Carmona-Gutierrez, D., Eisenberg, T., Michael, E., Kroemer, G., Tavernarakis, N., Sigrist, S. J. and Madeo, F. (2014)S. Spermidine protects against alpha-synuclein neurotoxicity. Cell Cycle [Epub ahead of print] PubMed ID: 25483063
As society ages, neurodegenerative disorders like Parkinson`s disease (PD) are increasing in pandemic proportions. While mechanistic understanding of PD is advancing, a treatment with well tolerable drugs is still elusive. This study shows that administration of the naturally occurring polyamine spermidine, which declines continuously during aging in various species, alleviates a series of PD-related degenerative processes in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, two established model systems for PD pathology. In the fruit fly, simple feeding with spermidine inhibits loss of climbing activity and early organismal death upon heterologous expression of human alpha-synuclein, which is thought to be the principal toxic trigger of PD. In this line, administration of spermidine rescued alpha-synuclein-induced loss of dopaminergic neurons, a hallmark of PD, in nematodes. Alleviation of PD-related neurodegeneration by spermidine was accompanied by induction of autophagy, suggesting that this cytoprotective process may be responsible for the beneficial effects of spermidine administration.
Wiemerslage, L. and Lee, D. (2014). Role of Drosophila calcium channel Cacophony in dopaminergic neurodegeneration and neuroprotection. Neurosci Lett 584C: 342-346. PubMed ID: 25445363
One of the most important questions in Parkinson's disease (PD) regards the selective vulnerability of a specific population of dopaminergic (DA) neurons. Recent reports identify Ca2+ channel as a potential source of this vulnerability. This work uses a Drosophila primary neuronal cell culture system as an in vitro PD model to explore the role of Ca2+ homeostasis in DA neurodegeneration and protection. The data showed that the Ca2+ chelator EGTA is neuroprotective against a PD toxin MPP+ (40mμM). The genetic tools available in Drosophila were used to manipulate Ca2+ channel activity. DA neurons lacking functional Ca2+ channels (i.e., cacophony mutant) are inherently protected against MPP+ toxicity. Furthermore, overexpression of wild type Ca2+ channels in DA neurons blocks the rescue effect of a D2 agonist quinpirole on DA neurodegeneration. The findings support the idea that Ca2+ is a source of vulnerability for DA neurons and that the modulation of Ca2+ levels in DA neurons could be a potential neuroprotective treatment.
Monday, December 29th
Zabidi, M. A., Arnold, C. D., Schernhuber, K., Pagani, M., Rath, M., Frank, O. and Stark, A. (2014). Enhancer--core-promoter specificity separates developmental and housekeeping gene regulation. Nature [Epub ahead of print]. PubMed ID: 25517091
Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. This study shows that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters-one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor-and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, two transcription factors - Dref and Trl - were identified that bind and activate housekeeping versus developmental enhancers, respectively. These results provide evidence for a sequence-encoded enhancer-core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.
Zehavi, Y., Sloutskin, A., Kuznetsov, O. and Juven-Gershon, T. (2014). The core promoter composition establishes a new dimension in developmental gene networks. Nucleus 5: 298-303. PubMed ID: 25482118
opmental processes are highly dependent on transcriptional regulation by RNA polymerase II, which initiates transcription at the core promoter. The dorsal-ventral gene regulatory network (GRN) includes multiple genes that are activated by different nuclear concentrations of the Dorsal transcription factor along the dorsal-ventral axis. Downstream core promoter element (DPE)-containing genes are conserved and highly prevalent among Dorsal target genes. Moreover, the DPE motif is functional in multiple Dorsal target genes, as mutation of the DPE results in the loss of transcriptional activity. Furthermore, analysis of hybrid enhancer-promoter constructs reveals that the core promoter composition plays a pivotal role in the transcriptional output. Importantly, in vivo evidence is provided that expression driven by the homeotic Antennapedia P2 promoter during Drosophila embryogenesis is dependent on the DPE. Taken together, it is proposed that transcriptional regulation results from the interplay between enhancers and core promoter composition, thus establishing a novel dimension in developmental GRNs.
Takayanagi, S., Toba, G., Lukacsovich, T., Ote, M., Sato, K. and Yamamoto, D. (2014). A fruitless upstream region that defines the species specificity in the male-specific muscle patterning in Drosophila. J Neurogenet: 1-7. PubMed ID: 25518733
The muscle of Lawrence (MOL) is a male-specific muscle present in the abdomen of some adult Drosophila species. Formation of the MOL depends on innervation by motoneurons that express fruitless, a neural male determinant. Drosophila melanogaster males carry a pair of MOLs in the 5th abdominal segment, whereas D. subobscura males carry a pair in both the 5th and 4th segments. It was hypothesized that the fru gene of D. subobscura but not that of D. melanogaster contains a cis element that directs the formation of the additional pair of MOLs. Successively extended 5' DNA fragments to the P1 promoter of D. subobscura or the corresponding fragments that are chimeric (i.e., containing both melanogaster and subobscura elements) were introduced into D. melanogaster and tested for their ability to induce the MOL to locate the hypothetical cis element. A 1.5-2-kb genomic fragment located 4-6-kb upstream of the P1 promoter in D. subobscura but not that of D. melanogaster was found to permit MOL formation in females, provided this fragment is grafted to the distal approximately 4-kb segment from D. melanogaster, demonstrating that this genomic fragment of D. subobscura contains a cis element for the MOL induction.
Zabet, N. R. and Adryan, B. (2014). Estimating binding properties of transcription factors from genome-wide binding profiles. Nucleic Acids Res. PubMed ID: 25432957
The binding of transcription factors (TFs) is essential for gene expression. One important characteristic is the actual occupancy of a putative binding site in the genome. In this study, an analytical model is proposed to predict genomic occupancy that incorporates the preferred target sequence of a TF in the form of a position weight matrix (PWM), DNA accessibility data (in the case of eukaryotes), the number of TF molecules expected to be bound specifically to the DNA and a parameter that modulates the specificity of the TF. Given actual occupancy data in the form of ChIP-seq profiles, copy number and specificity are backwards inferred for five Drosophila TFs during early embryonic development: Bicoid, Caudal, Giant, Hunchback and Kruppel. The results suggest that these TFs display thousands of molecules that are specifically bound to the DNA and that whilst Bicoid and Caudal display a higher specificity, the other three TFs (Giant, Hunchback and Kruppel) display lower specificity in their binding (despite having PWMs with higher information content). This study gives further weight to earlier investigations into TF copy numbers that suggest a significant proportion of molecules are not bound specifically to the DNA.
Sunday, December 28th
Bontonou, G., Shaik, H. A., Denis, B. and Wicker-Thomas, C. (2014). Acp70A regulates Drosophila pheromones through juvenile hormone induction. Insect Biochem Mol Biol. PubMed ID: 25484200
Mated Drosophila melanogaster females show a decrease in mating receptivity, enhanced ovogenesis, egg-laying and activation of juvenile hormone (JH) production. Components in the male seminal fluid, especially the sex peptide ACP70A stimulate these responses in females. This study demonstrates that ACP70A is involved in the down-regulation of female sex pheromones and hydrocarbon (CHC) production. Drosophila G10 females which express Acp70A under the control of the vitellogenin gene yp1, produced fewer pheromones and CHCs. There was a dose-dependent relationship between the number of yp1-Acp70A alleles and the reduction of these compounds. Similarly, a decrease in CHCs and diene pheromones was observed in da > Acp70A flies that ubiquitously overexpress Acp70A. Quantitative-PCR experiments showed that the expression of Acp70A in G10 females was the same as in control males and 5 times lower than in da > Acp70A females. Three to four days after injection with 4.8 pmol ACP70A, females from two different strains, exhibited a significant decrease in CHC and pheromone levels. Similar phenotypes were observed in ACP70A injected flies whose ACP70A receptor expression was knocked-down by RNAi and in flies which overexpress ACP70A N-terminal domain. These results suggest that the action of ACP70A on CHCs could be a consequence of JH activation. Female flies exposed to a JH analog had reduced amounts of pheromones, whereas genetic ablation of the corpora allata or knock-down of the JH receptor Met, resulted in higher amounts of both CHCs and pheromonal dienes. Mating had negligible effects on CHC levels, however pheromone amounts were slightly reduced 3 and 4 days post copulation. The physiological significance of ACP70A on female pheromone synthesis is discussed.
Dustin Rubinstein, C. and Wolfner, M. F. (2014). Reproductive hacking; A male seminal protein acts through intact reproductive pathways in female Drosophila. Fly (Austin) 8: 80-85. PubMed ID: 25483253
Seminal proteins are critical for reproductive success in all animals that have been studied. Although seminal proteins have been identified in many taxa, and female reproductive responses to receipt of these proteins have been documented in several, little is understood about the mechanisms by which seminal proteins affect female reproductive physiology. To explore this topic, this study investigated how a Drosophila seminal protein, ovulin, increases ovulation rate in mated females. Ovulation is a relatively simple physiological process, with known female regulators: previous studies have shown that ovulation rate is promoted by the neuromodulator octopamine (OA) in D. melanogaster and other insects. Ovulin was found to stimulate ovulation by increasing OA signaling in the female. This finding supports a model in which a male seminal protein acts through 'hacking' a well-conserved, regulatory system females use to adjust reproductive output, rather than acting downstream of female mechanisms of control or in parallel pathways altogether. Similarities are discussed between two forms of intersexual control of behavior through chemical communication: seminal proteins and pheromones.
Niepielko, M. G. and Yakoby, N. (2014). Evolutionary changes in TGFalpha distribution underlie morphological diversity in eggshells from Drosophila species. Development 141: 4710-4715. PubMed ID: 25468939
Drosophila eggshells display remarkable morphological diversity among species; however, the molecular origin of this structural diversification is mostly unknown. This study analyzed the dorsal ridge (DR), a lumen-like structure along the dorsal side of eggshells, from numerous Drosophila species. This structure varies in length and width across species, and is absent from D. melanogaster eggshells. DR formation with distinct spatiotemporal changes in epidermal growth factor receptor (EGFR) activation, which acts as a key receptor in eggshell patterning. Changes in the distribution of the TGFalpha-like ligand Gurken (GRK), a crucial ligand for axis formation, underlies EGFR activation and DR formation in D. willistoni. Furthermore, this study demonstrates that GRK from D. willistoni rescues a grk-null D. melanogaster fly and, remarkably, it is also sufficient to generate a DR-like structure on its eggshell.
Qian, Y., Ng, C. L. and Schulz, C. (2014). CSN maintains the germline cellular microenvironment and controls the level of stem cell genes via distinct CRLs in testes of Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 25459658
Stem cells and their daughters are often associated with and depend on cues from their cellular microenvironment. In Drosophila testes, each Germline Stem Cell (GSC) contacts apical hub cells and is enclosed by cytoplasmic extensions from two Cyst Stem Cells (CySCs). Each GSC daughter becomes enclosed by cytoplasmic extensions from two CySC daughters, called cyst cells. CySC fate depends on an Unpaired (Upd) signal from the hub cells, which activates the Janus Kinase and Signal Transducer and Activator of Transcription (Jak/STAT) pathway in the stem cells. Germline enclosure depends on Epidermal Growth Factor (EGF) signals from the germline to the somatic support cells. Expression of RNA-hairpins against subunits of the COnstitutively Photomorphogenic-9- (COP9-) signalosome (CSN; see CSN5) in somatic support cells disrupted germline enclosure. Furthermore, CSN-depleted somatic support cells in the CySC position next to the hub had reduced levels of the Jak/STAT effectors Zinc finger homeotic-1 (Zfh-1) and Chronologically inappropriate morphogenesis (Chinmo). Knockdown of CSN in the somatic support cells does not disrupt EGF and Upd signal transduction as downstream signal transducers, phosphorylated STAT (pSTAT) and phosphorylated Mitogen Activated Protein Kinase (pMAPK), were still localized to the somatic support cell nuclei. The CSN modifies fully formed Cullin RING ubiquitin ligase (CRL) complexes to regulate selective proteolysis. Reducing cullin2 (cul2) from the somatic support cells disrupted germline enclosure, while reducing cullin1 (cul1) from the somatic support cells led to a low level of Chinmo. It is proposed that different CRLs enable the responses of somatic support cells to Upd and EGF
Basiri, M. L., Ha, A., Chadha, A., Clark, N. M., Polyanovsky, A., Cook, B. and Avidor-Reiss, T. (2014). A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids. Curr Biol 24: 2622-2631. PubMed ID: 25447994
In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at the base of the cilium in a region known as the transition zone. The transition zone is closely associated with multiple structures of the ciliary base, including the centriole, axoneme, and ciliary membrane. However, the contribution of these structures to the ciliary gate remains unclear. This study reports that, in Drosophila spermatids, a conserved module of transition zone proteins mutated in Meckel-Gruber syndrome (MKS), including Cep290, Mks1, B9d1, and B9d2, comprise a ciliary gate that continuously migrates away from the centriole to compartmentalize the growing axoneme tip. Cep290 was shown to be essential for transition zone composition, compartmentalization of the axoneme tip, and axoneme integrity; MKS proteins also delimit a centriole-independent compartment in mouse spermatids. These findings demonstrate that the ciliary gate can migrate away from the base of the cilium, thereby functioning independently of the centriole and of a static interaction with the axoneme to compartmentalize the site of axoneme assembly.
Saturday, December 27
Cao, L., Wang, P., Gao, Y., Lin, X., Wang, F. and Wu, S. (2014). Ubiquitin E3 ligase dSmurf is essential for Wts protein turnover and Hippo signaling. Biochem Biophys Res Commun 454: 167-171. PubMed ID: 25450375
The Hippo pathway has been implicated in controlling organ size and tumorigenesis and the underlying molecular mechanisms have attracted intensive attentions. This work identified dSmurf as a new regulator of Wts, a core component of the Hippo pathway, in Drosophila. The data revealed that Wts and dSmurf colocalize to cytoplasm and physically form an immunoprecipitated complex in S2 cells. Sufficient knock-down of dSmurf increases the protein abundance of Wts and thus increases phosphorylation level at S168 of Yki, the key downstream target of Wts in the Hippo pathway. Genetic epistasis assays showed that halving dosage of dSmurf dominantly enhances the phenotype caused by overexpression of Wts and restrains Yki activity in Drosophila eyes. This works defines a novel role of dSmurf in animal development through modulating Wts turnover and thereby Hippo signal transduction, implying that targeting dSmurf may be a promising therapeutic strategy to manipulate the Hippo pathway in pathological conditions.
Vo, N., Horii, T., Yanai, H., Yoshida, H. and Yamaguchi, M. (2014). The Hippo pathway as a target of the Drosophila DRE/DREF transcriptional regulatory pathway. Sci Rep 4: 7196. PubMed ID: 25424907
The DRE/DREF transcriptional regulatory system has been demonstrated to activate a wide variety of genes with various functions. In Drosophila, the Hippo pathway is known to suppress cell proliferation by inducing apoptosis and cell cycle arrest through inactivation of Yorkie, a transcription co-activator. This study found that half dose reduction of the hippo (hpo) gene induces ectopic DNA synthesis in eye discs that is suppressed by overexpression of DREF. Half reduction of the hpo gene dose reduced apoptosis in DREF-overexpressing flies. Consistent with these observations, overexpression of DREF increased the levels of hpo and phosphorylated Yorkie in eye discs. Interestingly, the diap1-lacZ reporter was seen to be significantly decreased by overexpression of DREF. Luciferase reporter assays in cultured S2 cells revealed that one of two DREs identified in the hpo gene promoter region was responsible for promoter activity in S2 cells. Furthermore, endogenous hpo mRNA was reduced in DREF knockdown S2 cells, and chromatin immnunoprecipitation assays with anti-DREF antibodies proved that DREF binds specifically to the hpo gene promoter region containing DREs in vivo. Together, these results indicate that the DRE/DREF pathway is required for transcriptional activation of the hpo gene to positively control Hippo pathways.
Palmer, W. H., Jia, D. and Deng, W. M. (2014). Cis-interactions between Notch and its ligands block ligand-independent Notch activity. Elife 3. PubMed ID: 25486593
The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a two-fold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. This study shows that cells without both cis and trans ligands are able to mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis and trans ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically-induced situations of elevated trans-ligand-independent Notch signaling. It is concluded that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand independent activation, and a new function of cis-inhibition is proposed which buffers cells against accidental Notch activity.
Bell, G. P., Fletcher, G. C., Brain, R. and Thompson, B. J. (2014). Aurora kinases phosphorylate Lgl to induce mitotic spindle orientation in Drosophila epithelia. Curr Biol [Epub ahead of print]. PubMed ID: 25484300
The Lethal giant larvae (Lgl) protein was discovered in Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. This study shows that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and Aurora Bkinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. It is proposed that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. These findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions.
