What's hot today
Friday, October 31st, 2014
Koryakov, D. E. and Zhimulev, I. F. (2014). DNA replication in nurse cell polytene chromosomes of Drosophila melanogaster otu mutants. Chromosoma [Epub ahead of print]. PubMed ID: 25256561
Drosophila cell lines are used extensively to study replication timing, yet data about DNA replication in larval and adult tissues are extremely limited. To address this gap, DNA replication in polytene chromosomes from nurse cells of Drosophila melanogaster otu mutants was traced using bromodeoxyuridine incorporation. Importantly, nurse cells are of female germline origin, unlike the classical larval salivary glands, that are somatic. In contrast to salivary gland polytene chromosomes, where replication begins simultaneously across all puffs and interbands, replication in nurse cells is first observed at several specific chromosomal regions. For instance, in the chromosome 2L, these include the regions 31B-E and 37E and proximal parts of 34B and 35B, with the rest of the decondensed chromosomal regions joining replication process a little later. It was observed that replication timing of pericentric heterochromatin in nurse cells was shifted from late S phase to early and mid stages. Curiously, chromosome 4 may represent a special domain of the genome, as it replicates on its own schedule which is uncoupled from the rest of the chromosomes. Finally, it is report that SUUR protein, an established marker of late replication in salivary gland polytene chromosomes, does not always colocalize with late-replicating regions in nurse cells.
Sakurai, H., Takai, S., Kawamura, K., Ogura, Y., Yoshioka, Y. and Kawasaki, K. (2014). Drosophila RecQ5 is involved in proper progression of early spermatogenesis. Biochem Biophys Res Commun. PubMed ID: 25245292
RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes. However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. This study analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, recq5 mutant testes were compared with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.
Glowinski, C., Liu, R. H., Chen, X., Darabie, A. and Godt, D. (2014). Myosin VIIA regulates microvillus morphogenesis and interacts with cadherin Cad99C in Drosophila oogenesis. J Cell Sci. PubMed ID: 25236597
Microvilli and related actin-based protrusions permit multiple interactions between cells and their environment. How shape, length, and arrangement of microvilli are determined remains largely unclear. To address this issue and explore the cooperation of the two main components of a microvillus, the central F-actin bundle and the enveloping plasma membrane, expression and function of Myosin VIIA (Myo7A), which is encoded by crinkled (ck) and its interaction with cadherin Cad99C in the microvilli of the Drosophila follicular epithelium. Myo7A is present in the microvilli and terminal web of follicle cells, and associates with several other F-actin-rich structures in the ovary. Loss of Myo7A caused brush border defects and a reduction in the amount of the microvillus regulator Cad99C. This study shows that Myo7A and Cad99C form a molecular complex and that the cytoplasmic tail of Cad99C recruits Myo7A to microvilli. The data indicate that Myo7A regulates the structure and spacing of microvilli, and interacts with Cad99C in vivo. A comparison of the mutant phenotypes suggests that Myo7A and Cad99C have co-dependent and independent functions in microvilli.
Thursday, October 30th
Cao, W. and Edery, I. (2014). A novel pathway for sensory-mediated arousal involves splicing of an intron in the clock gene. Sleep [Epub ahead of print]. PubMed ID: 25325457
D. melanogaster is an excellent animal model to study how the circadian timing system and sleep regulate daily wake-sleep cycles. Splicing of a temperature-sensitive 3'-terminal intron (termed dmpi8) from the circadian clock gene period (per) regulates the distribution of daily activity in Drosophila. The role of dmpi8 splicing on daily behavior was further evaluated by analyzing sleep. Transgenic flies of the same genetic background but expressing either a wild-type recombinant per gene or one where the efficiency of dmpi8 splicing was increased were exposed to different temperatures in daily light-dark cycles and sleep parameters measured. In addition, transgenic flies were briefly exposed to a variety of sensory-mediated stimuli to measure arousal responses. Surprisingly, the effect of dmpi8 splicing on daytime activity levels does not involve a circadian role for per but is linked to adjustments in sensory-dependent arousal and sleep behavior. Genetically altered flies with high dmpi8 splicing efficiency remain aroused longer following short treatments with light and non-photic cues such as mechanical stimulation. It is proposed that the thermal regulation of dmpi8 splicing acts as a temperature-calibrated rheostat in a novel arousal mechanism, so that on warm days the inefficient splicing of the dmpi8 intron triggers an increase in quiescence by decreasing sensory-mediated arousal, thus ensuring flies minimize being active during the hot midday sun despite the presence of light in the environment, which is usually a strong arousal cue for diurnal animals.
Schinaman, J. M., Giesey, R. L., Mizutani, C. M., Lukacsovich, T. and Sousa-Neves, R. (2014). The KRUPPEL-Like transcription factor DATILOGRAFO is required in specific cholinergic neurons for sexual receptivity in Drosophila females. PLoS Biol 12: e1001964. PubMed ID: 25291190
Courtship is a widespread behavior in which one gender conveys to the other a series of cues about their species identity, gender, and suitability as mates. In many species, females decode these male displays and either accept or reject them. Despite the fact that courtship has been investigated for a long time, the genes and circuits that allow females to generate these mutually exclusive responses remain largely unknown. This study provides evidence that the Kruppel-like transcription factor datilografo (dati) is required for proper locomotion and courtship acceptance in adult Drosophila females. dati mutant females are completely unable to decode male courtship and almost invariably reject males. Molecular analyses reveal that dati is broadly expressed in the brain and its specific removal in excitatory cholinergic neurons recapitulates the female courtship behavioral phenotype but not the locomotor deficits, indicating that these are two separable functions. Clonal analyses in female brains identified three discrete foci where dati is required to generate acceptance. These include neurons around the antennal lobe, the lateral horn, and the posterior superior lateral protocerebrum. Together, these results show that dati is required to organize and maintain a relatively simple excitatory circuit in the brain that allows females to either accept or reject courting males.
Nowotny, T., de Bruyne, M., Berna, A. Z., Warr, C. G. and Trowell, S. C. (2014). Drosophila olfactory receptors as classifiers for volatiles from disparate real world applications. Bioinspir Biomim 9: 046007. PubMed ID: 25313522
Olfactory receptors evolved to provide animals with ecologically and behaviourally relevant information. The resulting extreme sensitivity and discrimination has proven useful to humans, who have therefore co-opted some animals' sense of smell. One aim of machine olfaction research is to replace the use of animal noses and one avenue of such research aims to incorporate olfactory receptors into artificial noses. This study investigate how well the olfactory receptors of the fruit fly, Drosophila melanogaster, perform in classifying volatile odourants that they would not normally encounter. A large number of in vivo recordings were collected from individual Drosophila olfactory receptor neurons in response to an ecologically relevant set of 36 chemicals related to wine ('wine set') and an ecologically irrelevant set of 35 chemicals related to chemical hazards ('industrial set'), each chemical at a single concentration. Resampled response sets were used to classify the chemicals against all others within each set, using a standard linear support vector machine classifier and a wrapper approach. Drosophila receptors appear highly capable of distinguishing chemicals that they have not evolved to process. In contrast to previous work with metal oxide sensors, Drosophila receptors achieved the best recognition accuracy if the outputs of all 20 receptor types were used.
Wednesday, October 29th
Beira, J. V., Springhorn, A., Gunther, S., Hufnagel, L., Pyrowolakis, G. and Vincent, J. P. (2014). The Dpp/TGFbeta-dependent corepressor Schnurri protects epithelial cells from JNK-induced apoptosis in Drosophila embryos. Dev Cell. PubMed ID: 25307481
Jun N-terminal kinase (JNK) often mediates apoptosis in response to cellular stress. However, during normal development, JNK signaling controls a variety of live cell behaviors, such as during dorsal closure in Drosophila embryos. During this process, the latent proapoptotic activity of JNK becomes apparent following Dpp signaling suppression, which leads to JNK-dependent transcriptional activation of the proapoptotic gene reaper. Dpp signaling also protects cells from JNK-dependent apoptosis caused by epithelial disruption. This study found that repression of reaper transcription by Dpp is mediated by Schnurri. Moreover, reporter gene analysis shows that a transcriptional regulatory module comprising AP-1 and Schnurri binding sites located upstream of reaper integrate the activities of JNK and Dpp. This arrangement allows JNK to control a migratory behavior without triggering apoptosis. Dpp plays a dual role during dorsal closure. It cooperates with JNK in stimulating cell migration and also prevents JNK from inducing apoptosis.
Nelson, C., Ambros, V. and Baehrecke, E. H. (2014). miR-14 regulates autophagy during developmental cell death by targeting ip3-kinase 2. Mol Cell. PubMed ID: 25306920
Macroautophagy (autophagy) is a lysosome-dependent degradation process that has been implicated in age-associated diseases. Autophagy is involved in both cell survival and cell death, but little is known about the mechanisms that distinguish its use during these distinct cell fates. This study identified the microRNA miR-14 as being both necessary and sufficient for autophagy during developmentally regulated cell death in Drosophila. Loss of miR-14 prevented induction of autophagy during salivary gland cell death, but had no effect on starvation-induced autophagy in the fat body. Moreover, misexpression of miR-14 was sufficient to prematurely induce autophagy in salivary glands, but not in the fat body. Importantly, miR-14regulates this context-specific autophagy through its target, Inositol 1,4,5-trisphosphate kinase 2 (Ip3k2), thereby affecting inositol 1,4,5-trisphosphate (IP3) signaling and calcium levels during salivary gland cell death. This study provides in vivo evidence of microRNA regulation of autophagy through modulation of IP3 signaling.
Zhan, L., Xie, Q. and Tibbetts, R. S. (2014). Opposing roles of p38 and JNK in a Drosophila model of TDP-43 proteinopathy reveal oxidative stress and innate immunity as pathogenic components of neurodegeneration. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25281658
Pathological aggregation and mutation of the 43-kDa TAR DNA-binding protein (TDP-43) are strongly implicated in the pathogenesis amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 neurotoxicity has been extensively modeled in mice, zebrafish, Caenorhabditis elegans and Drosophila, where selective expression of TDP-43 in motoneurons led to paralysis and premature lethality. Through a genetic screen aimed to identify genetic modifiers of TDP-43, this study found that the Drosophila dual leucine kinase Wallenda (Wnd) and its downstream kinases JNK and p38 influenced TDP-43 neurotoxicity. Reducing Wnd gene dosage or overexpressing its antagonist highwire partially rescued TDP-43-associated premature lethality. Downstream of Wnd, the JNK and p38 kinases played opposing roles in TDP-43-associated neurodegeneration. LOF alleles of the p38b gene as well as p38 inhibitors diminished TDP-43-associated premature lethality, whereas p38b GOF caused phenotypic worsening. In stark contrast, disruptive alleles of Basket (Bsk), the Drosophila homologue of JNK, exacerbated longevity shortening, whereas overexpression of Bsk extended lifespan. Among possible mechanisms, motoneuron-directed expression of TDP-43 was found to elicit oxidative stress and innate immune gene activation that were exacerbated by p38 GOF and Bsk LOF, respectively. A key pathologic role for innate immunity in TDP-43-associated neurodegeneration was further supported by the finding that genetic suppression of the Toll/Dif and Imd/Relish inflammatory pathways dramatically extended lifespan of TDP-43 transgenic flies. It is proposed that oxidative stress and neuroinflammation are intrinsic components of TDP-43-associated neurodegeneration and that the balance between cytoprotective JNK and cytotoxic p38 signaling dictates phenotypic outcome to TDP-43 expression in Drosophila.
Tuesday, October 28th
Green, A. A., Silver, P. A., Collins, J. J. and Yin, P.. Toehold switches: De-novo-designed regulators of gene expression. Cell 159: 2014. Full text
Efforts to construct synthetic networks in living cells have been hindered by the limited number of regulatory components that provide wide dynamic range and low crosstalk. This study reports a class of de-novo-designed prokaryotic riboregulators called toehold switches that activate gene expression in response to cognate RNAs with arbitrary sequences. Toehold switches provide a high level of orthogonality and can be forward engineered to provide average dynamic range above 400. Switches can be integrated into the genome to regulate endogenous genes; they can be used as sensors that respond to endogenous RNAs. This study exploits the orthogonality of toehold switches to regulate 12 genes independently and to construct a genetic circuit that evaluates 4-input AND logic. Toehold switches, with their wide dynamic range, orthogonality, and programmability, represent a versatile and powerful platform for regulation of translation, offering diverse applications in molecular biology, synthetic biology, and biotechnology.
Kedmi, A., Zehavi, Y., Glick, Y., Orenstein, Y., Ideses, D., Wachtel, C., Doniger, T., Waldman Ben-Asher, H., Munster, N., Thompson, J., Anderson, S., Avrahami, D., Yates, J. R., Shamir, R., Gerber, D., and Juven-Gershon, T. (2014). Drosophila TRF2 is a preferential core promoter regulator. Genes Dev. 28(19):2163-74. PubMed ID: 25223897
Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter. Core promoters, generally defined as the regions that direct transcription initiation, consist of functional core promoter motifs (such as the TATA-box, initiator [Inr], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. The known basal transcription factors that support TATA-dependent transcription are insufficient for
in vitro transcription of DPE-dependent promoters. In search of a transcription factor that supports DPE-dependent transcription, a biochemical complementation approach was used to identify the Drosophila TBP (TATA-box-binding protein)-related factor 2 (TRF2) as an enriched factor in the fractions that support DPE-dependent transcription. The short TRF2 isoform was demonstrated to preferentially activate DPE-dependent promoters. DNA microarray analysis reveals the enrichment of DPE promoters among short TRF2 upregulated genes. Using primer extension analysis and reporter assays, the importance of the DPE in transcriptional regulation of TRF2 target genes was demonstrated. It has been shown that, unlike TBP, TRF2 fails to bind DNA containing TATA-boxes. Using microfluidic affinity analysis, short TRF2-bound DNA oligos were found to be enriched for Inr and DPE motifs. Taken together, these findings highlight the role of short TRF2 as a preferential core promoter regulator (Kedmi, 2014).
Hsiao, H. C., Gonzalez, K. L., Catanese, D. J., Jordy, K. E., Matthews, K. S. and Bondos, S. E. (2014). The intrinsically disordered regions of the Drosophila melanogaster hox protein Ultrabithorax select interacting proteins based on partner topology. PLoS One 9: e108217. PubMed ID: 25286318
Interactions between structured proteins require a complementary topology and surface chemistry to form sufficient contacts for stable binding. However, approximately one third of protein interactions are estimated to involve intrinsically disordered regions of proteins. The dynamic nature of disordered regions before and, in some cases, after binding calls into question the role of partner topology in forming protein interactions. To understand how intrinsically disordered proteins identify the correct interacting partner proteins, this study evaluated interactions formed by the Drosophila melanogaster Hox transcription factor Ultrabithorax (Ubx), which contains both structured and disordered regions. Ubx binding proteins are enriched in specific folds: 23 of its 39 partners include one of 7 folds, out of the 1195 folds recognized by SCOP. For the proteins harboring the two most populated folds, DNA-RNA binding 3-helical bundles and alpha-alpha superhelices, the regions of the partner proteins that exhibit these preferred folds are sufficient for Ubx binding. Three disorder-containing regions in Ubx are required to bind these partners. These regions are either alternatively spliced or multiply phosphorylated, providing a mechanism for cellular processes to regulate Ubx-partner interactions. Indeed, partner topology correlates with the ability of individual partner proteins to bind Ubx spliceoforms. Partners bind different disordered regions within Ubx to varying extents, creating the potential for competition between partners and cooperative binding by partners. The ability of partners to bind regions of Ubx that activate transcription and regulate DNA binding provides a mechanism for partners to modulate transcription regulation by Ubx, and suggests that one role of disorder in Ubx is to coordinate multiple molecular functions in response to tissue-specific cues.
Malik, A. N., Vierbuchen, T., Hemberg, M., Rubin, A. A., Ling, E., Couch, C. H., Stroud, H., Spiegel, I., Farh, K. K., Harmin, D. A. and Greenberg, M. E. (2014). Genome-wide identification and characterization of functional neuronal activity-dependent enhancers. Nat Neurosci 17: 1330-1339. PubMed ID: 25195102
Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. This study characterize the enhancers that function across the genome to mediate activity-dependent transcription in mouse cortical neurons. The subset of enhancers enriched for monomethylation of histone H3 Lys4 (H3K4me1; see Drosophila Histone H3) and binding of the transcriptional coactivator CREBBP (also called CBP; see Drosophila Slow border cells) that shows increased acetylation of histone H3 Lys27 (H3K27ac) after membrane depolarization of cortical neurons functions to regulate activity-dependent transcription. A subset of these enhancers appears to require binding of FOS (see Drosophila Kayak), which was previously thought to bind primarily to promoters. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function and provide a resource of functional cis-regulatory elements that may give insight into the genetic variants that contribute to brain development and disease.
Librado, P. and Rozas, J. (2013). Uncovering the functional constraints underlying the genomic organization of the odorant-binding protein genes. Genome Biol Evol 5: 2096-2108. PubMed ID: 24148943Summary:
Animal olfactory systems have a critical role for the survival and reproduction of individuals. In insects, the odorant-binding proteins (OBPs; see Lush, for example) are encoded by a moderately sized gene family, and mediate the first steps of the olfactory processing. Most OBPs are organized in clusters of a few paralogs, which are conserved over time. Currently, the biological mechanism explaining the close physical proximity among OBPs is not yet established. This study conducted a comprehensive study aiming to gain insights into the mechanisms underlying the OBP genomic organization. A total of 31 conserved clusters were identified in Drosophila that include both OBP and other nonhomologous genes. These 31 clusters are maintained, on average, in 5.9 Drosophila species, comprise a mean of 8.3 genes and, more importantly, the study recover most of the OBP clusters defined in previous studies. The OBP clusters were found to be embedded within large conserved arrangements. These organizations also include other non-OBP genes, which often encode proteins integral to plasma membrane. Moreover, the conservation degree of such large clusters is related to the following: 1) the promoter architecture of the confined genes, 2) a characteristic transcriptional environment, and 3) the chromatin conformation of the chromosomal region. These results suggest that chromatin domains may restrict the location of OBP genes to regions having the appropriate transcriptional environment, leading to the OBP cluster structure. However, the appropriate transcriptional environment for OBP and the other neighbor genes is not dominated by reduced levels of expression noise. Indeed, the stochastic fluctuations in the OBP transcript abundance may have a critical role in the combinatorial nature of the olfactory coding process.
Monday, October 27th
Ozdemir, A., Ma, L., White, K. P. and Stathopoulos, A. (2014). Su(H)-mediated repression positions gene boundaries along the dorsal-central axis of Drosophila embryos. Dev Cell 31: 100-113. PubMed ID: 25313963
In Drosophila embryos, a nuclear gradient of the Dorsal (Dl) transcription factor directs differential gene expression along the dorsoventral (DV) axis, translating it into distinct domains that specify future mesodermal, neural, and ectodermal territories. However, the mechanisms used to differentially position gene expression boundaries along this axis are not fully understood. Using a combination of approaches, including mutant phenotype analyses and chromatin immunoprecipitation, this study shows that the transcription factor Suppressor of Hairless, Su(H), helps define dorsal boundaries for many genes expressed along the DV axis. Synthetic reporter constructs also provide molecular evidence that Su(H) binding sites support repression and act to counterbalance activation through Dl and the ubiquitous activator Zelda. This study highlights a role for broadly expressed repressors, like Su(H), and organization of transcription factor binding sites within cis-regulatory modules as important elements controlling spatial domains of gene expression to facilitate flexible positioning of boundaries across the entire DV axis.
