11 research outputs found
Funktionelle Analyse der RGG-Box von Mushroom body miniature und deren Rolle in der Neuroblastenproliferation in Drosophila melanogaster
Development of the central nervous system in Drosophila melanogaster relies on neural stem cells called neuroblasts. Neuroblasts divide asymmetrically to give rise to a new neuroblast as well as a small daughter cell which eventually generates neurons or glia cells. Between each division, neuroblasts have to re-grow to be able to divide again. In previous studies, it was shown that neuroblast proliferation, cell size and the number of progeny cells is negatively affected in larvae carrying a P-element induced disruption of the gene mushroom body miniature (mbm). This mbm null mutation called mbmSH1819 is homozygously lethal during pupation. It was furthermore shown that the nucleolar protein Mbm plays a role in the processing of ribosomal RNA (rRNA) as well as the translocation of ribosomal protein S6 (RpS6) in neuroblasts and that it is a transcriptional target of Myc. Therefore, it was suggested that Mbm might regulate neuroblast proliferation through a role in ribosome biogenesis.
In the present study, it was attempted to further elucidate these proposed roles of Mbm and to identify the protein domains that are important for those functions. Mbm contains an arginine/glycine rich region in which a di-RG as well as a di-RGG motif could be found. Together, these two motifs were defined as Mbmâs RGG-box. RGG-boxes can be found in many proteins of different families and they can either promote or inhibit protein-RNA as well as protein-protein interactions. Therefore, Mbmâs RGG-box is a likely candidate for a domain involved in rRNA binding and RpS6 translocation. It could be shown by deletion of the RGG-box, that MbmdRGG is unable to fully rescue survivability and neuroblast cell size defects of the null mutation mbmSH1819. Furthermore, Mbm does indeed rely on its RGG-box for the binding of rRNA in vitro and in mbmdRGG as well as mbmSH1819 mutants RpS6 is partially delocalized. Mbm itself also seems to depend on the RGG-box for correct localization since MbmdRGG is partially delocalized to the nucleus. Interestingly, protein synthesis rates are increased in mbmdRGG mutants, possibly induced by an increase in TOR expression. Therefore, Mbm might possess a promoting function in TOR signaling in certain conditions, which is regulated by its RGG-box. Moreover, RGG-boxes often rely on methylation by protein arginine methyltransferases (in Drosophila: Darts â Drosophila arginine methyltransferases) to fulfill their functions. Mbm might be symmetrically dimethylated within its RGG-box, but the results are very equivocal. In any case, Dart1 and Dart5 do not seem to be capable of Mbm methylation.
Additionally, Mbm contains two C2HC type zinc-finger motifs, which could be involved in rRNA binding. In an earlier study, it was shown that the mutation of the zinc-fingers, mbmZnF, does not lead to changes in neuroblast cell size, but that MbmZnF is delocalized to the cytoplasm. In the present study, mbmZnF mutants were included in most experiments. The results, however, are puzzling since mbmZnF mutant larvae exhibit an even lower viability than the mbm null mutants and MbmZnF shows stronger binding to rRNA than wild-type Mbm. This suggests an unspecific interaction of MbmZnF with either another protein, DNA or RNA, possibly leading to a dominant negative effect by disturbing other interaction partners. Therefore, it is difficult to draw conclusions about the zinc-fingersâ functions.
In summary, this study provides further evidence that Mbm is involved in neuroblast proliferation as well as the regulation of ribosome biogenesis and that Mbm relies on its RGG-box to fulfill its functions.Die Entwicklung des zentralen Nervensystems von Drosophila melanogaster beruht auf neuronalen Stammzellen genannt Neuroblasten. Neuroblasten teilen sich asymmetrisch und bringen dabei sowohl einen neuen Neuroblasten als auch eine kleinere Tochterzelle hervor, die wiederum letztlich Neuronen oder Gliazellen generiert. Zwischen jeder Zellteilung mĂŒssen die Neuroblasten wieder auf ihre ursprĂŒngliche GröĂe wachsen, sodass sie zur erneuten Teilung in der Lage sind. In vorhergehenden Studien konnte gezeigt werden, dass sowohl die Proliferation der Neuroblasten, deren ZellgröĂe als auch die Anzahl ihrer Tocherzellen reduziert ist in Larven, die eine P-Element-induzierte Unterbrechung des Gens mushroom body miniature (mbm) tragen. Diese mbm-Nullmutation, genannt mbmSH1819, ist homozygot letal wĂ€hrend des Puppenstadiums. Es konnte auĂerdem gezeigt werden, dass das nucleolĂ€re Protein Mbm eine Rolle in der Prozessierung ribosomaler RNA (rRNA), sowie der Translokation des ribosomalen Proteins S6 (RpS6) in Neuroblasten erfĂŒllt und dass seine Transkription durch Myc reguliert wird. Daher wurde geschlussfolgert, dass Mbm die Proliferation von Neuroblasten durch eine Funktion in der Ribosomenbiogenese regulieren könnte.
