What's hot today:
Current papers in developmental biology and gene function


Monday, January 16th

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Vaccaro, A., Issa, A. R., Seugnet, L., Birman, S. and Klarsfeld, A. (2017). Drosophila Clock is required in brain pacemaker neurons to prevent premature locomotor aging independently of its circadian function. PLoS Genet 13(1): e1006507. PubMed ID: 28072817
Circadian clocks control many self-sustained rhythms in physiology and behavior with approximately 24-hour periodicity. This study examined the effects of clock disruptions on locomotor aging and longevity in Drosophila. Lifespan was found to be similarly reduced in three arrhythmic mutants (ClkAR, cyc0 and tim0) and in wild-type flies under constant light, which stops the clock. In contrast, ClkAR mutants showed significantly faster age-related locomotor deficits (as monitored by startle-induced climbing) than cyc0 and tim0, or than control flies under constant light. Clk, but not Cyc, inactivation by RNA interference in the pigment-dispersing factor (PDF)-expressing central pacemaker neurons led to similar loss of climbing performance as ClkAR. Conversely, restoring Clk function in these cells was sufficient to rescue the ClkAR locomotor phenotype, independently of behavioral rhythmicity. Accelerated locomotor decline of the ClkAR mutant required expression of the PDF receptor and correlated to an apparent loss of dopaminergic neurons in the posterior protocerebral lateral 1 (PPL1) clusters. This neuronal loss was rescued when the ClkAR mutation was placed in an apoptosis-deficient background. Impairing dopamine synthesis in a single pair of PPL1 neurons that innervate the mushroom bodies accelerated locomotor decline in otherwise wild-type flies. These results therefore reveal a novel circadian-independent requirement for Clk in brain circadian neurons to maintain a subset of dopaminergic cells and avoid premature locomotor aging in Drosophila.
He, Q., Zhang, Y., Zhang, X., Xu, D., Dong, W., Li, S. and Wu, R. (2016). Nucleoporin Nup358 facilitates nuclear import of Methoprene-tolerant (Met) in an importin beta- and Hsp83-dependent manner. Insect Biochem Mol Biol 81: 10-18. PubMed ID: 27979731
The bHLH-PAS transcription factor, Methoprene-tolerant (Met)1, functions as a juvenile hormone (JH) receptor and transduces JH signals by directly binding to E-box like motifs in the regulatory regions of JH response genes. Nuclear localization of Met is crucial for its transcriptional activity. It has been shown that the chaperone protein Hsp83 facilitates JH-induced Met nuclear import in Drosophila. However, the exact molecular mechanisms of Met nuclear transport are not fully elucidated. Using DNA affinity chromatography, binding of the nucleoporin Nup358, in the presence of JH, has been detected to the JH response region (JHRR) sequences isolated from the Kruppel-homolog 1 (Kr-h1) promoter. This study demonstrated that Nup358 regulates JH-Hsp83-induced Met nuclear localization. RNAi-mediated knockdown of Nup358 expression in Drosophila fat body perturbs Met nuclear transport during the 3 h after initiation of wandering, when the JH titer is high. The accompanying reduced expression of the transport receptor importin β in Nup358 RNAi flies could be one of the reasons accounting for Met mislocalization. Furthermore, a tetratricopeptide repeat (TPR) domain at the N-terminal end of Nup358 interacts with Hsp83 and is indispensable for Met nuclear localization. Overexpression of the TPR domain in Drosophila fat body prevents Met nuclear localization resulting in a decrease in JHRR-driven reporter activity and Kr-h1 expression. These data show that Nup358 facilitates JH-induced Met nuclear transport in a manner dependent on importin β and Hsp83.
Moon, S., Kim, W., Kim, S., Kim, Y., Song, Y., Bilousov, O., Kim, J., Lee, T., Cha, B., Kim, M., Kim, H., Katanaev, V. L. and Jho, E. H. (2016). Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization. EMBO Rep [Epub ahead of print]. PubMed ID: 27979972
Hippo signaling controls organ size by regulating cell proliferation and apoptosis. Yes-associated protein (YAP; see Drosophila Yorkie) is a key downstream effector of Hippo signaling, and LATS-mediated phosphorylation of YAP at Ser127 inhibits its nuclear localization and transcriptional activity. This study reports that Nemo-like kinase (NLK; see Drosophila Nemo) phosphorylates YAP at Ser128 both in vitro and in vivo, which blocks interaction with 14-3-3 (see Drosophila 14-3-3) and enhances its nuclear localization. Depletion of NLK increases YAP phosphorylation at Ser127 and reduces YAP-mediated reporter activity. These results suggest that YAP phosphorylation at Ser128 and at Ser127 may be mutually exclusive. It was also found that with the increase in cell density, nuclear localization and the level of NLK are reduced, resulting in reduction in YAP phosphorylation at Ser128. Furthermore, knockdown of Nemo (the Drosophila NLK) in fruit fly wing imaginal discs results in reduced expression of the Yorkie (the Drosophila YAP) target genes expanded and DIAP1, while Nemo overexpression reciprocally increased the expression. Overall, these data suggest that NLK/Nemo acts as an endogenous regulator of Hippo signaling by controlling nuclear localization and activity of YAP/Yorkie.
Plaza-Menacho, I., Barnouin, K., Barry, R., Borg, A., Orme, M., Chauhan, R., Mouilleron, S., Martinez-Torres, R. J., Meier, P. and McDonald, N. Q. (2016). RET functions as a Dual-Specificity Kinase that requires allosteric inputs from juxtamembrane elements. Cell Rep 17(12): 3319-3332. PubMed ID: 28009299
Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. This study shows that the juxtamembrane (JM) segment enhances RET (see Drosophila Ret oncogene catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. This phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. These findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

Sunday, January 15th

Kale, A., Rimesso, G. and Baker, N. E. (2016). Local cell death changes the orientation of cell division in the developing Drosophila wing imaginal disc without using Fat or Dachsous as orienting signals. PLoS One 11(12): e0167637. PubMed ID: 28030539
Drosophila imaginal disc cells exhibit preferred cell division orientations according to location within the disc. These orientations are altered if cell death occurs within the epithelium, such as is caused by cell competition or by genotypes affecting cell survival. Both normal cell division orientations, and their orientations after cell death, depend on the Fat-Dachsous pathway of planar cell polarity (PCP). The hypothesis that cell death initiates a planar polarity signal was investigated. When clones homozygous for the pineapple eye (pie) mutation were made to initiate cell death, neither Dachsous nor Fat was required in pie cells for the re-orientation of nearby cells, indicating a distinct signal for this PCP pathway. Dpp and Wg were also not needed for pie clones to re-orient cell division. Cell shapes were evaluated in wild type and mosaic wing discs to assess mechanical consequences of cell loss. Although proximal wing disc cells and cells close to the dorso-ventral boundary were elongated in their preferred cell division axes in wild type discs, cell shapes in much of the wing pouch were symmetrical on average and did not predict their preferred division axis. Cells in pie mutant clones were slightly larger than their normal counterparts, consistent with mechanical stretching following cell loss, but no bias in cell shape was detected in the surrounding cells. These findings indicate that an unidentified signal influences PCP-dependent cell division orientation in imaginal discs.
Lebensohn, A.M., Dubey, R., Neitzel, L.R., Tacchelly-Benites, O., Yang, E., Marceau, C.D., Davis, E.M., Patel, B.B., Bahrami-Nejad, Z., Travaglini, K.J., Ahmed, Y., Lee, E., Carette, J.E. and Rohatgi, R. (2016). Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling. Elife [Epub ahead of print]. PubMed ID: 27996937
Evolutionary Homolog Study:
The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, this study conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, sensitizing and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling, and suppressors of constitutive signaling induced by loss of the tumor suppressor APC (see Drosophila Apc) or casein kinase 1α (see Drosophila CkIα) uncovered new regulatory features at many levels of the pathway. These include a requirement for the transcription factor TFAP4 (see Drosophila crp), a role for the DAX domain of AXIN2 (see Drosophila Axn) in controlling β-catenin activity, a contribution of GPI anchor biosynthetic enzymes and glypicans to R-spondin-potentiated signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost.

Recasens-Alvarez, C., Ferreira, A. and Milán, M. (2017). JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling. Nat Commun 8: 13815. PubMed ID: 28045022
A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. This study presents evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. The inhibitor of apoptosis dIAP1 and Cyclin A were identified as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. JAK/STAT was found to have an early role in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ.

Lee, Y. C., Zhou, Q., Chen, J. and Yuan, J. (2016). RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response. Curr Biol 26(24): 3257-3268. PubMed ID: 27818175
Evolutionary Homolog Study

ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. This study reports the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway (see Drosophila meiotic 41) via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, these data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control.