Tsikala, G., Karagogeos, D. and Strigini, M. (2014). Btk-dependent epithelial cell rearrangements contribute to the invagination of nearby tubular structures in the posterior spiracles of Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 25305143
The Drosophila respiratory system consists of two connected organs, the tracheae and the spiracles. Together they ensure the efficient delivery of air-borne oxygen to all tissues. The posterior spiracles consist internally of the spiracular chamber, an invaginated tube with filtering properties that connects the main tracheal branch to the environment, and externally of the stigmatophore, an extensible epidermal structure that covers the spiracular chamber. The primordia of both components are first specified in the plane of the epidermis and subsequently the spiracular chamber is internalized through the process of invagination accompanied by apical cell constriction. It has become clear that invagination processes do not always or only rely on apical constriction. This study shows here that in mutants for the src-like kinase Btk29A spiracle cells constrict apically but do not complete invagination, giving rise to shorter spiracular chambers. This defect can be rescued by using different GAL4 drivers to express Btk29A throughout the ectoderm, in cells of posterior segments only, or in the stigmatophore pointing to a non cell-autonomous role for Btk29A. This analysis suggests that complete invagination of the spiracular chamber requires Btk29A-dependent planar cell rearrangements of adjacent non-invaginating cells of the stigmatophore. These results highlight the complex physical interactions that take place among organ components during morphogenesis, which contribute to their final form and function.
Werner, M. E., Mitchell, J. W., Putzbach, W., Bacon, E., Kim, S. K. and Mitchell, B. J. (2014). Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1. J Cell Biol 206: 367-376. PubMed ID: 25070955
The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue from an inner layer to an outer layer. This study used the radial intercalation of cells into the skin of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue. A novel function was identified for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation. Specifically, it was shown that Par3 (see Drosophila Bazooka) and aPKC (see Drosophila aPKC) promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. A model is proposed in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule stability required for the active migration of cells into the outer epithelium.
Williams, S. E., Ratliff, L. A., Postiglione, M. P., Knoblich, J. A. and Fuchs, E. (2014). Par3-mInsc and Galphai3 cooperate to promote oriented epidermal cell divisions through LGN. Nat Cell Biol 16: 758-769. PubMed ID: 25016959
Asymmetric cell divisions allow stem cells to balance proliferation and differentiation. During embryogenesis, murine epidermis expands rapidly from a single layer of unspecified basal layer progenitors to a stratified, differentiated epithelium. Morphogenesis involves perpendicular (asymmetric) divisions and the spindle orientation protein LGN (see Drosophila Pins), but little is known about how the apical localization of LGN is regulated. Ghis study combined conventional genetics and lentiviral-mediated in vivo RNAi to explore the functions of the LGN-interacting proteins Par3, mInsc and Galphai3 (see Drosophila Bazooka, Insc, and Galphai). Whereas loss of each gene alone leads to randomized division angles, combined loss of Gnai3 and mInsc causes a phenotype of mostly planar divisions, akin to loss of LGN. These findings lend experimental support for the hitherto untested model that Par3-mInsc and Galphai3 act cooperatively to polarize LGN and promote perpendicular divisions. Finally, a developmental switch was uncovered between delamination-driven early stratification and spindle-orientation-dependent differentiation that occurs around E15, revealing a two-step mechanism underlying epidermal maturation.
Sunday, October 26th
Dodson, M. W., Leung, L. K., Lone, M., Lizzio, M. A. and Guo, M. (2014). Novel alleles of the Drosophila LRRK2 homolog reveal a crucial role in endolysosomal functions and autophagy in vivo. Dis Model Mech [Epub ahead of print]. PubMed ID: 25288684
Mutations in LRRK2 cause a dominantly inherited form of Parkinson's disease (PD), and are the most common known genetic determinant of PD. As inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD, understanding the consequences of inhibiting the normal cellular functions of LRRK2 is vital. Despite much interest, the physiologic functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. This study reports the characterization of multiple novel nonsense alleles in the Drosophila LRRK2 homolog lrrk. Using these alleles, this study shows that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in accumulation of autophagosomes, as well as enlarged early endosomes laden with mono-ubiquitinated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase Rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, these data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.
Ivatt, R. M., Sanchez-Martinez, A., Godena, V. K., Brown, S., Ziviani, E. and Whitworth, A. J. (2014). Genome-wide RNAi screen identifies the Parkinson disease GWAS risk locus SREBF1 as a regulator of mitophagy. Proc Natl Acad Sci U S A 111: 8494-8499. PubMed ID: 24912190
Genetic analysis of Parkinson disease (PD) has identified several genes whose mutation causes inherited parkinsonism, as well as risk loci for sporadic PD. PTEN-induced kinase 1 (PINK1) and parkin, linked to autosomal recessive PD, act in a common genetic pathway regulating the autophagic degradation of mitochondria, termed mitophagy. This study consisted of a genome-wide RNAi screen, in Drosophila cultured cells, as an unbiased approach to identify genes regulating the PINK1/Parkin pathway. Several genes were identified that have a conserved function in promoting mitochondrial translocation of Parkin and subsequent mitophagy, most notably Sterol regulatory element binding transcription factor 1 (SREBF1), F-box and WD40 domain protein 7 (FBXW7), and other components of the lipogenesis pathway. The relevance of mechanisms of autosomal recessive parkinsonism to sporadic PD has long been debated. However, with the recent identification of SREBF1 as a risk locus for sporadic PD, these findings suggest a common mechanistic link between autosomal recessive and sporadic PD, and underscore the importance of mitochondrial homeostasis.
Ashrafi, G., Schlehe, J. S., LaVoie, M. J. and Schwarz, T. L. (2014). Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin. J Cell Biol 206: 655-670. PubMed ID: 25154397
To minimize oxidative damage to the cell, malfunctioning mitochondria need to be removed by mitophagy. In neuronal axons, mitochondrial damage may occur in distal regions, far from the soma where most lysosomal degradation is thought to occur. This paper reports that PINK1 (see Drosophila PINK1) and Parkin (see Drosophila Parkin), two Parkinson's disease-associated proteins, mediate local mitophagy of dysfunctional mitochondria in neuronal axons. A subset of mitochondria in hippocampal axons was damaged to reduce cytotoxicity and mimic physiological levels of mitochondrial damage. Parkin was rapidly recruited to damaged mitochondria in axons followed by formation of LC3-positive autophagosomes and LAMP1-positive lysosomes. In PINK1(-/-) axons, damaged mitochondria did not accumulate Parkin and failed to be engulfed in autophagosomes. Similarly, initiation of mitophagy was blocked in Parkin(-/-) axons. These findings demonstrate that the PINK1-Parkin-mediated pathway is required for local mitophagy in distal axons in response to focal damage. Local mitophagy likely provides rapid neuroprotection against oxidative stress without a requirement for retrograde transport to the soma.
Demay, Y., Perochon, J., Szuplewski, S., Mignotte, B. and Gaumer, S. (2014). The PERK pathway independently triggers apoptosis and a Rac1/Slpr/JNK/Dilp8 signaling favoring tissue homeostasis in a chronic ER stress Drosophila model. Cell Death Dis 5: e1452. PubMed ID: 25299777
The endoplasmic reticulum (ER) has a major role in protein folding. The accumulation of unfolded proteins in the ER induces a stress, which can be resolved by the unfolded protein response (UPR). Chronicity of ER stress leads to UPR-induced apoptosis and in turn to an unbalance of tissue homeostasis. Although ER stress-dependent apoptosis is observed in a great number of devastating human diseases, how cells activate apoptosis and promote tissue homeostasis after chronic ER stress remains poorly understood. This study used the Drosophila wing imaginal disc as a model system. Presenilin overexpression induces chronic ER stress in vivo. In this novel model of chronic ER-stress, a PERK/ATF4-dependent apoptosis required downregulation of the antiapoptotic diap1 gene. PERK/ATF4 also activated the JNK pathway through Rac1 and Slpr activation in apoptotic cells, leading to the expression of Dilp8. This insulin-like peptide caused a developmental delay, which partially allowed the replacement of apoptotic cells. Thanks to a novel chronic ER stress model, these results establish a new pathway that both participates in tissue homeostasis and triggers apoptosis through an original regulation.
Saturday, October 25th
Vogler, G., Liu, J., Iafe, T. W., Migh, E., Mihaly, J. and Bodmer, R. (2014). Cdc42 and formin activity control non-muscle myosin dynamics during Drosophila heart morphogenesis. J Cell Biol 206: 909-922. PubMed ID: 25267295
During heart formation, a network of transcription factors and signaling pathways guide cardiac cell fate and differentiation, but the genetic mechanisms orchestrating heart assembly and lumen formation remain unclear. This study shows that the small GTPase Cdc42 is essential for Drosophila melanogaster heart morphogenesis and lumen formation. Cdc42 genetically interacts with the cardiogenic transcription factor tinman; with dDAAM which belongs to the family of actin organizing formins; and with zipper, which encodes nonmuscle myosin II. Zipper is required for heart lumen formation, and its spatiotemporal activity at the prospective luminal surface is controlled by Cdc42. Heart-specific expression of activated Cdc42, or the regulatory formins dDAAM and Diaphanous caused mislocalization of Zipper and induced ectopic heart lumina, as characterized by luminal markers such as the extracellular matrix protein Slit. Placement of Slit at the lumen surface depends on Cdc42 and formin function. Thus, Cdc42 and formins play pivotal roles in heart lumen formation through the spatiotemporal regulation of the actomyosin network.
Vizcarra, C. L., Bor, B. and Quinlan, M. E. (2014). The role of Formin tails in actin nucleation, processive elongation, and filament bundling. J Biol Chem [Epub ahead of print]. PubMed ID: 25246531
Formins are multi-domain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well-conserved Formin Homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous Autoinhibitory Domain (DAD), which is C-terminal to the Formin Homology-2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, this study found that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this story, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Further, when the tail is removed, it was found that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. These data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Due to its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.
Rasson, A. S., Bois, J. S., Pham, D. S., Yoo, H. and Quinlan, M. E. (2014). Filament assembly by Spire: Key residues and concerted actin binding. J Mol Biol [Epub ahead of print]. PubMed ID: 25234086
The most recently identified class of actin nucleators, WASp Homology domain 2 (WH2) -nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. This study used Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. The third WH2 domain in Spir (Spir-C or SC) was found to play a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. The native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. Two residues within SC were identifed that are critical for its activity. Using this information it was possible to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, evidence was found that SC binds actin filaments, in addition to monomers.
Baird, N. A., Douglas, P. M., Simic, M. S., Grant, A. R., Moresco, J. J., Wolff, S. C., Yates, J. R., Manning, G. and Dillin, A. (2014). HSF-1-mediated cytoskeletal integrity determines thermotolerance and life span. Science 346: 360-363. PubMed ID: 25324391
The conserved heat shock transcription factor-1 (HSF-1; see Drosophila Hsf) is essential to cellular stress resistance and life-span determination. The canonical function of HSF-1 is to regulate a network of genes encoding molecular chaperones that protect proteins from damage caused by extrinsic environmental stress or intrinsic age-related deterioration. In Caenorhabditis elegans, a modified HSF-1 strain was engineered that increased stress resistance and longevity without enhanced chaperone induction. This health assurance acted through the regulation of the calcium-binding protein PAT-10. Loss of pat-10 caused a collapse of the actin cytoskeleton, stress resistance, and life span. Furthermore, overexpression of pat-10 increased actin filament stability, thermotolerance, and longevity, indicating that in addition to chaperone regulation, HSF-1 has a prominent role in cytoskeletal integrity, ensuring cellular function during stress and aging.
Friday, October 24th
Kim, M. J. and O'Connor, M. B. (2014). Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction. PLoS One 9: e107443. PubMed ID: 25255438
Members of the TGF-beta superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. This study analyzed whether the Activin branch of the TGF-beta superfamily also contributes to NMJ development and function. Elimination of the Activin/TGF-beta type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. These results demonstrate that anterograde Activin/TGF-beta signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling via Glass bottom boat and Wishful thinking, these findings indicate that the two branches of the TGF-beta superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function.
Park, D., Li, P., Dani, A. and Taghert, P. H. (2014). Peptidergic cell-specific Synaptotagmins in Drosophila: localization to dense-core granules and regulation by the bHLH protein DIMMED. J Neurosci 34: 13195-13207. PubMed ID: 25253864
Bioactive peptides are packaged in large dense-core secretory vesicles, which mediate regulated secretion by exocytosis. In a variety of tissues, the regulated release of neurotransmitters and hormones is dependent on calcium levels and controlled by vesicle-associated synaptotagmin (SYT) proteins. Drosophila express seven SYT isoforms, of which two (SYT-α and SYT-β) were previously found to be enriched in neuroendocrine cells. This study shows that SYT-α and SYT-β tissue expression patterns are similar, though not identical. Furthermore, both display significant overlap with the bHLH transcription factor Dimm, a known neuroendocrine (NE) regulator. RNAi-mediated knockdown indicates that both SYT-α and SYT-β functions are essential in identified NE cells as these manipulations phenocopy loss-of-function states for the indicated peptide hormones. In Drosophila cell culture, both SYT-α and neuropeptide cargo form Dimm-dependent fluorescent puncta that are coassociated by super-resolution microscopy. Dimm is required to maintain SYT-α and SYT-β protein levels in Dimm-expressing cells in vivo. In neurons normally lacking all three proteins (Dimm-/SYT-α-/SYT-β-), Dimm misexpression conferred accumulation of endogenous SYT-α and SYT-β proteins. Furthermore transgenic SYT-α does not appreciably accumulate in nonpeptidergic neurons in vivo but does so if Dimm is comisexpressed. Among Drosophila syt genes, only syt-α and syt-β RNA levels are upregulated by Dimm overexpression. Together, these data suggest that SYT-α and SYT-β are important for NE cell physiology, that one or both are integral membrane components of the large dense-core vesicles, and that they are closely regulated by Dimm at a post-transcriptional level.
Dani, N., Zhu, H. and Broadie, K. (2014). Two protein N-acetylgalactosaminyl transferases regulate synaptic plasticity by activity-dependent regulation of integrin signaling. J Neurosci 34: 13047-13065. PubMed ID: 25253852
Using a Drosophila whole-genome transgenic RNAi screen for glycogenes regulating synapse function, two protein alpha-N-acetylgalactosaminyltransferases (pgant3 and pgant35A) were identified that regulate synaptic O-linked glycosylation (GalNAcalpha1-O-S/T). Loss of either pgant alone elevates presynaptic/postsynaptic molecular assembly and evoked neurotransmission strength, but synapses appear restored to normal in double mutants. Likewise, activity-dependent facilitation, augmentation, and posttetanic potentiation are all suppressively impaired in pgant mutants. In non-neuronal contexts, Pgant function regulates integrin signaling, and the synaptic Position Specific 2 (αPS2) integrin receptor Inflated and transmembrane tenascin ligand are both suppressively downregulated in pgant mutants. Channelrhodopsin-driven activity rapidly (<1 min) drives integrin signaling in wild-type synapses but is suppressively abolished in pgant mutants. Optogenetic stimulation in pgant mutants alters presynaptic vesicle trafficking and postsynaptic pocket size during the perturbed integrin signaling underlying synaptic plasticity defects. Critically, acute blockade of integrin signaling acts synergistically with pgant mutants to eliminate all activity-dependent synaptic plasticity.
Knodel, M. M., Geiger, R., Ge, L., Bucher, D., Grillo, A., Wittum, G., Schuster, C. M. and Queisser, G. (2014). Synaptic bouton properties are tuned to best fit the prevailing firing pattern. Front Comput Neurosci 8: 101. PubMed ID: 25249970
The morphology of presynaptic specializations can vary greatly ranging from classical single-release-site boutons in the central nervous system to boutons of various sizes harboring multiple vesicle release sites. Multi-release-site boutons can be found in several neural contexts, for example at the neuromuscular junction (NMJ) of body wall muscles of Drosophila larvae. These NMJs are built by two motor neurons forming two types of glutamatergic multi-release-site boutons with two typical diameters. However, it is unknown why these distinct nerve terminal configurations are used on the same postsynaptic muscle fiber. To systematically dissect the biophysical properties of these boutons, a full three-dimensional model of such boutons, their release sites and transmitter-harboring vesicles was developed, the local vesicle dynamics of various configurations during stimulation was analyzed. This study shows that the rate of transmission of a bouton is primarily limited by diffusion-based vesicle movements and that the probability of vesicle release and the size of a bouton affect bouton-performance in distinct temporal domains allowing for an optimal transmission of the neural signals at different time scales. A comparison of these in silico simulations with in vivo recordings of the natural motor pattern of both neurons revealed that the bouton properties resemble a well-tuned cooperation of the parameters release probability and bouton size, enabling a reliable transmission of the prevailing firing-pattern at diffusion-limited boutons. These findings indicate that the prevailing firing-pattern of a neuron may determine the physiological and morphological parameters required for its synaptic terminals.
Thursday, October 23rd
Chai, G., Zhou, L., Manto, M., Helmbacher, F., Clotman, F., Goffinet, A. M. and Tissir, F. (2014). Celsr3 is required in motor neurons to steer their axons in the hindlimb. Nat Neurosci 17: 1171-1179. PubMed ID: 25108913
The cadherin Celsr3 (Drosophila homolog: Starry night) regulates the directional growth and targeting of axons in the CNS, but whether it acts in collaboration with or in parallel to other guidance cues is unknown. Furthermore, the function of Celsr3 in the peripheral nervous system is still largely unexplored. This study shows that Celsr3 mediates pathfinding of motor axons innervating the hindlimb. In mice, Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall near the branch point. Mutant axons respond to repulsive ephrinA-EphA forward signaling and glial cell-derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA (see Drosophila Ephrins) reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor alpha1 (GFRalpha1) and Frizzled3 (Fzd3; see Drosophila Frizzled). The function of Celsr3 is Fzd3 dependent but Vangl2 independent. The results provide evidence that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb.