Friday, December 26th
Lee, G. J., Jun, J. W. and Hyun, S. (2014). MicroRNA miR-8 regulates multiple growth factor hormones produced from Drosophila fat cells. Insect Mol Biol. PubMed ID: 25492518
Metabolic organs such as the liver and adipose tissue produce several peptide hormones that influence metabolic homeostasis. Fat bodies, the Drosophila counterpart of liver and adipose tissues, have been thought to analogously secrete several hormones that affect organismal physiology, but their identity and regulation remain poorly understood. Previous studies have indicated that microRNA miR-8, functions in the fat body to non-autonomously regulate organismal growth, suggesting that fat body-derived humoral factors are regulated by imiR-8. This study found that several putative peptide hormones known to have mitogenic effects are regulated by imiR-8 in the fat body. Most members of the imaginal disc growth factors and two members of the adenosine deaminase-related growth factors are up-regulated in the absence of imiR-8. Drosophila insulin-like peptide 6 (Dilp6) and Imaginal morphogenesis protein-late 2 (Imp-L2), a binding partner of Dilp, are also up-regulated in the fat body of miR-8 null mutant larvae. The fat body-specific reintroduction of miR-8 into the miR-8 null mutants revealed six peptides that showed fat-body organ-autonomous regulation by miR-8. Amongst them, only Imp-L2 was found to be regulated by U-shaped, the miR-8 target for body growth. However, a rescue experiment by knockdown of Imp-L2 indicated that Imp-L2 alone does not account for miR-8's control over the insect's growth. These findings suggest that multiple peptide hormones regulated by miR-8 in the fat body may collectively contribute to Drosophila growth.
Ghosh, S., Wang, Y., Cook, J. A., Chhiba, L. and Vaughn, J. C. (2013). A molecular, phylogenetic and functional study of the mRNA truncated isoform during embryonic development reveals an editing-independent function. Open J Anim Sci 3: 20-30. PubMed ID: 25414802
Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3'-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition. 3'-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5'-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, RNAi technology was used, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.
Bradley, T., Cook, M. E. and Blanchette, M. (2015). SR proteins control a complex network of RNA-processing events. RNA 21(1):75-92. PubMed ID: 25414008
SR proteins are a well-conserved class of RNA-binding proteins that are essential for regulation of splice-site selection, and have also been implicated as key regulators during other stages of RNA metabolism. For many SR proteins, the complexity of the RNA targets and specificity of RNA-binding location are poorly understood. It is also unclear if general rules governing SR protein alternative pre-mRNA splicing (AS) regulation uncovered for individual SR proteins on few model genes, apply to the activity of all SR proteins on endogenous targets. Using RNA-seq, this study characterized the global AS regulation of the eight Drosophila SR protein family members. A majority of AS events are regulated by multiple SR proteins, and that all SR proteins can promote exon inclusion, but also exon skipping. Most coregulated targets exhibit cooperative regulation, but some AS events are antagonistically regulated. Additionally, it was found that SR protein levels can affect alternative promoter choices and polyadenylation site selection, as well as overall transcript levels. Cross-linking and immunoprecipitation coupled with high-throughput sequencing (iCLIP-seq), reveals that SR proteins bind a distinct and functionally diverse class of RNAs, which includes several classes of noncoding RNAs, uncovering possible novel functions of the SR protein family. Finally, it was found that SR proteins exhibit positional RNA binding around regulated AS events. Therefore, regulation of AS by the SR proteins is the result of combinatorial regulation by multiple SR protein family members on most endogenous targets, and SR proteins have a broader role in integrating multiple layers of gene expression regulation.
Wang, W., Yoshikawa, M., Han, B. W., Izumi, N., Tomari, Y., Weng, Z. and Zamore, P. D. (2014). The initial uridine of primary piRNAs does not create the tenth adenine that is the hallmark of secondary piRNAs. Mol Cell 56: 708-716. PubMed ID: 25453759
PIWI-interacting RNAs (piRNAs) silence transposons in animal germ cells. PIWI proteins bind and amplify piRNAs via the "Ping-Pong" pathway. Because PIWI proteins cleave RNAs between target nucleotides t10 and t11-the nucleotides paired to piRNA guide positions g10 and g11-the first ten nucleotides of piRNAs participating in the Ping-Pong amplification cycle are complementary. Drosophila piRNAs bound to the PIWI protein Aubergine typically begin with uridine (1U), while piRNAs bound to Argonaute3, which are produced by Ping-Pong amplification, often have adenine at position 10 (10A). The Ping-Pong model proposes that the 10A is a consequence of 1U. This study found that 10A is not caused by 1U. Instead, fly Aubergine as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs; during Ping-Pong amplification, this t1A subsequently becomes the g10A of a piRNA bound to Argonaute3.
Thursday, December 25th
Zheng, S., Villa, R., Wang, J., Feng, Y., Wang, J., Becker, P. B. and Ye, K. (2014). Structural basis of X chromosome DNA recognition by the MSL2 CXC domain during Drosophila dosage compensation. Genes Dev 28: 2652-2662. PubMed ID: 25452275
The male-specific lethal dosage compensation complex (MSL-DCC) selectively assembles on the X chromosome in Drosophila males and activates gene transcription by twofold through histone acetylation. An MSL recognition element (MRE) sequence motif nucleates the initial MSL association, but how it is recognized remains unknown. This study identified the CXC domain of MSL2 specifically recognizing the MRE motif and determined its crystal structure bound to specific and nonspecific DNAs. The CXC domain primarily contacts one strand of DNA duplex and employs a single arginine to directly read out dinucleotide sequences from the minor groove. The arginine is flexible when bound to nonspecific sequences. The core region of the MRE motif harbors two binding sites on opposite strands that can cooperatively recruit a CXC dimer. Specific DNA-binding mutants of MSL2 are impaired in MRE binding and X chromosome localization in vivo. These results reveal multiple dynamic DNA-binding modes of the CXC domain that target the MSL-DCC to X chromosomes.
Larracuente, A. M. (2014). The organization and evolution of the Responder satellite in species of the Drosophila melanogaster group: dynamic evolution of a target of meiotic drive. BMC Evol Biol 14: 233. PubMed ID: 25424548
Satellite DNA can make up a substantial fraction of eukaryotic genomes and has roles in genome structure and chromosome segregation. The rapid evolution of satellite DNA can contribute to genomic instability and genetic incompatibilities between species. Despite its ubiquity and its contribution to genome evolution, little about the dynamics of satellite DNA evolution. The Responder (Rsp) satellite DNA family is found in the pericentric heterochromatin of chromosome 2 of Drosophila melanogaster. Rsp is well-known for being the target of Segregation Distorter (SD), an autosomal meiotic drive system in D. melanogaster. This paper presents an evolutionary genetic analysis of the Rsp family of repeats in D. melanogaster and its closely-related species in the melanogaster group (D. simulans, D. sechellia, D. mauritiana, D. erecta, and D. yakuba) using a combination of available BAC sequences, whole genome shotgun Sanger reads, Illumina short read deep sequencing, and fluorescence in situ hybridization. Rsp repeats were shown to have euchromatic locations throughout the D. melanogaster genome, that Rsp arrays show evidence for concerted evolution, and that Rsp repeats exist outside of D. melanogaster, in the melanogaster group. The repeats in these species are considerably diverged at the sequence level compared to D. melanogaster, and have a strikingly different genomic distribution, even between closely-related sister taxa. It is concluded that the genomic organization of the Rsp repeat in the D. melanogaster genome is complex. It consists of large blocks of tandem repeats in the heterochromatin and small blocks of tandem repeats in the euchromatin. The discovery of heterochromatic Rsp-like sequences outside of D. melanogaster suggests that SD evolved after its target satellite and that the evolution of the Rsp satellite family is highly dynamic over a short evolutionary time scale (<240,000 years).
Jiang, L., Li, X. N. and Niu, D. K. (2014). Higher frequency of intron loss from the promoter proximally paused genes of Drosophila melanogaster. Fly (Austin) 8: 120-125. PubMed ID: 25483256
Although intron losses have been widely reported, it is not clear whether they are neutral and therefore random or driven by positive selection. Intron transcription and splicing are time-consuming and can delay the expression of its host gene. For genes that must be activated quickly to respond to physiological or stress signals, intron delay may be deleterious. Promoter proximally paused (PPP) genes are a group of rapidly expressed genes. To respond quickly to activation signals, they generally initiate transcription competently but stall after synthesizing a short RNA. In this study, performed in Drosophila melanogaster, the PPP genes were found to have a significantly higher rate of intron loss than control genes. However, further analysis did not find more significant shrinkage of intron size in PPP genes. Referring to previous studies on the rates of transcription and splicing and to the time saved by deletion of the introns from mouse gene Hes7, it is suggested that transcription delay is comparable to splicing delay only when the intron is 28.5 kb or larger, which is greater in size than 95% of vertebrate introns, 99.5% of Drosophila introns, and all the annotated introns of Saccharomyces cerevisiae and Arabidopsis thaliana. Delays in intron splicing are probably a selective force, promoting intron loss from quickly expressed genes. In other genes, it may have been an exaptation (a feature that is not considered an adaptation) during the emergency of developmental clocks.
Liu, H. Q., Li, Y., Irwin, D. M., Zhang, Y. P. and Wu, D. D. (2014). Integrative analysis of young genes, positively selected genes and lncRNAs in the development of Drosophila melanogaster. BMC Evol Biol 14: 241. PubMed ID: 25470998
Young genes and genes under positive selection commonly contribute to adaptive phenotypic evolution. Early developmental stages are very important for establishing phenotypes, which might be helpful for studying the evolutionary patterns of these rapidly evolving genes. This study performed a weighted gene co-expression network analysis to identify modules of co-expressed genes at different stages of Drosophila melanogaster development. Young genes, including duplicated, orphan, and young lncRNA genes, were found to be significantly enriched among modules associated with specific developmental stages. In addition, genes undergoing rapid amino acid sequence evolution driven by positive selection showed a similar proportion of essentiality with other genes, and enrichment in modules for specific developmental stages. This integrative analysis revealed important roles for the origin of new genes and rapid amino acid sequence evolution in development that may account for specific phenotype evolution in Drosophila melanogaster.
Wednesday, December 24th
Jiang, Y., Boll, W. and Noll, M. (2014). Pox neuro control of cell lineages that give rise to larval poly-innervated external sensory organs in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 25446278
The Pox neuro (Poxn) gene of Drosophila plays a crucial role in the development of poly-innervated external sensory (p-es) organs. However, how Poxn exerts this role has remained elusive. This paper analyzes the cell lineages of all larval p-es organs, namely of the kolbchen, papilla 6, and hair 3. Surprisingly, these lineages are distinct from any previously reported cell lineages of sensory organs. Unlike the well-established lineage of mono-innervated external sensory (m-es) organs and a previously proposed model of the p-es lineage, this study demonstrate that all wild-type p-es lineages exhibit the following features: the secondary precursor, pIIa, gives rise to all the three support cells - socket, shaft, and sheath, whereas the other secondary precursor, pIIb, is neuronal and gives rise to all neurons. It was further shown that in one of the p-es lineages, that of papilla 6, one cell undergoes apoptosis. By contrast in Poxn null mutants, all p-es lineages have a reduced number of cells and their pattern of cell divisions is changed to that of an m-es organ, with the exception of a lineage in a minority of mutant kolbchen that retains a second bipolar neuron. Indeed, the role of Poxn in p-es lineages is consistent with the specification of the developmental potential of secondary precursors and the regulation of cell division but not apoptosis.
Lee, L. H. and Godenschwege, T. A. (2014). Structure-function analyses of tyrosine phosphatase PTP69D in giant fiber synapse formation of Drosophila. Mol Cell Neurosci 64C: 24-31. PubMed ID: 25433167
PTP69D is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2) and has been shown to play a role in axon guidance of embryonic motoneurons as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. This study characterized the developmental role of PTP69D in the giant fiber (GF) neurons, two interneurons in the central nervous system (CNS) that control the escape response of the fly. These studies revealed that PTP69D has a function in synaptic terminal growth in the CNS. Missense mutations in the first immunoglobulin (Ig) domain and in the Cat1 domain, present in Ptp69D10 and Ptp69D20 mutants, respectively, did not affect axon guidance or targeting but resulted in stunted terminal growth of the GFs. Cell autonomous rescue experiments demonstrated a function for the Cat1 and the first Ig domain of PTP69D in the GFs but not in its postsynaptic target neurons. In addition, complementation studies and structure-function analyses revealed that for GF terminal growth Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domains, but not the fibronectin III or the membrane proximal region domains. In contrast, the fibronectin III but not the immunoglobulin domains were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, these studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function in photoreceptor targeting.
Nagel, K. I., Hong, E. J. and Wilson, R. I. (2014). Synaptic and circuit mechanisms promoting broadband transmission of olfactory stimulus dynamics. Nat Neurosci [Epub ahead of print]. PubMed ID: 25485755
Sensory stimuli fluctuate on many timescales. However, short-term plasticity causes synapses to act as temporal filters, limiting the range of frequencies that they can transmit. How synapses in vivo might transmit a range of frequencies in spite of short-term plasticity is poorly understood. The first synapse in the Drosophila olfactory system exhibits short-term depression, but can transmit broadband signals. This study describes two mechanisms that broaden the frequency characteristics of this synapse. First, two distinct excitatory postsynaptic currents transmit signals on different timescales. Second, presynaptic inhibition dynamically updates synaptic properties to promote accurate transmission of signals across a wide range of frequencies. Inhibition is transient, but grows slowly, and simulations reveal that these two features of inhibition promote broadband synaptic transmission. Dynamic inhibition is often thought to restrict the temporal patterns that a neuron responds to, but these results illustrate a different idea: inhibition can expand the bandwidth of neural coding.
Lin, C. Y., Chuang, C. C., Hua, T. E., Chen, C. C., Dickson, B. J., Greenspan, R. J. and Chiang, A. S. (2013). A comprehensive wiring diagram of the protocerebral bridge for visual information processing in the Drosophila brain. Cell Rep 3: 1739-1753. PubMed ID: 23707064
How the brain perceives sensory information and generates meaningful behavior depends critically on its underlying circuitry. The protocerebral bridge (PB) is a major part of the insect central complex (CX), a premotor center that may be analogous to the human basal ganglia. By deconstructing hundreds of PB single neurons and reconstructing them into a common three-dimensional framework, this study has constructed a comprehensive map of PB circuits with labeled polarity and predicted directions of information flow. The analysis reveals a highly ordered information processing system that involves directed information flow among CX subunits through 194 distinct PB neuron types. Circuitry properties such as mirroring, convergence, divergence, tiling, reverberation, and parallel signal propagation were observed; their functional and evolutional significance is discussed. This layout of PB neuronal circuitry may provide guidelines for further investigations on transformation of sensory (e.g., visual) input into locomotor commands in fly brains.
Tuesday, December 23rd
James, R. E., Hoover, K. M., Bulgari, D., McLaughlin, C. N., Wilson, C. G., Wharton, K. A., Levitan, E. S. and Broihier, H. T. (2014). Crimpy enables discrimination of presynaptic and postsynaptic pools of a BMP at the Drosophila neuromuscular junction. Dev Cell 31: 586-598. PubMed ID: 25453556
Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. This study presents evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, it is proposed that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal.
Matusek, T., Wendler, F., Poles, S., Pizette, S., D'Angelo, G., Furthauer, M. and Therond, P. P. (2014). The ESCRT machinery regulates the secretion and long-range activity of Hedgehog. Nature 516: 99-103. PubMed ID: 25471885
The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading. Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. This study shows that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT). In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, ESCRT activity in Hh-producing cells is required for long-range signalling. Evidence is provided that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction.
Slawson, J. B., Kuklin, E. A., Mukherjee, K., Pirez, N., Donelson, N. C. and Griffith, L. C. (2014). Regulation of dopamine release by CASK- modulates locomotor initiation in Drosophila. Front Behav Neurosci 8: 394. PubMed ID: 25477794
CASK is an evolutionarily conserved scaffolding protein that has roles in many cell types. In Drosophila, loss of the entire CASK gene or just the CASK-β transcript causes a complex set of adult locomotor defects. This study shows that the motor initiation component of this phenotype is due to loss of CASK-β in dopaminergic neurons and can be specifically rescued by expression of CASK-β within this subset of neurons. Functional imaging demonstrates that mutation of CASK-β disrupts coupling of neuronal activity to vesicle fusion. Consistent with this, locomotor initiation can be rescued by artificially driving activity in dopaminergic neurons. The molecular mechanism underlying this role of CASK-β in dopaminergic neurons involves interaction with Hsc70-4, a molecular chaperone previously shown to regulate calcium-dependent vesicle fusion. These data suggest that there is a novel CASK-β-dependent regulatory complex in dopaminergic neurons that serves to link activity and neurotransmitter release.
Gillingham, A. K., Sinka, R., Torres, I. L., Lilley, K. S. and Munro, S. (2014). Toward a comprehensive map of the effectors of Rab GTPases. Dev Cell 31: 358-373. PubMed ID: 25453831
The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, a comprehensive set of Drosophila Rabs was used for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. Over 25 interactions were confirmed for Rab2, Rab4, Rab5, Rab6, Rab7, Rab9, Rab18, Rab19, Rab30, and Rab39. These include tethering complexes, coiled-coiled proteins, motor linkers, Rab regulators, and several proteins linked to human disease.