Saturday, January 14th

Xiao, C. and Robertson, R.M. (2017). White-cGMP interaction promotes fast locomotor recovery from anoxia in adult Drosophila. PLoS One 12: e0168361. PubMed ID: 28060942
Increasing evidence indicates that the white (w) gene in Drosophila possesses extra-retinal functions in addition to its classical role in eye pigmentation. It has been previously shown that w+ promotes fast and consistent locomotor recovery from anoxia, but how w+ modulates locomotor recovery is largely unknown. This study shows that in the absence of w+, several PDE mutants, especially cyclic guanosine monophosphate (cGMP)-specific PDE mutants, display wildtype-like fast locomotor recovery from anoxia, and that during the night time, locomotor recovery is light-sensitive in white-eyed mutant w1118, and light-insensitive in PDE mutants under w1118 background. Data indicate the involvement of cGMP in the modulation of recovery timing and presumably, light-evoked cGMP fluctuation is associated with light sensitivity of locomotor recovery. This is further supported by the observations that w-RNAi-induced delay of locomotor recovery is completely eliminated by upregulation of cGMP through multiple approaches, including PDE mutation, simultaneous overexpression of an atypical soluble guanylyl cyclase Gyc88E, or sildenafil feeding. Lastly, prolonged sildenafil feeding promotes fast locomotor recovery from anoxia in w1118. Taken together, these data suggest that a White-cGMP interaction modulates the timing of locomotor recovery from anoxia.

Colinet, H., Renault, D. and Roussel, D. (2016). Cold acclimation allows Drosophila flies to maintain mitochondrial functioning under cold stress. Insect Biochem Mol Biol 80: 52-60. PubMed ID: 27903433
Environmental stress generally disturbs cellular homeostasis. Researchers have hypothesized that chilling injury is linked to a shortage of ATP. However, previous studies conducted on insects exposed to nonfreezing low temperatures presented conflicting results. This study investigated the mitochondrial bioenergetics of Drosophila melanogaster flies exposed to chronic cold stress (4 degrees C). Mitochondrial oxygen consumption was assessed while monitoring the rate of ATP synthesis at various times (0, 1, 2, and 3 days) during prolonged cold stress and at two assay temperatures (25 and 4 ° C). Organelle responses were compared between cold-susceptible and cold-acclimated phenotypes. Continuous exposure to low temperature provoked temporal declines in the rates of mitochondrial respiration and ATP synthesis. Respiratory control ratios (RCRs) suggested that mitochondria were not critically uncoupled. Nevertheless, after 3 days of continuous cold stress, a sharp decline in the mitochondrial ATP synthesis rate was observed in control flies when they were assayed at low temperature. This change was associated with reduced survival capacity in control flies. In contrast, cold-acclimated flies exhibited high survival and maintained higher rates of mitochondrial ATP synthesis and coupling (i.e., higher RCRs). Adaptive changes due to cold acclimation observed in the whole organism were thus manifested in isolated mitochondria. These observations suggest that cold tolerance is linked to the ability to maintain bioenergetics capacity under cold stress.
Croset, V., Schleyer, M., Arguello, J. R., Gerber, B. and Benton, R. (2016). A molecular and neuronal basis for amino acid sensing in the Drosophila larva. Sci Rep 6: 34871. PubMed ID: 27982028
Amino acids are important nutrients for animals, reflected in conserved internal pathways in vertebrates and invertebrates for monitoring cellular levels of these compounds. In mammals, sensory cells and metabotropic glutamate receptor-related taste receptors that detect environmental sources of amino acids in food are also well-characterised. By contrast, it is unclear how insects perceive this class of molecules through peripheral chemosensory mechanisms. This study investigate amino acid sensing in Drosophila melanogaster larvae, which feed ravenously to support their rapid growth. Larvae were shown to display diverse behaviours (attraction, aversion, neutral) towards different amino acids, which depend upon stimulus concentration. Some of these behaviours require IR76b, a member of the variant ionotropic glutamate receptor repertoire of invertebrate chemoreceptors. IR76b is broadly expressed in larval taste neurons, suggesting a role as a co-receptor. A subpopulation of these neurons were identified that displays physiological activation by some, but not all, amino acids, and which mediate suppression of feeding by high concentrations of at least a subset of these compounds. These data reveal the first elements of a sophisticated neuronal and molecular substrate by which these animals detect and behave towards external sources of amino acids.
Kang, M. J., Vasudevan, D., Kang, K., Kim, K., Park, J. E., Zhang, N., Zeng, X., Neubert, T. A., Marr, M. T., and Don Ryoo, H. (2016). 4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging. J Cell Biol. PubMed ID: 27979906
Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP (Thor) is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. This study shows that GCN2 and its downstream transcription factor, ATF4 (Cryptocephal), mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.

Friday, January 13th

Lai, Y. W., Chu, S. Y., Wei, J. Y., Cheng, C. Y., Li, J. C., Chen, P. L., Chen, C. H. and Yu, H. H. (2016). Drosophila microRNA-34 impairs axon pruning of mushroom body γ neurons by downregulating the expression of ecdysone receptor. Sci Rep 6: 39141. PubMed ID: 28008974
MicroRNA-34 (miR-34) is crucial for preventing chronic large-scale neurite degeneration in the aged brain of Drosophila melanogaster. This study investigated the role of miR-34 in two other types of large-scale axon degeneration in Drosophila: axotomy-induced axon degeneration in olfactory sensory neurons (OSNs) and developmentally related axon pruning in mushroom body (MB) neurons. Ectopically overexpressed miR-34 did not inhibit axon degeneration in OSNs following axotomy, whereas ectopically overexpressed miR-34 in differentiated MB neurons impaired γ axon pruning. Intriguingly, the miR-34-induced γ axon pruning defect resulted from downregulating the expression of ecdysone receptor B1 (EcR-B1) in differentiated MB γ neurons. Notably, the separate overexpression of EcR-B1 or a transforming growth factor- β receptor Baboon, whose activation can upregulate the EcR-B1 expression, in MB neurons rescued the miR-34-induced gamma axon pruning phenotype. Future investigations of miR-34 targets that regulate the expression of EcR-B1 in MB γ neurons are warranted to elucidate pathways that regulate axon pruning, and to provide insight into mechanisms that control large-scale axon degeneration in the nervous system.
Lin, C. J., Wen, J., Bejarano, F., Hu, F., Bortolamiol-Becet, D., Kan, L., Sanfilippo, P., Kondo, S. and Lai, E. C. (2016). Characterization of a TUTase/nuclease complex required for Drosophila gametogenesis. RNA [Epub ahead of print]. PubMed ID: 27974621
The 3' exoribonuclease Dis3L2 metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of non-coding RNAs. This study performed a structure-function analysis of the Drosophila TUTase Tailor, which inhibits biogenesis of splicing-derived miRNA hairpins. Tailor/Dis3L2 forms a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. Mutants of Tailor and Dis3L2 are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. Dis3L2 defects are more severe than Tailor, and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis causing complete male sterility. RNA-seq analysis from single and double mutant testes reveal aberrant gene expression programs, and suggest that non-coding RNAs may be preferentially affected by Dis3L2. Overall, these studies of a new tailing/trimming complex reveals unexpectedly specific requirements during gametogenesis.
Jones, B. C., Wood, J. G., Chang, C., Tam, A. D., Franklin, M. J., Siegel, E. R. and Helfand, S. L. (2016). A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan. Nat Commun 7: 13856. PubMed ID: 28000665
In gonadal tissues, the Piwi-interacting (piRNA) pathway preserves genomic integrity by employing 23-29 nucleotide (nt) small RNAs complexed with argonaute proteins to suppress parasitic mobile sequences of DNA called transposable elements (TEs). Although recent evidence suggests that the piRNA pathway may be present in select somatic cells outside the gonads, the role of a non-gonadal somatic piRNA pathway is not well characterized. This study reports a functional somatic piRNA pathway in the adult Drosophila fat body including the presence of the piRNA effector protein Piwi and canonical 23-29 nt long TE-mapping piRNAs. The piwi mutants exhibit depletion of fat body piRNAs, increased TE mobilization, increased levels of DNA damage and reduced lipid stores. These mutants are starvation sensitive, immunologically compromised and short-lived, all phenotypes associated with compromised fat body function. These findings demonstrate the presence of a functional non-gonadal somatic piRNA pathway in the adult fat body that affects normal metabolism and overall organismal health.
Kim, K., Hung, R. J. and Perrimon, N. (2016). miR-263a regulates ENaC to maintain osmotic and intestinal stem cell homeostasis in Drosophila. Dev Cell [Epub ahead of print]. PubMed ID: 28017617
Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. This study found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Evidence is provided that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC.