Finci, L. I., Kruger, N., Sun, X., Zhang, J., Chegkazi, M., Wu, Y., Schenk, G., Mertens, H. D., Svergun, D. I., Zhang, Y., Wang, J. H. and Meijers, R. (2014)S. The crystal structure of netrin-1 in complex with DCC reveals the bifunctionality of netrin-1 as a guidance cue. Neuron 83: 839-849. PubMed ID: 25123307
Netrin-1 (see Drosophila Netrins) is a guidance cue that can trigger either attraction or repulsion effects on migrating axons of neurons, depending on the repertoire of receptors available on the growth cone. How a single chemotropic molecule can act in such contradictory ways has long been a puzzle at the molecular level. This study presents the crystal structure of netrin-1 in complex with the Deleted in Colorectal Cancer (DCC) receptor (see Drosophila Frazzled). One netrin-1 molecule can simultaneously bind to two DCC molecules through a DCC-specific site and through a unique generic receptor binding site, where sulfate ions staple together positively charged patches on both DCC and netrin-1. Furthermore, UNC5A (see Drosophila Unc-5) can replace DCC on the generic receptor binding site to switch the response from attraction to repulsion. It is proposed that the modularity of binding allows for the association of other netrin receptors at the generic binding site, eliciting alternative turning responses.
Samuel, M. A., Voinescu, P. E., Lilley, B. N., de Cabo, R., Foretz, M., Viollet, B., Pawlyk, B., Sandberg, M. A., Vavvas, D. G. and Sanes, J. R. (2014). LKB1 and AMPK regulate synaptic remodeling in old age. Nat Neurosci 17: 1190-1197. PubMed ID: 25086610
Age-related decreases in neural function result in part from alterations in synapses. To identify molecular defects that lead to such changes, this study focused on the outer retina, in which synapses are markedly altered in old rodents and humans. The serine/threonine kinase LKB1 (see Drosophila Lkb1) and one of its substrates, AMPK (see Drosophila AMPKalpha), regulate this process. In old mice, synaptic remodeling was accompanied by specific decreases in the levels of total LKB1 and active (phosphorylated) AMPK. In the absence of either kinase, young adult mice developed retinal defects similar to those that occurred in old wild-type animals. LKB1 and AMPK function in rod photoreceptors where their loss leads to aberrant axonal retraction, the extension of postsynaptic dendrites and the formation of ectopic synapses. Conversely, increasing AMPK activity genetically or pharmacologically attenuates and may reverse age-related synaptic alterations. Together, these results identify molecular determinants of age-related synaptic remodeling and suggest strategies for attenuating these changes.
Magnusson, J. P., Goritz, C., Tatarishvili, J., Dias, D. O., Smith, E. M., Lindvall, O., Kokaia, Z. and Frisen, J. (2014). A latent neurogenic program in astrocytes regulated by Notch signaling in the mouse. Science 346: 237-241. PubMed ID: 25301628
Neurogenesis is restricted in the adult mammalian brain; most neurons are neither exchanged during normal life nor replaced in pathological situations. This study reports that stroke elicits a latent neurogenic program in striatal astrocytes in mice. Notch1 (see Drosophila Notch) signaling is reduced in astrocytes after stroke, and attenuated Notch1 signaling is necessary for neurogenesis by striatal astrocytes. Blocking Notch signaling triggers astrocytes in the striatum and the medial cortex to enter a neurogenic program, even in the absence of stroke, resulting in new neurons in a mouse striatum. Thus, under Notch signaling regulation, astrocytes in the adult mouse brain parenchyma carry a latent neurogenic program that may potentially be useful for neuronal replacement strategies.
Wednesday, October 22nd
Marchetti, G., Reichardt, I., Knoblich, J. A. and Besse, F. (2014). The TRIM-NHL protein Brat promotes axon maintenance by repressing src64B expression. J Neurosci 34: 13855-13864. PubMed ID: 25297111
The morphology and the connectivity of neuronal structures formed during early development must be actively maintained as the brain matures. Although impaired axon stability is associated with the progression of various neurological diseases, relatively little is known about the factors controlling this process. This study identified Brain tumor (Brat), a conserved member of the TRIM-NHL family of proteins, as a new regulator of axon maintenance in Drosophila CNS. Brat function is dispensable for the initial growth of Mushroom Body axons, but is required for the stabilization of axon bundles. Brat was found to repress the translation of src64B, an upstream regulator of a conserved Rho-dependent pathway previously shown to promote axon retraction. Furthermore, brat phenotypes are phenocopied by src64B overexpression, and partially suppressed by reducing the levels of src64B or components of the Rho pathway, suggesting that brat promotes axon maintenance by downregulating the levels of Src64B. Finally, Brat regulates brain connectivity via its NHL domain, but independently of its previously described partners Nanos, Pumilio, and d4EHP. Thus, these results uncover a novel post-transcriptional regulatory mechanism that controls the maintenance of neuronal architecture by tuning the levels of a conserved rho-dependent signaling pathway.
Xie, Y., Li, X., Zhang, X., Mei, S., Li, H., Urso, A. and Zhu, S. (2014). The Sp8 transcription factor Buttonhead prevents premature differentiation of intermediate neural progenitors. Elife 3 [Epub ahead of print]. PubMed ID: 25285448
Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. This study reports that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast (NB) lineages. Loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. Evidence is provided to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. It was further shown that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, this work reveals a critical mechanism that prevents premature differentiation and cell cycle exit ofDrosophila INPs.
Komori, H., Xiao, Q., Janssens, D. H., Dou, Y. and Lee, C. Y. (2014). Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor Buttonhead. Elife 3 [Epub ahead of print]. PubMed ID: 25285447
The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. This study reports that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btd) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity.
Beirowski, B., Babetto, E., Golden, J. P., Chen, Y. J., Yang, K., Gross, R. W., Patti, G. J. and Milbrandt, J. (2014). Metabolic regulator LKB1 is crucial for Schwann cell-mediated axon maintenance. Nat Neurosci 17: 1351-1361. PubMed ID: 25195104
Schwann cells (SCs) promote axonal integrity independently of myelination by poorly understood mechanisms. Current models suggest that SC metabolism is critical for this support function and that SC metabolic deficits may lead to axonal demise. The LKB1-AMP-activated protein kinase (AMPK) kinase pathway (see Drosophila AMP-activated protein kinase alpha subunit) targets several downstream effectors, including mammalian target of rapamycin (mTOR, see Drosophila Tor), and is a key metabolic regulator implicated in metabolic diseases. Through molecular, structural and behavioral characterization of SC-specific mutant mice this study found that LKB1 activity is central to axon stability, whereas AMPK and mTOR in SCs are largely dispensable. The degeneration of axons in LKB1 mutants was most dramatic in unmyelinated small sensory fibers, whereas motor axons were comparatively spared. LKB1 deletion in SCs led to abnormalities in nerve energy and lipid homeostasis and to increased lactate release. The latter acts in a compensatory manner to support distressed axons. LKB1 signaling is essential for SC-mediated axon support, a function that may be dysregulated in diabetic neuropathy.
Tuesday, October 21st
Wang, X. and Page-McCaw, A. (2014). A matrix metalloproteinase mediates long-distance attenuation of stem cell proliferation. J Cell Biol 206: 923-936. PubMed ID: 25267296
Ligand-based signaling can potentiate communication between neighboring cells and between cells separated by large distances. In the Drosophila melanogaster ovary, Wingless (Wg) promotes proliferation of follicle stem cells located approximately 50 microm or five cell diameters away from the Wg source. How Wg traverses this distance is unclear. This study finds that this long-range signaling requires Division abnormally delayed (Dally)-like (Dlp), a glypican known to extend the range of Wg ligand in the wing disc by binding Wg. Dlp-mediated spreading of Wg to follicle stem cells is opposed by the extracellular protease Mmp2, which cleaved Dlp in cell culture, triggering its relocalization such that Dlp no longer contacted Wg protein. Mmp2-deficient ovaries displayed increased Wg distribution, activity, and stem cell proliferation. Mmp2 protein is expressed in the same cells that produce Wg; thus, niche cells produce both a long-range stem cell proliferation factor and a negative regulator of its spreading. This system could allow for spatial control of Wg signaling to targets at different distances from the source.
Martorell, O., Barriga, F. M., Merlos-Suarez, A., Stephan-Otto Attolini, C., Casanova, J., Batlle, E., Sancho, E. and Casali, A. (2014). Iro/IRX transcription factors negatively regulate Dpp/TGF-beta pathway activity during intestinal tumorigenesis. EMBO Rep. [Epub ahead of print]. PubMed ID: 25296644
Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co-activation of these pathways in the adult Drosophila midgut induces 'tumor-like' overgrowths. This study shows that, in these clones and in CRC cell lines, Dpp/TGF-beta acts as a tumor suppressor. Moreover, it was discovers that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. This genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-beta. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-beta. In addition, Irx5 expression confers a growth advantage in the presence of TGF-beta, but is selected against in its absence. Together, these results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-beta activity. It is proposed that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-beta, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-beta pathway components.
Lam, V., Tokusumi, T., Tokusumi, Y. and Schulz, R. A. (2014). bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 25280996
The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to lead to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. This study demonstrates that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, bantam was determined to be a novel upstream component of the insulin signaling cascade in the PSC, and dMyc was unveiled as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche.
Porlan, E., Marti-Prado, B., Morante-Redolat, J. M., Consiglio, A., Delgado, A. C., Kypta, R., Lopez-Otin, C., Kirstein, M. and Farinas, I. (2014). MT5-MMP regulates adult neural stem cell functional quiescence through the cleavage of N-cadherin. Nat Cell Biol 16: 629-638. PubMed ID: 24952463
The identification of mechanisms that maintain stem cell niche architecture and homeostasis is fundamental to understanding of tissue renewal and repair. Cell adhesion is a well-characterized mechanism for developmental morphogenetic processes, but its contribution to the dynamic regulation of adult mammalian stem cell niches is still poorly defined. This study shows that N-cadherin-mediated anchorage of neural stem cells (NSCs) to ependymocytes in the adult murine subependymal zone modulates their quiescence. MT5-MMP was identified as a membrane-type metalloproteinase responsible for the shedding of the N-cadherin (see Drosophila Shotgun) ectodomain in this niche. MT5-MMP is co-expressed with N-cadherin in adult NSCs and ependymocytes and, whereas MT5-MMP-mediated cleavage of N-cadherin is dispensable for the regulation of NSC generation and identity, it is required for proper activation of NSCs under physiological and regenerative conditions. These results indicate that the proliferative status of stem cells can be dynamically modulated by regulated cleavage of cell adhesion molecules.
Monday, October 20th
Schuettengruber, B., Oded Elkayam, N., Sexton, T., Entrevan, M., Stern, S., Thomas, A., Yaffe, E., Parrinello, H., Tanay, A. and Cavalli, G. (2014). Cooperativity, specificity, and evolutionary stability of polycomb targeting in Drosophila. Cell Rep 9: 219-233. PubMed ID: 25284790
Metazoan genomes are partitioned into modular chromosomal domains containing active or repressive chromatin. In flies, Polycomb group (PcG) response elements (PREs) recruit Pho and other DNA-binding factors and act as nucleation sites for the formation of Polycomb repressive domains. The sequence specificity of PREs is not well understood. This study used comparative epigenomics and transgenic assays to show that Drosophila domain organization and PRE specification are evolutionarily conserved despite significant cis-element divergence within Polycomb domains, whereas cis-element evolution is strongly correlated with transcription factor binding divergence outside of Polycomb domains. Cooperative interactions of PcG complexes and their recruiting factor Pho stabilize Pho recruitment to low-specificity sequences. Consistently, Pho recruitment to sites within Polycomb domains is stabilized by PRC1. These data suggest that cooperative rather than hierarchical interactions among low-affinity sequences, DNA-binding factors, and the Polycomb machinery are giving rise to specific and strongly conserved 3D structures in Drosophila.
Oliveira, R. A., Kotadia, S., Tavares, A., Mirkovic, M., Bowlin, K., Eichinger, C. S., Nasmyth, K. and Sullivan, W. (2014). Centromere-independent accumulation of cohesin at ectopic heterochromatin sites induces chromosome stretching during anaphase. PLoS Biol 12: e1001962. PubMed ID: 25290697
Pericentric heterochromatin, while often considered as 'junk' DNA, plays important functions in chromosome biology. It contributes to sister chromatid cohesion, a process mediated by the cohesin complex that ensures proper genome segregation during nuclear division. Long stretches of heterochromatin are almost exclusively placed at centromere-proximal regions but it remains unclear if there is functional (or mechanistic) importance in linking the sites of sister chromatid cohesion to the chromosomal regions that mediate spindle attachment (the centromere). Using engineered chromosomes in Drosophila melanogaster, this study demonstrate that cohesin enrichment is dictated by the presence of heterochromatin rather than centromere proximity. This preferential accumulation is caused by an enrichment of the cohesin-loading factor (Nipped-B/NIPBL/Scc2) at dense heterochromatic regions. As a result, chromosome translocations containing ectopic pericentric heterochromatin embedded in euchromatin display additional cohesin-dependent constrictions. These ectopic cohesion sites, placed away from the centromere, disjoin abnormally during anaphase and chromosomes exhibit a significant increase in length during anaphase (termed chromatin stretching). These results provide evidence that long stretches of heterochromatin distant from the centromere, as often found in many cancers, are sufficient to induce abnormal accumulation of cohesin at these sites and thereby compromise the fidelity of chromosome segregation.
Petruk, S., Black, K. L., Kovermann, S. K., Brock, H. W. and Mazo, A. (2013). Stepwise histone modifications are mediated by multiple enzymes that rapidly associate with nascent DNA during replication. Nat Commun 4: 2841. PubMed ID: 24276476
The mechanism of epigenetic inheritance following DNA replication may involve dissociation of chromosomal proteins from parental DNA and reassembly on daughter strands in a specific order. This study investigated the behaviour of different types of chromosomal proteins using newly developed methods that allow assessment of the assembly of proteins during DNA replication. Unexpectedly, most chromatin-modifying proteins tested, including methylases, demethylases, acetyltransferases and a deacetylase, are found in close proximity to PCNA or associate with short nascent DNA. Histone modifications occur in a temporal order following DNA replication, mediated by complex activities of different enzymes. In contrast, components of several major nucleosome-remodelling complexes are dissociated from parental DNA, and are later recruited to nascent DNA following replication. Epigenetic inheritance of gene expression patterns may require many aspects of chromatin structure to remain in close proximity to the replication complex followed by reassembly on nascent DNA shortly after replication.
Seridi, L., Ryu, T. and Ravasi, T. (2014). Dynamic epigenetic control of highly conserved noncoding elements. PLoS One 9: e109326. PubMed ID: 25289637
Many noncoding genomic loci have remained constant over long evolutionary periods, suggesting that they are exposed to strong selective pressures. The molecular functions of these elements have been partially elucidated, but the fundamental reason for their extreme conservation is still unknown. To gain new insights into the extreme selection of highly conserved noncoding elements (HCNEs), this study used a systematic analysis of multi-omic data to study the epigenetic regulation of such elements during the development of Drosophila melanogaster. At the sequence level, HCNEs are GC-rich and have a characteristic oligomeric composition. They have higher levels of stable nucleosome occupancy than their flanking regions, and lower levels of mononucleosomes and H3.3, suggesting that these regions reside in compact chromatin. Furthermore, these regions showed remarkable modulations in histone modification and the expression levels of adjacent genes during development. Although HCNEs are primarily initiated late in replication, about 10% were related to early replication origins. Finally, HCNEs showed strong enrichment within lamina-associated domains. It is concluded that HCNEs have distinct and protective sequence properties, undergo dynamic epigenetic regulation, and appear to be associated with the structural components of the chromatin, replication origins, and nuclear matrix. These observations indicate that such elements are likely to have essential cellular functions, and offer insights into their epigenetic properties.
Sunday, October 19th
Korzelius, J., Naumann, S. K., Loza-Coll, M. A., Chan, J. S., Dutta, D., Oberheim, J., Glasser, C., Southall, T. D., Brand, A. H., Jones, D. L. and Edgar, B. A. (2014). Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells. EMBO J [Epub ahead of print]. PubMed ID: 25298397
Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). Loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1.
Amcheslavsky, A., Song, W., Li, Q., Nie, Y., Bragatto, I., Ferrandon, D., Perrimon, N. and Ip, Y. T. (2014). Enteroendocrine Cells Support Intestinal Stem-Cell-Mediated Homeostasis in Drosophila. Cell Rep 9(1):32-9. PubMed ID: 25263551
Intestinal stem cells in the adult Drosophila midgut are regulated by growth factors produced from the surrounding niche cells including enterocytes and visceral muscle. The role of the other major cell type, the secretory enteroendocrine cells, in regulating intestinal stem cells remains unclear. This study shows that newly eclosed scute loss-of-function mutant flies are completely devoid of enteroendocrine cells. These enteroendocrine cell-less flies have normal ingestion and fecundity but shorter lifespan. Moreover, in these newly eclosed mutant flies, the diet-stimulated midgut growth that depends on the insulin-like peptide 3 expression in the surrounding muscle is defective. The depletion of Tachykinin-producing enteroendocrine cells or knockdown of Tachykinin leads to a similar although less severe phenotype. These results establish that enteroendocrine cells serve as an important link between diet and visceral muscle expression of an insulin-like growth factor to stimulate intestinal stem cell proliferation and tissue growth.
Song, W., Veenstra, J. A. and Perrimon, N. (2014). Control of Lipid Metabolism by Tachykinin in Drosophila. Cell Rep 9(1):40-7. PubMed ID: 25263556
The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. This study demonstrates that Tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila and that TKs repress lipogenesis in enterocytes (ECs) associated with TKR99D receptor and protein kinase A (PKA) signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions.
Brankatschk, M., Dunst, S., Nemetschke, L. and Eaton, S. (2014). Delivery of circulating lipoproteins to specific neurons in the brain regulates systemic Insulin signaling. Elife 3 [Epub ahead of print]. PubMed ID: 25275323
The Insulin signaling pathway couples growth, development and lifespan to nutritional conditions. This study demonstrates a function for the Drosophila lipoprotein LTP in conveying information about dietary lipid composition to the brain to regulate Insulin signaling. When yeast lipids are present in the diet, free calcium levels rise in Blood Brain Barrier glial cells. This induces transport of LTP across the Blood Brain Barrier by two LDL receptor-related proteins: LRP1 and Megalin. LTP accumulates on specific neurons that connect to cells that produce Insulin-like peptides, and induces their release into the circulation. This increases systemic Insulin signaling and the rate of larval development on yeast-containing food compared with a plant-based food of similar nutritional content.
Saturday, October 18th
Katayama, N., Abbott, J. K., Kjaerandsen, J., Takahashi, Y. and Svensson, E. I. (2014). Sexual selection on wing interference patterns in Drosophila melanogaster. Proc Natl Acad Sci U S A. [Epub ahead of print] PubMed ID: 25294931
Animals with color vision use color information in intra- and interspecific communication, which in turn may drive the evolution of conspicuous colored body traits via natural and sexual selection. A recent study found that the transparent wings of small flies and wasps in lower-reflectance light environments display vivid and stable structural color patterns, called 'wing interference patterns' (WIPs). Such WIPs were hypothesized to function in sexual selection among small insects with wing displays, but this has not been experimentally verified. This study presents the first experimental evidence that WIPs in males of Drosophila melanogaster are targets of mate choice from females, and that two different color traits - saturation and hue - experience directional and stabilizing sexual selection, respectively. Using isogenic lines from the D. melanogaster Genetic Reference Panel, attractiveness of different male WIPs was compared against black and white visual backgrounds. Males with more vivid wings were shown to be more attractive to females than are males with dull wings. Wings with a large magenta area (i.e., intermediate trait values) were also preferred over those with a large blue or yellow area. These experimental results add a visual element to the Drosophila mating array, integrating sexual selection with elements of genetics and evo-devo, potentially applicable to a wide array of small insects with hyaline wings. The results further underscore that the mode of sexual selection on such visual signals can differ profoundly between different color components, in this case hue and saturation.