In der vorliegenden Studie wurde das Ziel verfolgt, weitere Hinweise auf diese möglichen Funktionen von Mbm zu finden und die ProteindomĂ€nen zu identifizieren, die dafĂŒr benötigt werden. Mbm beinhaltet einen Arginin/Glycin-reichen Abschnitt, der ein di-RG sowie ein di-RGG Motiv enthĂ€lt. Diese beiden Motive wurden zusammen zu Mbms RGG-Box definiert. RGG-Boxen finden sich in vielen Proteinen verschiedener Familien und sie können sich sowohl verstĂ€rkend als auch inhibierend auf Protein-RNA- sowie Protein-Protein-Interaktionen auswirken. Somit stellt Mbms RGG-Box einen vielversprechenden Kandidaten dar fĂŒr eine ProteindomĂ€ne, die in die rRNA-Bindung sowie die Translokation von RpS6 involviert ist. Es konnte gezeigt werden, dass Mbm mit deletierter RGG-Box (MbmdRGG) nicht in der Lage ist, die ĂberlebensfĂ€higkeit und die NeuroblastengröĂe der Nullmutation mbmSH1819 vollstĂ€ndig zu retten. Des Weiteren benötigt Mbm die RGG-Box, um rRNA in vitro zu binden und in mbmdRGG sowie mbmSH1819 Mutanten konnte eine partielle Delokalisation von RpS6 beobachtet werden. Die korrekte Lokalisation von Mbm selbst scheint auch von der RGG-Box abzuhĂ€ngen, da MbmdRGG teilweise in den Nukleus delokalisiert ist. Interessanterweise ist auĂerdem die Proteinsyntheserate in mbmdRGG Mutanten erhöht, was möglicherweise in einer Erhöhung der TOR-Expression begrĂŒndet ist. Somit könnte Mbm unter bestimmten Bedingungen eine verstĂ€rkende Funktion im TOR-Signalweg erfĂŒllen, die durch seine eigene RGG-Box reguliert wird. Des Weiteren sind RGG-Boxen hinsichtlich ihrer Funktion hĂ€ufig von der Methylierung durch Protein-Arginin-Methyltransferasen (in Drosophila: Darts â Drosophila arginine methyltransferases) abhĂ€ngig. Mbm könnte innerhalb seiner RGG-Box symmetrisch dimethyliert sein, allerdings sind die Ergebnisse in dieser Hinsicht sehr zweifelhaft. Jedenfalls scheinen Dart1 und Dart5 nicht imstande zu sein, Mbm zu methylieren.
AuĂerdem beinhaltet Mbm zwei Zink-Finger-Motive des C2HC-Typs, die in die Bindung von rRNA involviert sein könnten. Eine vorhergehende Studie konnte zeigen, dass die Mutation der Zink-Finger, mbmZnF, zwar nicht zu einer VerĂ€nderung der NeuroblastengröĂe fĂŒhrt, allerdings, dass MbmZnF ins Zytoplasma delokalisiert vorliegt. In der vorliegenden Studie wurden die mbmZnF Mutanten in die meisten Experimente mit einbezogen. Allerdings sind die Ergebnisse rĂ€tselhaft, da mbmZnF-mutierte Larven sogar eine geringere Ăberlebensrate zeigen als die mbm Nullmutanten und da MbmZnF eine stĂ€rkere BindungsaffinitĂ€t zu rRNA zeigt als wildtypisches Mbm. Dies weist auf eine unspezifische Interaktion zwischen MbmZnF und einem anderen Protein, RNA oder DNA hin, was einen dominant-negativen Effekt auslösen könnte, indem andere Interaktionspartner gestört werden. Somit gestaltet es sich schwierig, Schlussfolgerungen zur Funktion der Zink-Finger zu ziehen.
Zusammengefasst liefert die vorliegende Studie weitere Anhaltspunkte, dass Mbm in der Neuroblastenproliferation sowie der Regulation der Ribosomenbiogenese involviert ist und dass Mbm seine RGG-Box benötigt, um seine Funktionen zu erfĂŒllen
The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock
All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ⌠24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways
p38b promotes PER phosphorylation during the dark phase.