Thursday, Friday 12th

Nakajima, E., Shimaji, K., Umegawachi, T., Tomida, S., Yoshida, H., Yoshimoto, N., Izawa, S., Kimura, H. and Yamaguchi, M. (2016). The Histone deacetylase gene Rpd3 is required for starvation stress resistance. PLoS One 11(12): e0167554. PubMed ID: 27907135
Epigenetic regulation in starvation is important but not fully understood yet. This study identified the Rpd3 gene, a Drosophila homolog of histone deacetylase 1, as a critical epigenetic regulator for acquiring starvation stress resistance. Immunostaining analyses of Drosophila fat body revealed that the subcellular localization and levels of Rpd3 dynamically changed responding to starvation stress. In response to starvation stress, the level of Rpd3 rapidly increased, and it accumulated in the nucleolus in what appeared to be foci. These observations suggest that Rpd3 plays a role in regulation of rRNA synthesis in the nucleolus. The RT-qPCR and ChIP-qPCR analyses clarified that Rpd3 binds to the genomic region containing the rRNA promoters and activates rRNA synthesis in response to starvation stress. Polysome analyses revealed that the amount of polysomes was decreased in Rpd3 knockdown flies under starvation stress compared with the control flies. Since the autophagy-related proteins are known to be starvation stress tolerance proteins, autophagy activity was examined, and it was reduced in Rpd3 knockdown flies. Taken together, it is concluded that Rpd3 accumulates in the nucleolus in the early stage of starvation, upregulates rRNA synthesis, maintains the polysome amount for translation, and finally increases stress tolerance proteins, such as autophagy-related proteins, to acquire starvation stress resistance.
Cicconi, A., Micheli, E., Verni, F., Jackson, A., Gradilla, A. C., Cipressa, F., Raimondo, D., Bosso, G., Wakefield, J. G., Ciapponi, L., Cenci, G., Gatti, M., Cacchione, S. and Raffa, G. D. (2016). The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27940556
Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. It has been suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. This study used electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. It was also shown that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, Ver-depleted telomeres were found to form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, these findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin.
Nguyen, S. C., Yu, S., Oberlick, E. and Wu, C. T. (2016). An unexpected regulatory cascade governs a core function of the Drosophila PRC1 chromatin protein Su(z)2. Genetics [Epub ahead of print]. PubMed ID: 27881472
Polycomb group (PcG) proteins are major chromatin-bound factors that can read and modify chromatin states to maintain gene silencing throughout development. This study focused on a close homolog of the PcG protein Posterior sex combs in order to better understand how these proteins affect regulation. This homolog, called Suppressor 2 of zeste, or Su(z)2 is comprised of two regions: the N-terminal "homology region" (HR), which serves as a hub for protein interactions, and the C-terminal region (CTR), which is believed to harbor the core activity of compacting chromatin. This paper describes classical genetic studies to dissect the structure of Su(z)2 Surprisingly, it was found that the CTR is dispensable for viability. Furthermore, the core activity of Su(z)2 seems to reside in the HR instead of the CTR. Remarkably, the data also suggest a regulatory cascade between CTR and HR of Su(z)2, which, in turn, may help prioritize the myriad of PcG interactions that occur with the HR.
Babour, A., Shen, Q., Dos-Santos, J., Murray, S., Gay, A., Challal, D., Fasken, M., Palancade, B., Corbett, A., Libri, D., Mellor, J. and Dargemont, C. (2016). The chromatin remodeler ISW1 is a quality control factor that surveys nuclear mRNP biogenesis. Cell 167(5): 1201-1214 e1215. PubMed ID: 27863241
Evolutionary Homolog Study
Chromatin dynamics play an essential role in regulating DNA transaction processes, but it is unclear whether transcription-associated chromatin modifications control the mRNA ribonucleoparticles (mRNPs) pipeline from synthesis to nuclear exit. This study identified the yeast ISW1 chromatin remodeling complex (see Drosophili ISWI) as an unanticipated mRNP nuclear export surveillance factor that retains export-incompetent transcripts near their transcription site. This tethering activity of ISW1 requires chromatin binding and is independent of nucleosome sliding activity or changes in RNA polymerase II processivity. Combination of in vivo UV-crosslinking and genome-wide RNA immunoprecipitation assays show that Isw1 and its cofactors interact directly with premature mRNPs. These results highlight that the concerted action of Isw1 and the nuclear exosome ensures accurate surveillance mechanism that proofreads the efficiency of mRNA biogenesis.

Wednesday, January 11th

Tokusumi, T., Tokusumi, Y., Brahier, M. S., Lam, V., Stoller-Conrad, J. R., Kroeger, P. T., Jr. and Schulz, R. A. (2016). Screening and analysis of Janelia FlyLight project enhancer-Gal4 strains identifies multiple gene enhancers active during hematopoiesis in normal and wasp-challenged Drosophila larvae. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27913635
A GFP expression screen has been conducted on greater than one thousand Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and Delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to our knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process.
Cannavo, E., Koelling, N., Harnett, D., Garfield, D., Casale, F. P., Ciglar, L., Gustafson, H. E., Viales, R. R., Marco-Ferreres, R., Degner, J. F., Zhao, B., Stegle, O., Birney, E. and Furlong, E. E. (2016). Genetic variants regulating expression levels and isoform diversity during embryogenesis. Nature [Epub ahead of print]. PubMed ID: 28024300
Embryonic development is driven by tightly regulated patterns of gene expression, despite extensive genetic variation among individuals. Studies of expression quantitative trait loci (eQTL) indicate that genetic variation frequently alters gene expression in cell-culture models and differentiated tissues. However, the extent and types of genetic variation impacting embryonic gene expression, and their interactions with developmental programs, remain largely unknown.This study assessed the effect of genetic variation on transcriptional (expression levels) and post-transcriptional (3' RNA processing) regulation across multiple stages of metazoan development, using 80 inbred Drosophila wild isolates, identifying thousands of developmental-stage-specific and shared QTL. Given the small blocks of linkage disequilibrium in Drosophila, near base-pair resolution was obtained, resolving causal mutations in developmental enhancers and validated transcription-factor-binding sites and RNA motifs. This fine-grain mapping uncovered extensive allelic interactions within enhancers that have opposite effects, thereby buffering their impact on enhancer activity. QTL affecting 3' RNA processing identify new functional motifs leading to transcript isoform diversity and changes in the lengths of 3' untranslated regions. These results highlight how developmental stage influences the effects of genetic variation and uncover multiple mechanisms that regulate and buffer expression variation during embryogenesis.
Weasner, B. M., Weasner, B. P., Neuman, S. D., Bashirullah, A. and Kumar, J. P. (2016). Retinal expression of the Drosophila eyes absent gene is controlled by several cooperatively acting cis-regulatory elements. PLoS Genet 12(12): e1006462. PubMed ID: 27930646
The eyes absent (eya) gene of the fruit fly, Drosophila melanogaster, is a member of an evolutionarily conserved gene regulatory network that controls eye formation in all seeing animals. The loss of eya leads to the complete elimination of the compound eye while forced expression of eya in non-retinal tissues is sufficient to induce ectopic eye formation. Within the developing retina eya is expressed in a dynamic pattern and is involved in tissue specification/determination, cell proliferation, apoptosis, and cell fate choice. This study explores the mechanisms by which eya expression is spatially and temporally governed in the developing eye. Multiple cis-regulatory elements function cooperatively to control eya transcription and spacing between a pair of enhancer elements is important for maintaining correct gene expression. Lastly, it was shown that the loss of eya expression in sine oculis (so) mutants is the result of massive cell death and a progressive homeotic transformation of retinal progenitor cells into head epidermis.
Arnold, C. D., Zabidi, M. A., Pagani, M., Rath, M., Schernhuber, K., Kazmar, T. and Stark, A. (2016). Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution. Nat Biotechnol. PubMed ID: 28024147
Gene expression is controlled by enhancers that activate transcription from the core promoters of their target genes. Although a key function of core promoters is to convert enhancer activities into gene transcription, whether and how strongly they activate transcription in response to enhancers has not been systematically assessed on a genome-wide level. This study describes self-transcribing active core promoter sequencing (STAP-seq), a method to determine the responsiveness of genomic sequences to enhancers, and apply it to the Drosophila melanogaster genome. Candidate fragments at the position of the core promoter (also called minimal promoter) were cloned in reporter plasmids with or without a strong enhancer, the resulting library was transfected into cells, and the transcripts that initiated from each candidate were quantified for each setup by deep sequencing. In the presence of a single strong enhancer, the enhancer responsiveness of different sequences differs by several orders of magnitude, and different levels of responsiveness are associated with genes of different functions. Sequence features were identified that predict enhancer responsiveness and how different core promoters are employed for the regulation of gene expression is discussed.