Yun, L. and Agrawal, A. F. (2014). Variation in the strength of inbreeding depression across environments: Effects of stress and density dependence. Evolution [Epub ahead of print]. PubMed ID: 25213285Summary:
In what types of environments should one expect to find strong inbreeding depression? Previous studies indicate that inbreeding depression, δ, is positively correlated with the stressfulness of the environment in which it is measured. However, it remains unclear why stress, per se, should increase δ. Only 'competitive stress' should have a logical connection to δ. Through competition for resources, better quality (outbred) individuals make the environment worse for lower quality (inbred) individuals, accentuating the differences between them. For this reason, inbreeding depression would be expected to be stronger in environments where the fitness of individuals is more sensitive to the presence of conspecifics (i.e., where fitness is more density dependent). Indeed, some studies suggest a role for competition within environments but this idea has not been tested in the context of understanding variation in δ across environments. Using Drosophila melanogaster, this study estimated δ for viability in 22 different environments. These environments were simultaneously characterized for (1) stressfulness and (2) density dependence. Though stress and density dependence are moderately correlated with each other, inbreeding depression is much more strongly correlated with density dependence. These results suggest that mean selection across the genome is stronger in environments where competition is intense, rather than in environments that are stressful for other reasons.
Malagon, J. N., Ahuja, A., Sivapatham, G., Hung, J., Lee, J., Munoz-Gomez, S. A., Atallah, J., Singh, R. S. and Larsen, E. (2014). Evolution of Drosophila sex comb length illustrates the inextricable interplay between selection and variation. Proc Natl Acad Sci U S A 111(39):E4103-9. PubMed ID: 25197080
In spite of the diversity of possible biological forms observed in nature, a limited range of morphospace is frequently occupied for a given trait. Several mechanisms have been proposed to explain this bias in the distribution of phenotypes including selection, drift, and developmental constraints. Despite extensive work on phenotypic bias, the underlying developmental mechanisms explaining why particular regions of morphological space remain unoccupied are poorly understood. To address this issue, the sex comb, a group of modified bristles used in courtship that shows marked morphological diversity among Drosophila species, was studied. In many Drosophila species including Drosophila melanogaster, the sex comb rotates 90 degrees to a vertical position during development. This study analyzed the effect of changing D. melanogaster sex comb length on the process of rotation. Artificial selection was found to change the number of bristles per comb without a proportional change in the space available for rotation. As a result, when increasing sex comb length, rather than displaying a similar straight vertical shape observed in other Drosophila species, long sex combs bend because rotation is blocked by a neighboring row of bristles. These results show ways in which morphologies that would be favored by natural selection are apparently impossible to achieve developmentally. These findings highlight the potential role of development in modifying selectable variation in the evolution of Drosophila sex comb length.
Jackson, B. C., Campos, J. L. and Zeng, K. (2014). The effects of purifying selection on patterns of genetic differentiation between Drosophila melanogaster populations. Heredity (Edinb) [Epub ahead of print]. PubMed ID: 25227256
Using the data provided by the Drosophila Population Genomics Project, this study investigated factors that affect the genetic differentiation between Rwandan and French populations of D. melanogaster. By examining within-population polymorphisms, it was shown that sites in long introns (especially those >2000 bp) have significantly lower pi (nucleotide diversity) and more low-frequency variants (as measured by Tajima's D, minor allele frequencies, and prevalence of variants that are private to one of the two populations) than short introns, suggesting a positive relationship between intron length and selective constraint. A similar analysis of protein-coding polymorphisms shows that 0-fold (degenerate) sites in more conserved genes are under stronger purifying selection than those in less conserved genes. There is limited evidence that selection on codon bias has an effect on differentiation (as measured by the fixation index or FST) at 4-fold (degenerate) sites, and 4-fold sites and sites in 8-30 bp of short introns 65 bp have comparable FST values. Consistent with the expected effect of purifying selection, sites in long introns and 0-fold sites in conserved genes are less differentiated than those in short introns and less conserved genes, respectively. Genes in non-crossover regions (for example, the fourth chromosome) have very high FST values at both 0-fold and 4-fold degenerate sites, which is probably because of the large reduction in within-population diversity caused by tight linkage between many selected sites. These analyses also reveal subtle statistical properties of FST, which arise when information from multiple single nucleotide polymorphisms is combined and can lead to the masking of important signals of selection.
Friday, October 17th
Camp, D., He, B. H., Li, S., Althaus, I. W., Holtz, A. M., Allen, B. L., Charron, F. and van Meyel, D. J. (2014). Ihog and Boi elicit Hh signaling via Ptc but do not aid Ptc in sequestering the Hh ligand. Development 141(20):3879-88. PubMed ID: 25231763
Hedgehog (Hh) proteins are secreted molecules essential for tissue development in vertebrates and invertebrates. Hh reception via the 12-pass transmembrane protein Patched (Ptc) elicits intracellular signaling through Smoothened (Smo). Hh binding to Ptc is also proposed to sequester the ligand, limiting its spatial range of activity. In Drosophila, Interference hedgehog (Ihog) and Brother of ihog (Boi) are two conserved and redundant transmembrane proteins that are essential for Hh pathway activation. How Ihog and Boi activate signaling in response to Hh remains unknown; each can bind both Hh and Ptc and so it has been proposed that they are essential for both Hh reception and sequestration. Using genetic epistasis this study established that Ihog and Boi, and their orthologs in mice, act upstream or at the level of Ptc to allow Hh signal transduction. In the Drosophila developing wing model it was found that through Hh pathway activation Ihog and Boi maintain the boundary between the anterior and posterior compartments. The contributions of Ptc was dissociated from those of Ihog/Boi, and, surprisingly, it was found that cells expressing Ptc can retain and sequester the Hh ligand without Ihog and Boi, but that Ihog and Boi cannot do so without Ptc. Together, these results reinforce the central role for Ptc in Hh binding in vivo and demonstrate that, although Ihog and Boi are dispensable for Hh sequestration, they are essential for pathway activation because they allow Hh to inhibit Ptc and thereby relieve its repression of Smo.
Caine, C., Kasherov, P., Silber, J. and Lalouette, A. (2014). Mef2 interacts with the Notch pathway during adult muscle development in Drosophila melanogaster. PLoS One 9: e108149. PubMed ID: 25247309
Myogenesis of indirect flight muscles (IFMs) in Drosophila melanogaster follows a well-defined cellular developmental scheme. During embryogenesis, a set of cells, the Adult Muscle Precursors (AMPs), are specified. These cells will become proliferating myoblasts during the larval stages which will then give rise to the adult IFMs. Although the cellular aspect of this developmental process is well studied, the molecular biology behind the different stages is still under investigation. In particular, the interactions required during the transition from proliferating myoblasts to differentiated myoblasts ready to fuse to the muscle fiber. It has been previously shown that the Notch pathway is active in proliferating myoblasts, and that this pathway is inhibited in developing muscle fibers. Furthermore, the Myocyte enhancing factor 2 (Mef2), Vestigial (Vg) and Scalloped (Sd) transcription factors are necessary for IFM development and that Vg is required for Notch pathway repression in differentiating fibers. This study examined the interactions between Notch and Mef2 and mechanisms by which the Notch pathway is inhibited during differentiation. Mef2 was shown to be capable of inhibiting the Notch pathway in non myogenic cells. A previous screen for Mef2 potential targets identified Delta a component of the Notch pathway. Dl is expressed in Mef2 and Sd-positive developing fibers. These results show that Mef2 and possibly Sd regulate a Dl enhancer specifically expressed in the developing IFMs and that Mef2 is required for Dl expression in developing IFMs.
Harwood, B. N., Draper, I. and Kopin, A. S. (2014). Targeted inactivation of the rickets receptor in muscle compromises Drosophila viability. J Exp Biol [Epub ahead of print]. PubMed ID: 25278473
Bursicon is a hormone that modulates wing expansion, cuticle hardening, and melanization in Drosophila melanogaster. Bursicon activity is mediated through its cognate G protein-coupled receptor, Rickets. This study developed a membrane tethered Bursicon construct that enables spatial modulation of Rickets mediated physiology in transgenic flies. Ubiquitous expression of tethered Bursicon throughout development results in arrest at the pupal stage. The few organisms that eclose fail to undergo wing expansion. These phenotypes suggest that expression of tethered Bursicon inhibits Rickets mediated function. Consistent with this hypothesis, this study showed in vitro that sustained stimulation of Rickets by tethered Bursicon leads to receptor desensitization. Furthermore, tissue specific expression of the tethered Bursicon inhibitor unraveled a critical role for Rickets in a subset of adult muscles. Taken together, these finds highlight the utility of membrane tethered inhibitors as important genetic/pharmacological tools to dissect the tissue specific roles of GPCRs in vivo.
Baqri, R. M., Pietron, A. V., Gokhale, R. H., Turner, B. A., Kaguni, L. S., Shingleton, A. W., Kunes, S. and Miller, K. E. (2014). Mitochondrial chaperone TRAP1 activates the mitochondrial UPR and extends healthspan in Drosophila. Mech Ageing Dev [Epub ahead of print]. PubMed ID: 25265088
The molecular mechanisms influencing healthspan are unclear but mitochondrial function, resistance to oxidative stress and proteostasis are recurring themes. Tumor necrosis factor Receptor Associated Protein 1 (TRAP1), the mitochondrial analog of Hsp75, regulates levels of reactive oxygen species in vitro and is found expressed at higher levels in tumor cells where it is thought to play a pro-survival role. While TRAP1-directed compartmentalized protein folding is a promising target for cancer therapy, its role at the organismal level is unclear. This study reports that overexpression of TRAP1 in Drosophila extends healthspan by enhancing stress resistance, locomotor activity and fertility while depletion of TRAP1 has the opposite effect, with little effect on lifespan under both conditions. In addition, modulating TRAP1 expression promotes the nuclear translocation of homeobox protein Dve and increases expression of genes associated with the mitochondrial unfolded protein response (UPRmt), indicating an activation of this proteostasis pathway. Notably, independent genetic knockdown of components of the UPRmt pathway dampen the enhanced stress resistance observed in TRAP1 overexpression flies. Together these studies suggest that TRAP1 regulates healthspan, potentially through activation of the UPRmt.
Thursday, October 16th
Dzhindzhev, N. S., Tzolovsky, G., Lipinszki, Z., Schneider, S., Lattao, R., Fu, J., Debski, J., Dadlez, M. and Glover, D. M. (2014). Plk4 Phosphorylates Ana2 to Trigger Sas6 Recruitment and Procentriole Formation. Curr Biol. PubMed ID: 25264260
Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4; see SAK) counterpart in other organisms. Although Plk4's centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. This study shows that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 (see Drosophila Ana2) to enable it to bind and recruit its Sas6 (see Drosophila Sas-6) partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent.
Poulton, J. S., Cuningham, J. C. and Peifer, M. (2014). Acentrosomal Drosophila epithelial cells exhibit abnormal cell division, leading to cell death and compensatory proliferation. Dev Cell 30: 731-745. PubMed ID: 25241934
Mitotic spindles are critical for accurate chromosome segregation. Centrosomes, the primary microtubule nucleating centers of animal cells, play key roles in forming and orienting mitotic spindles. However, the survival of Drosophila without centrosomes suggested they are dispensable in somatic cells, challenging the canonical view. Fly wing disc epithelia were used as a model to resolve these conflicting hypotheses, revealing that centrosomes play vital roles in spindle assembly, function, and orientation. Many acentrosomal cells exhibit prolonged spindle assembly, chromosome missegregation, DNA damage, misoriented divisions, and eventual apoptosis. It was found that multiple mechanisms buffer the effects of centrosome loss, including alternative microtubule nucleation pathways and the spindle assembly checkpoint. Apoptosis of acentrosomal cells is mediated by JNK signaling, which also drives compensatory proliferation to maintain tissue integrity and viability. These data reveal the importance of centrosomes in fly epithelia and demonstrate the robust compensatory mechanisms at the cellular and organismal level.
>Kong, D., Farmer, V., Shukla, A., James, J., Gruskin, R., Kiriyama, S. and Loncarek, J. (2014). Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles. J Cell Biol 206: 855-865. PubMed ID: 25246616
Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. Controlled Plk1 activity (see Drosophila Polo) is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.
Pefani, D. E., Latusek, R., Pires, I., Grawenda, A. M., Yee, K. S., Hamilton, G., van der Weyden, L., Esashi, F., Hammond, E. M. and O'Neill, E. (2014). RASSF1A-LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2. Nat Cell Biol 16: 962-971. PubMed ID: 25218637
Genomic instability is a key hallmark of cancer leading to tumour heterogeneity and therapeutic resistance. BRCA2 has a fundamental role in error-free DNA repair but also sustains genome integrity by promoting RAD51 (see Drosophila Rad51-like) nucleofilament formation at stalled replication forks. CDK2 (see Drosophila Cdc2) phosphorylates BRCA2 (pS3291-BRCA2) to limit stabilizing contacts with polymerized RAD51; however, how replication stress modulates CDK2 activity and whether loss of pS3291-BRCA2 regulation results in genomic instability of tumours are not known. This study demonstrates that the Hippo pathway kinase LATS1 (see Drosophila Warts interacts with CDK2 in response to genotoxic stress to constrain pS3291-BRCA2 and support RAD51 nucleofilaments, thereby maintaining genomic fidelity during replication stalling. It was also shown that LATS1 forms part of an ATR-mediated response to replication stress that requires the tumour suppressor RASSF1A. Importantly, perturbation of the ATR-RASSF1A-LATS1 signalling axis leads to genomic defects associated with loss of BRCA2 function and contributes to genomic instability and 'BRCA-ness' in lung cancers.
Wednesday, October 15th
Collins, B., Kaplan, H. S., Cavey, M., Lelito, K. R., Bahle, A. H., Zhu, Z., Macara, A. M., Roman, G., Shafer, O. T. and Blau, J. (2014). Differentially timed extracellular signals synchronize pacemaker neuron clocks. PLoS Biol 12: e1001959. PubMed ID: 25268747
Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, the minimal clock neural circuit in Drosophila larvae were studied, manipulating either the four master pacemaker neurons (LNvs) or two dorsal clock neurons (DN1s). Unexpectedly, it was found that the PDF Receptor (PdfR) is required in both LNvs and DN1s to maintain synchronized LNv clocks. It was also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LNvs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LNvs severely dampened Timeless clock protein oscillations, it is concluded that the master pacemaker LNvs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LNvs. Finally it was found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LNvs. It is proposed that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species.
Chang, S. and Wang, Z. J. (2014). Predicting fruit fly's sensing rate with insect flight simulations. Proc Natl Acad Sci U S A 111: 11246-11251. PubMed ID: 25049376
Without sensory feedback, flies cannot fly. Exactly how various feedback controls work in insects is a complex puzzle to solve. What do insects measure to stabilize their flight? How often and how fast must insects adjust their wings to remain stable? To gain insights into algorithms used by insects to control their dynamic instability, a simulation tool was developed to study free flight. To stabilize flight, a control algorithm was constructed that modulates wing motion based on discrete measurements of the body-pitch orientation. These simulations give theoretical bounds on both the sensing rate and the delay time between sensing and actuation. Interpreting the findings together with experimental results on fruit flies' reaction time and sensory motor reflexes, it is conjectured that fruit flies sense their kinematic states every wing beat to stabilize their flight. A candidate for such a control i proposed involving the fly's haltere and first basalar motor neuron. The framework for these simulation and discrete control algorithms is applicable to studies of both natural and man-made fliers.
Lin, S., Owald, D., Chandra, V., Talbot, C., Huetteroth, W. and Waddell, S. (2014). Neural correlates of water reward in thirsty Drosophila. Nat Neurosci [Epub ahead of print]. PubMed ID: 25262493
Drinking water is innately rewarding to thirsty animals. In addition, the consumed value can be assigned to behavioral actions and predictive sensory cues by associative learning. This study shows that thirst converts water avoidance into water-seeking in naive Drosophila melanogaster. Thirst also permitted flies to learn olfactory cues paired with water reward. Water learning required water taste and <40 water-responsive dopaminergic neurons that innervate a restricted zone of the mushroom body gamma lobe. These water learning neurons are different from those that are critical for conveying the reinforcing effects of sugar. Naive water-seeking behavior in thirsty flies did not require water taste but relied on another subset of water-responsive dopaminergic neurons that target the mushroom body beta' lobe. Furthermore, these naive water-approach neurons were not required for learned water-seeking. These results therefore demonstrate that naive water-seeking, learned water-seeking and water learning use separable neural circuitry in the brain of thirsty flies.
Saumweber, T., Cano, C., Klessen, J., Eichler, K., Fendt, M. and Gerber, B. (2014). Immediate and punitive impact of mechanosensory disturbance on olfactory behaviour of larval Drosophila. Biol Open. PubMed ID: 25260919
The ability to respond to and to learn about mechanosensory disturbance is widespread among animals. Using Drosophila larvae, this study describes how the frequency of mechanosensory disturbance ('buzz') affects three aspects of behaviour: free locomotion, innate olfactory preference, and potency as a punishment. It is reported that (1) during 2-3 seconds after buzz onset the larvae slowed down and then turned, arguably to escape this situation; this was seen for buzz frequencies of 10, 100, and 1000 Hz, (2) innate olfactory preference was reduced when tested in the presence of the buzz; this effect was strongest for the 100 Hz frequency, (3) after odour-buzz associative training, escape from the buzz-associated odour was observed; this effect was apparent for 10 and 100, but not for 1000 Hz. The paper discusses the multiple behavioural effects of mechanosensation and stress that the immediate effects on locomotion and the impact as punishment differ in their frequency-dependence. Similar dissociations between immediate, reflexive behavioural effects and reinforcement potency were previously reported for sweet, salty and bitter tastants. It should be interesting to see how these features map onto the organization of sensory, ascending pathways.
Tuesday, October 14th
Wang, Z., Linden, L. M., Naegeli, K. M., Ziel, J. W., Chi, Q., Hagedorn, E. J., Savage, N. S. and Sherwood, D. R. (2014). UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity. J Cell Biol 206: 619-633. PubMed ID: 25154398
The receptor deleted in colorectal cancer (DCC; see Drosophila Frazzled) directs dynamic polarizing activities in animals toward its extracellular ligand netrin (see Drosophila Netrins). How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, this study has found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. Endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40-binding protein MADD-2 (a TRIM family protein; see Drosophla Trim9) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, these data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. It is proposed that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.
He, M., Subramanian, R., Bangs, F., Omelchenko, T., Liem, K. F., Jr., Kapoor, T. M. and Anderson, K. V. (2014). The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip compartment.