Monday, December 22nd
Garcia, J. D., Dewey, E. B. and Johnston, C. A. (2014). Dishevelled binds the Discs large 'Hook' domain to activate GukHolder-dependent spindle positioning in Drosophila. PLoS One 9: e114235. PubMed ID: 25461409
Communication between cortical cell polarity cues and the mitotic spindle ensures proper orientation of cell divisions within complex tissues. Defects in mitotic spindle positioning have been linked to various developmental disorders and have recently emerged as a potential contributor to tumorigenesis. Despite the importance of this process to human health, the molecular mechanisms that regulate spindle orientation are not fully understood. Moreover, it remains unclear how diverse cortical polarity complexes might cooperate to influence spindle positioning. Spindle orientation roles have been identified for Dishevelled (Dsh), a key regulator of planar cell polarity, and Discs large (Dlg), a conserved apico-basal cell polarity regulator, effects which were previously thought to operate within distinct molecular pathways. This study identified a novel direct interaction between the Dsh-PDZ domain and the alternatively spliced "I3-insert" of the Dlg-Hook domain, thus establishing a potential convergent Dsh/Dlg pathway. Furthermore, a Dlg sequence motif that is necessary for the Dsh interaction was identified that shares homology to the site of Dsh binding in the Frizzled receptor. Expression of Dsh enhanced Dlg-mediated spindle positioning similar to deletion of the Hook domain. This Dsh-mediated activation was dependent on the Dlg-binding partner, GukHolder (GukH). These results suggest that Dsh binding may regulate core interdomain conformational dynamics previously described for Dlg. Together, these results identify Dlg as an effector of Dsh signaling and demonstrate a Dsh-mediated mechanism for the activation of Dlg/GukH-dependent spindle positioning. Cooperation between these two evolutionarily-conserved cell polarity pathways could have important implications to both the development and maintenance of tissue homeostasis in animals.
Issman-Zecharya, N. and Schuldiner, O. (2014). The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation. Dev Cell 31: 461-473. PubMed ID: 25458013
Developmental axon pruning is essential for wiring the mature nervous system, but its regulation remains poorly understood. This study shows that the endosomal-lysosomal pathway regulates developmental pruning of Drosophila mushroom body γ neurons. The UV radiation resistance-associated gene (Uvrag) functions together with all core components of the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex to promote pruning via the endocytic pathway. By studying several PI(3)P binding proteins, this study found that Hrs, a subunit of the ESCRT-0 complex, required for multivesicular body (MVB) maturation, is essential for normal pruning progression. Thus, the existence of an inhibitory signal that needs to be downregulated is hypothesized. Finally, the data suggest that the Hedgehog receptor, Patched, is the source of this inhibitory signal likely functioning in a Smo-independent manner. Taken together, this in vivo study demonstrates that the PI3K-cIII complex is essential for downregulating Patched via the endosomal-lysosomal pathway to execute axon pruning.
Auer, J. S., Nagel, A. C., Schulz, A., Wahl, V. and Preiss, A. (2014). MAPK-dependent phosphorylation modulates the activity of Suppressor of Hairless in Drosophila. Cell Signal 27: 115-124. PubMed ID: 25452105
Cell differentiation strictly depends on the epidermal growth factor receptor (EGFR)- and Notch-signalling pathways, which are closely intertwined. This study addresses the molecular cross talk at the level of Suppressor of Hairless [Su(H)]. The Drosophila transcription factor Su(H) mediates Notch signalling at the DNA level: in the presence of signalling input Su(H) assembles an activator complex on Notch target genes and a repressor complex in its absence. Su(H) contains a highly conserved mitogen activated protein kinase (MAPK) target sequence. Evidence is provided that Su(H) is phosphorylated in response to MAPK activity. Mutation of the Su(H) MAPK-site modulated the Notch signalling output: whereas a phospho-deficient Su(H)MAPK-ko isoform provoked a stronger Notch signalling activity, a phospho-mimetic Su(H)MAPK-ac mutant resulted in its attenuation. In vivo assays in Drosophila cell culture as well as in flies support the idea that Su(H) phosphorylation affects the dynamics of repressor or activator complex formation or the transition from the one into the other complex. In summary, the phosphorylation of Su(H) attenuates Notch signalling in vivo in several developmental settings. Consequently, a decrease of EGFR signal causes an increase of Notch signalling intensity. Hence, the antagonistic relationship between EGFR- and Notch-signalling pathways may involve a direct modification of Su(H) by MAPK in several developmental contexts of fly development. The high sequence conservation of the MAPK target site in the mammalian Su(H) homologues supports the idea that EGFR signalling impacts on Notch activity in a similar way in humans as well.
Kwon, H. J., Waghmare, I., Verghese, S., Singh, A., Singh, A. and Kango-Singh, M. (2014). Drosophila C-terminal Src kinase regulates growth via the Hippo signaling pathway. Dev Biol [Epub ahead of print]. PubMed ID: 25446534
The Hippo signaling pathway is involved in regulating tissue size by inhibiting cell proliferation and promoting apoptosis. Aberrant Hippo pathway function is often detected in human cancers and correlates with poor prognosis. The Drosophila C-terminal Src kinase (d-Csk) is a genetic modifier of warts (wts), a tumor-suppressor gene in the Hippo pathway, and interacts with the Src oncogene. Reduction in d-Csk expression and the consequent activation of Src are frequently seen in several cancers including hepatocellular and colorectal tumors. Previous studies show that d-Csk regulates cell proliferation and tissue size during development. Given the similarity in the loss-of-function phenotypes of d-Csk and wts, the interactions of d-Csk with the Hippo pathway were investigated. Multiple lines of evidence are presented suggesting that d-Csk regulates growth via the Hippo signaling pathway. Loss of dCsk caused increased
Yki activity, and genetic epistasis places dCsk downstream of Dachs. Furthermore, dCsk requires Yki for its growth regulatory functions, suggesting that dCsk is another upstream member of the network of genes that interact to regulate Wts and its effector Yki in the Hippo signaling pathway.
Sunday, December 21st
Sullivan, J. M., Broadhead, G. T., Sumner, C. J., Lloyd, T. E., Macleod, G. T., Bellen, H. J. and Venkatachalam, K. (2014). A TRPV channel in Drosophila motor neurons regulates presynaptic resting Ca(2+) levels, synapse growth, and synaptic transmission. Neuron 84: 764-777. PubMed ID: 25451193
Presynaptic resting Ca2+ influences synaptic vesicle (SV) release probability. This study reports that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca2+] by promoting Ca2+ release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. The absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, these findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [2+].
Inagaki, H. K., Panse, K. M. and Anderson, D. J. (2014). Independent, reciprocal neuromodulatory control of sweet and bitter taste sensitivity during starvation in Drosophila. Neuron 84: 806-820. PubMed ID: 25451195
An organism's behavioral decisions often depend upon the relative strength of appetitive and aversive sensory stimuli, the relative sensitivity to which can be modified by internal states like hunger. However, whether sensitivity to such opposing influences is modulated in a unidirectional or bidirectional manner is not clear. Starved flies exhibit increased sugar and decreased bitter sensitivity. It is widely believed that only sugar sensitivity changes, and that this masks bitter sensitivity. This study used gene- and circuit-level manipulations to show that sweet and bitter sensitivity are independently and reciprocally regulated by starvation in Drosophila. Orthogonal neuromodulatory cascades were detected that oppositely control peripheral taste sensitivity for each modality. Moreover, these pathways are recruited at increasing hunger levels, such that low-risk changes (higher sugar sensitivity) precede high-risk changes (lower sensitivity to potentially toxic resources). In this way, state-intensity-dependent, reciprocal regulation of appetitive and aversive peripheral gustatory sensitivity permits flexible, adaptive feeding decisions.
Ryglewski, S., Kadas, D., Hutchinson, K., Schuetzler, N., Vonhoff, F. and Duch, C. (2014). Dendrites are dispensable for basic motoneuron function but essential for fine tuning of behaviorE. Proc Natl Acad Sci U S A. PubMed ID: 25453076
Dendrites are highly complex 3D structures that define neuronal morphology and connectivity and are the predominant sites for synaptic input. Defects in dendritic structure are highly consistent correlates of brain diseases. However, the precise consequences of dendritic structure defects for neuronal function and behavioral performance remain unknown. This study probed dendritic function by using genetic tools to selectively abolish dendrites in identified Drosophila wing motoneurons without affecting other neuronal properties. These motoneuron dendrites were unexpectedly found to be dispensable for synaptic targeting, qualitatively normal neuronal activity patterns during behavior, and basic behavioral performance. However, significant performance deficits in sophisticated motor behaviors, such as flight altitude control and switching between discrete courtship song elements, scale with the degree of dendritic defect. These observations provide the first direct evidence that complex dendrite architecture is critically required for fine-tuning and adaptability within robust, evolutionarily constrained behavioral programs that are vital for mating success and survival. It is speculated that the observed scaling of performance deficits with the degree of structural defect is consistent with gradual increases in intellectual disability during continuously advancing structural deficiencies in progressive neurological disorders.
Zhang, L., Huang, Y., Chen, J. Y., Ding, Y. Q. and Song, N. N. (2015). DSCAM and DSCAML1 regulate the radial migration and callosal projection in developing cerebral cortex. Brain Res 1594: 61-70. PubMed ID: 25451118
Down syndrome cell adhesion molecule (Dscam) is essential for self-avoidance and tiling of dendritic development in sensory neurons in Drosophila. Recent studies also show that DSCAM together with its closely related protein DSCAML1 functions in dendritic self-avoidance of a certain types of interneuron in mammalian retina. However, the functions of these DSCAMs in developing mammalian cerebral cortex are not well understood. This study reduced the expression of DSCAM or DSCAML1 in mouse cortical neurons by RNA interference both in vitro and in vivo. Knockdown of DSCAM or DSCAML1 was found to increase the complexity of proximal dendritic branching, and impedes the axon growth in cultured neurons. In vivo knockdown experiments showed that both DSCAM and DSCAML1 contribute to normal radial migration and callosal projection during the postnatal development. This results indicate an important role of DSCAM and DSCAML1 in the development of cortical neural network.
Saturday, December 20th
Coyne, A. N., Siddegowda, B. B., Estes, P. S., Johannesmeyer, J., Kovalik, T., Daniel, S. G., Pearson, A., Bowser, R. and Zarnescu, D. C. (2014). Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of Amyotrophic Lateral Sclerosis. J Neurosci 34: 15962-15974. PubMed ID: 25429138
TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. This study provides evidence that Drosophila TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where higher levels of transcript were found compared with controls. TDP-43 also leads to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulates the translation of futsch mRNA. futsch overexpression was shown to be neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal cords in a manner similar to these observations in Drosophila motor neurons. Together, theseresults suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo.
Pogson, J. H., Ivatt, R. M., Sanchez-Martinez, A., Tufi, R., Wilson, E., Mortiboys, H. and Whitworth, A. J. (2014). The Complex I subunit NDUFA10 selectively rescues Drosophila pink1 mutants through a mechanism independent of mitophagy. PLoS Genet 10: e1004815. PubMed ID: 25412178
Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson's disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and has recently been suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, a screen was conducted in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen was ND42. In Drosophila pink1 mutants, transgenic overexpression of ND42 or its co-chaperone sicily was sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily failed to rescue any Drosophila parkin mutant phenotypes. It was also found that knockdown of the human homologue, NDUFA10, only minimally affecting CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. These findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy.
Joardar, A., Menzl, J., Podolsky, T. C., Manzo, E., Estes, P. S., Ashford, S. and Zarnescu, D. C. (2014). PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25432537
Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. A Drosophila model has been developed of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, a drug screening strategy was designed based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1,200 FDA approved compounds, the PPARgamma agonist pioglitazone was found to be neuroprotective in Drosophila. This study shows that pioglitazone can rescue TDP-43 dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS it was found that pioglitazone is also neuroprotective when FUS, but not SOD1, is expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models show no increase in lifespan, which is consistent with recent clinical trials. Using a pharmacogenetic approach, this study showed that the predicted Drosophila PPARγ homologs, E75 and E78 are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, a set of metabolites was identified that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, these data provide evidence that modulating PPARγ activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, these data also identifies several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials.
Jackel, S., Summerer, A. K., Thommes, C. M., Pan, X., Voigt, A., Schulz, J. B., Rasse, T. M., Dormann, D., Haass, C. and Kahle, P. J. (2014). Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila. Neurobiol Dis 74C: 76-88. PubMed ID: 25447237
Inclusions containing Fused in Sarcoma (FUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline-tyrosine nuclear localization sequence (PY-NLS) of FUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of FUS is mediated by transportin, which binds the PY-NLS and the last arginine/glycine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of FUS in an in vivo model, different transgenic Drosophila lines were generated expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming FUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, the Drosophila PRMT proteins (DART1-9) were screened, and it was found that knockdown of Dart1 led to a reduction in methylation of hFUS P525L and aggravated its phenotype. These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS autoregulation are conserved between humans and Drosophila. In addition to the well-known neurodegenerative effects of FUS loss-of function, these data suggest toxic potential of overexpressed FUS in the nucleus and of insoluble FUS.
Friday, December 19th
Nordman, J. T., Kozhevnikova, E. N., Verrijzer, C. P., Pindyurin, A. V., Andreyeva, E. N., Shloma, V. V., Zhimulev, I. F. and Orr-Weaver, T. L. (2014). DNA copy-number control through inhibition of replication fork progression. Cell Rep 9: 841-849. PubMed ID: 25437540
Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein Suppressor of Under-Replication (SuUR) through an unknown mechanism. This study demonstrates that SuUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. These results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression.
Djabrayan, N. J., Cruz, J., de Miguel, C., Franch-Marro, X. and Casanova, J. (2014). Specification of differentiated adult progenitors via inhibition of endocycle entry in the Drosophila trachea. Cell Rep 9: 859-865. PubMed ID: 25437542
A population of Drosophila adult tracheal progenitor cells arises from differentiated cells of the larval main trachea that retain the ability to reenter the cell cycle and give rise to the multiple adult tracheal cell types. These progenitors are unique to the second tracheal metamere as homologous cells from other segments, express fizzy-related (fzr), the Drosophila homolog of CDH1 protein of the APC complex, and enter endocycle and do not contribute to adult trachea. This study examined the mechanisms for their quiescence and show that they reenter the cell cycle by expression of string/cdc25 through ecdysone. Furthermore, preventing endocycle entry is both necessary and sufficient for these tracheal cells to exhibit markers of adult progenitors, thus modifying their genetic program. Finally, Hox-mediated regulation of fzr expression was shown to be responsible for progenitor identity and thus specifies a group of differentiated cells with facultative stem cell features.
Stephenson, R., Hosler, M. R., Gavande, N. S., Ghosh, A. K. and Weake, V. M. (2014). Characterization of a Drosophila ortholog of the Cdc7 kinase: A role for Cdc7 in endoreplication independent of Chiffon. J Biol Chem [Epub ahead of print]. PubMed ID: 25451925
Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-Replication Complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G1/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has been characterized as of yet. This paper reports the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact, and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification, but still undergo endocycling. These results show that Cdc7 interacts with Chiffon to form a functional Dbf4-Dependent Kinase (DDK) complex, and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, it was shown that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.
Asano, E., Hasegawa, H., Hyodo, T., Ito, S., Maeda, M., Chen, D., Takahashi, M., Hamaguchi, M. and Senga, T. (2014). SHCBP1 is required for midbody organization and cytokinesis completion. Cell Cycle 13: 2744-2751. PubMed ID: 25486361
The centralspindlin complex, which is composed of MKLP1 and MgcRacGAP, is one of the crucial factors involved in cytokinesis initiation. Centralspindlin is localized at the middle of the central spindle during anaphase and then concentrates at the midbody to control abscission. A number of proteins that associate with centralspindlin have been identified. These associating factors regulate furrowing and abscission in coordination with centralspindlin. A recent study identified a novel centralspindlin partner, called Nessun Dorma, which is essential for germ cell cytokinesis in Drosophila melanogaster. SHCBP1 is a human ortholog of Nessun Dorma that associates with human centralspindlin. This report analyzes the interaction of SHCBP1 with centralspindlin in detail and determined the regions that are required for the interaction. In addition, it was demonstrated that the central region is necessary for the SHCBP1 dimerization. Both MgcRacGAP and MKLP1 are degraded once cells exit mitosis. Similarly, endogenous and exogenous SHCBP1 were degraded with mitosis progression. Interestingly, SHCBP1 expression was significantly reduced in the absence of centralspindlin, whereas centralspindlin expression was not affected by SHCBP1 knockdown. Finally, it was demonstrated that SHCBP1 depletion promotes midbody structure disruption and inhibits abscission, a final stage of cytokinesis. This study gives novel insight into the role of SHCBP in cytokinesis completion.