Tuesday, January 10th

Jussen, D., von Hilchen, J. and Urbach, R. (2016). Genetic regulation and function of epidermal growth factor receptor signalling in patterning of the embryonic Drosophila brain. Open Biol 6(12). PubMed ID: 27974623
The specification of distinct neural cell types in central nervous system development crucially depends on positional cues conferred to neural stem cells in the neuroectoderm. This study investigated the regulation and function of the epidermal growth factor receptor (EGFR) signalling pathway in early development of the Drosophila brain. Localized EGFR signalling in the brain neuroectoderm was found to rely on a neuromere-specific deployment of activating (Spitz, Vein) and inhibiting (Argos) ligands. Activated EGFR controls the spatially restricted expression of all dorsoventral (DV) patterning genes in a gene- and neuromere-specific manner. Further, a novel role of DV genes-ventral nervous system defective (vnd), intermediate neuroblast defective (ind), Nkx6-in regulating the expression of vein and argos, which feed back on EGFR, indicating that EGFR signalling stands not strictly atop the DV patterning genes. Within this network of genetic interactions, Vnd acts as a positive EGFR feedback regulator. Further, it was shown that EGFR signalling becomes dependent on single-minded-expressing midline cells in the posterior brain (tritocerebrum), but remains midline-independent in the anterior brain (deuto- and protocerebrum). Finally, it was demonstrated that activated EGFR controls the proper formation of brain neuroblasts by regulating the number, survival and proneural gene expression of neuroectodermal progenitor cells. These data demonstrate that EGFR signalling is crucially important for patterning and early neurogenesis of the brain.
Pilaz, L. J., Lennox, A. L., Rouanet, J. P. and Silver, D. L. (2016). Dynamic mRNA transport and local translation in radial glial progenitors of the developing brain. Curr Biol 26(24): 3383-3392. PubMed ID: 27916527
Evolutionary Homolog Study
In the developing brain, neurons are produced from neural stem cells termed radial glia. Radial glial progenitors span the neuroepithelium, extending long basal processes to form endfeet hundreds of micrometers away from the soma. Basal structures influence neuronal migration, tissue integrity, and proliferation. Yet, despite the significance of these distal structures, their cell biology remains poorly characterized, impeding understanding of how basal processes and endfeet influence neurogenesis. This study used live imaging of embryonic brain tissue to visualize, for the first time, rapid mRNA transport in radial glia, revealing that the basal process is a highway for directed molecular transport. RNA- and mRNA-binding proteins, including the syndromic autism protein FMRP (see Drosophila Fmr1), move in basal processes at velocities consistent with microtubule-based transport, accumulating in endfeet. An ex vivo tissue preparation was developed to mechanically isolate radial glia endfeet from the soma, and photoconvertible proteins were used to demonstrate that mRNA is locally translated. Using RNA immunoprecipitation and microarray analyses of endfeet, FMRP-bound transcripts, which encode signaling and cytoskeletal regulators, were discovered including many implicated in autism and neurogenesis. FMRP controls transport and localization of one target, Kif26a. These discoveries reveal a rich, regulated local transcriptome in radial glia, far from the soma, and establish a tractable mammalian model for studying mRNA transport and local translation in vivo. It is concluded that cytoskeletal and signaling events at endfeet may be controlled through translation of specific mRNAs transported from the soma, exposing new mechanistic layers within stem cells of the developing brain.
Barber, A. F., Erion, R., Holmes, T. C. and Sehgal, A. (2016). Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells. Genes Dev 30(23): 2596-2606. PubMed ID: 27979876
Circadian clocks regulate much of behavior and physiology, but the mechanisms by which they do so remain poorly understood. While cyclic gene expression is thought to underlie metabolic rhythms, little is known about cycles in cellular physiology. This study found that Drosophila insulin-producing cells (IPCs), which are located in the pars intercerebralis and lack an autonomous circadian clock, are functionally connected to the central circadian clock circuit via DN1 neurons. Insulin mediates circadian output by regulating the rhythmic expression of a metabolic gene (sxe2) in the fat body. Patch clamp electrophysiology reveals that IPCs display circadian clock-regulated daily rhythms in firing event frequency and bursting proportion under light:dark conditions. The activity of IPCs and the rhythmic expression of sxe2 are additionally regulated by feeding, as demonstrated by night feeding-induced changes in IPC firing characteristics and sxe2 levels in the fat body. These findings indicate circuit-level regulation of metabolism by clock cells in Drosophila and support a role for the pars intercerebralis in integrating circadian control of behavior and physiology.
Chang, A. E., Vaughan, A. G. and Wilson, R. I. (2016). A mechanosensory circuit that mixes opponent channels to produce selectivity for complex stimulus features. Neuron 92(4): 888-901. PubMed ID: 27974164
Johnston's organ is the largest mechanosensory organ in Drosophila; it analyzes movements of the antenna due to sound, wind, gravity, and touch. Different Johnston's organ neurons (JONs) encode distinct stimulus features. Certain JONs respond in a sustained manner to steady displacements, and these JONs subdivide into opponent populations that prefer push or pull displacements. This study describes neurons in the brain (aPN3 neurons) that combine excitation and inhibition from push/pull JONs in different ratios. Consequently, different aPN3 neurons are sensitive to movement in different parts of the antenna's range, at different frequencies, or at different amplitude modulation rates. A model was used to show how the tuning of aPN3 neurons can arise from rectification and temporal filtering in JONs, followed by mixing of JON signals in different proportions. These results illustrate how several canonical neural circuit components-rectification, opponency, and filtering-can combine to produce selectivity for complex stimulus features.

Monday, January 9th

Jenny, F. H. and Basler, K. (2016). Drosophila DDX3/Belle exerts its function outside of the Wnt/Wingless signaling pathway. PLoS One 11(12): e0166862. PubMed ID: 28030561
The helicases human DDX3 and Drosophila Belle (Bel) are part of a well-defined subfamily of the DEAD-box helicases. Individual subfamily-members perform a myriad of functions in nuclear and cytosolic RNA metabolism. It has also been reported that DDX3X is involved in cell signaling, including IFN-alpha and IFN-beta inducing pathways upon viral infection as well as in Wnt signaling. This study used a collection of EMS-induced bel alleles recovered from a Wingless (Wg) suppressor screen to analyze the role of the Drosophila homolog of DDX3 in Wg/Wnt signaling. These EMS alleles, as well as a P-element induced null allele and RNAi-mediated knock down of bel, all suppressed the phenotype of ectopic Wg signaling in the eye. However, they did not affect the expression of known Wg target genes like senseless, Distalless or wingful/Notum. Ectopic Wg signaling in eye imaginal discs induces apoptosis by increasing grim expression. Mutations in bel revert grim expression to wild-type levels. Together, these results indicate that Bel does not function as a core component in the Drosophila Wg pathway, and that mutations affecting its helicase function suppress the effects of ectopic Wg signaling downstream of the canonical pathway.
Heintz, C., et al. (2016). Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans. Nature [Epub ahead of print]. PubMed ID: 27919065
Evolutionary Homolog Study
Ageing is driven by a loss of transcriptional and protein homeostasis and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses. However, the role of splicing homeostasis in healthy ageing remains unclear. This study demonstrates that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, this study found defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP; see Drosophila SF1). SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. It was also demonstrated that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.
Takemura, M. and Nakato, H. (2016). Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration. J Cell Sci [Epub ahead of print]. PubMed ID: 27888216
Stem cell division is activated to trigger regeneration in response to tissue damage. The molecular mechanisms by which this stem cell mitotic activity is properly repressed at the end of regeneration are poorly understood. This study shows that a specific modification of heparan sulfate (HS) is critical in regulating Drosophila intestinal stem cell (ISC) division during normal midgut homeostasis and regeneration. Loss of the extracellular HS endosulfatase Sulf1 results in increased ISC division during normal homeostasis, which is caused by upregulation of mitogenic signaling including the JAK/STAT, EGFR, and Hedgehog pathways. Using a regeneration model, this study found that ISCs failed to properly halt division at the termination stage in Sulf1 mutants, showing that Sulf1 is required for terminating ISC division at the end of regeneration. It is proposed that post-transcriptional regulation of mitogen signaling by HS structural modifications provides a novel regulatory step for precise temporal control of stem cell activity during regeneration.
Vaughen, J. and Igaki, T. (2016). Slit-Robo repulsive signaling extrudes tumorigenic cells from epithelia. Dev Cell 39: 683-695. PubMed ID: 27997825
Cells dynamically interact throughout animal development to coordinate growth and deter disease. For example, cell-cell competition weeds out aberrant cells to enforce homeostasis. In Drosophila, tumorigenic cells mutant for the cell polarity gene scribble (scrib) are actively eliminated from epithelia when surrounded by wild-type cells. While scrib cell elimination depends critically on JNK signaling, JNK-dependent cell death cannot sufficiently explain scrib cell extirpation. Thus, how JNK executed cell elimination remained elusive. This study shows that repulsive Slit-Robo2-Ena signaling exerts an extrusive force downstream of JNK to eliminate scrib cells from epithelia by disrupting E-cadherin. While loss of Slit-Robo2-Ena in scrib cells potentiates scrib tumor formation within the epithelium, Robo2-Ena hyperactivation surprisingly triggers luminal scrib tumor growth following excess extrusion. This extrusive signaling is amplified by a positive feedback loop between Slit-Robo2-Ena and JNK. These observations provide a potential causal mechanism for Slit-Robo dysregulation in numerous human cancers.