Mammalian Hedgehog (Hh) signal transduction requires a primary cilium, a microtubule-based organelle, and the Gli-Sufu complexes that mediate Hh signalling, which are enriched at cilia tips. Kif7, a kinesin-4 family protein and homolog of Drosophila Costa, is a conserved regulator of the Hh signalling pathway and a human ciliopathy protein. This study shows that Kif7 localizes to the cilium tip, the site of microtubule plus ends, where it limits cilium length and controls cilium structure. Purified recombinant Kif7 binds the plus ends of growing microtubules in vitro, where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. Kif7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of Kif7 in the mammalian Hh pathway is to control cilium architecture and to create a single cilium tip compartment, where Gli-Sufu activity can be correctly regulated.
Romano, D., Nguyen, L. K., Matallanas, D., Halasz, M., Doherty, C., Kholodenko, B. N. and Kolch, W. (2014). Protein interaction switches coordinate Raf-1 and MST2/Hippo signalling. Nat Cell Biol 16: 673-684. PubMed ID: 24929361
Signal transduction requires the coordination of activities between different pathways. In mammalian cells, Raf-1 (see Drosophila Raf) regulates the MST-LATS and MEK-ERK pathways. A complex circuitry of competing protein interactions coordinates the crosstalk between the ERK and MST pathways. Combining mathematical modelling and experimental validation this study shows that competing protein interactions can cause steep signalling switches through phosphorylation-induced changes in binding affinities. These include Akt (see Drosophila Akt) phosphorylation of MST2 (a STE20-family member and purported Hpo ortholog) and a feedback phosphorylation of Raf-1 Ser 259 by LATS1, which enables Raf-1 to suppress both MST2 and MEK signalling. Mutation of Raf-1 Ser 259 stimulates both pathways, simultaneously driving apoptosis and proliferation, whereas concomitant MST2 downregulation switches signalling to cell proliferation, transformation and survival. Thus, competing protein interactions provide a versatile regulatory mechanism for signal distribution through the dynamic integration of graded signals into switch-like responses.
Li, M. Y., Lai, P. L., Chou, Y. T., Chi, A. P., Mi, Y. Z., Khoo, K. H., Chang, G. D., Wu, C. W., Meng, T. C. and Chen, G. C. (2014). Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation. Oncogene. PubMed ID: 25263444
Epidermal growth factor receptor (EGFR; see Drosophila Egfr) regulates multiple signaling cascades essential for cell proliferation, growth and differentiation. Using a genetic approach, this study found that Drosophila FERM and PDZ domain-containing protein tyrosine phosphatase, dPtpmeg, negatively regulates border cell migration and inhibits the EGFR/Ras/mitogen-activated protein kinase signaling pathway during wing morphogenesis. EGFR pathway substrate 15 (Eps15) was further identified as a target of dPtpmeg and its human homolog PTPN3. Eps15 is a scaffolding adaptor protein known to be involved in EGFR endocytosis and trafficking. Interestingly, PTPN3-mediated tyrosine dephosphorylation of Eps15 promotes EGFR for lipid raft-mediated endocytosis and lysosomal degradation. PTPN3 and the Eps15 tyrosine phosphorylation-deficient mutant suppress non-small-cell lung cancer cell growth and migration in vitro and reduce lung tumor xenograft growth in vivo. Moreover, depletion of PTPN3 impairs the degradation of EGFR and enhances proliferation and tumorigenicity of lung cancer cells. Taken together, these results indicate that PTPN3 may act as a tumor suppressor in lung cancer through its modulation of EGFR signaling.
Ribeiro, S. A., D'Ambrosio, M. V. and Vale, R. D. (2014). Induction of focal adhesions and motility in Drosophila S2 cells. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25273555
Focal adhesions are dynamic structures that interact with the extracellular matrix on the cell exterior and actin filaments on the cell interior, enabling cells to adhere and crawl along surfaces. This study describes a system for inducing the formation of focal adhesions in normally non-ECM-adherent, non-motile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a non-polarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. A subset of non-motile, non-polarized cells also exhibited focal adhesions that rapidly assembled and disassembled around the cell perimeter. Such cooperative and dynamic fluctuations of focal adhesions were decreased by RNAi depletion of myosin II and focal adhesion kinase (FAK), suggesting that this behavior requires force and focal adhesion maturation. These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility.
Monday, October 13th
Takino, K., Ohsawa, S. and Igaki, T. (2014). Loss of Rab5 drives non-autonomous cell proliferation through TNF and Ras signaling in Drosophila. Dev Biol 395(1):19-28. PubMed ID: 25224221
Deregulation of the endocytic machinery has been implicated in human cancers. However, the mechanism by which endocytic defects drive cancer development remains to be clarified. This study found through a genetic screen in Drosophila that loss of Rab5, a protein required for early endocytic trafficking, drives non-autonomous cell proliferation in imaginal epithelium. The genetic data indicate that dysfunction of Rab5 leads to cell-autonomous accumulation of Eiger (a TNF homolog) and EGF receptor (EGFR), which causes activation of downstream JNK and Ras signaling, respectively. JNK signaling and its downstream component Cdc42 cooperate with Ras signaling to induce upregulation of a secreted growth factor Upd (an IL-6 homolog) through inactivation of the Hippo pathway. Such non-autonomous tissue growth triggered by Rab5 defect could contribute to epithelial homeostasis as well as cancer development within heterogeneous tumor microenvironment.
Rodrigues-Campos, M. and Thompson, B. J. (2014). The ubiquitin ligase FbxL7 regulates the Dachsous-Fat-Dachs system in Drosophila. Development [Epub ahead of print]. PubMed ID: 25256343
The atypical cadherins Dachsous (Ds) and Fat (Ft) are required to control the size and shape of tissues and organs in animals. In Drosophila, a key effector of Ds and Ft is the atypical myosin Dachs, which becomes planar polarised along the proximal-distal axis in developing epithelia to regulate tissue size via the Hippo pathway and tissue shape via modulating tension at junctions. How Ds and Ft control Dachs polarisation remains unclear. This study identified a ubiquitin ligase, FbxL7, as a novel component of the Ds-Ft-Dachs system that is required to control the level and localisation of Dachs. Loss of FbxL7 results in accumulation of Dachs, similar to loss of Ft. Overexpression of FbxL7 causes downregulation of Dachs, similar to overexpression of the Ft intracellular domain. In addition to regulating Dachs, FbxL7 also influences Ds in a similar manner. GFP-tagged FbxL7 localises to the plasma membrane in a Ft-dependent manner and is planar polarised. It is proposed that Ft recruits FbxL7 to the proximal side of the cell to help restrict Ds and Dachs to the distal side of the cell.
LeBon, L., Lee, T. V., Jafar-Nejad, H., Sprinzak, D. and Elowitz, M. B. (2014). Fringe proteins modulate Notch-ligand and interactions to specify signaling states. Elife 3. PubMed ID: 25255098
The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases (see Drosophyla Fringe). A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. This study analyzed same-cell (cis) Notch-ligand interactions for Notch1 (see Drosophila Notch), Dll1, and Jag1 (see Drosophila Delta), and their dependence on Fringe protein expression in mammalian cells. Dll1 and Jag1 were found to induce cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, it was shown how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation.
Wang, J., Wang, S. and Li, S. (2014). Sumoylation modulates 20-hydroxyecdysone signaling by maintaining USP protein levels in Drosophila. Insect Biochem Mol Biol 54C: 80-88. PubMed ID: 25240618
The nuclear receptor complex for the insect steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer of EcR and USP. It has been shown that Drosophila EcR and USP can be sumoylated in mammalian cells, but it is unknown whether EcR-USP sumoylation naturally occurs in Drosophila. In Drosophila cells, USP, but not EcR, was sumoylated by Smt3, the only Drosophila SUMO protein. The presence of EcR enhanced USP sumoylation, which is further enhanced by 20E treatment. In addition to the Lys20 sumoylation site, five potential acceptor lysine residues in USP were predicted and verified. Mutation of the USP sumoylation sites or reduction of smt3 expression by RNAi attenuated 20E-induced reporter activity. Moreover, in the salivary glands, reducing smt3 expression by RNAi decreased 20E-induced reporter activity, gene expression, and autolysosome formation. Importantly, at least partially, the smt3 RNAi-mediated reduction in 20E signaling resulted from decreased protein levels of USP. In conclusion, sumoylation modulates 20E signaling by maintaining USP protein levels in Drosophila.
Gagliardi, M., Hernandez, A., McGough, I. J. and Vincent, J. P. (2014). Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal beta-Catenin/Armadillo. J Cell Sci [Epub ahead of print]. PubMed ID: 25236598
A key step in canonical Wnt signalling pathway is the inhibition of GSK3beta (see Drosophila Shaggy), which results in the accumulation of nuclear beta-Catenin (see Drosophila Armadillo) and hence target gene regulation. Evidence suggests that endocytosis is required for signalling yet its role and the molecular understanding remains unclear. A recent and controversial model suggests that endocytosis contributes to Wnt signalling by causing the sequestration of the ligand-receptor complex, including LRP6 and GSK3 to multivesicular bodies (MVBs) thus preventing GSK3beta from accessing beta-Catenin. This study used specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in Human and Drosophila cells. However, no evidence was found that, in Drosophila cells or imaginal discs, LRP6/Arrow traffics to MVBs or that MVBs are required for Wnt/Wingless signalling. Moreover, it was shown that activation of signalling by chemical blockade of GSK3beta is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand-receptor complex. It is proposed that, through an unknown mechanism, endocytosis boosts the resting pool of beta-Catenin upon which GSK3beta normally acts.
Sunday, October 12th
Fukaya, T., Iwakawa, H. O. and Tomari, Y. (2014). MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila. Mol Cell 56: 67-78. PubMed ID: 25280104
miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. Previously studies have shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. This study examined the assembly of translation initiation factors on the target mRNA under repression. Ago1-RISC was shown to induce dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block the assembly of the eIF4F complex during translation initiation.
Ott, K. M., Nguyen, T. and Navarro, C. (2014). The DExH box helicase domain of Spindle-E is necessary for retrotransposon silencing and axial patterning during Drosophila oogenesis. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25239103
Transposable selfish genetic elements have the potential to cause debilitating mutations as they replicate and reinsert within the genome. Therefore, it is critical to keep the cellular levels of these elements low. This is especially true in the germline where these mutations could affect the viability of the next generation. A class of small non-coding RNAs, the Piwi-associated RNAs, are responsible for silencing transposable elements in the germline of most organisms. Several proteins have been identified as playing essential roles in piRNA generation and transposon silencing. However, for the most part their function in piRNA generation is currently unknown. One of these proteins is the Drosophila DExH box/Tudor domain protein Spindle-E whose activity is necessary for the generation of most germline piRNAs. This study molecularly and phenotypically characterized 14 previously identified spindle-E alleles. Of the alleles that express detectable Spindle-E protein five were found to have mutations in the DExH box domain. Additionally, it was found that processes that depend on piRNA function, including Aubergine localization, Dynein motor movement and retrotransposon silencing, were severely disrupted in alleles with DExH box domain mutations. The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, while alleles with mutations in other regions of Spindle-E did not affect these processes as much. From these data it is concluded that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.
Sytnikova, Y. A., Rahman, R., Chirn, G. W., Clark, J. P. and Lau, N. C. (2014). Transposable element dynamics and PIWI regulation impacts lncRNA and gene expression diversity in Drosophila ovarian cell cultures. Genome Res [Epub ahead of print]. PubMed ID: 25267525
Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposable elements (TEs) from mobilizing in gonadal cells. To determine the spectrum of piRNA-regulated targets that may extend beyond TEs, a genome-wide survey was conducted for transcripts associated with PIWI and for transcripts affected by PIWI knockdown in Drosophila Ovarian Somatic Sheet (OSS) cells, a follicle cell line expressing the Piwi pathway. Despite the immense sequence diversity amongst OSS cell piRNAs, this analysis indicates TE transcripts were the major transcripts associated with and directly regulated by PIWI. However, several coding genes were indirectly regulated by PIWI via an adjacent de novo TE insertion that generated a nascent TE transcript. Interestingly, it was noticed that PIWI-regulated genes in OSS cells greatly differed from genes affected in a related follicle cell culture, Ovarian Somatic Cells (OSCs). Therefore, this study characterized the distinct genomic TE insertions across four OSS and OSC lines and discovered dynamic TE landscapes in gonadal cultures that were defined by a subset of active TEs. Particular de novo TEs appeared to stimulate the expression of novel candidate long non-coding RNAs (lncRNAs) in a cell-lineage specific manner, and some of these TE-associated lncRNAs were associated with PIWI and overlapped PIWI-regulated genes. These analyses of OSCs and OSS cells demonstrate that despite having a Piwi pathway to suppress endogenous mobile elements, gonadal cell TE landscapes can still dramatically change and create transcriptome diversity.
Fricke, C., Green, D., Smith, D., Dalmay, T. and Chapman, T. (2014). MicroRNAs influence reproductive responses by females to male Sex peptide in Drosophila melanogaster/B>. Genetics [Epub ahead of print]. PubMed ID: 25245794
Across taxa, female behavior and physiology changes significantly following the receipt of ejaculate molecules during mating. For example, receipt of Sex peptide (SP) in female Drosophila significantly alters female receptivity, egg production, lifespan, hormone levels, immunity, sleep and feeding patterns. These changes are underpinned by distinct tissue- and time-specific changes in diverse sets of mRNAs. However, little is yet known about the regulation of these gene expression changes, and hence the potential role of microRNAs (miRNAs), in female post-mating responses. A preliminary screen of genomic responses in females to receipt of SP suggested that there were changes in the expression of several miRNAs. This study tested directly whether females lacking four of the candidate miRNAs highlighted (miR-279, miR-317, miR-278 and miR-184) showed altered fecundity, receptivity and lifespan responses to receipt of SP, when mated once or continually to SP null or control males. The results showed that miRNA-lacking females mated to SP null males exhibited altered receptivity, but not reproductive output, in comparison to controls. However, these effects interacted significantly with the genetic background of the miRNA-lacking females. No significant survival effects were observed in miRNA-lacking females housed continually with SP null or control males. However, continual exposure to control males that transferred SP resulted in significantly higher variation in miRNA-lacking female lifespan than did continual exposure to SP null males. The results provide the first insight into the effects and importance of miRNAs in regulating post-mating responses in females.
Oas, S. T., Bryantsev, A. L. and Cripps, R. M. (2014). Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila. J Cell Biol 206: 895-908. PubMed ID: 25246617
Drosophila flight muscles are distinct from other skeletal muscles, such as jump muscles, and express several uniquely spliced muscle-associated transcripts. This study sought to identify factors mediating splicing differences between the flight and jump muscle fiber types. The ribonucleic acid-binding protein Arrest (Aret) was found to be expressed in flight muscles: in founder cells, Aret accumulates in a novel intranuclear compartment that was termed the Bruno body, and after the onset of muscle differentiation, Aret disperses in the nucleus. Down-regulation of the aret gene leads to ultrastructural changes and functional impairment of flight muscles, and transcripts of structural genes expressed in the flight muscles become spliced in a manner characteristic of jump muscles. Aret also potently promotes flight muscle splicing patterns when ectopically expressed in jump muscles or tissue culture cells. Genetically, aret is located downstream of exd (extradenticle), hth (homothorax), and salm (spalt major), transcription factors that control fiber identity. These observations provide insight into a transcriptional and splicing regulatory network for muscle fiber specification.
Saturday, October 11th
Avet-Rochex, A., Carvajal, N., Christoforou, C. P., Yeung, K., Maierbrugger, K. T., Hobbs, C., Lalli, G., Cagin, U., Plachot, C., McNeill, H. and Bateman, J. M. (2014). Unkempt is negatively regulated by mTOR and uncouples neuronal differentiation from growth control. PLoS Genet 10: e1004624. PubMed ID: 25210733
Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. The insulin receptor (InR)/mTOR pathway has been identified as a critical regulator of the timing of neuronal differentiation in the Drosophila eye. This pathway has also been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway transcriptional targets of mTOR were screened for neuronal differentiation phenotypes in photoreceptor neurons. The conserved gene unkempt (unk), which encodes a zinc finger/RING domain containing protein, was identified as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. The gene headcase (hdc) was also identified as a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, Unkempt-like was shown to be is expressed in the developing mouse retina and in neural stem/progenitor cells, suggesting that the role of Unk in neurogenesis may be conserved in mammals.
Tanaka-Matakatsu, M., Miller, J. and Du, W. (2014). The homeodomain of Eyeless regulates cell growth and antagonizes the paired domain-dependent retinal differentiation function. Protein Cell [Epub ahead of print]. PubMed ID: 25234589
Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. This study used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. Ey was shown to promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhances Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. The third helix of the Ey HD was shown to directly interact with the RED subdomain in Ey PD, and deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, it is speculated that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.
Parvy, J. P., Wang, P., Garrido, D., Maria, A., Blais, C., Poidevin, M. and Montagne, J. (2014). Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster. Development 141(20):3955-65. PubMed ID: 25252945
In most animals, steroid hormones are crucial regulators of physiology and developmental life transitions. Steroid synthesis depends on extrinsic parameters and autoregulatory processes to fine-tune the dynamics of hormone production. In Drosophila, transient increases of the steroid prohormone ecdysone, produced at each larval stage, are necessary to trigger moulting and metamorphosis. Binding of the active ecdysone (20-hydroxyecdysone) to its receptor (EcR) is followed by the sequential expression of the nuclear receptors E75, DHR3 and βFtz-f1, representing a model for steroid hormone signalling. This study combined genetic and imaging approaches to investigate the precise role of this signalling cascade within the prothoracic gland (PG), where ecdysone synthesis takes place. These receptors were shown to operate through an apparent unconventional hierarchy in the PG to control ecdysone biosynthesis. At metamorphosis onset, DHR3 emerges as the downstream component that represses steroidogenic enzymes and requires an early effect of EcR for this repression. To avoid premature repression of steroidogenesis, E75 counteracts DHR3 activity, whereas EcR and betaFtz-f1 act early in development through a forward process to moderate DHR3 levels. These findings suggest that within the steroidogenic tissue, a given 20-hydroxyecdysone peak induces autoregulatory processes to sharpen ecdysone production and to confer competence for ecdysteroid biosynthesis at the next developmental phase, providing novel insights into steroid hormone kinetics.
Wernitznig, S., Rind, F. C., Polt, P., Zankel, A., Pritz, E., Kolb, D., Bock, E. and Leitinger, G. (2014). Synaptic connections of first stage visual neurons in the locust Schistocerca gregaria extend evolution of tetrad synapses back 200 million years. J Comp Neurol [Epub ahead of print]. PubMed ID: 25255709
The small size of some insects, and the crystalline regularity of their eyes have made them ideal for large-scale reconstructions of visual circuits. In phylogenetically recent muscomorph flies, like Drosophila, precisely coordinated output to different motion-processing pathways is delivered by photoreceptors (R cells), targeting four different postsynaptic cells at each synapse (tetrad). Tetrads were linked to evolution of aerial agility. This study adopted serial block-face scanning electron microscopy (SBEM) to reconstruct circuits for vision in the larger brain of a locust - a phylogenetically old, flying insect. Locust lamina monopolar cells, L1 and L2, were the main targets of the R cell pathway, L1 and L2 each feed a different circuit, only L1 providing feedback onto R cells. Unexpectedly, 40 % of all locust R cell synapses onto both L1 and L2 were tetrads, revealing the emergence of tetrads in an arthropod group present 200 million years before muscomorph flies appeared, coinciding with the early evolution of flight.