Thursday, December 18th
Fenk, L. M., Poehlmann, A. and Straw, A. D. (2014). Asymmetric processing of visual motion for simultaneous object and background responses. Curr Biol 24(24):2913-9. PubMed ID: 25454785
Visual object fixation and figure-ground discrimination in Drosophila are robust behaviors requiring sophisticated computation by the visual system, yet the neural substrates remain unknown. Recent experiments in walking flies revealed object fixation behavior mediated by circuitry independent from the motion-sensitive T4-T5 cells required for wide-field motion responses. In tethered flight experiments under closed-loop conditions, this study found similar results for one feedback gain, whereas intact T4-T5 cells were necessary for robust object fixation at a higher feedback gain and in figure-ground discrimination tasks. Dynamical models were implemented based on neurons downstream of T4-T5 cells-one a simple phenomenological model and another, physiologically more realistic model-and found that both predict key features of stripe fixation and figure-ground discrimination and are consistent with a classical formulation. Fundamental to both models is motion asymmetry in the responses of model neurons, whereby front-to-back motion elicits stronger responses than back-to-front motion. When a bilateral pair of such model neurons, based on well-understood horizontal system cells, downstream of T4-T5, is coupled to turning behavior, asymmetry leads to object fixation and figure-ground discrimination in the presence of noise. Furthermore, the models also predict fixation in front of a moving background, a behavior previously suggested to require an additional pathway. Thus, the models predict several aspects of object responses on the basis of neurons that are also thought to serve a key role in background stabilization.
Alekseyenko, O. V., Chan, Y. B., Fernandez Mde, L., Bulow, T., Pankratz, M. J. and Kravitz, E. A. (2014). Single serotonergic neurons that modulate aggression in Drosophila. Curr Biol 24: 2700-2707. PubMed ID: 25447998
Monoamine serotonin (5HT) has been linked to aggression for many years across species. However, elaboration of the neurochemical pathways that govern aggression has proven difficult because monoaminergic neurons also regulate other behaviors. There are approximately 100 serotonergic neurons in the Drosophila nervous system, and they influence sleep, circadian rhythms, memory, and courtship. In the Drosophila model of aggression, the acute shut down of the entire serotonergic system yields flies that fight less, whereas induced activation of 5HT neurons promotes aggression. Using intersectional genetics, the population of 5HT neurons that can be reproducibly manipulated were restricted to identify those that modulate aggression. Although similar approaches were used recently to find aggression-modulating dopaminergic and Fru(M)-positive peptidergic neurons, the downstream anatomical targets of the neurons that make up aggression-controlling circuits remain poorly understood. This study identified a symmetrical pair of serotonergic PLP neurons that are necessary for the proper escalation of aggression. Silencing these neurons reduced aggression in male flies, and activating them increased aggression in male flies. GFP reconstitution across synaptic partners (GRASP) analyses suggested that 5HT-PLP neurons form contacts with 5HT1A receptor-expressing neurons in two distinct anatomical regions of the brain. Activation of these 5HT1A receptor-expressing neurons, in turn, caused reductions in aggression. These studies, therefore, suggest that aggression may be held in check, at least in part, by inhibitory input from 5HT1A receptor-bearing neurons, which can be released by activation of the 5HT-PLP neurons.
Zhu, E. Y., Guntur, A. R., He, R., Stern, U. and Yang, C. H. (2014). Egg-laying demand induces aversion of UV light in Drosophila females. Curr Biol 24: 2797-2804. PubMed ID: 25455037
Drosophila melanogaster females are highly selective about the chemosensory quality of their egg-laying sites, an important trait that promotes the survival and fitness of their offspring. How egg-laying females respond to UV light is not known, however. UV is a well-documented phototactic cue for adult Drosophila, but it is an aversive cue for larvae. This study shows that female flies exhibit UV aversion in response to their egg-laying demand. First, females exhibit egg-laying aversion of UV: they prefer to lay eggs on dark sites when choosing between UV-illuminated and dark sites. Second, they also exhibit movement aversion of UV: positional tracking of single females suggests that egg-laying demand increases their tendency to turn away from UV. Genetic manipulations of the retina suggest that egg-laying and movement aversion of UV are both mediated by the inner (R7) and not the outer (R1-R6) photoreceptors. Finally, the Dm8 amacrine neurons, a synaptic target of R7 photoreceptors and a mediator of UV spectral preference, were shown to be dispensable for egg-laying aversion but essential for movement aversion of UV. This study suggests that egg-laying demand can temporarily convert UV into an aversive cue for female Drosophila and that R7 photoreceptors recruit different downstream targets to control different egg-laying-induced behavioral modifications.
Kohsaka, H., Takasu, E., Morimoto, T. and Nose, A. (2014). A group of segmental premotor interneurons regulates the speed of axial locomotion in Drosophila larvae. Curr Biol 24: 2632-2642. PubMed ID: 25438948
Animals control the speed of motion to meet behavioral demands. Yet, the underlying neuronal mechanisms remain poorly understood. This study shows that a class of segmentally arrayed local interneurons (period-positive median segmental interneurons, or PMSIs) regulates the speed of peristaltic locomotion in Drosophila larvae. PMSIs formed glutamatergic synapses on motor neurons and, when optogenetically activated, inhibited motor activity, indicating that they are inhibitory premotor interneurons. Calcium imaging showed that PMSIs are rhythmically active during peristalsis with a short time delay in relation to motor neurons. Optogenetic silencing of these neurons elongated the duration of motor bursting and greatly reduced the speed of larval locomotion. These results suggest that PMSIs control the speed of axial locomotion by limiting, via inhibition, the duration of motor outputs in each segment. Similar mechanisms are found in the regulation of mammalian limb locomotion, suggesting that common strategies may be used to control the speed of animal movements in a diversity of species.
Reinhart, M., Carney, T., Clark, A. G. and Fiumera, A. C. (2014). Characterizing male-female interactions using natural genetic variation in Drosophila melanogaster. J Hered 106(1):67-79. PubMed ID: 25425680
Drosophila melanogaster females commonly mate with multiple males establishing the opportunity for pre- and postcopulatory sexual selection. Traits impacting sexual selection can be affected by a complex interplay of the genotypes of the competing males, the genotype of the female, and compatibilities between the males and females. This study scored males from 96 2nd and 94 3rd chromosome substitution lines for traits affecting reproductive success when mated with females from 3 different genetic backgrounds. The traits included male-induced female refractoriness, male remating ability, the proportion of offspring sired under competitive conditions and male-induced female fecundity. Significant effects of male line, female genetic background, and strong male by female interactions. Some males appeared to be 'generalists' and performed consistently across the different females; other males appeared to be 'specialists' and performed very well with a particular female and poorly with others. 'Specialist' males did not, however, prefer to court those females with whom they had the highest reproductive fitness. Using 143 polymorphisms in male reproductive genes, several genes were mapped that had consistent effects across the different females including a derived, high fitness allele in Acp26Aa that may be the target of adaptive evolution. Also, a polymorphism upstream of Protein ejaculatory bulb II (PebII) was identified that may interact with the female genetic background to affect male-induced refractoriness to remating. These results suggest that natural variation in PebII might contribute to the observed male-female interactions.
Wednesday, December 17th
Li, Q., Li, S., Mana-Capelli, S., Roth Flach, R. J., Danai, L. V., Amcheslavsky, A., Nie, Y., Kaneko, S., Yao, X., Chen, X., Cotton, J. L., Mao, J., McCollum, D., Jiang, J., Czech, M. P., Xu, L. and Ip, Y. T. (2014). The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila. Dev Cell 31: 291-304. PubMed ID: 25453828
Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. This study shows that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP.
Stine, R. R., Greenspan, L. J., Ramachandran, K. V. and Matunis, E. L. (2014). Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis. PLoS Genet 10: e1004713. PubMed ID: 25375180
Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). This study shows a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. This work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche.
Kao, S. H., Tseng, C. Y., Wan, C. L., Su, Y. H., Hsieh, C. C., Pi, H. and Hsu, H. J. (2014). Aging and insulin signaling differentially control normal and tumorous germline stem cells. Aging Cell [Epub ahead of print]. PubMed ID: 25470527
Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. This study reports that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. These results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging.
Andersen, J., Urban, N., Achimastou, A., Ito, A., Simic, M., Ullom, K., Martynoga, B., Lebel, M., Goritz, C., Frisen, J., Nakafuku, M. and Guillemot, F. (2014). A transcriptional mechanism integrating inputs from extracellular signals to activate hippocampal stem cells. Neuron 83: 1085-1097. PubMed ID: 25189209
The activity of adult stem cells is regulated by signals emanating from the surrounding tissue. Many niche signals have been identified, but it is unclear how they influence the choice of stem cells to remain quiescent or divide. This study shows that when stem cells of the adult hippocampus receive activating signals, they first induce the expression of the transcription factor Ascl1 (Drosophila homolog: Achaete) and only subsequently exit quiescence. Moreover, lowering Ascl1 expression reduces the proliferation rate of hippocampal stem cells, and inactivating Ascl1 blocks quiescence exit completely, rendering them unresponsive to activating stimuli. Ascl1 promotes the proliferation of hippocampal stem cells by directly regulating the expression of cell-cycle regulatory genes. Ascl1 is similarly required for stem cell activation in the adult subventricular zone. These results support a model whereby Ascl1 integrates inputs from both stimulatory and inhibitory signals and converts them into a transcriptional program activating adult neural stem cells.
Tuesday, December 16th
Kunst, M., Hughes, M. E., Raccuglia, D., Felix, M., Li, M., Barnett, G., Duah, J. and Nitabach, M. N. (2014). Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila. Curr Biol 24: 2652-2664. PubMed ID: 25455031
Imbalances in amount and timing of sleep are harmful to physical and mental health. Therefore, the study of the underlying mechanisms is of great biological importance. Proper timing and amount of sleep are regulated by both the circadian clock and homeostatic sleep drive. However, very little is known about the cellular and molecular mechanisms by which the circadian clock regulates sleep. This study describes a novel role for Diuretic hormone 31 (DH31), the fly homolog of the vertebrate neuropeptide calcitonin gene-related peptide, as a circadian wake-promoting signal that awakens the fly in anticipation of dawn. Analysis of loss-of-function and gain-of-function Drosophila mutants demonstrates that DH31 suppresses sleep late at night. DH31 is expressed by a subset of dorsal circadian clock neurons that also express the receptor for the circadian neuropeptide pigment-dispersing factor (PDF). PDF secreted by the ventral pacemaker subset of circadian clock neurons acts on PDF receptors in the DH31-expressing dorsal clock neurons to increase DH31 secretion before dawn. Activation of PDF receptors in DH31-positive DN1 specifically affects sleep and has no effect on circadian rhythms, thus constituting a dedicated locus for circadian regulation of sleep. This study has identified a novel signaling molecule (DH31) as part of a neuropeptide relay mechanism for circadian control of sleep. The results indicate that outputs of the clock controlling sleep and locomotor rhythms are mediated via distinct neuronal pathways.
Mauthner, S. E., Hwang, R. Y., Lewis, A. H., Xiao, Q., Tsubouchi, A., Wang, Y., Honjo, K., Skene, J. H., Grandl, J. and Tracey, W. D., Jr. (2014). Balboa binds to Pickpocket in vivo and is required for mechanical nociception in Drosophila larvae. Curr Biol [Epub ahead of print]. PubMed ID: 25454784
The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the degenerin/epithelial sodium channel (DEG/ENaC) family. PPK is specifically expressed in nociceptive, class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses. In a genome-wide genetic screen for other ion channel subunits required for mechanical nociception, this study identified a gene that was named balboa (also known as CG8546, ppk26). Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK. Moreover, laser-capture isolation of RNA from larval neurons and microarray analyses reveal that balboa is also highly enriched in nociceptive neurons. The requirement for Balboa and PPK in mechanical nociception behaviors and their specific expression in larval nociceptors led to an hypothesis that these DEG/ENaC subunits form an ion channel complex in vivo. In nociceptive neurons, Balboa::GFP proteins distribute uniformly throughout dendrites but remarkably localize to discrete foci when ectopically expressed in other neuron subtypes (where PPK is not expressed). Indeed, ectopically coexpressing ppk transforms this punctate Balboa::GFP expression pattern to the uniform distribution observed in its native cell type. Furthermore, ppk-RNAi in class IV neurons alters the broad Balboa::GFP pattern to a punctate distribution. Interestingly, this interaction is mutually codependent as balboa-RNAi eliminates Venus::PPK from the sensory dendrites of nociceptors. Finally, using a GFP-reconstitution approach in transgenic larvae, in vivo physical interactions among PPK and Balboa subunits were directly detected. Combined, these results indicate a critical mechanical nociception function for heteromeric PPK and Balboa channels in vivo.
Qi, C. and Lee, D. (2014). Pre- and postsynaptic role of Dopamine D2 Receptor DD2R in Drosophila olfactory associative learning. Biology (Basel) 3: 831-845. PubMed ID: 25422852
Dopaminergic neurons in Drosophila play critical roles in diverse brain functions such as motor control, arousal, learning, and memory. Using genetic and behavioral approaches, it has been firmly established that proper dopamine signaling is required for olfactory classical conditioning (e.g., aversive and appetitive learning). Dopamine mediates its functions through interaction with its receptors. There are two different types of dopamine receptors in Drosophila: D1-like (dDA1, DAMB) and D2-like receptors (DD2R). Currently, no study has attempted to characterize the role of DD2R in Drosophila learning and memory. Using a DD2R-RNAi transgenic line, this study has examined the role of DD2R, expressed in dopamine neurons (i.e., the presynaptic DD2R autoreceptor), in larval olfactory learning. The function of postsynaptic DD2R expressed in mushroom body (MB) was also studied as MB is the center for Drosophila learning, with a function analogous to that of the mammalian hippocampus. These results showed that suppression of presynaptic DD2R autoreceptors impairs both appetitive and aversive learning. Similarly, postsynaptic DD2R in MB neurons appears to be involved in both appetitive and aversive learning. The data confirm, for the first time, that DD2R plays an important role in Drosophila olfactory learning.
Kavlie, R. G., Fritz, J. L., Nies, F., Gopfert, M. C., Oliver, D., Albert, J. T. and Eberl, D. F. (2014). Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing. J Comp Physiol A Neuroethol Sens Neural Behav Physiol [Epub ahead of print]. PubMed ID: 25412730
In mammals, the membrane-based protein Prestin confers unique electromotile properties to cochlear outer hair cells, which contribute to the cochlear amplifier. Like mammals, the ears of insects, such as those of Drosophila melanogaster, mechanically amplify sound stimuli and have also been reported to express Prestin homologs. To determine whether the D. melanogaster Prestin homolog (dpres) is required for auditory amplification, dpres mutant flies were generated and analyzed. dpres is robustly expressed in the fly's antennal ear. However, dpres mutant flies show normal auditory nerve responses, and intact non-linear amplification. Thus it is concluded that, in D. melanogaster, auditory amplification is independent of Prestin. This finding resonates with prior phylogenetic analyses, which suggest that the derived motor function of mammalian Prestin replaced, or amended, an ancestral transport function. Indeed, this study shows that dpres encodes a functional anion transporter. Interestingly, the acquired new motor function in the phylogenetic lineage leading to birds and mammals coincides with loss of the mechanotransducer channel NompC (=TRPN1), which has been shown to be required for auditory amplification in flies. The advent of Prestin (or loss of NompC, respectively) may thus mark an evolutionary transition from a transducer-based to a Prestin-based mechanism of auditory amplification.
Monday, December 15th
Zobel, T., Brinkmann, K., Koch, N., Schneider, K., Seemann, E., Fleige, A., Qualmann, B., Kessels, M. M. and Bogdan, S. (2014). Cooperative functions of the two F-BAR proteins Cip4 and Nostrin in regulating E-cadherin in epithelial morphogenesis. J Cell Sci [Epub ahead of print]. PubMed ID: 25413347
F-BAR proteins are prime candidates to regulate membrane curvature and dynamics during different developmental processes. This study analyzed nostrin (nost), a novel Drosophila F-BAR protein related to Cip4. Genetic analyses revealed a strong synergism between nost and cip4 functions. While single mutant flies are viable and fertile, combined loss of nost and cip4 results in reduced viability and fertility. Double mutant escaper flies show enhanced wing polarization defects and females exhibit strong egg chamber encapsulation defects. Live-imaging analysis suggests that the observed phenotypes are caused by an impaired E-cadherin membrane turnover. Simultaneous knock-down of Cip4 and Nostrin strongly increases the formation of tubular E-cadherin vesicles at adherens junctions. Cip4 and Nostrin localize at distinct membrane subdomains. Both proteins prefer similar membrane curvatures but seem to form different membrane coats and do not heterooligomerize. These data suggest an important synergistic function of both F-BAR proteins in membrane dynamics. A cooperative recruitment model is proposed in which first Cip4 promotes membrane invagination and early actin-based endosomal motility while Nostrin makes contact with microtubules through the kinesin Khc-73 for trafficking of recycling endosomes.
Milton, C. C., Grusche, F. A., Degoutin, J. L., Yu, E., Dai, Q., Lai, E. C. and Harvey, K. F. (2014). The Hippo pathway regulates hematopoiesis in Drosophila melanogaster. Curr Biol 24: 2673-2680. PubMed ID: 25454587
The Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, as well as in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing), and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals. This study defines the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely, and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell-autonomous and non-cell-autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell-autonomous and Notch-pathway-dependent fashion.