Sunday, January 8th

Phillips, M. A., Long, A. D., Greenspan, Z. S., Greer, L. F., Burke, M. K., Villeponteau, B., Matsagas, K. C., Rizza, C. L., Mueller, L. D. and Rose, M. R. (2016). Genome-wide analysis of long-term evolutionary domestication in Drosophila melanogaster. Sci Rep 6: 39281. PubMed ID: 28004838
Experimental evolutionary genomics now allows biologists to test fundamental theories concerning the genetic basis of adaptation. This laboratory conducted one of the longest laboratory evolution experiments with any sexually-reproducing metazoan, Drosophila melanogaster. Next-generation resequencing data from this experiment was conducted to examine genome-wide patterns of genetic variation over an evolutionary time-scale that approaches 1,000 generations. Measures of variation within and differentiation between populations were compared to simulations based on a variety of evolutionary scenarios. This analysis yielded no clear evidence of hard selective sweeps, whereby natural selection acts to increase the frequency of a newly-arising mutation in a population until it becomes fixed. Evidence was found for selection acting on standing genetic variation, as independent replicate populations exhibit similar population-genetic dynamics, without obvious fixation of candidate alleles under selection. A hidden-Markov model test for selection also found widespread evidence for selection. More genetic variation was found genome-wide, and less differentiation was found between replicate populations genome-wide, than arose in any of the simulated evolutionary scenarios.
Griffin, P. C., Hangartner, S. B., Fournier-Level, A. and Hoffmann, A. A. (2016).. Genomic trajectories to desiccation resistance: Convergence and divergence among replicate selected Drosophila lines. Genetics [Epub ahead of print]. PubMed ID: 28007884
Adaptation to environmental stress is critical for long-term species persistence. With climate change and other anthropogenic stressors compounding natural selective pressures, understanding the nature of adaptation is as important as ever in evolutionary biology. This study investigated this issue in a set of replicated Drosophila lines selected for increased desiccation resistance, a classical physiological trait that has been closely linked to Drosophila species distributions. Pooled whole-genome sequencing was used to compare the genetic basis of their selection responses. While selected SNPs in replicates of the same treatment (desiccation-selection or lab adaptation) tended to change frequency in the same direction, suggesting some commonality in the selection response, candidate SNP and gene lists often differed among replicates. Three of the five desiccation-selection replicates showed significant overlap at the gene and network level. All five replicates showed enrichment for ovary-expressed genes, suggesting maternal effects on the selected trait. Divergence between pairs of replicate lines for desiccation-candidate SNPs was greater than between pairs of control lines. This difference also far exceeded the divergence between pairs of replicate lines for neutral SNPs. Overall, while there was overlap in the direction of allele frequency changes and the network and functional categories affected by desiccation selection, replicates showed unique responses at all levels likely reflecting hitchhiking effects, and highlighting the challenges in identifying candidate genes from these types of experiments when traits are likely to be polygenic.
Appel, M., Scholz, C. J., Kocabey, S., Savage, S., Konig, C. and Yarali, A. (2016). Independent natural genetic variation of punishment- versus relief-memory. Biol Lett 12(12). PubMed ID: 28003518
A painful event establishes two opponent memories: cues that are associated with pain onset are remembered negatively, whereas cues that coincide with the relief at pain offset acquire positive valence. Such punishment- versus relief-memories are conserved across species, including humans, and the balance between them is critical for adaptive behaviour with respect to pain and trauma. In the fruit fly, Drosophila melanogaster as a study case, this study found that both punishment- and relief-memories display natural variation across wild-derived inbred strains, but they do not covary, suggesting a considerable level of dissociation in their genetic effectors. This provokes the question whether there may be heritable inter-individual differences in the balance between these opponent memories in man, with potential psycho-clinical implications.
Nozawa, M., Onizuka, K., Fujimi, M., Ikeo, K. and Gojobori, T. (2016). Accelerated pseudogenization on the neo-X chromosome in Drosophila miranda. Nat Commun 7: 13659. PubMed ID: 27897175
Y chromosomes often degenerate via the accumulation of pseudogenes and transposable elements. By contrast, little is known about X-chromosome degeneration. This study compared the pseudogenization process between genes on the neo-sex chromosomes in Drosophila miranda and their autosomal orthologues in closely related species. The pseudogenization rate on the neo-X is much lower than the rate on the neo-Y, but appears to be higher than the rate on the orthologous autosome in D. pseudoobscura. Genes under less functional constraint and/or genes with male-biased expression tend to become pseudogenes on the neo-X, indicating the accumulation of slightly deleterious mutations and the feminization of the neo-X. A weak trend was found that the genes with female-benefit/male-detriment effects identified in D. melanogaster are pseudogenized on the neo-X, implying the masculinization of the neo-X. These observations suggest that both X and Y chromosomes can degenerate due to a complex suite of evolutionary forces.

Saturday, January 7th

Zhang, B., Li, Q., Chu, X., Sun, S. and Chen, S. (2016). Salidroside reduces tau hyperphosphorylation via up-regulating GSK-3β phosphorylation in a tau transgenic Drosophila model of Alzheimer's disease. Transl Neurodegener 5: 21. PubMed ID: 27933142
Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease that causes substantial public health care burdens. Intensive efforts have been made to find effective and safe treatment against AD. The plant product Salidroside (Sal) is the main effective component of Rhodiola rosea L., which has several pharmacological activities. The objective of this study was to investigate the efficacy of Sal in the treatment of AD transgenic Drosophila and the associated mechanisms. Microtubule associated protein tau transgenic Drosophila line (TAU) was used in which tau protein is expressed in the central nervous system and eyes by the Gal4/UAS system. After feeding flies with Sal, the lifespan and locomotor activity were recorded. The appearance of vacuoles in the mushroom body was examined using immunohistochemistry, and the levels of total glycogen synthase kinase 3β (t-GSK-3β), phosphorylated GSK-3β (p-GSK-3β), t-tau and p-tau was detected in the brain by western blot analysis. The results showed that the longevity was improved in salidroside-fed Drosophila groups as well as the locomotor activity. Less vacuoles in the mushroom body, upregulated level of p-GSK-3β and downregulated p-tau were detected following Sal treatment. These data presented the evidence that Sal was capable of reducing the neurodegeneration in tau transgenic Drosophila and inhibiting neuronal loss. The neuroprotective effects of Sal were associated with its up-regulation of the p-GSK-3β and down-regulation of the p-tau.
Hatkevich, T., Kohl, K. P., McMahan, S., Hartmann, M. A., Williams, A. M. and Sekelsky, J. (2016). Bloom syndrome helicase promotes meiotic crossover patterning and homolog disjunction. Curr Biol [Epub ahead of print]. PubMed ID: 27989672
In most sexually reproducing organisms, crossover formation between homologous chromosomes is necessary for proper chromosome disjunction during meiosis I. During meiotic recombination, a subset of programmed DNA double-strand breaks (DSBs) are repaired as crossovers, with the remainder becoming noncrossovers. Whether a repair intermediate is designated to become a crossover is a highly regulated decision that integrates several crossover patterning processes, both along chromosome arms (interference and the centromere effect) and between chromosomes (crossover assurance). Because the mechanisms that generate crossover patterning have remained elusive for over a century, it has been difficult to assess the relationship between crossover patterning and meiotic chromosome behavior. This study showse that meiotic crossover patterning is lost in Drosophila melanogaster mutants that lack the Bloom syndrome helicase. In the absence of interference and the centromere effect, crossovers are distributed more uniformly along chromosomes. Crossovers even occur on the small chromosome 4, which normally never has meiotic crossovers. Regulated distribution of crossovers between chromosome pairs is also lost, resulting in an elevated frequency of homologs that do not receive a crossover, which in turn leads to elevated nondisjunction.
Xu, S., Stern, M. and McNew, J. A. (2016). Beneficial effects of rapamycin in a Drosophila model for hereditary spastic paraplegia. J Cell Sci [Epub ahead of print]. PubMed ID: 27909242
The locomotor deficits in the hereditary spastic paraplegias (HSPs) reflect degeneration of upper motor neurons, but the mechanisms underlying this neurodegeneration are unknown. This study established a Drosophila model for the HSP atlastin (atl), which encodes an ER fusion protein. Neuronal atl loss causes degeneration of specific thoracic muscles that is preceded by other pathologies including accumulation of aggregates containing poly-ubiquitin (poly-UB), increased generation of reactive oxygen species, and activation of the JNK/Foxo stress response pathway. Inhibiting the Tor kinase, either genetically or by administering rapamycin, at least partially reversed many of these pathologies. atl loss from muscle also triggers muscle degeneration and rapamycin-sensitive locomotor deficits and poly-UB aggregate accumulation. These results indicate that atl loss triggers muscle degeneration both cell autonomously and nonautonomously.
Delabaere, L., et al. (2016). Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells. Aging Cell [Epub ahead of print]. PubMed ID: 28000382
Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability. This study shows that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, it is proposed that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.