Friday, October 10th
Chakrabarti, S., Poidevin, M. and Lemaitre, B. (2014). The Drosophila MAPK p38c regulates oxidative stress and lipid homeostasis in the intestine. PLoS Genet 10: e1004659. PubMed ID: 25254641
The p38 mitogen-activated protein (MAP) kinase signaling cassette has been implicated in stress and immunity in evolutionarily diverse species. In response to a wide variety of physical, chemical and biological stresses p38 kinases phosphorylate various substrates, transcription factors of the ATF family and other protein kinases, regulating cellular adaptation to stress. The Drosophila genome encodes three p38 kinases named p38a, p38b and p38c. This study analyzed the role of p38c in the Drosophila intestine. The p38c gene is expressed in the midgut and upregulated upon intestinal infection. p38c mutant flies are more resistant to infection with the lethal pathogen Pseudomonas entomophila but are more susceptible to the non-pathogenic bacterium Erwinia carotovora. This phenotype was linked to a lower production of Reactive Oxygen Species (ROS) in the gut of p38c mutants, whereby the transcription of the ROS-producing enzyme Dual oxidase (Duox) is reduced in p38c mutant flies. This genetic analysis shows that p38c functions in a pathway with Mekk1 and Licorne (Mkk3) to induce the phosphorylation of Atf-2, a transcription factor that controls Duox expression. Interestingly, p38c deficient flies accumulate lipids in the intestine while expressing higher levels of antimicrobial peptide and metabolic genes. The role of p38c in lipid metabolism is mediated by the Atf3 transcription factor. This observation suggests that p38c and Atf3 function in a common pathway in the intestine to regulate lipid metabolism and immune homeostasis. Collectively, this study demonstrates that p38c plays a central role in the intestine of Drosophila. It also reveals that many roles initially attributed to p38a are in fact mediated by p38c.
Martinez, J., Longdon, B., Bauer, S., Chan, Y. S., Miller, W. J., Bourtzis, K., Teixeira, L. and Jiggins, F. M. (2014). Symbionts commonly provide broad spectrum resistance to viruses in insects: a comparative analysis of wolbachia strains. PLoS Pathog 10: e1004369. PubMed ID: 25233341
In the last decade, bacterial symbionts have been shown to play an important role in protecting hosts against pathogens. Wolbachia, a widespread symbiont in arthropods, can protect Drosophila and mosquito species against viral infections. This study investigated antiviral protection in 19 Wolbachia strains originating from 16 Drosophila species after transfer into the same genotype of Drosophila simulans. Approximately half of the strains protected against two RNA viruses. Given that 40% of terrestrial arthropod species are estimated to harbour Wolbachia, as many as a fifth of all arthropods species may benefit from Wolbachia-mediated protection. The level of protection against two distantly related RNA viruses - DCV and FHV - was strongly genetically correlated, which suggests that there is a single mechanism of protection with broad specificity. Furthermore, Wolbachia is making flies resistant to viruses, as increases in survival can be largely explained by reductions in viral titer. Variation in the level of antiviral protection provided by different Wolbachia strains is strongly genetically correlated to the density of the bacteria strains in host tissues. No support was found for two previously proposed mechanisms of Wolbachia-mediated protection - activation of the immune system and upregulation of the methyltransferase Dnmt2. The large variation in Wolbachia's antiviral properties highlights the need to carefully select Wolbachia strains introduced into mosquito populations to prevent the transmission of arboviruses.
Nystrand, M. and Dowling, D. K. (2014). Transgenerational interactions involving parental age and immune status affect female reproductive success in Drosophila melanogaster. Proc Biol Sci 281. PubMed ID: 25253454
It is well established that the parental phenotype can influence offspring phenotypic expression, independent of the effects of the offspring's own genotype. Nonetheless, the evolutionary implications of such parental effects remain unclear, partly because previous studies have generally overlooked the potential for interactions between parental sources of non-genetic variance to influence patterns of offspring phenotypic expression. This study tested for such interactions, subjecting male and female Drosophila melanogaster of two different age classes to an immune activation challenge or a control treatment. Flies were then crossed in all age and immune status combinations, and the reproductive success of their immune- and control-treated daughters measured. Daughters produced by two younger parents were found to exhibit reduced reproductive success relative to those of other parental age combinations. Furthermore, immune-challenged daughters exhibited higher reproductive success when produced by immune-challenged relative to control-treated mothers, a pattern consistent with transgenerational immune priming. Finally, a complex interplay between paternal age and parental immune statuses influenced daughter's reproductive success. These findings demonstrate the dynamic nature of age- and immune-mediated parental effects, traceable to both parents, and regulated by interactions between parents and between parents and offspring.
Chtarbanova, S., Lamiable, O., Lee, K. Z., Galiana, D., Troxler, L., Meignin, C., Hetru, C., Hoffmann, J. A., Daeffler, L. and Imler, J. L. (2014). Drosophila C virus systemic infection leads to intestinal obstruction. J Virol. PubMed ID: 25253354
Drosophila C virus (DCV) is a positive-sense RNA virus belonging to the Dicistroviridae family. This natural pathogen of the model organism Drosophila melanogaster is commonly used to investigate antiviral host-defense in flies, which involves both RNA interference and inducible responses. Although lethality is routinely used as readout for the efficiency of the antiviral immune response in these studies, virus-induced pathologies in flies are still poorly understood. This study characterize the pathogenesis associated with systemic DCV infection. Comparison of the transcriptome of flies infected with DCV or two other positive-sense RNA viruses, Flock House virus and Sindbis virus, reveals that DCV infection, unlike those of the other two viruses, represses the expression of a large number of genes. Several of these genes are specifically expressed in the midgut, and are also repressed by starvation. Systemic DCV infection triggers a nutritional stress in Drosophila, which results from intestinal obstruction with accumulation of peritrophic matrix at the entry of the midgut, and accumulation of the food ingested in the crop, a blind muscular food storage organ. The related virus Cricket paralysis virus (CrPV), which efficiently grows in Drosophila, does not trigger this pathology. DCV, but not CrPV, infects the smooth muscles surrounding the crop, causing extensive cytopathology and strongly reducing the rate of contractions. It is concluded that the pathogenesis associated with systemic DCV infection results from the tropism of the virus for an important organ within the foregut of Dipteran insects, the crop. DCV is one of the few identified natural viral pathogens affecting Drosophila. As such, it is an important virus for the deciphering of host-virus interactions in insects. The pathogenesis associated with DCV infection in flies results from the tropism of the virus for an essential but as yet poorly characterized organ in the digestive tract, the crop. These results may have relevance for other members of the Dicistroviridae, some of which are pathogenic to beneficial or pest insect species.
Bronkhorst, A. W., van Cleef, K. W., Venselaar, H. and van Rij, R. P. (2014). A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 25274730
Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. The dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. This study shows that RNAi is suppressed in IIV-6-infected cells, and RNAi suppressor activity was mapped to the viral protein 340R. Using biochemical assays, it was revealed that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, 340R was shown to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, these findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.
Thursday, October 9th
Clohisey, S. M., Dzhindzhev, N. S. and Ohkura, H. (2014). Kank is an EB1 interacting protein that localises to muscle-tendon attachment sites in Drosophila. PLoS One 9: e106112. PubMed ID: 25203404
Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. This study reports the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. This study found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. The mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells.
Wang, L., Sexton, T. R., Venard, C., Giedt, M., Guo, Q., Chen, Q. and Harrison, D. A. (2014). Pleiotropy of the Drosophila JAK pathway cytokine Unpaired 3 in development and aging. Dev Biol [Epub ahead of print]. PubMed ID: 25245869
The Janus kinase (JAK) pathway is an essential, highly re-utilized developmental signaling cascade found in most metazoans. In vertebrates, the JAK intracellular cascade mediates signaling by dozens of cytokines and growth factors. In Drosophila, the Unpaired (Upd) family, encoded by three tandemly duplicated genes, is the only class of ligands associated with JAK stimulation. Unpaired has a central role in activation of JAK for most pathway functions, while Unpaired 2 regulates body size through insulin signaling. This study shows that the third member of the family, unpaired 3 (upd3), overlaps upd in expression in some tissues and is essential for a subset of JAK-mediated developmental functions. First, consistent with the known requirements of JAK signaling in gametogenesis, this study found that mutants of upd3 show an age-dependent impairment of fertility in both sexes. In oogenesis, graded JAK activity stimulated by Upd specifies the fates of the somatic follicle cells. As upd3 mutant females age, defects arise that can be attributed to perturbations of the terminal follicle cells, which require the highest levels of JAK activation. Therefore, in oogenesis, the activities of Upd and Upd3 both appear to quantitatively contribute to specification of those follicle cell fates. Furthermore, the sensitization of upd3 mutants to age-related decline in fertility can be used to investigate reproductive senescence. Second, loss of Upd3 during imaginal development results in defects of adult structures, including reduced eye size and abnormal wing and haltere posture. The outstretched wing and small eye phenotypes resemble classical alleles referred to as outstretched (os) mutations that have been previously ascribed to upd. However, this study shows that os alleles affect expression of both upd and upd3 and map to untranscribed regions, suggesting that they disrupt regulatory elements shared by both genes. Thus the upd region serves as a genetically tractable model for coordinate regulation of tandemly duplicated gene families that are commonly found in higher eukaryotes.
Wells, A. R., Zou, R. S., Tulu, U. S., Sokolow, A. C., Crawford, J. M., Edwards, G. S. and Kiehart, D. P. (2014). Complete canthi removal reveals that forces from the amnioserosa are alone sufficient to drive dorsal closure in Drosophila. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25253724
Drosophila's dorsal closure provides an excellent model system to analyze biomechanical processes during morphogenesis. During native closure, the amnioserosa, flanked by two lateral epidermal sheets, forms an eye-shaped opening with canthi at each corner. The dynamics of amnioserosa cells and actomyosin purse strings in the leading edges of epidermal cells promote closure while the bulk of the lateral epidermis opposes closure. Canthi maintain purse string curvature (necessary for their dorsal-ward forces) and zipping at the canthi shortens leading edges, ensuring a continuous epithelium at closure completion. The requirement for intact canthi during closure was investigated with laser dissection approaches. Dissection of one or both canthi resulted in tissue recoil and flattening of each purse string. Following recoil and a temporary pause, closure resumed at approximately native rates until slowing near the completion of closure. Thus, the amnioserosa alone can drive closure following dissection of one or both canthi, requiring neither substantial purse string curvature nor zipping during the bulk of closure. How the embryo coordinates multiple, large forces (each of which is orders of magnitude greater than the net force) during native closure, and is also resilient to multiple perturbations are key, extant questions.
Haack, T., Schneider, M., Schwendele, B. and Renault, A. (2014). Drosophila heart cell movement to the midline occurs through both cell autonomous migration and dorsal closure. Dev Biol [Epub ahead of print]. PubMed ID: 25224224
The Drosophila heart is a linear organ formed by the movement of bilaterally specified progenitor cells to the midline and adherence of contralateral heart cells. This movement occurs through the attachment of heart cells to the overlying ectoderm which is undergoing dorsal closure. Therefore heart cells are thought to move to the midline passively. Through live imaging experiments and analysis of mutants that affect the speed of dorsal closure, heart cells in Drosophila were found to be autonomously migratory and part of their movement to the midline is independent of the ectoderm. This means that heart formation in flies is more similar to that in vertebrates than previously thought. It was also shown that defects in dorsal closure can result in failure of the amnioserosa to properly degenerate, which can physically hinder joining of contralateral heart cells leading to a broken heart phenotype.
Thursday, October 8th
Gunesdogan, U., Jackle, H. and Herzig, A. (2014). Histone supply regulates S phase timing and cell cycle progression. Elife 3: e02443. PubMed ID: 25205668
Eukaryotes package DNA into nucleosomes that contain a core of histone proteins. During DNA replication, nucleosomes are disrupted and re-assembled with newly synthesized histones and DNA. Despite much progress, it is still unclear why higher eukaryotes contain multiple core histone genes, how chromatin assembly is controlled, and how these processes are coordinated with cell cycle progression. This study used a histone null mutation of Drosophila melanogaster, called Df(2L)HisC, which lacks all genes encoding the canonical histones, to show that histone supply levels, provided by a defined number of transgenic histone genes, regulate the length of S phase during the cell cycle. Lack of de novo histone supply not only extends S phase, but also causes a cell cycle arrest during G2 phase, and thus prevents cells from entering mitosis. The results suggest a novel cell cycle surveillance mechanism that monitors nucleosome assembly without involving the DNA repair pathways and exerts its effect via suppression of CDC25 phosphatase String expression.
Delabaere, L., Orsi, G. A., Sapey-Triomphe, L., Horard, B., Couble, P. and Loppin, B. (2014). The Spartan ortholog Maternal haploid is required for paternal chromosome integrity in the Drosophila zygote. Curr Biol 24(19): 2281-2287. PubMed ID: 25242033
The animal sperm nucleus is characterized by an extremely compacted organization of its DNA after the global replacement of histones with sperm-specific nuclear basic proteins, such as protamines. In the absence of DNA repair activity in the mature gamete, the integrity of the paternal genome is potentially challenged by the unique topological constraints exerted on sperm DNA. In addition, the maintenance of paternal DNA integrity during the rapid remodeling of sperm chromatin at fertilization has long been regarded as a maternal trait. However, little is known about the nature of the egg proteins involved in this essential aspect of zygote formation. Previous studies characterized the unique phenotype of the classical Drosophila maternal effect mutant maternal haploid (mh), which specifically affects the integration of paternal chromosomes in the zygote. This study has shown that MH is the fly ortholog of the recently identified human DVC1/Spartan protein, a conserved regulator of DNA damage tolerance. Like Spartan, MH protein is involved in the resistance to UV radiation and recruits the p97/TER94 segregase to stalled DNA replication forks in somatic cells. In the zygote, it was found that the mh phenotype is consistent with perturbed or incomplete paternal DNA replication. Remarkably, however, the specific accumulation of MH in the male pronucleus before the first S phase suggests that this maternal protein is required to maintain paternal DNA integrity during nuclear decondensation or to set the paternal chromatin landscape in preparation of the first zygotic cycle.
Adhikari, D., Diril, M. K., Busayavalasa, K., Risal, S., Nakagawa, S., Lindkvist, R., Shen, Y., Coppola, V., Tessarollo, L., Kudo, N. R., Kaldis, P. and Liu, K. (2014). Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II. J Cell Biol 206: 843-853. PubMed ID: 25246615
In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A; see Drosophila Microtubule star) activity. PP2A suppression in turn leads to high levels of Cdk1 (see Drosophila cdc2) substrate phosphorylation. This study used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. These results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.
Kuo, H. K., McMahan, S., Rota, C. M., Kohl, K. P. and Sekelsky, J. (2014). Drosophila FANCM helicase prevents spontaneous mitotic crossovers generated by the MUS81 and SLX1 nucleases. Genetics [Epub ahead of print]. PubMed ID: 25205745
Several helicases function during repair of double-strand breaks (DSBs) and handling of blocked or stalled replication forks to promote pathways that prevent formation of crossovers. Among these are the Bloom syndrome helicase BLM and the Fanconi anemia group M (FANCM) helicase. To better understand functions of these helicases, this study compared phenotypes of Drosophila melanogaster Blm and Fancm mutants. As previously reported for BLM, FANCM has roles in responding to several types of DNA damage, in preventing mitotic and meiotic crossovers, and in promoting the synthesis-dependent strand annealing pathway for repair of a double-strand gap. In most assays, the phenotype of Fancm mutants is less severe than that of Blm mutants, and the phenotype of Blm Fancm double mutants is more severe than either single mutant, indicating both overlapping and unique functions. It is thought that mitotic crossovers arise when structure-selective nucleases cleave DNA intermediates that would normally be unwound or disassembled by these helicases. When BLM is absent, three nucleases believed to function as Holliday junction resolvases - MUS81-MMS4, MUS312-SLX1, and GEN - become essential. In contrast, no single resolvase is essential in mutants lacking FANCM, although simultaneous loss of GEN and either of the others is lethal in Fancm mutants. Since Fancm mutants can tolerate loss of a single resolvase, this study could show that spontaneous mitotic crossovers that occur when FANCM is missing are dependent on MUS312 and either MUS81 or SLX1.
Tuesday, October 7th
Ueno, T. and Kume, K. (2014). Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral assays. Front Behav Neurosci 8: 303. PubMed ID: 25232310
Dopamine mediates diverse functions such as motivation, reward, attention, learning/memory and sleep/arousal. Recent studies using model organisms including the fruit fly, have elucidated various physiological functions of dopamine, and identified specific neural circuits for these functions. Flies with mutations in the Drosophila dopamine transporter (dDAT) gene show enhanced dopamine signaling, and short sleep and memory impairment phenotypes. However, understanding the mechanism by which dopamine signaling causes these phenotypes requires an understanding of the dynamics of dopamine release. This study reports the effects of dDAT expression on behavioral traits. This study shows that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. dDAT expression in other cell types such as Kenyon cells and glial cells can also rescue the short sleep phenotype of dDAT mutants. dDAT mutants also show a down-regulation of the D1-like dopamine receptor dDA1, and this phenotype is rescued when dDAT is expressed in the same cell types in which it rescues sleep. On the other hand, dDAT overexpression in mushroom bodies, which are the target of memory forming dopamine neurons, abolishes olfactory aversive memory. These data demonstrate that expression of extrasynaptic dopamine transporters can rescue some aspects of dopamine signaling in dopamine transporter mutants. These results provide novel insights into regulatory systems that modulate dopamine signaling.
Dissel, S., Hansen, C. N., Ozkaya, O., Hemsley, M., Kyriacou, C. P. and Rosato, E. (2014). The logic of circadian organization in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 25220056
In the fruit fly Drosophila melanogaster, interlocked negative transcription/translation feedback loops provide the core of the circadian clock that generates rhythmic phenotypes. Although the current molecular model portrays the oscillator as cell autonomous, cross-talk among clock neurons is essential for robust cycling behavior. Nevertheless, the functional organization of the neuronal network remains obscure. This study shows that shortening or lengthening of the circadian period of locomotor activity can be obtained either by targeting different groups of clock cells with the same genetic manipulation or by challenging the same group of cells with activators and repressors of neuronal excitability. Based on these observations this study interprets circadian rhythmicity as an emerging property of the circadian network and an initial model is proposed for its architectural design.