Koyama, T., Rodrigues, M. A., Athanasiadis, A., Shingleton, A. W. and Mirth, C. K. (2014). Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis. Elife 3 [Epub ahead of print]. PubMed ID: 25421296
Despite their fundamental importance for body size regulation, the mechanisms that stop growth are poorly understood. In Drosophila melanogaster, growth ceases in response to a peak of the molting hormone ecdysone that coincides with a nutrition-dependent checkpoint, critical weight. Previous studies indicate that insulin/insulin-like growth factor signaling (IIS)/Target of Rapamycin (TOR) signaling in the prothoracic glands (PGs) regulates ecdysone biosynthesis and critical weight. This study elucidates a mechanism through which this occurs. This study shows that Forkhead Box class O (FoxO), a negative regulator of IIS/TOR, directly interacts with Ultraspiracle (Usp), part of the ecdysone receptor. While overexpressing FoxO in the PGs delays ecdysone biosynthesis and critical weight, disrupting FoxO-Usp binding reduces these delays. Further, feeding ecdysone to larvae eliminates the effects of critical weight. Thus, nutrition controls ecdysone biosynthesis partially via FoxO-Usp prior to critical weight, ensuring that growth only stops once larvae have achieved a target nutritional status.
Gautam, N. K., Verma, P. and Tapadia, M. G. (2014). Ecdysone regulates morphogenesis and function of malpighian tubules in Drosophila melanogaster through EcR-B2 isoform. Dev Biol [Epub ahead of print]. PubMed ID: 25476260
Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. This study conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and beta-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na+/K+-ATPase pump. B2 isoform of Ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development.
Sunday, December 14th
Dong, B., Miao, G. and Hayashi, S. (2014). A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila. Development 141: 4104-4109. PubMed ID: 25336738
The apical extracellular matrix plays a central role in epithelial tube morphogenesis. In the Drosophila tracheal system, Serpentine (Serp), a secreted chitin deacetylase expressed by the tracheal cells plays a key role in regulating tube length. This study shows that the fly fat body, which is functionally equivalent to the mammalian liver, also contributes to tracheal morphogenesis. Serp is expressed by the fat body, and the secreted Serp is taken up by the tracheal cells and translocated to the lumen to functionally support normal tracheal development. This process is defective in rab9 and shrub/vps32 mutants and in wild-type embryos treated with a secretory pathway inhibitor, leading to an abundant accumulation of Serp in the fat body. Fat body-derived Serp reaches the tracheal lumen after establishment of epithelial barrier function and is retained in the lumen in a chitin synthase-dependent manner. These results thus reveal that the fat body, a mesodermal organ, actively contributes to tracheal development.
Saxena, A., Denholm, B., Bunt, S., Bischoff, M., VijayRaghavan, K. and Skaer, H. (2014). Epidermal growth factor signalling controls Myosin II planar polarity to orchestrate convergent extension movements during Drosophila tubulogenesis. PLoS Biol 12: e1002013. PubMed ID: 25460353
Most epithelial tubes arise as small buds and elongate by regulated morphogenetic processes including oriented cell division, cell rearrangements, and changes in cell shape. Through live analysis of Drosophila renal tubule morphogenesis this study shows that tissue elongation results from polarised cell intercalations around the tubule circumference, producing convergent-extension tissue movements. Using genetic techniques, it was demonstrated that the vector of cell movement is regulated by localised Epidermal growth factor (EGF) signalling from the distally placed tip cell lineage, which sets up a distal-to-proximal gradient of pathway activation to planar polarise cells, without the involvement for PCP gene activity. Time-lapse imaging at subcellular resolution shows that the acquisition of planar polarity leads to asymmetric pulsatile Myosin II accumulation in the basal, proximal cortex of tubule cells, resulting in repeated, transient shortening of their circumferential length. This repeated bias in the polarity of cell contraction allows cells to move relative to each other, leading to a reduction in cell number around the lumen and an increase in tubule length. Physiological analysis demonstrates that animals whose tubules fail to elongate exhibit abnormal excretory function, defective osmoregulation, and lethality.
Haralalka, S., Shelton, C., Cartwright, H. N., Guo, F., Trimble, R., Kumar, R. P. and Abmayr, S. M. (2014). Live imaging provides new insights on dynamic F-Actin filopodia and differential endocytosis during myoblast fusion in Drosophila. PLoS One 9: e114126. PubMed ID: 25474591
The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 (see Drosophila Arp2/3 component Arpc1) pathways. This study utilized the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. Differential endocytosis of cell surface components was observed at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube.
Umetsu, D., Dunst, S. and Dahmann, C. (2014). An RNA interference screen for genes required to shape the anteroposterior compartment boundary in Drosophila identifies the Eph receptor. PLoS One 9: e114340. PubMed ID: 25473846
The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. This study reports on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, a single novel candidate was identified that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. These results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.
Saturday, December 13th
Guo, Y., Wang, Y., Wang, Q. and Wang, Z. (2014). The role of PPK26 in Drosophila larval mechanical nociception. Cell Rep 9: 1183-1190. PubMed ID: 25457610
In Drosophila larvae, the class IV dendritic arborization (da) neurons are polymodal nociceptors. This study shows that ppk26 (CG8546) plays an important role in mechanical nociception in class IV da neurons. Immunohistochemical and functional results demonstrate that ppk26 is specifically expressed in class IV da neurons. Larvae with mutant ppk26 showed severe behavioral defects in a mechanical nociception behavioral test but responded to noxious heat stimuli comparably to wild-type larvae. In addition, functional studies suggest that ppk26 and ppk (also called ppk1 or pickpocket) function in the same pathway, whereas Piezo functions in a parallel pathway. Consistent with these functional results, Ppk and Ppk26 are interdependent on each other for their cell surface localization. This work indicates that Ppk26 and Ppk might form heteromeric DEG/ENaC channels that are essential for mechanotransduction in class IV da neurons.
Gorczyca, D. A., Younger, S., Meltzer, S., Kim, S. E., Cheng, L., Song, W., Lee, H. Y., Jan, L. Y. and Jan, Y. N. (2014). Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila. Cell Rep 9: 1446-1458. PubMed ID: 25456135
A major gap in understanding of sensation is how a single sensory neuron can differentially respond to a multitude of different stimuli (polymodality), such as propio- or nocisensation. The prevailing hypothesis is that different stimuli are transduced through ion channels with diverse properties and subunit composition. In a screen for ion channel genes expressed in polymodal nociceptive neurons, this study identified Ppk26, a member of the trimeric degenerin/epithelial sodium channel (DEG/ENaC) family, as being necessary for proper locomotion behavior in Drosophila larvae in a mutually dependent fashion with coexpressed Ppk1 (Pickpocket), another member of the same family. Mutants lacking Ppk1 and Ppk26 were defective in mechanical, but not thermal, nociception behavior. Mutants of Piezo, a channel involved in mechanical nociception in the same neurons, did not show a defect in locomotion, suggesting distinct molecular machinery for mediating locomotor feedback and mechanical nociception.
Zhang, J., Tanenhaus, A. K., Davis, J. C., Hanlon, B. M. and Yin, J. C. (2014). Spatio-temporal in vivo recording of dCREB2 dynamics in Drosophila long-term memory processing. Neurobiol Learn Mem [Epub ahead of print]. PubMed ID: 25460038
CREB (cAMP response element-binding protein) is an evolutionarily conserved transcription factor, playing key roles in synaptic plasticity, intrinsic excitability and long-term memory (LTM) formation. The Drosophila homologue of mammalian CREB, dCREB2, is also important for LTM. However, the spatio-temporal nature of dCREB2 activity during memory consolidation is poorly understood. Using an in vivo reporter system, this study examined dCREB2 activity continuously in specific brain regions during LTM processing. Two brain regions that have been shown to be important for Drosophila LTM are the ellipsoid body (EB) and the mushroom body (MB). dCREB2 reporter activity is persistently elevated in EB R2/R4m neurons, but not neighboring R3/R4d neurons, following LTM-inducing training. In multiple subsets of MB neurons, dCREB2 reporter activity is suppressed immediately following LTM-specific training, and elevated during late windows. In addition, heterogeneous responses were observed across different subsets of neurons in MB αβ lobe during LTM processing. All of these changes suggest that dCREB2 functions in both the EB and MB for LTM formation, and that this activity contributes to the process of systems consolidation.
Raccuglia, D. and Mueller, U. (2014). Temporal integration of cholinergic and GABAergic inputs in isolated insect mushroom body neurons exposes pairing-specific signal processing. J Neurosci 34: 16086-16092. PubMed ID: 25429149
GABAergic modulation of neuronal activity plays a crucial role in physiological processes including learning and memory in both insects and mammals. During olfactory learning in honeybees (Apis mellifera) and Drosophila melanogaster the temporal relation between excitatory cholinergic and inhibitory GABAergic inputs critically affects learning. However, the cellular mechanisms of temporal integration of these antagonistic inputs are unknown. To address this question, this study used calcium imaging of isolated honeybee and Drosophila Kenyon cells (KCs), which are targets of cholinergic and GABAergic inputs during olfactory learning. In the whole population of honeybee KCs, pairing of acetylcholine (ACh) and γ-aminobutyric acid (GABA) was found to reduce the ACh-induced calcium influx, and depending on their temporal sequence, induces different forms of neuronal plasticity. After ACh-GABA pairing the calcium influx of a subsequent excitatory stimulus is increased, while GABA-ACh pairing affects the decay time leading to elevated calcium levels during the late phase of a subsequent excitatory stimulus. In an exactly defined subset of Drosophila KCs implicated in learning similar pairing-specific differences were found. Specifically the GABA-ACh pairing splits the KCs in two functional subgroups: one is only weakly inhibited by GABA and shows no neuronal plasticity and the other subgroup is strongly inhibited by GABA and shows elevated calcium levels during the late phase of a subsequent excitatory stimulus. These findings provide evidence that insect KCs are capable of contributing to temporal processing of cholinergic and GABAergic inputs, which provides a neuronal mechanism of the differential temporal role of GABAergic inhibition during learning.
Friday, December 12th
Li, S., Jiang, C., Pan, J., Wang, X., Jin, J., Zhao, L., Pan, W., Liao, G., Cai, X., Li, X., Xiao, J., Jiang, J. and Wang, P. (2014). Regulation of c-Myc protein stability by proteasome activator REGgamma. Cell Death Differ [Epub ahead of print]. PubMed ID: 25412630
c-Myc is a key transcriptional factor that has a prominent role in cell growth, differentiation and tumor development. Its protein levels are tightly controlled by ubiquitin-proteasome pathway and frequently deregulated in various cancers. This study reports that the 11S proteasomal activator REGgamma is a novel regulator of c-Myc abundance in cells. Overexpression of wild-type REGgamma, but not inactive mutants including N151Y and G250S, significantly promoted the degradation of c-Myc. Depletion of REGgamma markedly increased the protein stability of c-Myc. REGgamma interacts with the C-terminal region of c-Myc and regulates c-Myc protein turnover. Functionally, REGgamma negatively regulates c-Myc-mediated cell proliferation. Interestingly, depletion of the Drosophila Reg homolog (dReg) in developing wings induced the upregulation of Drosophila Myc, which contributes to cell death. Collectively, these results suggest that REGgamma proteasome has a conserved role in the regulation of Myc abundance in both mammalian cells and Drosophila.
Huang, H., Lu-Bo, Y. and Haddad, G. G. (2014). A Drosophila ABC transporter regulates lifespan. PLoS Genet 10: e1004844. PubMed ID: 25474322
MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. This study investigated the biological functions of a Drosophila homolog of human MRP4, dMRP4. dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, it was demonstrated that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. Impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling.
Chatterjee, D., Katewa, S. D., Qi, Y., Jackson, S. A., Kapahi, P. and Jasper, H. (2014). Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25472843
Metabolic adaptation to changing dietary conditions is critical to maintain homeostasis of the internal milieu. In metazoans, this adaptation is achieved by a combination of tissue-autonomous metabolic adjustments and endocrine signals that coordinate the mobilization, turnover, and storage of nutrients across tissues. To understand metabolic adaptation comprehensively, detailed insight into these tissue interactions is necessary. This study characterize the tissue-specific response to fasting in adult flies and identified an endocrine interaction between the fat body and liver-like oenocytes that regulates the mobilization of lipid stores. Using tissue-specific expression profiling, it was confirmed that oenocytes in adult flies play a central role in the metabolic adaptation to fasting. Furthermore, it was found that fat body-derived Drosophila insulin-like peptide 6 (dILP6) induces lipid uptake in oenocytes, promoting lipid turnover during fasting and increasing starvation tolerance of the animal. Selective activation of insulin/IGF signaling in oenocytes by a fat body-derived peptide represents a previously unidentified regulatory principle in the control of metabolic adaptation and starvation tolerance.
Zeng, Z., de Gorter, D. J., Kowalski, M., Ten Dijke, P. and Shimmi, O. (2014). Ter94/VCP is a novel component involved in BMP signaling. PLoS One 9: e114475. PubMed ID: 25469707
Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor (TGF)-beta family, transduce their signal through multiple components downstream of their receptors. Even though the components involved in the BMP signaling pathway have been intensely studied, many molecules mediating BMP signaling remain to be addressed. To identify novel components that participate in BMP signaling, RNA interference (RNAi)-based screening was established by detecting phosphorylated Mad (pMad) in Drosophila S2 cells. Ter94, a member of the family of AAA ATPases, was identified as a novel mediator of BMP signaling, which is required for the phosphorylation of Mad in Drosophila S2 cells. Moreover, the mammalian orthlog of Ter94 valosin-containing protein (VCP) plays a critical role in the BMP-Smad1/5/8 signaling pathway in mammalian cells. Genetic evidence suggests that Ter94 is involved in the dorsal-ventral patterning of the Drosophila early embryo through regulating Decapentaplegic (Dpp)/BMP signals. Taken together, these data suggest that Ter94/VCP appears to be an evolutionarily conserved component that regulates BMP-Smad1/5/8 signaling.
Thursday, June 11th
Meyer, S. N., Amoyel, M., Bergantinos, C., de la Cova, C., Schertel, C., Basler, K. and Johnston, L. A. (2014). An ancient defense system eliminates unfit cells from developing tissues during cell competition. Science 346: 1258236. PubMed ID: 25477468
Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. This study reports that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. Toll-related receptors (TRRs) and the cytokine Spatzle (Spz) lead to NFkappaB-dependent apoptosis. Null mutations in Toll-3, Toll-8, or Toll-9 suppress elimination of loser cells, increasing loser clone size and cell number per clone, but do not alter control clones. Diverse 'loser' cells require different TRRs and NFkappaB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. These findings demonstrate a functional repurposing of components of TRRs and NFkappaB signaling modules in the surveillance of cell fitness during development.
Li, Y. X. and Dijkers, P. F. (2014). Specific Calcineurin isoforms are involved in Drosophila Toll immune signaling. J Immunol [Epub ahead of print]. PubMed ID: 25429067
Because excessive or inadequate responses can be detrimental, immune responses to infection require appropriate regulation. Networks of signaling pathways establish versatility of immune responses. Drosophila melanogaster is a powerful model organism for dissecting conserved innate immune responses to infection. For example, the Toll pathway, which promotes activation of NF-kappaB transcription factors Dorsal/Dorsal-related immune factor (Dif), was first identified in Drosophila. Together with the IMD pathway, acting upstream of NF-kappaB transcription factor calcineurin A1, acts on Relish during infection. However, it is not known whether there is a role for calcineurin in Dorsal/Dif immune signaling. This article demonstrates involvement of specific calcineurin isoforms, protein phosphatase at 14D (Pp2B-14D)/calcineurin A at 14F (CanA-14F), in Toll-mediated immune signaling. These isoforms do not affect IMD signaling. In cell culture, pharmacological inhibition of calcineurin or RNA interference against homologous calcineurin isoforms Pp2B-14D/CanA-14F, but not against isoform calcineurin A1, decreased Toll-dependent Dorsal/Dif activity. A Pp2B-14D gain-of-function transgene promoted Dorsal nuclear translocation and Dorsal/Dif activity. In vivo, Pp2B-14D/CanA-14F RNA interference attenuated the Dorsal/Dif-dependent response to infection without affecting the Relish-dependent response. Altogether, these data identify a novel input, calcineurin, in Toll immune signaling and demonstrate involvement of specific calcineurin isoforms in Drosophila NF-kappaB signaling.
Ferreira, A. G., Naylor, H., Esteves, S. S., Pais, I. S., Martins, N. E. and Teixeira, L. (2014). The Toll-Dorsal pathway is required for resistance to viral oral infection in Drosophila. PLoS Pathog 10: e1004507. PubMed ID: 25473839
Pathogen entry route can have a strong impact on the result of microbial infections in different hosts, including insects. Drosophila melanogaster has been a successful model system to study the immune response to systemic viral infection. This study investigated the role of the Toll pathway in resistance to oral viral infection in D. melanogaster. Several Toll pathway components, including Spatzle, Toll, Pelle and the NF-κB-like transcription factor Dorsal, are required to resist oral infection with Drosophila C virus. Furthermore, in the fat body Dorsal is translocated from the cytoplasm to the nucleus and a Toll pathway target gene reporter is upregulated in response to Drosophila C Virus infection. This pathway also mediates resistance to several other RNA viruses (Cricket paralysis virus, Flock House virus, and Nora virus). Compared with control, viral titres are highly increased in Toll pathway mutants. The role of the Toll pathway in resistance to viruses in D. melanogaster is restricted to oral infection since no phenotype was associated with systemic infection. It was also shown that Wolbachia and other Drosophila-associated microbiota do not interact with the Toll pathway-mediated resistance to oral infection. This study therefore identified the Toll pathway as a new general inducible pathway that mediates strong resistance to viruses with a route-specific role. These results contribute to a better understanding of viral oral infection resistance in insects, which is particularly relevant in the context of transmission of arboviruses by insect vectors.