Friday, January 6th

Allen, A. M., Anreiter, I., Neville, M. C. and Sokolowski, M. B. (2016). Feeding-related traits are affected by dosage of the foraging gene in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28007892
Nutrient acquisition and energy storage are critical parts of achieving metabolic homeostasis. The foraging gene in Drosophila melanogaster has previously been implicated in multiple feeding-related and metabolic traits. Before foraging's functions can be further dissected, a precise genetic null mutant is needed to definitively map its amorphic phenotypes. This study used homologous recombination to precisely delete foraging, generating the for0 null allele, and used recombineering to re-integrate a full copy of the gene, generating the {forBAC} rescue allele. Total loss of foraging expression in larvae results in reduced larval path length and food intake behavior, while conversely showing an increase in triglyceride levels. Furthermore, varying foraging gene dosage demonstrates a linear dose-response on these phenotypes in relation to foraging gene expression levels. These experiments have unequivocally proven a causal, dose-dependent relationship between the foraging gene and its pleiotropic influence on these feeding-related traits. In that regard, this analysis of foraging's transcription start sites, termination sites, and splicing patterns using RACE and full length cDNA sequencing, revealed 4 independent promoters, pr1-4, that produce 21 transcripts with 9 distinct ORFs. The use of alternative promoters and alternative splicing at the foraging locus creates diversity and flexibility in the regulation of gene expression, and ultimately function. Future studies will exploit these genetic tools to precisely dissect the isoform- and tissue-specific requirements of foraging's functions and shed light on the genetic control of feeding-related traits involved in energy homeostasis.
Kubrak, O. I., Kucerova, L., Theopold, U., Nylin, S. and Nassel, D. R. (2016). Characterization of reproductive dormancy in male Drosophila melanogaster. Front Physiol 7: 572. PubMed ID: 27932997
Insects are known to respond to seasonal and adverse environmental changes by entering dormancy, also known as diapause. In some insect species, including Drosophila melanogaster, dormancy occurs in the adult organism and postpones reproduction. This adult dormancy has been studied in female flies where it is characterized by arrested development of ovaries, altered nutrient stores, lowered metabolism, increased stress and immune resistance and drastically extended lifespan. Male dormancy, however, has not been investigated in D. melanogaster, and its physiology is poorly known in most insects. This study shows that unmated 3-6 h old male flies placed at low temperature (11 ° C) and short photoperiod (10 Light:14 Dark) enter a state of dormancy with arrested spermatogenesis and development of testes and male accessory glands. Over 3 weeks of diapause a dynamic increase is seen in stored carbohydrates and an initial increase and then a decrease in lipids. An up-regulated expression of genes involved in metabolism, stress responses and innate immunity is also noted. Interestingly, it was found that male flies that entered reproductive dormancy do not attempt to mate females kept under non-diapause conditions (25 ° C, 12L:12D), and conversely non-diapausing males do not mate females in dormancy. In summary, this study shows that male D. melanogaster can enter reproductive dormancy. However, our data suggest that dormant male flies deplete stored nutrients faster than females, studied earlier, and that males take longer to recover reproductive capacity after reintroduction to non-diapause conditions.
Zhan, Y. P., Liu, L. and Zhu, Y. (2016). Taotie neurons regulate appetite in Drosophila. Nat Commun 7: 13633. PubMed ID: 27924813
The brain has an essential role in maintaining a balance between energy intake and expenditure of the body. Deciphering the processes underlying the decision-making for timely feeding of appropriate amounts may improve our understanding of physiological and psychological disorders related to feeding control. This study identified a group of appetite-enhancing neurons in a behavioural screen for flies with increased appetite. Manipulating the activity of these neurons, which were name Taotie neurons, induces bidirectional changes in feeding motivation. Long-term stimulation of Taotie neurons results in flies with highly obese phenotypes. Furthermore, it was shown that the in vivo activity of Taotie neurons in the neuroendocrine region reflects the hunger/satiety states of un-manipulated animals, and that appetitive-enhancing Taotie neurons control the secretion of insulin, a known regulator of feeding behaviour. Thus, this study reveals a new set of neurons regulating feeding behaviour in the high brain regions that represents physiological hunger states and control feeding behaviour in Drosophila.
Zhang, R., Wang, B., Grossi, G., Falabella, P., Liu, Y., Yan, S., Lu, J., Xi, J. and Wang, G. (2016). Molecular Basis of Alarm Pheromone Detection in Aphids. Curr Biol. PubMed ID: 27916525
Evolutionary Homolog Study
The sesquiterpene (E)-beta-farnesene (EBF) is the alarm pheromone for many species of aphids. When released from aphids attacked by parasitoids or predators, it alerts nearby conspecifics to escape by walking away and dropping off the host plan. The reception of alarm pheromone in aphids is accomplished through a highly sensitive chemosensory system. This study demonstrates that ApisOR5, a member of the large superfamily of odorant receptors, is expressed in large placoid sensillum neurons on the sixth antennal segment and confers response to EBF when co-expressed with Orco, an obligate odorant receptor co-receptor, in parallel heterologous expression systems. In addition, the repellent behavior of Acyrthosiphon pisum to EBF disappears after knocking down ApisOR5 by RNAi as well as two A. pisum odorant-binding proteins known to bind EBF (ApisOBP3 and ApisOBP7). Furthermore, other odorants that can also activate ApisOR5, such as geranyl acetate, significantly repel A. pisum, as does EBF. Taken together, these data lead to the conclusion that ApisOR5 is essential to EBF reception in A. pisum. The characterization of the EBF receptor allows high-throughput screening of aphid repellents, providing the necessary information to develop new strategies for aphid control.

Thursday, January 5th

Sengupta, S., Rath, U., Yao, C., Zavortink, M., Wang, C., Girton, J., Johansen, K.M. and Johansen, J. (2016). Digitor/dASCIZ has multiple roles in Drosophila development. PLoS One 11: e0166829. PubMed ID: 27861562
This study provides evidence that the spindle matrix protein Skeletor in Drosophila interacts with the human ASCIZ (also known as ATMIN and ZNF822) ortholog, Digitor. This interaction was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. The study also confirms a previously documented function of Digitor as a regulator of Dynein light chain/Cut up expression. Digitor was shown to be a nuclear protein that localizes to interband and developmental puff chromosomal regions during interphase but redistributes to the spindle region during mitosis. Its mitotic localization and physical interaction with Skeletor suggests the possibility that Digitor plays a direct role in mitotic progression as a member of the spindle matrix complex. Furthermore, a true null Digitor allele results in complete pupal lethality when homozygous, indicating that Digitor is an essential gene. Digitor plays critical roles in regulation of metamorphosis and organogenesis as well as in the DNA damage response. In the Digitor null mutant larvae there are greatly elevated levels of γH2Av, indicating accumulation of DNA double-strand breaks. Furthermore, reduced levels of Digitor decrease the resistance to paraquat-induced oxidative stress resulting in increased mortality in a stress test paradigm. It was shown that an early developmental consequence of the absence of Digitor is reduced third instar larval brain size although overall larval development appears otherwise normal at this stage. Altogether the data shows that Digitor is a nuclear protein that performs multiple roles in Drosophila larval and pupal development.

Kulkarni, A., Khan, Y. and Ray, K. (2016). Heterotrimeric kinesin-2, together with kinesin-1, steers vesicular acetylcholinesterase movements toward the synapse. Faseb j. [Epub ahead of print]. PubMed ID: 27920150
Acetylcholinesterase (AChE), which is implicated in the pathophysiology of neurological disorders, is distributed along the axon and enriched at the presynaptic basal lamina. It hydrolyses the neurotransmitter acetylcholine, which inhibits synaptic transmission. Aberrant AChE activity and ectopic axonal accumulation of the enzyme are associated with neurodegenerative disorders, such as Alzheimer's disease. The molecular mechanism that underlies AChE transport is still unclear. This study shows that expression of Drosophila AChE tagged with photoactivable green fluorescent protein and m-Cherry (GPAC) in cholinergic neurons compensates for the RNA interference-mediated knockdown of endogenous AChE activity. GPAC-AChE, which is enriched in the neuropil region of the brain, moves in the apparently vesicular form in axons with an anterograde bias in Drosophila larvae. Two anterograde motors, kinesin-1 and kinesin-2, propel distinct aspects of GPAC-AChE movements. Total loss of kinesin-2 reduces the density of anterograde traffic and increases bidirectional movements of GPAC-AChE vesicles without altering their speed. A partial loss of kinesin-1 reduces both the density and speed of anterograde GPAC-AChE traffic and enhances the pool of stationary vesicles. Together, these results suggest that combining activity of a relatively weak kinesin-2 with that of a stronger kinesin-1 motor could steer AChE-containing vesicles toward synapse, and provides a molecular basis for the observed subcellular distribution of the enzyme.
Qu, Y., Hahn, I., Webb, S., Pearce, S. P. and Prokop, A. (2016). Periodic actin structures in neuronal axons are required to maintain microtubules. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27881663
Axons are the cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly-spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily-conserved, ubiquitous, highly-ordered feature of axons, the function of PMS is unknown. This paper examined PMS abundance, organisation and function, combining versatile Drosophila genetics with super-resolution microscopy and various functional readouts. Analyses with 11 different actin regulators and 3 actin-targeting drugs suggest PMS to contain short actin filaments which are depolymerisation resistant and sensitive to spectrin, adducin and nucleator deficiency - consistent with microscopy-derived models proposing PMS as specialised cortical actin. Upon actin removal gaps were observed in microtubule bundles, reduced microtubule polymerisation and reduced axon numbers suggesting a role of PMS in microtubule organisation. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilising protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerisation contributes to their maintenance in axons. Potential implications are discussed of this novel PMS function along axon shafts for axon maintenance and regeneration.
Radford, S. J., Go, A. M. and McKim, K. S. (2016). Cooperation between kinesin motors promotes spindle symmetry and chromosome organization in oocytes. Genetics [Epub ahead of print]. PubMed ID: 27932541
The oocyte spindle in most animal species is assembled in the absence of the microtubule-organizing centers called centrosomes. Without the organization provided by centrosomes, acentrosomal meiotic spindle organization may rely heavily on the bundling of microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-14 NCD and the plus-end directed microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-6 Subito are known to be required for oocyte spindle organization in Drosophila melanogaster How multiple microtubule-bundling kinesins interact to produce a functional acentrosomal spindle is not known. In addition, there have been few studies on the meiotic function of one of the most important microtubule-bundlers in mitotic cells, the kinesin-5 KLP61F. This study found that the kinesin-5 KLP61F is required for spindle and centromere symmetry in oocytes. The asymmetry observed in the absence of KLP61F depends on NCD, the kinesin-12 KLP54D, and the microcephaly protein ASP. In contrast, KLP61F and Subito work together in maintaining a bipolar spindle. It is proposed that the prominent central spindle, stabilized by Subito, provides the framework for the coordination of multiple microtubule-bundling activities. The activities of several proteins, including NCD, KLP54D, and ASP, generate asymmetries within the acentrosomal spindle, while KLP61F and Subito balance these forces resulting in the capacity to accurately segregate chromosomes.