>Edelsparre, A. H., Vesterberg, A., Lim, J. H., Anwari, M. and Fitzpatrick, M. J. (2014). Alleles underlying larval foraging behaviour influence adult dispersal in nature. Ecol Lett 17: 333-339. PubMed ID: 24386971
The dispersal and migration of organisms have resulted in the colonisation of nearly every possible habitat and ultimately the extraordinary diversity of life. Animal dispersal tendencies are commonly heterogeneous (e.g. long vs. short) and non-random suggesting that phenotypic and genotypic variability between individuals can contribute to population-level heterogeneity in dispersal. Using laboratory and field experiments, this study demonstrates that natural allelic variation in a gene underlying a foraging polymorphism in larval fruit flies (for), also influences their dispersal tendencies as adults. Rover flies (forR) ; higher foraging activity) have consistently greater dispersal tendencies and are more likely to disperse longer distances than sitter flies (fors) ; lower foraging activity). Increasing for expression in the brain and nervous system increases dispersal in sitter flies. This study supports the notion that variation in dispersal can be driven by intrinsic variation in food-dependent search behaviours and confirms that single gene pleiotropic effects can contribute to population-level heterogeneity in dispersal.
Kimura, K. I., Sato, C., Yamamoto, K. and Yamamoto, D. (2014). From the back or front: the courtship position is a matter of smell and sight in Drosophila melanogaster males. J Neurogenet [Epub ahead of print]. PubMed ID: 25257899
During courtship, Drosophila melanogaster males usually position themselves behind the target female. To decipher the sensory cues that guide the males to the courting position, male locomotion traces were quantitatively analyzed within a circular observation chamber, at the center of which an immobilized virgin female was placed as a courtship target. Wild-type males preferentially stayed behind the female under not only daylight but also dark conditions, indicating that vision is dispensable for orientation by males. By contrast, olfaction-defective Or83b2 homozygous males often positioned themselves in front of a female to court under dark but not daylight conditions. It is concluded that vision and olfaction redundantly guide the male fly to move behind the female to perform courtship actions. The visual and olfactory features that provide a male with cues for differentiating between the front and back end of a female are yet to be determined.
Grewal, J. S., et al. (2014). Complex and non-redundant signals from individual odor receptors that underlie chemotaxis behavior in Drosophila melanogaster larvae. Biol Open. PubMed ID: 25238759
The rules by which odor receptors encode odors and allow behavior are still largely unexplored. Although large data sets of electrophysiological responses of receptors to odors have been generated, few hypotheses have been tested with behavioral assays. This study used a data set on odor responses of Drosophila larval odor receptors coupled with chemotaxis behavioral assays to examine rules of odor coding. Using mutants of odor receptors, it was found that odor receptors with similar electrophysiological responses to odors across concentrations play non-redundant roles in odor coding at specific odor concentrations. It was also found that high affinity receptors for odors determine behavioral response thresholds, but the rules for determining peak behavioral responses are more complex. While receptor mutants typically show loss of attraction to odors, some receptor mutants result in increased attraction at specific odor concentrations. The odor receptor mutants were rescued using transgenic expression of odor receptors, validating assignment of phenotypes to the alleles. Vapor pressures alone cannot fully explain behavior in the assay. Finally, some odors that did not elicit strong electrophysiological responses are associated with behavioral phenotypes upon examination of odor receptor mutants. This result is consistent with the role of sensory neurons in lateral inhibition via local interneurons in the antennal lobe. Taken together, these results suggest a complexity of odor coding rules even in a simple olfactory sensory system.
Monday, October 6th
Colin, J., Garibal, J., Clavier, A., Rincheval-Arnold, A., Gaumer, S., Mignotte, B. and Guenal, I. (2014). The Drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the beta-TrCP homologue slimb. Apoptosis 19: 1444-1456. PubMed ID: 25208640
The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, this study identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. Uba1 was demonstrated to regulate apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, these apoptotic processes were shown to involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of beta-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.
Liang, C. J., Chang, Y. C., Chang, H. C., Wang, C. K., Hung, Y. C., Lin, Y. E., Chan, C. C., Chen, C. H., Chang, H. Y. and Sang, T. K. (2014). Derlin-1 regulates mutant VCP-linked pathogenesis and endoplasmic reticulum stress-induced apoptosis. PLoS Genet 10: e1004675. PubMed ID: 25255315
Mutations in VCP (Valosin-containing protein), an AAA ATPase critical for ER-associated degradation, are linked to IBMPFD (Inclusion body myopathy with Paget disease and frontotemporal dementia). Using a Drosophila IBMPFD model, this study identified the ER protein Derlin-1 as a modifier of pathogenic TER94 (the fly VCP homolog) mutants. Derlin-1 binds to TER94 directly, and this interaction is essential for Derlin-1 overexpression to suppress the pathogenic TER94-induced neurodegeneration. Derlin-1 overexpression reduces the elevated ATPase activity of pathogenic TER94, implying that IBMPFD is caused by ATPase hyper-activation. Under physiological condition, Derlin-1 expression is increased upon ER stress to recruit TER94 to the ER. However, in response to severe ER stress, Derlin-1 is required for activating apoptosis to eliminate damaged cells. This pro-apoptotic response is mimicked by Derlin-1 overexpression, which elicits acute ER stress and triggers apoptosis via a novel C-terminal motif (alpha). As this Derlin-1-dependent cell death is negated by TER94 overexpression, it is proposed that while Derlin-1 and VCP work cooperatively in ER stress response, their imbalance has a role in removing cells suffering prolonged ER stress.
Luan, Z., Reddig, K. and Li, H. S. (2014). Loss of Na/K-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration. Exp Neurol 261C: 791-801. PubMed ID: 25205229
The activity of Na+/K+-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na+/K+-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na+/K+-ATPase deficiency to in vivo neurodegeneration has been lacking. This study used Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na+/K+ ATPase. Loss of ATPα, an α subunit of Na+/K+-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K+. Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na+/K+-ATPase β subunit, partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na+/K+-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na+/K+ ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na+/K+ ATPase deficiency-mediated neurodegeneration.
Ulgherait, M., Rana, A., Rera, M., Graniel, J. and Walker, D. W. (2014). AMPK modulates tissue and organismal aging in a non-cell-autonomous manner. Cell Rep [Epub ahead of print]. PubMed ID: 25199830
AMPK exerts prolongevity effects in diverse species; however, the tissue-specific mechanisms involved are poorly understood. This study shows that upregulation of AMPK in the adult Drosophila nervous system induces autophagy both in the brain and also in the intestinal epithelium. Induction of autophagy is linked to improved intestinal homeostasis during aging and extended lifespan. Neuronal upregulation of the autophagy-specific protein kinase Atg1 is both necessary and sufficient to induce these intertissue effects during aging and to prolong the lifespan. Furthermore, upregulation of AMPK in the adult intestine induces autophagy both cell autonomously and non-cell-autonomously in the brain, slows systemic aging, and prolongs the lifespan. The organism-wide response to tissue-specific AMPK/Atg1 activation is linked to reduced insulin-like peptide levels in the brain and a systemic increase in 4E-BP expression. Together, these results reveal that localized activation of AMPK and/or Atg1 in key tissues can slow aging in a non-cell-autonomous manner.
Sunday, October 5th
Xu, N., Emelyanov, A. V., Fyodorov, D. V. and Skoultchi, A. I. (2014). Drosophila linker histone H1 coordinates STAT-dependent organization of heterochromatin and suppresses tumorigenesis caused by hyperactive JAK-STAT signaling. Epigenetics Chromatin 7: 16. PubMed ID: 25177369
Within the nucleus of eukaryotic cells, chromatin is organized into compact, silent regions called heterochromatin and more loosely packaged regions of euchromatin where transcription is more active. Although the existence of heterochromatin has been known for many years, the cellular factors responsible for its formation have only recently been identified. Two key factors involved in heterochromatin formation in Drosophila are the H3 lysine 9 methyltransferase Su(var)3-9 and heterochromatin protein 1 (HP1). The linker histone H1 also plays a major role in heterochromatin formation in Drosophila by interacting with Su(var)3-9 and helping to recruit it to heterochromatin. Drosophila STAT (Signal transducer and activator of transcription) (STAT92E) has also been shown to be involved in the maintenance of heterochromatin, but its relationship to the H1-Su(var)3-9 heterochromatin pathway is unknown. STAT92E is also involved in tumor formation in flies. Hyperactive Janus kinase (JAK)-STAT signaling due to a mutation in Drosophila JAK (Hopscotch) causes hematopoietic tumors. This study shows that STAT92E is a second partner of H1 in the regulation of heterochromatin structure. H1 physically interacts with STAT92E and regulates its ectopic localization in the chromatin. Mis-localization of STAT92E due to its hyperphosphorylation or H1 depletion disrupts heterochromatin integrity. The contribution of the H1-STAT pathway to heterochromatin formation is mechanistically distinct from that of H1 and Su(var)3-9. The recruitment of STAT92E to chromatin by H1 also plays an important regulatory role in JAK-STAT induced tumors in flies. Depleting the linker histone H1 in flies carrying the oncogenic hopscotch (Tum-l) allele enhances tumorigenesis, and H1 overexpression suppresses tumorigenesis. These results suggest the existence of two independent pathways for heterochromatin formation in Drosophila, one involving Su(var)3-9 and HP1 and the other involving STAT92E and HP1. The H1 linker histone directs both pathways through physical interactions with Su(var)3-9 and STAT92E, as well with HP1. The physical interaction of H1 and STAT92E confers a regulatory role on H1 in JAK-STAT signaling. H1 serves as a molecular reservoir for STAT92E in chromatin, enabling H1 to act as a tumor suppressor and oppose an oncogenic mutation in the JAK-STAT signaling pathway.
Blanchard, D. P., Georlette, D., Antoszewski, L. and Botchan, M. R. (2014). Chromatin reader L(3)mbt requires the Myb-MuvB/DREAM transcriptional regulatory complex for chromosomal recruitment. Proc Natl Acad Sci U S A. PubMed ID: 25249635
Lethal malignant brain tumors (lmbt) result from the loss of the conserved transcriptional repressor l(3)mbt, in Drosophila melanogaster. Similar mutations in the human homolog L3MBTL1 correlate with some cancers. The protein's C-terminal MBT repeats bind mono and dimethylated histones in vitro, which could influence recruitment of L3MBTL1 to its target sites. The L(3)mbt chromatin targeting mechanism, however, is controversial and several studies suggest insufficiency or a minor role for histone methylation in determining the site specificity for recruitment. This study reports that L(3)mbt colocalizes with core members of the Myb-MuvB/DREAM (MMB/DREAM; see Myb oncogene-like) transcriptional regulatory complex genome-wide, and that L(3)mbt-mediated repression requires this complex in salivary glands and larval brains. Loss of l(3)mbt or of MMB components through mutation cause similar spurious expression of genes, including the transposon regulatory gene piwi, in terminally differentiated cells. The DNA-binding MMB core component Mip120 (Lin54) is required for L(3)mbt recruitment to chromosomes, whereas Mip130 (Lin9) (an MMB core protein) and E2f2 (an MMB transcriptional repressor) are not, but are essential for repression. Cytolocalization experiments suggest the presence of site-specific differential composition of MMB in polytene chromosomes where some loci were bound by a Myb-containing or alternatively, an E2f2 and L(3)mbt form of the complex.
Liu, K., Ding, L., Li, Y., Yang, H., Zhao, C., Lei, Y., Han, S., Tao, W., Miao, D., Steller, H., Welsh, M. J. and Liu, L. (2014). Neuronal necrosis is regulated by a conserved chromatin-modifying cascade. Proc Natl Acad Sci U S A. PubMed ID: 25201987
Neuronal necrosis induced by calcium overload causes devastating brain dysfunction in diseases such as stroke and brain trauma. It has been considered a stochastic event lacking genetic regulation, and pharmacological means to suppress neuronal necrosis are lacking. Using a Drosophila model of calcium overloading, this study found JIL-1/mitogen- and stress-activated protein kinase 1/2 is a regulator of neuronal necrosis through phosphorylation of histone H3 serine 28 (H3S28ph). Further, its downstream events were identified, including displacement of polycomb repressive complex 1 (PRC1) and activation of Trithorax (Trx). To test the role of JIL-1/PRC1/Trx cascade in mammals, the necrosis induced by glutamate was studied in rat cortical neuron cultures and rodent models of brain ischemia; the cascade was found to be activated in these conditions and inhibition of the cascade suppresses necrosis in vitro and in vivo. Together, this research demonstrates that neuronal necrosis is regulated by a chromatin-modifying cascade, and this discovery may provide potential therapeutic targets and biomarkers for neuronal necrosis.
Emelyanov, A. V., Rabbani, J., Mehta, M., Vershilova, E., Keogh, M. C., Fyodorov, D. V. (2014). Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization. Genes Dev 28: 2027-2040. PubMed ID: 25228646
Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. This study identified four Drosophila protamine chaperones that mediate the dissociation of protamine-DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. This study showed TAP/p32 to be required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, this study identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells.
Theofel, I., Bartkuhn, M., Hundertmark, T., Boettger, T., Gartner, S. M., Leser, K., Awe, S., Schipper, M., Renkawitz-Pohl, R. and Rathke, C. (2014). tBRD-1 selectively controls gene activity in the Drosophila testis and interacts with two new members of the Bromodomain and Extra-Terminal (BET) Family. PLoS One 9: e108267. PubMed ID: 25251222
Multicellular organisms have evolved specialized mechanisms to control transcription in a spatial and temporal manner. Gene activation is tightly linked to histone acetylation on lysine residues that can be recognized by bromodomains. Previously, the testis-specifically expressed bromodomain protein tBRD-1 was identified in Drosophila. Expression of tBRD-1 is restricted to highly transcriptionally active primary spermatocytes. tBRD-1 is essential for male fertility and proposed to act as a co-factor of testis-specific TATA box binding protein-associated factors (tTAFs) for testis-specific transcription. This study performed microarray analyses to compare the transcriptomes of tbrd-1 mutant testes and wild-type testes. The data confirmed that tBRD-1 controls gene activity in male germ cells. Additionally, comparing the transcriptomes of tbrd-1 and tTAF mutant testes revealed a subset of common target genes. Two new members of the bromodomain and extra-terminal (BET) family, tBRD-2 and tBRD-3, were also characterized. In contrast to other members of the BET family in animals, both possess only a single bromodomain, a characteristic feature of plant BET family members. Immunohistology techniques not only revealed that tBRD-2 and tBRD-3 partially co-localize with tBRD-1 and tTAFs in primary spermatocytes, but also that their proper subcellular distribution was impaired in tbrd-1 and tTAF mutant testes. Treating cultured male germ cells with inhibitors showed that localization of tBRD-2 and tBRD-3 depends on the acetylation status within primary spermatocytes. Yeast two-hybrid assays and co-immunoprecipitations using fly testes protein extracts demonstrated that tBRD-1 is able to form homodimers as well as heterodimers with tBRD-2, tBRD-3, and tTAFs. These data reveal for the first time the existence of single bromodomain BET proteins in animals, as well as evidence for a complex containing tBRDs and tTAFs that regulates transcription of a subset of genes with relevance for spermiogenesis.
Saturday, October 4th
Alic, N., Giannakou, M. E., Papatheodorou, I., Hoddinott, M. P., Andrews, T. D., Bolukbasi, E. and Partridge, L. (2014). Interplay of dFOXO and two ETS-family transcription factors determines lifespan in Drosophila melanogaster. PLoS Genet 10: e1004619. PubMed ID: 25232726
Forkhead box O (FoxO) transcription factors (TFs) are key drivers of complex transcriptional programmes that determine animal lifespan. FoxOs regulate a number of other TFs, but how these TFs in turn might mediate the anti-ageing programmes orchestrated by FoxOs in vivo is unclear. This study identified an E-twenty six (ETS)-family transcriptional repressor, Anterior open (Aop), as regulated by the single Drosophila melanogaster FoxO (dFoxO) in the adult gut. Aop, the functional orthologue of the human Etv6/Tel protein, binds numerous genomic sites also occupied by dFOXO and counteracts the activity of an ETS activator, Pointed (Pnt), to prevent the lifespan-shortening effects of co-activation of dFoxO and Pnt. This detrimental synergistic effect of dFoxO and Pnt appears to stem from a mis-regulation of lipid metabolism. At the same time, Aop activity in another fly organ, the fat body, has further beneficial roles, regulating genes in common with dfoxo, such as the secreted, non-sensory, odorant binding protein (Obp99b), and robustly extending lifespan. This study reveals a complex interplay between evolutionarily conserved ETS factors and dFOXO, the functional significance of which may extend well beyond animal lifespan.
Cabrero, P., Terhzaz, S., Romero, M. F., Davies, S. A., Blumenthal, E. M., Dow, J. A. (2014). Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Proc Natl Acad Sci U S A. PubMed ID: 25228763
Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. This study resolved this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin (see Leucokinin) stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a-mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a-independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia.
Li, Z., Ni, J. D., Huang, J. and Montell, C. (2014). Requirement for Drosophila SNMP1 for rapid activation and termination of pheromone-induced activity. PLoS Genet 10: e1004600. PubMed ID: 25255106
Pheromones are used for conspecific communication by many animals. In Drosophila, the volatile male-specific pheromone 11-cis vaccenyl acetate (cVA) supplies an important signal for gender recognition. Sensing of cVA by the olfactory system depends on multiple components, including an olfactory receptor (Or67d), the co-receptor Orco, and an odorant binding protein (Lush). In addition, a CD36 related protein, Sensory neuron membrane protein 1 (Snmp1) is also involved in cVA detection. Loss of Snmp1 has been reported to eliminate cVA responsiveness, and to greatly increase spontaneous activity of Or67d-expressing olfactory receptor neurons (ORNs). This study found the snmp11 mutation did not abolish cVA responsiveness or cause high spontaneous activity. The cVA responses in snmp1 mutants displayed a delayed onset, and took longer to reach peak activity than wild-type. Most strikingly, loss of Snmp1 caused a dramatic delay in signal termination. The profound impairment in signal inactivation accounted for the previously reported 'spontaneous activity,' which represented continuous activation following transient exposure to environmental cVA. This study introduced the silk moth receptor (BmOR1) in OR67d ORNs of snmp11 flies and found that the ORNs showed slow activation and deactivation kinetics in response to the BmOR1 ligand (bombykol). The bombykol receptor complex was expressed in Xenopus oocytes in the presence or absence of the silk moth SNMP1 (BmSNMP), and it was found that addition of BmSNMP accelerated receptor activation and deactivation. These results thus clarify SNMP1 as an important player required for the rapid kinetics of the pheromone response in insects.