<p>To analyze daily phosphorylation of PER in flies that express the dominant-negative form of p38b in clock neurons and photoreceptor cells, we performed Western blots on head extracts after 4 days entrainment to LD 12â¶12 cycles. According to our behavioral data, timing of PER accumulation was not affected in experimental flies (<i>UAS-p38b<sup>DN-S</sup>;cry-Gal4/+;+</i>) in comparison with their respective control (A). However, regarding the degree of PER phosphorylation we observed differences at all time points when we compared both genotypes. For better comparison Western blots were repeated and samples of control and <i>UAS-p38b<sup>DN-S</sup>;cry-Gal4/+;+</i> flies were plotted side by side for each ZT (B). Interestingly, flies with impaired p38 signaling indeed had less phosphorylated PER, showing the largest differences to the controls at the end of the night. Western blots were repeated 4 times and always gave similar results. Bars above the blots depict the light regime of the LD 12â¶12. The â<i>C</i>â refers to respective control, <i>DNS</i> to <i>UAS-p38b<sup>DN-S</sup>;cry-Gal4/+;+</i>.</p
Locomotor activity rhythms of <i>p38b</i> and <i>p38a</i> null mutants and hypomorphic double mutant flies.
<p>Both <i>p38</i> null mutants, <i>p38b<sup>Î45</sup></i> (upper panels in A) and <i>p38a<sup>Î1</sup></i> (upper panels in B), displayed wildtype-like behavior with activity bouts around lights-on and lights-off when recorded in LD 12â¶12. Even if evening activity onset of <i>p38a<sup>Î1</sup></i>seems to be delayed compared to <i>w<sup>1118</sup></i>, this delay did not result in a longer free-running period under constant darkness (lower panels in B). Similarly, flies, lacking the <i>p38b</i> gene, also showed comparable free-running rhythms as their respective controls (lower panels in A). Activity data in C show two representative single actograms of a double mutant strain with a hypomorphic <i>p38b</i> allele (<i>p38b<sup>Î25</sup>;p38a<sup>Î1</sup></i>). Since these flies are hardly viable and die within 3â6 days after emergence of the pupa, flies were already entrained to LD12â¶12 during pupal stage and subsequently monitored in DD conditions after eclosion. Even if periodogram analysis was not possible due to the short recording period, <i>p38b<sup>Î25</sup>;p38a<sup>Î1</sup></i> flies clearly showed a long free-running period when kept in constant darkness (C). For recording and processing of activity data as well as for figure labeling see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004565#pgen-1004565-g003" target="_blank">Figure 3</a>.</p
Daily oscillations of nuclear PER in s-LN<sub>v</sub>s and l-LN<sub>v</sub>s of flies expressing a dominant negative form of p38b in these cells.
<p>Flies were entrained in LD 12â¶12, dissected every one to two hours and staining intensity of nucleus and cell body was measured as described in Material and Methods. Nuclear PER staining intensities were normalized to total staining and tested for statistically significance. Expression of the dominant negative form of p38b phase delayed nuclear accumulation of PER in the s-LN<sub>v</sub>s (A) and l-LN<sub>v</sub>s (B). Arrows indicate the maxima of nuclear PER staining that occurred significantly later in <i>UAS-p38b<sup>DN-S</sup>;Pdf-Gal4/+;+</i> flies than in control flies. This delay in nuclear PER accumulation in PDF-positive clock neurons is well consistent with the shifted evening activity in these flies. Bars above the graphs depict the light regime of the LD 12â¶12.</p
p38b phosphorylates PER <i>in vitro</i>.