Kim, M. J. and Choe, K. M. (2014). Basement membrane and cell integrity of self-tissues in maintaining Drosophila immunological tolerance. PLoS Genet 10: e1004683. PubMed ID: 25329560
The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood. This study established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane. Genes for the two collagen IV subunits (see viking and Collagen type IV) and the four laminin subunits (see Laminin A) were nocked down individually via UAS-RNAi using ubiquitous and tissue-specific GAL4 drivers.
The basement membrane was found to endow otherwise susceptible target tissues with self-tolerance that prevents autoimmunity, and it was further demonstrated that laminin is a key component for both structural maintenance and the self-tolerance checkpoint function of the basement membrane. Moreover, cell integrity, as determined by cell-cell interaction and apicobasal polarity, was found to function as a second discrete checkpoint. Target tissues became vulnerable to blood cell encapsulation and subsequent melanization only after loss of both the basement membrane and cell integrity.
Wednesday, December 10th
Gambetta, M. C. and Muller, J. (2014). O-GlcNAcylation prevents aggregation of the Polycomb group repressor Polyhomeotic. Dev Cell [Epub ahead of print]. PubMed ID: 25468754
The glycosyltransferase Ogt adds O-linked N-Acetylglucosamine (O-GlcNAc) moieties to nuclear and cytosolic proteins. Drosophila embryos lacking Ogt protein arrest development with a remarkably specific Polycomb phenotype, arising from the failure to repress Polycomb target genes. The Polycomb protein Polyhomeotic (Ph), an Ogt substrate, forms large aggregates in the absence of O-GlcNAcylation both in vivo and in vitro. O-GlcNAcylation of a serine/threonine (S/T) stretch in Ph is critical to prevent nonproductive aggregation of both Drosophila and human Ph via their C-terminal sterile alpha motif (SAM) domains in vitro. Full Ph repressor activity in vivo requires both the SAM domain and O-GlcNAcylation of the S/T stretch. Ph mutants lacking the S/T stretch reproduce the phenotype of ogt mutants, suggesting that the S/T stretch in Ph is the key Ogt substrate in Drosophila. It is proposed that O-GlcNAcylation is needed for Ph to form functional, ordered assemblies via its SAM domain.
Fereres, S., Simon, R., Mohd-Sarip, A., Verrijzer, C. P. and Busturia, A. (2014). dRYBP counteracts chromatin-dependent activation and repression of transcription. PLoS One 9: e113255. PubMed ID: 25415640
Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities this study analyzed the function of Drosophila Ring and YY1 Binding Protein (dRYBP), a conserved PcG- and trxG-associated protein. dRYBP is ubiquitylated and binds ubiquitylated proteins. Additionally dRYBP was shown to maintain H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further it was shown that dRYBP interacts with the repressive SCE (Ring) and dKDM2 (Lysine (K)-specific demethylase 2) proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, the results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. These findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development.
Pathak, R. U., Srinivasan, A. and Mishra, R. K. (2014). Genome-wide mapping of matrix attachment regions in Drosophila melanogaster. BMC Genomics 15: 1022. PubMed ID: 25424749
Eukaryotic genome acquires functionality upon proper packaging within the nucleus. This process is facilitated by the structural framework of Nuclear Matrix, a nucleo-proteinaceous meshwork. Matrix Attachment Regions (MARs) in the genome serve as anchoring sites to this framework. This study reports direct sequencing of the MAR preparation from Drosophila melanogaster embryos and identifies >7350 MARs. This amounts to ~2.5% of the fly genome and often coincides with AT rich non-coding regions. Significant association of MARs with the origins of replication, transcription start sites, paused RNA Polymerase II sites and exons, but not introns, of highly expressed genes. Sequence motifs and repeats were identified that constitute MARs. These data reveal the contact points of genome to the nuclear architecture and provide a link between nuclear functions and genomic packaging.
Gartner, S. M., Rothenbusch, S., Buxa, M. K., Theofel, I., Renkawitz, R., Rathke, C. and Renkawitz-Pohl, R. (2014). The HMG-box-containing proteins tHMG-1 and tHMG-2 interact during the histone-to-protamine transition in Drosophila spermatogenesis. Eur J Cell Biol. PubMed ID: 25464903
Spermatogenesis is accompanied by a remarkable reorganization of the chromatin in post-meiotic stages, characterized by a near genome-wide displacement of histones by protamines and a transient expression of transition proteins. In Drosophila, the Transition-protein-like 94D (Tpl94D) contains an HMG-box domain and is expressed during chromatin reorganization. This study searched for additional HMG-box-containing proteins with a similar expression pattern. Two proteins specifically expressed in the testis, tHMG-1 (CG12104) and tHMG-2 (CG30356), whose expression levels were highest during the histone-to-protamine transition. Protein-protein interaction studies revealed that tHMG-1 and tHMG-2 form heterodimers in vivo. Tpl94D, tHMG-1 and tHMG-2 were shown to localize to chromatin of the male germ line, with the most abundant levels observed during post-meiotic chromatin reorganization. Analysis of a tpl94D mutant showed that the C-terminal region of Tpl94D is dispensable for fertility. These data strongly suggested either that the truncated protein, which still contains the N-terminal HMG-box domain, is functional or that other proteins act in functional redundancy with Tpl94D during spermiogenesis. A thmg-1/thmg-2 null mutant also had no detectable specific phenotype, but hmgz, which encodes the major somatic HMG-box-containing protein HMGZ, was transcriptionally up-regulated. These results showed that Drosophila spermatogenesis is characterized by continuous and overlapping expression of different HMG-box-containing proteins. It is hypothesized that the mechanism of chromatin reorganization is a process highly secured by redundancies.
Tuesday, December 9th
Ma, Q., Wawersik, M. and Matunis, E. L. (2014). The Jak-STAT target Chinmo prevents sex transformation of adult stem cells in the Drosophila testis niche. Dev Cell 31: 474-486. PubMed ID: 25453558
Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). This study found that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor DoublesexM (DsxM) within CySCs and their progeny, and ectopic expression of DsxM in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of DsxM. The Dsx homolog DMRT1 prevents the male-to-female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. This work indicates that sex maintenance occurs in adult somatic stem cells and that this highly conserved process is governed by effectors of niche signals
Matsuoka, S., Gupta, S., Suzuki, E., Hiromi, Y. and Asaoka, M. (2014). gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary. PLoS One 9: e113423. PubMed ID: 25420147
In order to sustain lifelong production of gametes, many animals have evolved a stem cell-based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. This study shows that a novel germline factor, Gone early (Goe; CG9634), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases (see Neprilysin 4). At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell-cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment.
Castanieto, A., Johnston, M. J. and Nystul, T. G. (2014). EGFR signaling promotes the identity of follicle stem cells via maintenance of partial cell polarity. Elife 3. PubMed ID: 25437306
Epithelial stem cells divide asymmetrically, such that one daughter replenishes the stem cell pool and the other differentiates. This study found that in the epithelial follicle stem cell (FSC) lineage of the Drosophila ovary, EGFR signaling functions specifically in the FSCs to promote the unique partially polarized state of the FSC, establish apical-basal polarity throughout the lineage, and promote FSC maintenance in the niche. In addition, a novel connection was identified between EGFR signaling and the cell polarity regulator, LKB1, which indicates that EGFR signals through both the Ras-Raf-MEK-Erk pathway and through the LKB1-AMPK pathway to suppress apical identity. The development of apical-basal polarity is the earliest visible difference between FSCs and their daughters, and these findings demonstrate that the EGFR-mediated regulation of apical-basal polarity is essential for the segregation of stem cell and daughter cell fates.
Cetera, M., Ramirez-San Juan, G. R., Oakes, P. W., Lewellyn, L., Fairchild, M. J., Tanentzapf, G., Gardel, M. L. and Horne-Badovinac, S. (2014). Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber. Nat Commun 5: 5511. PubMed ID: 25413675 elongation
Tissues use numerous mechanisms to change shape during development. The Drosophila egg chamber is an organ-like structure that elongates to form an elliptical egg. During elongation the follicular epithelial cells undergo a collective migration that causes the egg chamber to rotate within its surrounding basement membrane. Rotation coincides with the formation of a 'molecular corset', in which actin bundles in the epithelium and fibrils in the basement membrane are all aligned perpendicular to the elongation axis. This study shows that rotation plays a critical role in building the actin-based component of the corset. Rotation begins shortly after egg chamber formation and requires lamellipodial protrusions at each follicle cell's leading edge. During early stages, rotation is necessary for tissue-level actin bundle alignment, but it becomes dispensable after the basement membrane is polarized. This work highlights how collective cell migration can be used to build a polarized tissue organization for organ morphogenesis.
Kronja, I., Whitfield, Z. J., Yuan, B., Dzeyk, K., Kirkpatrick, J., Krijgsveld, J. and Orr-Weaver, T. L. (2014). Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition. Proc Natl Acad Sci U S A. PubMed ID: 25349405
The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, proteome remodeling has been described during Drosophila egg activation. This study describes quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. Muskelin, found to be up-regulated during oocyte maturation, was shown to be required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. This study found down-regulation of this class of proteins to be mediated partially by APC/CORT, a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C).
Monday, December 8th
Chen, W., Liu, Z., Li, T., Zhang, R., Xue, Y., Zhong, Y., Bai, W., Zhou, D. and Zhao, Z. (2014). Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle. Nat Commun 5: 5549. PubMed ID: 25417916
MicroRNA-mediated post-transcriptional regulations are increasingly recognized as important components of the circadian rhythm. This study identified microRNA let-7, part of the Drosophila let-7-Complex, as a regulator of circadian rhythms mediated by a circadian regulatory cycle. Overexpression of let-7 in clock neurons lengthens circadian period and its deletion attenuates the morning activity peak as well as molecular oscillation. Let-7 regulates the circadian rhythm via repression of Clockwork Orange (Cwo). Conversely, upregulated cwo in cwo-expressing cells can rescue the phenotype of let-7-Complex overexpression. Moreover, circadian Prothoracicotropic hormone (PTTH) and Clock-regulated 20-OH ecdysteroid signalling contribute to the circadian expression of let-7 through the 20-OH Ecdysteroid receptor. Thus, this study has found a regulatory cycle involving PTTH, a direct target of Clock, and PTTH-driven miRNA let-7.
Majzoub, K., Hafirassou, M. L., Meignin, C., Goto, A., Marzi, S., Fedorova, A., Verdier, Y., Vinh, J., Hoffmann, J. A., Martin, F., Baumert, T. F., Schuster, C. and Imler, J. L. (2014). RACK1 controls IRES-mediated translation of viruses. Cell 159: 1086-1095. PubMed ID: 25416947
Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, this study identified the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. It was further shown that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. These findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.
Vaughn, J. C., Ghosh, S. and Chen, J. (2013). A phylogenetic study of splicing assembly chaperone RNP-4F associated U4-/U6-snRNA secondary structure. Open J Anim Sci 3: 36-48. PubMed ID: 25419488
The rnp-4f gene in Drosophila melanogaster encodes nuclear protein RNP-4F. This encoded protein is represented by homologs in other eukaryotic species, where it has been shown to function as an intron splicing assembly factor. Here, RNP-4F is believed to initially bind to a recognition sequence on U6-snRNA, serving as a chaperone to facilitate its association with U4-snRNA by intermolecular hydrogen bonding. RNA conformations are a key factor in spliceosome function, so that elucidation of changing secondary structures for interacting snRNAs is a subject of considerable interest and importance. Among the five snRNAs which participate in removal of spliceosomal introns, there is a growing consensus that U6-snRNA is the most structurally dynamic and may constitute the catalytic core. Previous studies by others have generated potential secondary structures for free U4- and U6-snRNAs, including the Y-shaped U4-/U6-snRNA model. These models were based on study of RNAs from relatively few species, and the popular Y-shaped model remains to be systematically re-examined with reference to the many new sequences generated by recent genomic sequencing projects. This study utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure. This general model is supported by observation of abundant compensatory base mutations in every stem, and incorporates more of the nucleotides into base-paired associations than in previous models, thus being more energetically stable. the eukaryotic phylogenetic tree was extensively sampled to its deepest roots, but no genes potentially encoding either U4- or U6-snRNA were found in the Giardia and Trichomonas data-bases. The results support the hypothesis that nuclear introns in these most deeply rooted eukaryotes may represent evolutionary intermediates, sharing characteristics of both group II and spliceosomal introns. An unexpected result of this study was discovery of a potential competitive binding site for Drosophila splicing assembly factor RNP-4F to a 5'-UTR regulatory region within its own premRNA, which may play a role in negative feedback control.
Lu, Z. and Matera, A. G. (2014). Developmental analysis of spliceosomal snRNA isoform expression. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25416704
Pre-mRNA splicing is a critical step in eukaryotic gene expression that contributes to proteomic, cellular and developmental complexity. Small nuclear (sn)RNAs are core spliceosomal components, however, the extent to which differential expression of snRNA isoforms regulates splicing is completely unknown. This is partly due to difficulties in the accurate analysis of the spatial and temporal expression patterns of snRNAs. This study used high throughput RNA-sequencing (RNA-seq) data to profile expression of four major snRNAs throughout Drosophila development. This analysis shows that individual isoforms of each snRNA have distinct expression patterns in the embryo, larva and pharate adult stages. Expression of these isoforms is more heterogeneous during embryogenesis, and as development progresses, a single isoform from each snRNA subtype gradually dominates expression. Despite the lack of stable snRNA orthologous groups during evolution, this developmental switching of snRNA isoforms also occurs in distantly related vertebrate species, such as Xenopus, mouse and human. These results indicate that expression of snRNA isoforms is regulated, and lay the foundation for functional studies of individual snRNA isoforms.
Sunday, December 7th
Carvalho, L., Jacinto, A. and Matova, N. (2014). The Toll/NF-kappaB signaling pathway is required for epidermal wound repair in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 25427801
The Toll/NF-kappaB pathway, first identified in studies of dorsal-ventral polarity in the early Drosophila embryo, is well known for its role in the innate immune response. This study revealed that the Toll/NF-kappaB pathway is essential for wound closure in late Drosophila embryos. Toll mutants and Dif dorsal (NF-kappaB) double mutants are unable to repair epidermal gaps. Dorsal is activated on wounding, and Dif and Dorsal are required for the sustained down-regulation of E-cadherin, an obligatory component of the adherens junctions (AJs), at the wound edge. This remodeling of the AJs promotes the assembly of an actin-myosin cable at the wound margin; contraction of the actin cable, in turn, closes the wound. In the absence of Toll or Dif and dorsal, both E-cadherin down-regulation and actin-cable formation fail, thus resulting in open epidermal gaps. Given the conservation of the Toll/NF-kappaB pathway in mammals and the epithelial expression of many components of the pathway, this function in wound healing is likely to be conserved in vertebrates.
Wang, S. J., Tsai, A., Wang, M., Yoo, S., Kim, H. Y., Yoo, B., Chui, V., Kisiel, M., Stewart, B., Parkhouse, W., Harden, N. and Krieger, C. (2014). Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction. Biol Open [Epub ahead of print]. PubMed ID: 25416060
Adducin is a ubiquitously expressed actin- and spectrin-binding protein involved in cytoskeleton organization, and is regulated through phosphorylation of the myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain by Protein kinase C (PKC). The Drosophila adducin, Hu-li tai shao (Hts), has been shown to play a role in larval neuromuscular junction (NMJ) growth. This study finds that the predominant isoforms of Hts at the NMJ contain the MARCKS-homology domain, which is important for interactions with Discs large (Dlg) and phosphatidylinositol 4,5-bisphosphate (PIP2). Through the use of Proximity Ligation Assay (PLA), this study shows that the adducin-like Hts isoforms are in complexes with Dlg and PIP2 at the NMJ. Evidence is provided that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle. It was also shown that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain. These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila.
Fullard, J. F. and Baker, N. E. (2014). A modifier screen in Drosophila melanogaster implicates cytoskeletal regulators, Jun N-terminal kinase, and Yorkie in Draper signaling. Genetics [Epub ahead of print]. PubMed ID: 25395664
The Drosophila melanogaster homolog of the ced-1 gene from Caenorhabditis elegans is draper, which encodes a cell surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, a genetic screen of chromosomal deficiencies was performed to identify loci that dominantly modify the phenotype of over-expression of Draper isoform II, which suppresses differentiation of the posterior crossvein in the wing. The existence of 43 genetic modifiers of Draper II was deduced. 24 of the 37 suppressor loci and 3 of the 6 enhancer loci have been identified. A further 5 suppressors and 2 enhancers were identified from mutations in functionally related genes. These studies indicated positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott-Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in the engulfment process downstream of Draper activation. The relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador-Warts-Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are further investigated and discussed.