Gao, H., Wu, X., Simon, L. and Fossett, N. (2014). Antioxidants maintain E-cadherin levels to limit Drosophila prohemocyte differentiation. PLoS One 9: e107768. PubMed ID: 25226030
Mitochondrial reactive oxygen species (ROS) regulate a variety of biological processes by networking with signal transduction pathways to maintain homeostasis and support adaptation to stress. In this capacity, ROS have been shown to promote the differentiation of progenitor cells, including mammalian embryonic and hematopoietic stem cells and Drosophila hematopoietic progenitors (prohemocytes). However, many questions remain about how ROS alter the regulatory machinery to promote progenitor differentiation. This study provides evidence for the hypothesis that ROS reduce E-cadherin levels to promote Drosophila prohemocyte differentiation. Specifically, it was shown that knockdown of the antioxidants, Superoxide dismutatase 2 and Catalase reduce E-cadherin protein levels prior to the loss of Odd-skipped-expressing prohemocytes. Additionally, over-expression of E-cadherin limits prohemocyte differentiation resulting from paraquat-induced oxidative stress. Furthermore, two established targets of ROS, Enhancer of Polycomb and Fos, control the level of E-cadherin protein expression. Finally, knockdown of either Superoxide dismutatase 2 or Catalase was shown to lead to an increase in the E-cadherin repressor, Serpent. As a result, antioxidants and targets of ROS can control E-cadherin protein levels, and over-expression of E-cadherin can ameliorate the prohemocyte response to oxidative stress. Collectively, these data strongly suggest that ROS promote differentiation by reducing E-cadherin levels. In mammalian systems, ROS promote embryonic stem cell differentiation, whereas E-cadherin blocks differentiation. However, it is not known if elevated ROS reduce E-cadherin to promote embryonic stem cell differentiation. Thus, these findings may have identified an important mechanism by which ROS promote stem/progenitor cell differentiation.
Wang, J., Binks, T., Warr, C. G. and Burke, R. (2014). Vacuolar-type H-ATPase subunits and the neurogenic protein Big brain are required for optimal copper and zinc uptake. Metallomics [Epub ahead of print]. PubMed ID: 25209718
Copper and zinc homeostasis in polarized epithelial cells requires the correct localization and regulation of membrane-bound transport proteins at the apical and basolateral cell membranes. This study has identified a subunit of the vacuolar-type H+-ATPase (V-ATPase) complex, vhaPPA1-2, and the Drosophila aquaporin homolog Big brain (Bib), as being required for the correct localization of the copper uptake transporters Ctr1A and Ctr1B and the zinc uptake protein dZip89B and hence necessary for optimal copper and zinc accumulation in vivo. Knockdown of vhaPPA1-2 or bib resulted in cuticle hypo-pigmentation phenotypes typical of copper deficiency in the fly and induction of midgut Ctr1B expression, a known response to low cellular copper levels. Furthermore, midgut-specific knockdown of bib increased tolerance to elevated dietary zinc levels. Ctr1A, Ctr1B and dZip89B are normally localized to the apical plasma membrane. Upon knockdown of vhaPPA1-2 or bib, this localization was strongly disrupted as was that of the generic plasma membrane marker CD8-GFP, indicating that these two genes are not acting specifically on metal ion homeostasis but rather are necessary for general apical membrane protein localization in polarized epithelial cells. These results suggest that metal ion transport is particularly sensitive to disturbances in cellular protein localization processes.
Friday, October 3rd
Parsons, L. M., Portela, M., Grzeschik, N. A., Richardson, H. E. (2014) . Lgl regulates Notch signaling via endocytosis, independently of the apical aPKC-Par6-Baz polarity complex. Curr Biol 24(18): 2073-84. PubMed ID: 25220057
The Drosophila melanogaster junctional neoplastic tumor suppressor, Lethal-2-giant larvae (Lgl), is a regulator of apicobasal cell polarity and tissue growth. Previous studies have shown in the developing Drosophila eye epithelium that, without affecting cell polarity, depletion of Lgl results in ectopic cell proliferation and blockage of developmental cell death due to deregulation of the Hippo signaling pathway. This study shows that Notch signaling is increased in lgl-depleted eye tissue, independently of Lgl’s function in apicobasal cell polarity. The upregulation of Notch signaling is ligand dependent and correlates with accumulation of cleaved Notch. Concomitant with higher cleaved Notch levels in lgl− tissue, early endosomes, recycling endosomes, early multivesicular bodies, and acidified vesicles, but not late endosomal markers, accumulate. Colocalization studies revealed that Lgl associates with early to late endosomes and lysosomes. Upregulation of Notch signaling in lgl− tissue requires dynamin- and Rab5-mediated endocytosis and vesicle acidification but is independent of Hrs/Stam or Rab11 activity. Furthermore, Lgl regulates Notch signaling independently of the aPKC-Par6-Baz apical polarity complex. Altogether, these data show that Lgl regulates endocytosis to restrict vesicle acidification and prevent ectopic ligand-dependent Notch signaling. This Lgl function is independent of the aPKC-Par6-Baz polarity complex and uncovers a novel attenuation mechanism of ligand-activated Notch signaling during Drosophila eye development.
de Vreede, G., Schoenfeld, J. D., Windler, S. L., Morrison, H., Lu, H. and Bilder, D. (2014). The Scribble module regulates retromer-dependent endocytic trafficking during epithelial polarization. Development 141: 2796-2802. PubMed ID: 25005475
Scribble (Scrib) module proteins are major regulators of cell polarity, but how they influence membrane traffic is not known. Endocytosis is also a key regulator of polarity through roles that remain unclear. This study links Scrib to a specific arm of the endocytic trafficking system. Drosophila mutants that block AP-2-dependent endocytosis share many phenotypes with Scrib module mutants, but Scrib module mutants show intact internalization and endolysosomal transport. However, defective traffic of retromer pathway cargo is seen, and retromer components show strong genetic interactions with the Scrib module. The Scrib module is required for proper retromer localization to endosomes and promotes appropriate cargo sorting into the retromer pathway via both aPKC-dependent and -independent mechanisms. It is proposed that the Scrib module regulates epithelial polarity by influencing endocytic itineraries of Crumbs and other retromer-dependent cargo.
Pradhan-Sundd, T. and Verheyen, E. M. (2014). The role of Bro1- domain-containing protein Myopic in endosomal trafficking of Wnt/Wingless. Dev Biol 392: 93-107. PubMed ID: 24821423
Wingless (Wg) proteins are secreted-lipid-modified glycoproteins involved in tissue patterning and cell-fate specification. Wg secretion is regulated by a specialized mechanism involving a repertoire of proteins including Wntless (Wls). This study shows that the Bro1-domain-containing protein Myopic (Mop) is indispensable for endosomal trafficking of Wg and Wls. Reductions in Mop leads to trapping of Wg and Wls in the early endosomes. Overexpression of the endosomal sorting protein Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) rescues the trafficking defect caused by the mop loss of function. The vertebrate homolog of Mop, Histidine domain-containing protein tyrosine phosphatase (HDPTP), was also found to have a conserved role in Wnt trafficking. This study highlights the importance of early endosomal trafficking for Wg secretion, and identifies a novel role for Mop in Wg signaling.
Lorincz, P., Lakatos, Z., Maruzs, T., Szatmari, Z., Kis, V. and Sass, M. (2014). Atg6/UVRAG/Vps34-containing lipid kinase complex is required for receptor downregulation through endolysosomal degradation and epithelial polarity during Drosophila wing development. Biomed Res Int 2014: 851349. PubMed ID: 25006588
Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UV-resistance associated gene (UVRAG). The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells was performed in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. Atg6 was shown to be required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, these results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation.
Thursday, October 2nd
Panda, D., Pascual-Garcia, P., Dunagin, M., Tudor, M., Hopkins, K. C., Xu, J., Gold, B., Raj, A., Capelson, M., Cherry, S. (2014). Nup98 promotes antiviral gene expression to restrict RNA viral infection in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 25197089
In response to infection, the innate immune system rapidly activates an elaborate and tightly orchestrated gene expression program to induce critical antimicrobial genes. While many key players in this program have been identified in disparate biological systems, it is clear that there are additional uncharacterized mechanisms at play. Previous studies revealed that a rapidly-induced antiviral gene expression program is active against disparate human arthropod-borne viruses in Drosophila. Moreover, one-half of this program is regulated at the level of transcriptional pausing. This study found that Nup98, a virus-induced gene, was antiviral against a panel of viruses both in cells and adult flies since its depletion significantly enhanced viral infection. Mechanistically, it was found that Nup98 promotes antiviral gene expression in Drosophila at the level of transcription. Expression profiling revealed that the virus-induced activation of 36 genes was abrogated upon loss of Nup98; and this study found that a subset of these Nup98-dependent genes were antiviral. These Nup98-dependent virus-induced genes are Cdk9-dependent and translation-independent suggesting that these are rapidly induced primary response genes. Biochemically, it was demonstrated that Nup98 is directly bound to the promoters of virus-induced genes, and that it promotes occupancy of the initiating form of RNA polymerase II at these promoters, which are rapidly induced on viral infection to restrict human arboviruses in insects.
Huang, Y., McNeil, G. P. and Jackson, F. R. (2014). Translational regulation of the DOUBLETIME/CKIdelta/epsilon kinase by LARK contributes to circadian period modulation. PLoS Genet 10: e1004536. PubMed ID: 25211129
The Drosophila homolog of Casein Kinase I delta/epsilon, Doubletime (Dbt), is required for Wnt, Hedgehog, Fat and Hippo signaling as well as circadian clock function. Extensive studies have established a critical role of Dbt in circadian period determination. However, how Dbt expression is regulated remains largely unexplored. This study shows that translation of dbt transcripts are directly regulated by a rhythmic RNA-binding protein (RBP) called LARK (known as RBM4 in mammals). LARK promotes translation of specific alternative dbt transcripts in clock cells, in particular the dbt-RC transcript. Translation of dbt-RC exhibits circadian changes under free-running conditions, indicative of clock regulation. Translation of a newly identified transcript, dbt-RE, is induced by light in a LARK-dependent manner and oscillates under light/dark conditions. Altered LARK abundance affects circadian period length, and this phenotype can be modified by different dbt alleles. Increased LARK delays nuclear degradation of the Period (Per) clock protein at the beginning of subjective day, consistent with the known role of Dbt in Per dynamics. Taken together, these data support the idea that LARK influences circadian period and perhaps responses of the clock to light via the regulated translation of Dbt. This study is the first to investigate translational control of the Dbt kinase, revealing its regulation by LARK and a novel role of this RBP in Drosophila circadian period modulation.
Le Thomas, A., Marinov, G. K. and Aravin, A. A. (2014). A Transgenerational Process Defines piRNA Biogenesis in Drosophila virilis. Cell Rep 8(6):1617-23. PubMed ID: 25199836
Piwi-interacting (pi)RNAs repress diverse transposable elements in germ cells of Metazoa and are essential for fertility in both invertebrates and vertebrates. The precursors of piRNAs are transcribed from distinct genomic regions, the so-called piRNA clusters; however, how piRNA clusters are differentiated from the rest of the genome is not known. To address this question, piRNA biogenesis was studied in two D. virilis strains that show differential ability to generate piRNAs from several genomic regions. That active piRNA biogenesis was found to correlate with high levels of histone 3 lysine 9 trimethylation (H3K9me3) over genomic regions that give rise to piRNAs. Furthermore, piRNA biogenesis in the progeny requires the transgenerational inheritance of an epigenetic signal, presumably in the form of homologous piRNAs that are generated in the maternal germline and deposited into the oocyte. The inherited piRNAs enhance piRNA biogenesis through the installment of H3K9me3 on piRNA clusters.
Yatsenko, A. S., Marrone, A. K. and Shcherbata, H. R. (2014). miRNA-based buffering of the cobblestone-lissencephaly-associated extracellular matrix receptor dystroglycan via its alternative 3'-UTR. Nat Commun 5: 4906. PubMed ID: 25232965
Many proteins are expressed dynamically during different stages of cellular life and the accuracy of protein amounts is critical for cell endurance. Therefore, cells should have a perceptive system that notifies about fluctuations in the amounts of certain components and an executive system that efficiently restores their precise levels. At least one mechanism that evolution has employed for this task is regulation of 3'-UTR length for microRNA targeting. This study shows that in Drosophila the microRNA complex miR-310s (see miR-310) acts as an executive mechanism to buffer levels of the muscular dystrophy-associated extracellular matrix receptor dystroglycan via its alternative 3'-UTR. miR-310s gene expression fluctuates depending on dystroglycan amounts and nitric oxide signalling, which perceives dystroglycan levels and regulates microRNA gene expression. Aberrant levels of dystroglycan or deficiencies in miR-310s and nitric oxide signalling result in cobblestone brain appearance, resembling human lissencephaly type II phenotype.
Wednesday, October 1st
Weng, K. A., Jeffreys, C. A. and Bickel, S. E. (2014). Rejuvenation of meiotic cohesion in oocytes during prophase I Is required for chiasma maintenance and accurate chromosome segregation. PLoS Genet 10: e1004607. PubMed ID: 25211017
Chromosome segregation errors in human oocytes are the leading cause of birth defects, and the risk of aneuploid pregnancy increases dramatically as women age. Accurate segregation demands that sister chromatid cohesion remain intact for decades in human oocytes, and gradual loss of the original cohesive linkages established in fetal oocytes is proposed to be a major cause of age-dependent segregation errors. This study demonstrates that maintenance of meiotic cohesion in Drosophila oocytes during prophase I requires an active rejuvenation program, and provide mechanistic insight into the molecular events that underlie rejuvenation. Gal4/UAS inducible knockdown of the cohesion establishment factor Eco after meiotic S phase, but before oocyte maturation, causes premature loss of meiotic cohesion, resulting in destabilization of chiasmata and subsequent missegregation of recombinant homologs. Reduction of individual cohesin subunits or the cohesin loader Nipped B during prophase I leads to similar defects. These data indicate that loading of newly synthesized replacement cohesin rings by Nipped B and establishment of new cohesive linkages by the acetyltransferase Eco must occur during prophase I to maintain cohesion in oocytes. Moreover, it was shown that rejuvenation of meiotic cohesion does not depend on the programmed induction of meiotic double strand breaks that occurs during early prophase I, and is therefore mechanistically distinct from the DNA damage cohesion re-establishment pathway identified in G2 vegetative yeast cells. This work provides the first evidence that new cohesive linkages are established in Drosophila oocytes after meiotic S phase, and that these are required for accurate chromosome segregation. If such a pathway also operates in human oocytes, meiotic cohesion defects may become pronounced in a woman's thirties, not because the original cohesive linkages finally give out, but because the rejuvenation program can no longer supply new cohesive linkages at the same rate at which they are lost.
Li, W., Klovstad, M. and Schupbach, T. (2014). Repression of Gurken translation by a meiotic checkpoint in Drosophila oogenesis is suppressed by a reduction in the dose of eIF1A. Development [Epub ahead of print]. PubMed ID: 25231760
In Drosophila melanogaster, the anteroposterior (AP) and dorsoventral (DV) axes of the oocyte and future embryo are established through the localization and translational regulation of gurken (grk) mRNA. This process involves binding of specific factors to the RNA during transport and a dynamic remodeling of the grk-containing ribonucleoprotein (RNP) complexes once they have reached their destination within the oocyte. In ovaries of spindle-class females, an activated DNA damage checkpoint causes inefficient Grk translation and ventralization of the oocyte. In a screen for modifiers of the oocyte DV patterning defects, a mutation was identified in the eIF1A gene as a dominant suppressor. Reducing the function of eIF1A in spnB ovaries suppresses the ventralized eggshell phenotype by restoring Grk expression. This suppression is not the result of more efficient DNA damage repair or of disrupted checkpoint activation, but is coupled to an increase in the amount of grk mRNA associated with polysomes. In spnB ovaries, the activated meiotic checkpoint blocks Grk translation by disrupting the accumulation of grk mRNA in a translationally competent RNP complex that contains the translational activator Oo18 RNA-binding protein (Orb); this regulation involves the translational repressor Squid (Sqd). It is further proposed that reduction of eIF1A allows more efficient Grk translation possibly because of the presence of specific structural features in the grk 5'UTR.
Strochlic, T. I., Stavrides, K. P., Thomas, S. V., Nicolas, E., O'Reilly, A. M. and Peterson, J. R. (2014). Ack kinase regulates CTP synthase filaments during Drosophila oogenesis. EMBO Rep [Epub ahead of print]. PubMed ID: 25223282
The enzyme CTP synthase (CTPS) dynamically assembles into macromolecular filaments in bacteria, yeast, Drosophila, and mammalian cells, but the role of this morphological reorganization in regulating CTPS activity is controversial. During Drosophila oogenesis, CTPS filaments are transiently apparent in ovarian germline cells during a period of intense genomic endoreplication and stockpiling of ribosomal RNA. This study demonstrates that CTPS filaments are catalytically active and that their assembly is regulated by the non-receptor tyrosine kinase DAck, the Drosophila homologue of mammalian Ack1 (activated cdc42-associated kinase 1), which was found to also localize to CTPS filaments. Egg chambers from flies deficient in DAck or lacking DAck catalytic activity exhibit disrupted CTPS filament architecture and morphological defects that correlate with reduced fertility. Furthermore, ovaries from these flies exhibit reduced levels of total RNA, suggesting that DAck may regulate CTP synthase activity. These findings highlight an unexpected function for DAck and provide insight into a novel pathway for the developmental control of an essential metabolic pathway governing nucleotide biosynthesis.
Touret, F., Guiguen, F. and Terzian, C. (2014). Wolbachia influences the maternal transmission of the gypsy endogenous retrovirus in Drosophila melanogaster. MBio 5(5). PubMed ID: 25182324
The endosymbiotic bacteria of the genus Wolbachia are present in most insects and are maternally transmitted through the germline. Moreover, these intracellular bacteria exert antiviral activity against insect RNA viruses, as in Drosophila melanogaster, which could explain the prevalence of Wolbachia bacteria in natural populations. Wolbachia is maternally transmitted in D. melanogaster through a mechanism that involves distribution at the posterior pole of mature oocytes and then incorporation into the pole cells of the embryos. In parallel, maternal transmission of several endogenous retroviruses is well documented in D. melanogaster. Notably, gypsy retrovirus is expressed in permissive follicle cells and transferred to the oocyte and then to the offspring by integrating into their genomes. This study shows that the presence of Wolbachia wMel reduces the rate of gypsy insertion into the ovo gene. However, the presence of Wolbachia does not modify the expression levels of gypsy RNA and envelope glycoprotein from either permissive or restrictive ovaries. Moreover, Wolbachia affects the pattern of distribution of the retroviral particles and the gypsy envelope protein in permissive follicle cells. Altogether, these results enlarge the knowledge of the antiviral activity of Wolbachia to include reducing the maternal transmission of endogenous retroviruses in D. melanogaster. Animals have established complex relationships with bacteria and viruses that spread horizontally among individuals or are vertically transmitted, i.e., from parents to offspring. It is well established that members of the genus Wolbachia, maternally inherited symbiotic bacteria present mainly in arthropods, reduce the replication of several RNA viruses transmitted horizontally. This study demonstrates that Wolbachia diminishes the maternal transmission of gypsy, an endogenous retrovirus in Drosophila. It is hypothesized that gypsy cannot efficiently integrate into the germ cells of offspring during embryonic development in the presence of Wolbachia because both are competitors for localization to the posterior pole of the egg. More generally, it would be of interest to analyze the influence of Wolbachia on vertically transmitted exogenous viruses, such as some arboviruses.
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