Wednesday, January 4th

Marie, P.P., Ronsseray, S. and Boivin, A. (2016). From embryo to adult: piRNA-mediated silencing throughout germline development in Drosophila. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27932388
In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development, has not been addressed. This study shows that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, rhino-dependent piRNAs are detected indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the "ping-pong" amplification step. Moreover, Heterochromatin Protein 1a (HP1a) is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, these data show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development.

Li, S., Li, Y., Shen, L., Jin, P., Chen, L. and Ma, F. (2016). miR-958 inhibits Toll signaling and Drosomycin expression via directly targeting Toll and Dif in Drosophila melanogaster. Am J Physiol Cell Physiol [Epub ahead of print]. PubMed ID: 27974298
Drosophila melanogaster is widely used as a model system to study innate immunity and signaling pathways related to innate immunity, including the Toll signaling pathway. Although this pathway is well-studied, the precise mechanisms of post-transcriptional regulation of key components of the Toll signaling pathway by microRNAs (miRNAs) remain obscure. This study used an in silico strategy in combination with the Gal80ts-Gal4 driver system and identified microRNA-958 (miR-958) as a candidate Toll pathway regulating miRNA in Drosophila. Overexpression of miR-958 significantly reduces the expression of Drosomycin, a key antimicrobial peptide involved in Toll signaling and the innate immune response. It was demonstrated in vitro and in vivo that miR-958 targets the Toll and Dif genes, key components of the Toll signaling pathway, to negatively regulate Drosomycin expression. In addition, a miR-958-sponge rescues the expression of Toll and Dif, resulting in increased expression of Drosomycin. These results not only reveal a novel function and modulation pattern of miR-958, but also provide a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity.

Lence, T., Akhtar, J., Bayer, M., Schmid, K., Spindler, L., Ho, C. H., Kreim, N., Andrade-Navarro, M. A., Poeck, B., Helm, M. and Roignant, J. Y. (2016). m6A modulates neuronal functions and sex determination in Drosophila. Nature 540(7632): 242-247. PubMed ID: 27919077
N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. This study carried out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. The member of the split ends protein family, Spenito, was identified as a novel bona fide subunit of the methyltransferase complex. Important roles of this complex were demonstrated in neuronal functions and sex determination, and the nuclear YT521-B protein was implicated as a main m6A effector in these processes. Altogether, this work substantially extends knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal.
Gaspar, I., Sysoev, V., Komissarov, A. and Ephrussi, A. (2016). An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs. Embo J [Epub ahead of print]. PubMed ID: 28028052
Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport-competent mRNPs. The posterior-targeting kinesin-1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. Kinesin-1 recruitment requires the DmTropomyosin1-I/C isoform, an atypical RNA-binding tropomyosin that binds directly to dimerizing oskar 3'UTRs. The isoform is on of 17 predicted mRNA isoforms and 13 distinct polypeptides encoded by the TM1 gene. Finally, a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin-1, and that the motor is activated during mid-oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo-dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo-transport processes and between the cargo-associated dynein and kinesin-1.

Tuesday, January 3rd

Yuan, I., Leontiou, I., Amin, P., May, K. M., Soper Ni Chafraidh, S., Zlamalova, E. and Hardwick, K. G. (2016). Generation of a spindle checkpoint arrest from synthetic signaling assemblies. Curr Biol [Epub ahead of print]. PubMed ID: 28017606
Evolutionary Homolog Study

The spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules and ensuring high-fidelity chromosome segregation. The checkpoint is activated by unattached kinetochores, and Mps1 kinase phosphorylates KNL1 on conserved MELT motifs to generate a binding site for the Bub3-Bub1 complex (see Drosophila Bub3). This leads to dynamic kinetochore recruitment of Mad proteins, a conformational change in Mad2, and formation of the mitotic checkpoint complex (MCC: Cdc20-Mad3-Mad2: see Drosophila Cdc20). MCC formation inhibits the anaphase-promoting complex/cyclosome (Cdc20-APC/C), thereby preventing the proteolytic destruction of securin and cyclin and delaying anaphase onset. What happens at kinetochores after Mps1-dependent Bub3-Bub1 recruitment remains mechanistically unclear, and it is not known whether kinetochore proteins other than KNL1 have significant roles to play in checkpoint signaling and MCC generation. This study took a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1Spc7 and Mps1Mph1 is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest in fission yeast. A Mad1-Bub1 complex is formed during synthetic checkpoint signaling. Analysis of bub3Δ mutants demonstrates that Bub3 acts to suppress premature checkpoint signaling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analyzing several mutants that affect localization of checkpoint complexes, it is concluded that spindle checkpoint arrest can be independent of their kinetochore, spindle pole, and nuclear envelope localization.

Mochida, S., Rata, S., Hino, H., Nagai, T. and Novak, B. (2016). Two bistable switches govern M phase entry. Curr Biol 26(24): 3361-3367. PubMed ID: 27889260
Evolutionary Homolog Study

The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex (see Drosophila Cdk1). Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible. Cdk1 auto-activation was found to be dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). This study shows, in Xenopus egg extracts, that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway (see Drosophila Greatwall) is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, a sharp Cdk1 threshold was found for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. These results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry.

Duranteau, M., Montagne, J. J. and Rahmani, Z. (2016). A novel mutation in the N-terminal domain of Drosophila BubR1 affects the spindle assembly checkpoint function of BubR1. Biol Open [Epub ahead of print]. PubMed ID: 27742609
The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism that ensures accurate segregation of chromosomes into two daughter cells. BubR1, a key component of the SAC, play also a role in the mitotic timing since depletion of BubR1 leads to an accelerated mitosis. Unlike what was reported in mammalian cells, mutation of the KEN1-box domain of Drosophila BubR1 (bubR1-KEN1 mutant) that affects the binding of BubR1 to Cdc20, the activating co-factor of the APC/C, did not accelerate the mitotic timing despite resulting in a defective SAC. This study shows that a mutation in a novel Drosophila short sequence (bubR1-KAN mutant) leads to an accelerated mitotic timing as well as SAC failure. Moreover, the data indicate that the level of Fzy, the Drosophila homolog of Cdc20, recruited to kinetochores is diminished in bubR1-KEN1 mutant cells and further diminished in bubR1-KAN mutant cells. Altogether, these data show that this newly identified Drosophila BubR1 KAN motif is required for a functional SAC and suggest that it may play an important role on Cdc20/Fzy kinetochore recruitment.
Vo, N., Anh Suong, D. N., Yoshino, N., Yoshida, H., Cotterill, S. and Yamaguchi, M. (2016). Novel roles of HP1a and Mcm10 in DNA replication, genome maintenance and photoreceptor cell differentiation. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27903903
Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs. Proximity Ligation Assay (PLA) demonstrated that HP1a is in close proximity to DNA replication proteins including Mcm10, RFC140 and DNA polymerase 255 kDa subunit in S-phase. This was further confirmed by co-immunoprecipitation assay. The PLA signals between Mcm10 and HP1a are specifically observed in the mitotic cycling cells, but not in the endocycling cells. Interestingly, many cells in the posterior regions of eye imaginal discs carrying a double knockdown of Mcm10 and HP1a induced ectopic DNA synthesis and DNA damage without much of ectopic apoptosis. Therefore, the G1-S checkpoint may be affected by knockdown of both proteins. This event was also the case with other HP family proteins such as HP4 and HP6. In addition, both Mcm10 and HP1a are required for differentiation of photoreceptor cells R1, R6 and R7. Further analyses on several developmental genes involved in the photoreceptor cell differentiation suggest that a role of both proteins is mediated by regulation of the lozenge gene.

Monday, January 2nd

Şahin, A., Held, A., Bredvik, K., Major, P., Achilli, T.M., Kerson, A.G., Wharton, K., Stilwell, G. and Reenan, R. (2016). Human SOD1 ALS mutations in a Drosophila knock-in model cause severe phenotypes and reveal dosage-sensitive gain and loss of function components. Genetics [Epub ahead of print]. PubMed ID: 27974499
Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc Superoxide Dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, this study genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical and behavioral phenotypes. Contrary to previous transgenic models, ALS-like phenotypes recapitulate without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R and H71Y) exhibits neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) is phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes are recessive, a gain of function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which fail to show severe locomotor defects or nerve degeneration. The study concludes that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.