Bhabha, G., Cheng, H. C., Zhang, N., Moeller, A., Liao, M., Speir, J. A., Cheng, Y. and Vale, R. D. (2014). Allosteric communication in the Dynein motor domain. Cell 159: 857-868. PubMed ID: 25417161
Dyneins power microtubule motility using ring-shaped, AAA-containing motor domains. This study report X-ray and electron microscopy (EM) structures of yeast dynein bound to different ATP analogs, which collectively provide insight into the roles of dynein's two major ATPase sites, AAA1 and AAA3, in the conformational change mechanism. ATP binding to AAA1 triggers a cascade of conformational changes that propagate to all six AAA domains and cause a large movement of the "linker," dynein's mechanical element. In contrast to the role of AAA1 in driving motility, nucleotide transitions in AAA3 gate the transmission of conformational changes between AAA1 and the linker, suggesting that AAA3 acts as a regulatory switch. Further structural and mutational studies also uncover a role for the linker in regulating the catalytic cycle of AAA1. Together, these results reveal how dynein's two major ATP-binding sites initiate and modulate conformational changes in the motor domain during motility.
Saturday, December 6th
Couturier, L., Trylinski, M., Mazouni, K., Darnet, L. and Schweisguth, F. (2014). A fluorescent tagging approach in Drosophila reveals late endosomal trafficking of Notch and Sanpodo. J Cell Biol 207(3): 351-63. PubMed ID: 25365996
Signaling and endocytosis are highly integrated processes that regulate cell fate. In the Drosophila melanogaster sensory bristle lineages, Numb inhibits the recycling of Notch and its trafficking partner Sanpodo (Spdo) to regulate cell fate after asymmetric cell division. This paper used a dual GFP/Cherry tagging approach to study the distribution and endosomal sorting of Notch and Spdo in living pupae. The specific properties of GFP, i.e., quenching at low pH, and Cherry, i.e., slow maturation time, revealed distinct pools of Notch and Spdo: cargoes exhibiting high GFP/low Cherry fluorescence intensities localized mostly at the plasma membrane and early/sorting endosomes, whereas low GFP/high Cherry cargoes accumulated in late acidic endosomes. These properties were used to show that Spdo is sorted toward late endosomes in a Numb-dependent manner. This dual-tagging approach should be generally applicable to study the trafficking dynamics of membrane proteins in living cells and tissues.
Chabu, C. and Xu, T. (2014). Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth. Development 141(24):4729-39. PubMed ID: 25411211
Oncogenic mutations in Ras deregulate cell death and proliferation to cause cancer in a significant number of patients. Although normal Ras signaling during development has been well elucidated in multiple organisms, it is less clear how oncogenic Ras exerts its effects. Furthermore, cancers with oncogenic Ras mutations are aggressive and generally resistant to targeted therapies or chemotherapy. This study identified the exocytosis component Sec15 as a synthetic suppressor of oncogenic Ras in an in vivo Drosophila mosaic screen. Oncogenic Ras elevates exocytosis and promotes the export of the pro-apoptotic ligand Eiger (Drosophila TNF). This blocks tumor cell death and stimulates overgrowth by activating the JNK-JAK-STAT non-autonomous proliferation signal from the neighboring wild-type cells. Inhibition of Eiger/TNF exocytosis or interfering with the JNK-JAK-STAT non-autonomous proliferation signaling at various steps suppresses oncogenic Ras-mediated overgrowth. These findings highlight important cell-intrinsic and cell-extrinsic roles of exocytosis during oncogenic growth and provide a new class of synthetic suppressors for targeted therapy approaches.
Schreij, A. M., Chaineau, M., Ruan, W., Lin, S., Barker, P. A., Fon, E. A. and McPherson, P. S. (2014). LRRK2 localizes to endosomes and interacts with clathrin-light chains to limit Rac1 activation. EMBO Rep [Epub ahead of print]. PubMed ID: 25427558
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of dominant-inherited Parkinson's disease (PD), and yet the physiological functions of LRRK2 are not fully understood. Various components of the clathrin machinery have been recently found mutated in familial forms of PD. This study provides molecular insight into the association of LRRK2 with the clathrin machinery. Through its GTPase domain, LRRK2 binds directly to clathrin-light chains (CLCs). Using genome-edited HA-LRRK2 cells, LRRK2 was localized to endosomes on the degradative pathway, where it partially co-localizes with CLCs. Knockdown of CLCs and/or LRRK2 enhances the activation of the small GTPase Rac1, leading to alterations in cell morphology, including the disruption of neuronal dendritic spines. In Drosophila, a minimal rough eye phenotype caused by overexpression of Rac1, is dramatically enhanced by loss of function of CLC and LRRK2 homologues, confirming the importance of this pathway in vivo. These data identify a new pathway in which CLCs function with LRRK2 to control Rac1 activation on endosomes, providing a new link between the clathrin machinery, the cytoskeleton and PD.
Fernandes, A. C., Uytterhoeven, V., Kuenen, S., Wang, Y. C., Slabbaert, J. R., Swerts, J., Kasprowicz, J., Aerts, S. and Verstreken, P. (2014). Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration. J Cell Biol 207: 453-462. PubMed ID: 25422373
Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. This paper studied Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome; endosome-to-lysosome trafficking was identified as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, synaptic vesicle-associated proteins were shown to be younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, it was found that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. These findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.
Wu, F., Hu, X., Bian, X., Liu, X. and Hu, J. (2014). Comparison of human and Drosophila atlastin GTPases. Protein Cell [Epub ahead of print]. PubMed ID: 25407413
Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a class of atlastin (ATL) GTPases. Purified Drosophila ATL is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. This study determined the preliminary crystal structure of the cytosolic segment of Drosophila ATL in a GDP-bound state. The structure reveals a GTPase domain dimer with the subsequent three-helix bundles associating with their own GTPase domains and pointing in opposite directions. This conformation is similar to that of human ATL1, to which GDP and high concentrations of inorganic phosphate, but not GDP only, were included. Drosophila ATL restored ER morphology defects in mammalian cells lacking ATLs, and measurements of nucleotide-dependent dimerization and GTPase activity were comparable for Drosophila ATL and human ATL1. However, purified and reconstituted human ATL1 exhibited no in vitro fusion activity. When the cytosolic segment of human ATL1 was connected to the transmembrane (TM) region and C-terminal tail (CT) of Drosophila ATL, the chimera still exhibited no fusion activity, though its GTPase activity was normal. These results suggest that GDP-bound ATLs may adopt multiple conformations and the in vitro fusion activity of ATL cannot be achieved by a simple collection of functional domains.
Friday, December 5th
Smith-Vikos, T., de Lencastre, A., Inukai, S., Shlomchik, M., Holtrup, B. and Slack, F. J. (2014). MicroRNAs mediate dietary-restriction-induced longevity through PHA-4/FOXA and SKN-1/Nrf transcription factors. Curr Biol 24: 2238-2246. PubMed ID: 25242029
Dietary restriction (DR) has been shown to prolong longevity across diverse taxa, yet the mechanistic relationship between DR and longevity remains unclear. MicroRNAs (miRNAs) control aging-related functions such as metabolism and lifespan through regulation of genes in insulin signaling, mitochondrial respiration, and protein homeostasis. A network analysis was conducted of aging-associated miRNAs connected to transcription factors PHA-4/FOXA and SKN-1/Nrf (homologs of Drosophila Forkhead and Cap'n'collar respectively), which are both necessary for DR-induced lifespan extension in Caenorhabditis elegans. This network analysis has revealed extensive regulatory interactions between PHA-4, SKN-1, and miRNAs and points to two aging-associated miRNAs, miR-71 and miR-228, as key nodes of this network. This study shows that miR-71 and miR-228 are critical for the response to DR in C. elegans. DR induces the expression of miR-71 and miR-228, and the regulation of these miRNAs depends on PHA-4 and SKN-1. In turn, PHA-4 and SKN-1 were shown to be negatively regulated by miR-228, whereas miR-71 represses PHA-4. This study has discovered new links in an important pathway connecting DR to aging. By interacting with PHA-4 and SKN-1, miRNAs transduce the effect of dietary-restriction-mediated lifespan extension in C. elegans. Given the conservation of miRNAs, PHA-4, and SKN-1 across phylogeny, these interactions are likely to be conserved in more-complex species.
He, F., James, A., Raje, H., Ghaffari, H. and DiMario, P. (2014). Deletion of Drosophila Nopp140 induces subcellular ribosomopathies. Chromosoma [Epub ahead of print]. PubMed ID: 25384888
The nucleolar and Cajal body phosphoprotein of 140 kDa (Nopp140) is considered a ribosome assembly factor, but its precise functions remain unknown. To approach this problem, the Nopp140 gene was deleted in Drosophila using FLP-FRT recombination. Genomic PCR, reverse transcriptase-PCR (RT-PCR), and immunofluorescence microscopy confirmed the loss of Nopp140, its messenger RNA (mRNA), and protein products from all tissues examined. Nopp140-/- larvae arrested in the second instar stage and most died within 8 days. While nucleoli appeared intact in Nopp140-/- cells, the C/D small nucleolar ribonucleoprotein (snoRNP) methyltransferase, Fibrillarin, redistributed to the nucleoplasm in variable amounts depending on the cell type; RT-PCRs showed that 2'-O-methylation of ribosomal RNA (rRNA) in Nopp140-/- cells was reduced at select sites within both the 18S and 28S rRNAs. Ultrastructural analysis showed that Nopp140-/- cells were deficient in cytoplasmic ribosomes, but instead contained abnormal electron-dense cytoplasmic granules. Immunoblot analysis showed a loss of RpL34, and metabolic labeling showed a significant drop in protein translation, supporting the loss of functional ribosomes. Northern blots showed that pre-RNA cleavage pathways were generally unaffected by the loss of Nopp140, but that R2 retrotransposons that naturally reside within the 28S region of normally silent heterochromatic Drosophila ribosomal DNA (rDNA) genes were selectively expressed in Nopp140-/- larvae. Unlike copia elements and the related R1 retrotransposon, R2 expression appeared to be preferentially dependent on the loss of Nopp140 and not on environmental stresses. The phenotypes described in this study are believed to define novel intracellular ribosomopathies resulting from the loss of Nopp140.
Chen, D., Wu, C., Zhao, S., Geng, Q., Gao, Y., Li, X., Zhang, Y. and Wang, Z. (2014). Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila. PLoS Genet 10: e1004797. PubMed ID: 25412508
In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, a mutation was isolated in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. Tut was found to bind preferentially a long isoform of mei-P26 3'UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, this study demonstrates that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Both in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage.
Baggio, F., Bratic, A., Mourier, A., Kauppila, T. E., Tain, L. S., Kukat, C., Habermann, B., Partridge, L. and Larsson, N. G. (2014). Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 25428350
Members of the pentatricopeptide repeat domain (PPR) protein family bind RNA and are important for post-transcriptional control of organelle gene expression in unicellular eukaryotes, metazoans and plants. They also have a role in human pathology, as mutations in the leucine-rich PPR-containing (LRPPRC) gene cause severe neurodegeneration. The mammalian LRPPRC protein and its Drosophila melanogaster homolog DmLRPPRC1 (also known as Bicoid stability factor) have been shown to be necessary for mitochondrial translation by controlling stability and polyadenylation of mRNAs. This study reports characterization of DmLRPPRC2 (CG14786), a second fruit fly homolog of LRPPRC, and shows that it has a predominant mitochondrial localization and interacts with a stem-loop interacting RNA binding protein (CG3021/DmSLIRP2). Ubiquitous downregulation of DmLrpprc2 expression causes respiratory chain dysfunction, developmental delay and shortened lifespan. Unexpectedly, decreased DmLRPPRC2 expression does not globally affect steady-state levels or polyadenylation of mitochondrial transcripts. However, some mitochondrial transcripts abnormally associate with the mitochondrial ribosomes and some products are dramatically overproduced and other ones decreased, which, in turn, results in severe deficiency of respiratory chain complexes. The function of DmLRPPRC2 thus seems to be to ensure that mitochondrial transcripts are presented to the mitochondrial ribosomes in an orderly fashion to avoid poorly coordinated translation.
Thursday, December 4th
Austin, C. L., Manivannan, S. N. and Simcox, A. (2014). TGF-alpha ligands can substitute for the neuregulin Vein in Drosophila development. Development 141: 4110-4114. PubMed ID: 25336739
ErbB receptors, including the epidermal growth factor receptor (Egfr), are activated by EGF ligands to govern cell proliferation, survival, migration and differentiation. The different EGF-induced cell responses in development are regulated by deployment of multiple ligands. These inputs, however, engage only a limited number of intracellular pathways and are thought to elicit specific responses by regulating the amplitude or duration of the intracellular signal. The single Drosophila Egfr has four ligands: three of the TGF-alpha-type and a single neuregulin-like called vein (vn). This study used mutant combinations and gene replacement to determine the constraints of ligand specificity in development. Mutant analysis revealed extensive ligand redundancy in embryogenesis and wing development. Surprisingly, it was found that the essential role of vn in development could be largely replaced by expression of any TGF-alpha ligand, including spitz (spi), in the endogenous vn pattern. vn mutants die as white undifferentiated pupae, but the rescued individuals showed global differentiation of adult body parts. Spi is more potent than Vn, and the best morphological rescue occurred when Spi expression was reduced to achieve an intracellular signaling level comparable to that produced by Vn. These results show that the developmental repertoire of a strong ligand like Spi is flexible and at the appropriate level can emulate the activity of a weak ligand like Vn. These findings align with a model whereby cells respond similarly to an equivalent quantitative level of an intracellular signal generated by two distinct ligands regardless of ligand identity.
Miller, S. W., Rebeiz, M., Atanasov, J. E. and Posakony, J. W. (2014). Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function. Proc Natl Acad Sci U S A. PubMed ID: 25404315
Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. This study shows that the utilization of dual, or 'shadow', enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P; Scute, for example) proteins and basic helix-loop-helix (bHLH) repressor (R; Hairy/Enhancer of split (Hes) class) factors (a 'P+R' regulatory code), NP-specific enhancer modules were identified associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, P sites in these modules were shown to mediate activation by proneural factors in 'proneural cluster' territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. These results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), it was demonstrated that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event.
Anderson, E., Devenney, P. S., Hill, R. E. and Lettice, L. A. (2014). Mapping the Shh long-range regulatory domain. Development 141: 3934-3943. PubMed ID: 25252942
Coordinated gene expression controlled by long-distance enhancers is orchestrated by DNA regulatory sequences involving transcription factors and layers of control mechanisms. The Shh (see Drosophila Hedgehog) gene and well-established regulators are an example of genomic composition in which enhancers reside in a large desert extending into neighbouring genes to control the spatiotemporal pattern of expression. Exploiting the local hopping activity of the Sleeping Beauty transposon, the lacZ reporter gene was dispersed throughout the Shh region to systematically map the genomic features responsible for expression activity. Enhancer activities were found to be retained inside a genomic region that corresponds to the topological associated domain (TAD) defined by Hi-C. This domain of approximately 900 kb is in an open conformation over its length and is generally susceptible to all Shh enhancers. Similar to the distal enhancers, an enhancer residing within the Shh second intron activates the reporter gene located at distances of hundreds of kilobases away, suggesting that both proximal and distal enhancers have the capacity to survey the Shh topological domain to recognise potential promoters. The widely expressed Rnf32 gene lying within the Shh domain evades enhancer activities by a process that may be common among other housekeeping genes that reside in large regulatory domains. Finally, the boundaries of the Shh TAD do not represent the absolute expression limits of enhancer activity, as expression activity is lost stepwise at a number of genomic positions at the verges of these domains.
Ip, C. K., Fossat, N., Jones, V., Lamonerie, T. and Tam, P. P. (2014). Head formation: OTX2 regulates Dkk1 and Lhx1 activity in the anterior mesendoderm. Development 141: 3859-3867. PubMed ID: 25231759
The Otx2 (see Drosophila Ocelliless/Orthodenticle) gene encodes a paired-type homeobox transcription factor that is essential for the induction and the patterning of the anterior structures in the mouse embryo. Otx2 knockout embryos fail to form a head. Whereas previous studies have shown that Otx2 is required in the anterior visceral endoderm and the anterior neuroectoderm for head formation, its role in the anterior mesendoderm (AME) has not been assessed specifically. This study shows that tissue-specific ablation of Otx2 in the AME phenocopies the truncation of the embryonic head of the Otx2 null mutant. Expression of Dkk1 and Lhx1, two genes that are also essential for head formation, is disrupted in the AME of the conditional Otx2-deficient embryos. Consistent with the fact that Dkk1 is a direct target of OTX2, it was shown that OTX2 can interact with the H1 regulatory region of Dkk1 to activate its expression. Cross-species comparative analysis, RT-qPCR, ChIP-qPCR and luciferase assays have revealed two conserved regions in the Lhx1 locus to which OTX2 can bind to activate Lhx1 expression. Abnormal development of the embryonic head in Otx2;Lhx1 and Otx2;Dkk1 compound mutant embryos highlights the functional intersection of Otx2, Dkk1 and Lhx1 in the AME for head formation.
Wednesday, December 3rd
Jepson, J. E., Shahidullah, M., Liu, D., le Marchand, S. J., Liu, S., Wu, M. N., Levitan, I. B., Dalva, M. B. and Koh, K. (2014). Regulation of synaptic development and function by the Drosophila PDZ protein Dyschronic. Development 141: 4548-4557. PubMed ID: 25359729
Synaptic scaffold proteins control the localization of ion channels and receptors, and facilitate molecular associations between signaling components that modulate synaptic transmission and plasticity. This study defines novel roles for a recently described scaffold protein, Dsychronic (DYSC), at the Drosophila larval neuromuscular junction. DYSC is the Drosophila homolog of whirlin/DFNB31, a PDZ domain protein linked to Usher syndrome, the most common form of human deaf-blindness. DYSC is expressed presynaptically and is often localized adjacent to the active zone, the site of neurotransmitter release. Loss of DYSC results in marked alterations in synaptic morphology and cytoskeletal organization. Moreover, active zones are frequently enlarged and misshapen in dysc mutants. Electrophysiological analyses further demonstrate that dysc mutants exhibit substantial increases in both evoked and spontaneous synaptic transmission. Previous study have shown that DYSC binds to and regulates the expression of the Slowpoke (SLO) BK potassium channel. Consistent with this, slo mutant larvae exhibit similar alterations in synapse morphology, active zone size and neurotransmission, and simultaneous loss of dysc and slo does not enhance these phenotypes, suggesting that dysc and slo act in a common genetic pathway to modulate synaptic development and output. These data expand understanding of the neuronal functions of DYSC and uncover non-canonical roles for the SLO potassium channel at Drosophila synapses.
Andlauer, T. F., Scholz-Kornehl, S., Tian, R., Kirchner, M., Babikir, H. A., Depner, H., Loll, B., Quentin, C., Gupta, V. K., Holt, M. G., Dipt, S., Cressy, M., Wahl, M. C., Fiala, A., Selbach, M., Schwarzel, M. and Sigrist, S. J. (2014). Drep-2 is a novel synaptic protein important for learning and memory. Elife 3 [Epub ahead of print]. PubMed ID: 25392983
CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. This study reports that the CIDE-N protein DNA fragmentation factor-related protein 2 (Drep-2) is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation.
Piguel, N. H., et al. (2014). Scribble1/AP2 complex coordinates NMDA receptor endocytic recycling. Cell Rep. PubMed ID: 25310985
The appropriate trafficking of glutamate receptors to synapses is crucial for basic synaptic function and synaptic plasticity. It is now accepted that NMDA receptors (NMDARs; see Drosophila NMDA receptors) internalize and are recycled at the plasma membrane but also exchange between synaptic and extrasynaptic pools; these NMDAR properties are also key to governing synaptic plasticity. Scribble1 (see Drosophila Scribbled) is a large PDZ protein required for synaptogenesis and synaptic plasticity. This study shows that the level of Scribble1 is regulated in an activity-dependent manner and that Scribble1 controls the number of NMDARs at the plasma membrane. Notably, Scribble1 prevents GluN2A subunits from undergoing lysosomal trafficking and degradation by increasing their recycling to the plasma membrane following NMDAR activation. Finally, it was shown that a specific YxxR motif on Scribble1 controls these mechanisms through a direct interaction with AP2 (see Drosophila AP2). Altogether, these findings define a molecular mechanism to control the levels of synaptic NMDARs via Scribble1 complex signaling.
Adams, P. J., Ben-Johny, M., Dick, I. E., Inoue, T. and Yue, D. T. (2014). Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation. Cell 159: 608-622. PubMed ID: 25417111
The Ca(2+)-free form of calmodulin (apoCaM) often appears inert, modulating target molecules only upon conversion to its Ca(2+)-bound form. This schema has appeared to govern voltage-gated Ca(2+) channels, where apoCaM has been considered a dormant Ca(2+) sensor, associated with channels but awaiting the binding of Ca(2+) ions before inhibiting channel opening to provide vital feedback inhibition. Using single-molecule measurements of channels and chemical dimerization to elevate apoCaM, this study found that apoCaM binding on its own markedly upregulates opening, rivaling the strongest forms of modulation.
Examining adult mammalian heart cells this study found that CaM-mediated feedback on cardiac L-type Ca2+ channels is the dominant control factor in controlling the cardiac and neuronal action potential duration, a vital excitability parameter whose prolongation drives heart failure and neuronal Ca2+ overload. Upon Ca(2+) binding to this CaM, inhibition may simply reverse the initial upregulation. As RNA-edited and -spliced channel variants show different affinities for apoCaM, the apoCaM-dependent control mechanisms may underlie the functional diversity of these variants and explain an elongation of neuronal action potentials by apoCaM. More broadly, voltage-gated Na channels adopt this same modulatory principle. ApoCaM thus imparts potent and pervasive ion-channel regulation.
Luo, S., Schaefer, A. M., Dour, S. and Nonet, M. L. (2014). The conserved LIM domain-containing focal adhesion protein ZYX-1 regulates synapse maintenance in Caenorhabditis elegans. Development 141: 3922-3933. PubMed ID: 25252943
This study describes the identification of zyxin (see Drosophila Xyxin) as a regulator of synapse maintenance in mechanosensory neurons in C. elegans. zyx-1 mutants lacked PLM mechanosensory synapses as adult animals. However, most PLM synapses initially formed during development but were subsequently lost as the animals developed. Vertebrate zyxin regulates cytoskeletal responses to mechanical stress in culture. This work provides in vivo evidence in support of such a role for zyxin. In particular, zyx-1 mutant synaptogenesis phenotypes were suppressed by disrupting locomotion of the mutant animals, suggesting that zyx-1 protects mechanosensory synapses from locomotion-induced forces. In cultured cells, zyxin is recruited to focal adhesions and stress fibers via C-terminal LIM domains and modulates cytoskeletal organization via the N-terminal domain. The synapse-stabilizing activity was mediated by a short isoform of ZYX-1 containing only the LIM domains. Consistent with this notion, PLM synaptogenesis was independent of α-actinin and ENA-VASP, both of which bind to the N-terminal domain of zyxin. These results demonstrate that the LIM domain moiety of zyxin functions autonomously to mediate responses to mechanical stress and provide in vivo evidence for a role of zyxin in neuronal development.
Tuesday, December 2nd
Abe, T., Yamazaki, D., Murakami, S., Hiroi, M., Nitta, Y., Maeyama, Y. and Tabata, T. (2014). The NAV2 homolog Sickie regulates F-actin-mediated axonal growth in Drosophila mushroom body neurons via the non-canonical Rac-Cofilin pathway. Development [Epub ahead of print]. PubMed ID: 25411210
The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. This study demonstrates that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. Phenotypic similarities and genetic interactions were observed among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. It is proposed that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner.
Gao, P., Postiglione, M. P., Krieger, T. G., Hernandez, L., Wang, C., Han, Z., Streicher, C., Papusheva, E., Insolera, R., Chugh, K., Kodish, O., Huang, K., Simons, B. D., Luo, L., Hippenmeyer, S. and Shi, S. H. (2014). Deterministic progenitor behavior and unitary production of neurons in the neocortex. Cell 159: 775-788. PubMed ID: 25417155
Radial glial progenitors (RGPs) are responsible for producing nearly all neocortical neurons. To gain insight into the patterns of RGP division and neuron production, excitatory neuron genesis was quantitatively analyzed in the mouse neocortex using Mosaic Analysis with Double Markers, which provides single-cell resolution of progenitor division patterns and potential in vivo. RGPs were found to progress through a coherent program in which their proliferative potential diminishes in a predictable manner. Upon entry into the neurogenic phase, individual RGPs produce ~8-9 neurons distributed in both deep and superficial layers, indicating a unitary output in neuronal production. Removal of OTX1 (see Drosophila Orthodenticle), a transcription factor transiently expressed in RGPs, results in both deep- and superficial-layer neuron loss and a reduction in neuronal unit size. Moreover, ~1/6 of neurogenic RGPs proceed to produce glia. These results suggest that progenitor behavior and histogenesis in the mammalian neocortex conform to a remarkably orderly and deterministic program.
Morimoto-Suzki, N., Hirabayashi, Y., Tyssowski, K., Shinga, J., Vidal, M., Koseki, H. and Gotoh, Y. (2014). The polycomb component Ring1B regulates the timed termination of subcerebral projection neuron production during mouse neocortical development. Development 141: 4343-4353. PubMed ID: 25344075
In the developing neocortex, neural precursor cells (NPCs) sequentially generate various neuronal subtypes in a defined order. Although the precise timing of the NPC fate switches is essential for determining the number of neurons of each subtype and for precisely generating the cortical layer structure, the molecular mechanisms underlying these switches are largely unknown. This study shows that epigenetic regulation through Ring1B (see Drosophila Ring), an essential component of polycomb group (PcG) complex proteins, plays a key role in terminating NPC-mediated production of subcerebral projection neurons (SCPNs). The level of histone H3 residue K27 trimethylation at and Ring1B binding to the promoter of Fezf2, a fate determinant of SCPNs, increased in NPCs as Fezf2 expression decreased. Moreover, deletion of Ring1B in NPCs, but not in postmitotic neurons, prolonged the expression of Fezf2 and the generation of SCPNs that were positive for CTIP2. These results indicate that Ring1B mediates the timed termination of Fezf2 expression and thereby regulates the number of SCPNs.
Vue, T. Y., Kim, E. J., Parras, C. M., Guillemot, F. and Johnson, J. E. (2014). Ascl1 controls the number and distribution of astrocytes and oligodendrocytes in the gray matter and white matter of the spinal cord. Development 141: 3721-3731. PubMed ID: 25249462
Glia constitute the majority of cells in the mammalian central nervous system and are crucial for neurological function. However, there is an incomplete understanding of the molecular control of glial cell development. This study found that the transcription factor Ascl1 (Mash1; see Drosophila Achaete), which is best known for its role in neurogenesis, also functions in both astrocyte and oligodendrocyte lineages arising in the mouse spinal cord at late embryonic stages. Clonal fate mapping in vivo reveals heterogeneity in Ascl1-expressing glial progenitors and shows that Ascl1 defines cells that are restricted to either gray matter (GM) or white matter (WM) as astrocytes or oligodendrocytes. Conditional deletion of Ascl1 post-neurogenesis shows that Ascl1 is required during oligodendrogenesis for generating the correct numbers of WM but not GM oligodendrocyte precursor cells, whereas during astrocytogenesis Ascl1 functions in balancing the number of dorsal GM protoplasmic astrocytes with dorsal WM fibrous astrocytes. Thus, in addition to its function in neurogenesis, Ascl1 marks glial progenitors and controls the number and distribution of astrocytes and oligodendrocytes in the GM and WM of the spinal cord.
Monday, December 1st
Sarma, K., Cifuentes-Rojas, C., Ergun, A., Del Rosario, A., Jeon, Y., White, F., Sadreyev, R. and Lee, J. T. (2014). ATRX Directs Binding of PRC2 to Xist RNA and Polycomb Targets. Cell 159: 869-883. PubMed ID: 25417162
X chromosome inactivation (XCI) in the mouse depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). PRC2 is also targeted to other sites throughout the genome to effect transcriptional repression. Using XCI as a model, this study applied an unbiased proteomics approach to isolate Xist and PRC2 regulators; ATRX was identified. ATRX unexpectedly functions as a high-affinity RNA-binding protein that directly interacts with RepA/Xist RNA to promote loading of PRC2 in vivo. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X chromosome. Moreover, epigenomic profiling reveals that genome-wide targeting of PRC2 depends on ATRX, as loss of ATRX leads to spatial redistribution of PRC2 and derepression of Polycomb responsive genes. Thus, ATRX is a required specificity determinant for PRC2 targeting and function.
Markenscoff-Papadimitriou, E., Allen, W. E., Colquitt, B. M., Goh, T., Murphy, K. K., Monahan, K., Mosley, C. P., Ahituv, N. and Lomvardas, S. (2014). Enhancer interaction networks as a means for singular olfactory receptor expression. Cell 159: 543-557. PubMed ID: 25417106
The transcriptional activation of one out of ~2800 mammalian olfactory receptor (OR) alleles is a poorly understood process. This study identified a plethora of putative OR enhancers, and their in vivo activity was studied in olfactory neurons. Distinguished by an unusual epigenetic signature, candidate OR enhancers are characterized by extensive interchromosomal interactions associated with OR transcription and share a similar pattern of transcription factor footprints. In particular, the role of the transcription factor bromodomain PHD finger transcription facto (Bptf) was establised as a facilitator of both enhancer interactions and OR transcription. These observations agree with the model whereby OR transcription occurs in the context of multiple interacting enhancers. Disruption of these interchromosomal interactions results in weak and multigenic OR expression, suggesting that the rare coincidence of numerous enhancers over a stochastically chosen OR may account for the singularity and robustness in OR transcription.
Milon, B., Sun, Y., Chang, W., Creasy, T., Mahurkar, A., Shetty, A., Nurminsky, D. and Nurminskaya, M. (2014). Map of open and closed chromatin domains in Drosophila genome. BMC Genomics 15: 988. PubMed ID: 25407537
Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification. This study developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin "states", individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, diverse "exceptions" from the conventional rules were detected. A profound paucity of chromatin modifications was found in a major fraction of closed chromatin, and a number of loci were detected where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, it was observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin.These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.
Gilliland, W. D., Colwell, E. M., Osiecki, D. M., Park, S., Lin, D., Rathnam, C. and Barbash, D. A. (2014). Normal segregation of a foreign-species chromosome during Drosophila female meiosis despite extensive heterochromatin divergence. Genetics [Epub ahead of print]. PubMed ID: 25406466
The abundance and composition of heterochromatin changes rapidly between species and contributes to hybrid incompatibility and reproductive isolation. Heterochromatin differences may also destabilize chromosome segregation and cause meiotic drive, the non-Mendelian segregation of homologous chromosomes. This study used a range of genetic and cytological assays to examine the meiotic properties of a Drosophila simulans chromosome 4 (sim-IV) introgressed into Drosophila melanogaster. These two species differ by ~12-13% at synonymous sites and several genes essential for chromosome segregation have experienced recurrent adaptive evolution since their divergence. Furthermore, their chromosome 4s are visibly different due to heterochromatin divergence, including in the AATAT pericentromeric satellite DNA. A visible imbalance was found in the positioning of the two chromosome 4s in sim-IV/mel-IV heterozygote, and this finding was also replicated with a D. melanogaster 4 containing a heterochromatic deletion. These results demonstrate that heterochromatin abundance can have a visible effect on chromosome positioning during meiosis. Despite this effect, however, it was found that sim-IV segregates normally in both diplo and triplo 4 D. melanogaster females, and does not experience elevated non-disjunction. It is concluded that segregation abnormalities and a high level of meiotic drive are not inevitable byproducts of extensive heterochromatin divergence.
Moschetti, R., Celauro, E., Cruciani, F., Caizzi, R. and Dimitri, P. (2014). On the evolution of Yeti, a Drosophila melanogaster heterochromatin gene. PLoS One 9: e113010. PubMed ID: 25405891
Constitutive heterochromatin is a ubiquitous and still unveiled component of eukaryotic genomes, within which it comprises large portions. Although constitutive heterochromatin is generally considered to be transcriptionally silent, it contains a significant variety of sequences that are expressed, among which about 300 single-copy coding genes have been identified by genetic and genomic analyses in the last decades. This study reports the results of the evolutionary analysis of Yeti, an essential gene of Drosophila melanogaster located in the deep pericentromeric region of chromosome 2R. By FISH, it was shown that Yeti maintains a heterochromatin location in both D. simulans and D. sechellia species, closely related to D. melanogaster, while in the more distant species e.g., D. pseudoobscura and D. virilis, it is found within euchromatin, in the syntenic chromosome Muller C, that corresponds to the 2R arm of D. melanogaster chromosome 2. Thus, over evolutionary time, Yeti has been resident on the same chromosomal element, but it progressively moved closer to the pericentric regions. Moreover, in silico reconstruction of the Yeti gene structure in 19 Drosophila species and in 5 non-drosophilid dipterans shows a rather stable organization during evolution. Accordingly, by PCR analysis and sequencing, it was found that the single intron of Yeti does not undergo major intraspecies or interspecies size changes, unlike the introns of other essential Drosophila heterochromatin genes, such as light and Dbp80. This implicates diverse evolutionary forces in shaping the structural organization of genes found within heterochromatin. Finally, the results of dS - dN tests show that Yeti is under negative selection both in heterochromatin and euchromatin, and indicate that the change in genomic location did not affected significantly the molecular evolution of the gene. Together, the results of this work contribute to understanding of the evolutionary dynamics of constitutive heterochromatin in the genomes of higher eukaryotes.
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