Ramaker, J. M., Cargill, R. S., Swanson, T. L., Quirindongo, H., Cassar, M., Kretzschmar, D. and Copenhaver, P. F. (2016). Amyloid precursor proteins are dynamically trafficked and processed during neuronal development. Front Mol Neurosci 9: 130. PubMed ID: 27932950
Proteolytic processing of the Amyloid Precursor Protein (APP) produces β-amyloid (Aβ) peptide fragments that accumulate in Alzheimer's Disease (AD), but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. Insects express only a single APP-related protein (APP-Like, or APPL) that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. This study investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, it was shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, it was found that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate APPL-dependent responses within the nervous system. Lastly, targeted expression of the double-tagged constructs (combined with time-lapse imaging) revealed that APP family proteins are subject to complex patterns of trafficking and processing that vary dramatically between different neuronal subtypes. In combination, these results provide a new perspective on how the regulation of APP family proteins can be modulated to accommodate a variety of cell type-specific responses within the embryonic and adult nervous system.
Pomatto, L. C., Carney, C., Shen, B., Wong, S., Halaszynski, K., Salomon, M. P., Davies, K. J. and Tower, J. (2016). The mitochondrial Lon protease is required for age-specific and sex-specific adaptation to oxidative stress. J Curr Biol [Epub ahead of print]. PubMed ID: 27916526
Multiple human diseases involving chronic oxidative stress show a significant sex bias, including neurodegenerative diseases, cancer, immune dysfunction, diabetes, and cardiovascular disease. This study reports that Drosophila females but not males adapt to hydrogen peroxide stress, whereas males but not females adapt to paraquat (superoxide) stress. Stress adaptation in each sex requires the conserved mitochondrial Lon protease and is associated with sex-specific expression of Lon protein isoforms and proteolytic activity. Adaptation to oxidative stress is lost with age in both sexes. Transgenic expression of transformer gene during development transforms chromosomal males into pseudo-females and confers the female-specific pattern of Lon isoform expression, Lon proteolytic activity induction, and H2O2 stress adaptation; these effects were also observed using adult-specific transformation. Conversely, knockdown of transformer in chromosomal females eliminates the female-specific Lon isoform expression, Lon proteolytic activity induction, and H2O2 stress adaptation and produces the male-specific paraquat (superoxide) stress adaptation. Sex-specific expression of alternative Lon isoforms was also observed in mouse tissues. The results develop Drosophila melanogaster as a model for sex-specific stress adaptation regulated by the Lon protease, with potential implications for understanding sexual dimorphism in human disease.
Yedlapudi, D., Joshi, G. S., Luo, D., Todi, S. V. and Dutta, A. K. (2016). Inhibition of α-synuclein aggregation by multifunctional dopamine agonists assessed by a novel in vitro assay and an in vivo Drosophila synucleinopathy model. Sci Rep 6: 38510. PubMed ID: 27917933
Aggregation of α synuclein (α-syn) leading to dopaminergic neuronal death has been recognized as one of the main pathogenic factors in the initiation and progression of Parkinson's disease (PD). Consequently, α-syn has been targeted for the development of therapeutics for PD. This study developed a novel assay to screen compounds with α-syn modulating properties by mimicking recent findings from in vivo animal studies involving intrastriatal administration of pre-formed fibrils in mice, resulting in increased α-syn pathology accompanying the formation of Lewy-body (LB) type inclusions. In vitro generated α-syn pre-formed fibrils induce seeding of α-syn monomers to produce aggregates in a dose-and time-dependent manner under static conditions in vitro. These aggregates were toxic towards rat pheochromocytoma cells (PC12). Multifunctional dopamine agonists D-519 and D-520 exhibited significant neuroprotection in this assay, while their parent molecules did not. The neuroprotective properties of these compounds were further evaluated in a Drosophila model of synucleinopathy. Both of the compounds showed protective properties in fly eyes against the toxicity caused by α-syn. Thus, the in vitro results on modulation of aggregation and toxicity of α-syn by a novel assay were further validated with the in vivo experiments.

Sunday, January 1st

Sabeva, N., Cho, R. W., Vasin, A., Gonzalez, A., Littleton, J. T. and Bykhovskaia, M. (2016). Complexin mutants reveal partial segregation between recycling pathways that drive evoked and spontaneous neurotransmission. J Neurosci [Epub ahead of print]. PubMed ID: 27913592
Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. Synaptic vesicle recycling pathways were examined at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. Loading of the lipophilic dye FM1-43 was combined with photoconversion, electron microscopy (EM), and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. The total number of recycling vesicles was found to be equal to those retrieved through spontaneous and evoked pools, suggesting retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with EM analysis indicated spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, while vesicles recycled following evoked release involve a larger intra-terminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion.
Gokhale, A., Hartwig, C., Freeman, A. H., Das, R., Zlatic, S. A., Vistein, R., Burch, A., Carrot, G., Lewis, A. F., Nelms, S., Dickman, D. K., Puthenveedu, M. A., Cox, D. N. and Faundez, V. (2016). The proteome of BLOC-1 genetic defects identifies the Arp2/3 actin polymerization complex to function downstream of the schizophrenia susceptibility factor Dysbindin at the synapse. J Neurosci 36(49): 12393-12411. PubMed ID: 27927957
Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. This idea was tested by determining the proteome sensitive to genetic defects in a locus encoding dysbindin (see Drosophila Dysbindin), a protein required for synapse biology and implicated in schizophrenia risk. Quantitative mass spectrometry was applied to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). 491 proteins sensitive to dysbindin and BLOC-1 loss of function were found. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex (see Drosophila Arpc1), as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, it was demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. These results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects.
Laugks, U., Hieke, M. and Wagner, N. (2016). MAN1 restricts BMP signaling during synaptic growth in Drosophila. Cell Mol Neurobiol [Epub ahead of print]. PubMed ID: 27848060
Bone morphogenic protein (BMP) signaling is crucial for coordinated synaptic growth and plasticity. This study shows that the nuclear LEM-domain protein MAN1 is a negative regulator of synaptic growth at Drosophila larval and adult neuromuscular junctions (NMJs). Loss of MAN1 is associated with synaptic structural defects, including floating T-bars, membrane attachment defects, and accumulation of vesicles between perisynaptic membranes and membranes of the subsynaptic reticulum. In addition, MAN1 mutants accumulate more heterogeneously sized vesicles and multivesicular bodies in larval and adult synapses, the latter indicating that MAN1 may function in synaptic vesicle recycling and endosome-to-lysosome trafficking. Synaptic overgrowth in MAN1 is sensitive to BMP signaling levels, and loss of key BMP components attenuate BMP-induced synaptic overgrowth. Based on these observations, it is proposed that MAN1 negatively regulates accumulation and distribution of BMP signaling components to ensure proper synaptic growth and integrity at larval and adult NMJs.
Yoon, E. J., Jeong, Y. T., Lee, J. E., Moon, S. J. and Kim, C. H. (2016). Tubby domain superfamily protein is required for the formation of the 7S SNARE complex in Drosophila. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 27888110
Tubby domain superfamily protein (TUSP) is a distant member of the Tubby-like protein (TULP) family. Although other TULPs play important roles in sensation, metabolism, and development, the molecular functions of TUSP are completely unknown. This study explored the function of TUSP in the Drosophila nervous system where it is expressed in all neurons. Tusp mutant flies exhibit a temperature-sensitive paralysis. This paralysis can be rescued by tissue-specific expression of Tusp in the giant fibers and peripherally synapsing interneurons of the giant fiber system, a well-characterized neuronal circuit that mediates rapid escape behavior in flies. Consistent with this paralytic phenotype, a profound reduction was observed in the assembly of the ternary 7S SNARE complex (see synaptobrevin, syntaxin, and SNAP-25) that is required for neurotransmitter release despite seeing no changes in the expression of each individual SNARE complex component. Together, these data suggest TUSP is a novel regulator of SNARE assembly and, therefore, of neurotransmitter release.

Kauwe, G., Tsurudome, K., Penney, J., Mori, M., Gray, L., Calderon, M. R., Elazouzzi, F., Chicoine, N., Sonenberg, N. and Haghighi, A. P. (2016). Acute fasting regulates retrograde synaptic enhancement through a 4E-BP-dependent mechanism.Neuron [Epub ahead of print]. PubMed ID: 27916456
While beneficial effects of fasting on organismal function and health are well appreciated, little is known about the molecular details of how fasting influences synaptic function and plasticity. Genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, the findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. These results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits.
Nagel, B. M., Bechtold, M., Rodriguez, L. G. and Bogdan, S. (2016). Drosophila WASH is required for integrin-mediated cell adhesion, cell motility and lysosomal neutralization. J Cell Sci [Epub ahead of print]. PubMed ID: 27884932
The Wiskott-Aldrich Syndrome Protein and SCAR Homologue (WASH) is a conserved actin nucleation promoting factor controlling Arp2/3 complex activity in endosomal sorting and recycling. Previous studies have identified WASH as an essential regulator in Drosophila development. This study shows that homozygous wash mutant flies are viable and fertile. Drosophila WASH has conserved functions in integrin receptor recycling and lysosome neutralization. WASH generates actin patches on endosomes and lysosomes mediating both functions. Consistently, loss of WASH function results in cell spreading and cell migration defects of macrophages, and an increased lysosomal acidification that affects efficient phagocytic and autophagic clearance. WASH physically interacts with the vacuolar ATPase subunit Vha55 that is crucial to establish and maintain lysosome acidification. As a consequence, starved flies lacking WASH function show a dramatic increase in acidic autolysosomes causing a reduced lifespan. Thus, these data highlight a conserved role for WASH in the endocytic sorting and recycling of membrane proteins like integrins and the V-ATPase that increase the likelihood of survival under nutrient deprivation.

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