<p>To test whether p38b phosphorylates PER <i>in vitro</i>, either non-radioactive kinase assays followed by urea-PAGE (AâD) or radioactive kinase assays with autoradiography (EâF) were performed. AâD: Non-radioactive kinase assays were conducted with poly-histidine tagged p38b (His<sub>6</sub>-p38b) and two truncated GST-tagged PER isoforms, GST-PER<sup>1â700</sup> (A,B) and GST-PER<sup>658â1218</sup> (C,D). Samples were subsequently separated with urea-PAGE and visualized by Coomassie staining (A,C). To further confirm PER's position in the gel two samples of the same gel were additionally blotted onto nitrocellulose membrane and immunolabeled using an anti-PER antibody and a secondary fluorescent antibody (B,D). While GST-PER<sup>1â700</sup> without kinase did not shift within 60 minutes, the addition of His<sub>6</sub>-p38b induced a downward shift of GST-PER<sup>1â700</sup> indicating phosphorylation of PER (A; dotted line). Immunoblots with anti-PER further confirmed the size as well as the shift of the GST-PER<sup>1â700</sup> band (B). In addition to GST-PER<sup>1â700</sup>, GST-PER<sup>658â1218</sup> also displayed band shifts after incubation with His<sub>6</sub>-p38b (C). This was most prominent after 60 minutes, when addition of His<sub>6</sub>-p38b resulted in two distinct shifted bands (black arrowheads), which could be additionally confirmed by Western blots (D). Time scale below graphs represents minutes after addition of His<sub>6</sub>-p38b, the âCâ refers to control and represents substrate samples without kinase and ATP. (E) Radioactive <i>in vitro</i> kinase assays were conducted with the indicated GST-PER fusion proteins and GST-p38b. Control reactions were performed in the absence of GST-p38b or with GST in combination with GST-p38b. Coomassie staining proved loading of the indicated protein combinations. Below, phosphorylation of GST-PER proteins was detected by autoradiography. (F) For quantitative analysis five independent <i>in vitro</i> kinase assay experiments were performed and analyzed. For each reaction within a single experiment, autoradiography signal intensities were normalized to the corresponding Coomassie stained protein band. Values in the graph are shown as percentages of GST-PER<sup>658â1218</sup> phosphorylation (100%; * p<0.05, ** p<0.005).</p
Rhythmicity and period length of all investigated genotypes in constant darkness (DD) according to Ï<sup>2</sup>-periodogram analysis.
<p><i>n</i> indicates the number of tested flies per genotype that survived locomotor recordings. Power and period values were averaged over all rhythmic flies for each genotype.</p>1<p>Flies with power values <20 were defined as arrhythmic.</p>2<p>Power is a measure of rhythmicity and is given in % of variance.</p
Daily p38 mRNA (A) and protein expression (BâD) in <i>Canton S</i> wildtype.
<p>A: Quantitative real-time PCR on head extracts revealed constant mRNA expression throughout the day with allover higher levels of <i>p38b</i> compared to <i>p38a</i> (p<0.001). B: Antibody staining with anti-p-p38 on adult brains displayed rhythmic phosphorylation of p38 in DN<sub>1a</sub>s in LD with significant higher p-p38 levels occurring during the night than in the day (p<0.05). C: A highly significant reduction of active p38 in DN<sub>1a</sub>s at CT6 compared to CT18 in DD indicates a clock-controlled activation of p38 (p<0.001) D: Only a 15 minute light pulse (LP) during subjective night (CT18) and not during the subjective day (CT6) leads to a reduction in active p38 in DN<sub>1a</sub>s, suggesting a clock-dependent photic reduction of active p38. The âCâ in D indicates control brains without 15 minute light pulse (LP). Error bars show SEM. Significant differences (p<0.05) are indicated by *, highly significant differences (p<0.001) by **.</p
p38 MAPK expression pattern in adult male <i>Canton S</i> brains.
<p>p38 MAPK distribution within the circadian clock was investigated immunohistochemically with an antibody directed against <i>Drosophila</i> p38b (AâC) and against phosphorylated human p38 (DâG). AâC: Staining with anti-p38b (green) in <i>Canton S</i> wildtype brains was visible in many cell bodies close to the lateral clock neurons, but co-labeling with anti-VRI (magenta) and anti-PDF (blue) revealed clear p38b expression in the l-LN<sub>v</sub>s (white stars in B3) and the s-LN<sub>v</sub>s (white stars in C3). B1âB4 represents a close-up of l-LN<sub>v</sub>s, C1âC4 a shows close-up of s-LN<sub>v</sub>s. Furthermore, we found staining in the entire cortex including the region of the dorsal neurons (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004565#pgen.1004565.s001" target="_blank">Fig. S1</a>). DâG: Staining with anti-p-p38 (green) was restricted to fewer neurons, but revealed again staining in the entire cortex that was stronger at night (F, ZT21) than during the day (D, ZT9). Double-labeling with anti-VRI (magenta) and anti-p-p38 antibody (green) revealed active p38 only in 2 clock neurons, the DN<sub>1a</sub>s (white stars in G2). Also in these cells, p-p38 staining intensity depended on the time of day, showing a higher level of active p38 at ZT 21 (G2, white stars) than at ZT9 (E2). E1âE3 and G1âG3 represent a close-up of DN<sub>1a</sub>s. Scale barâ=â10 ”m.</p
Locomotor activity rhythms of flies expressing a dominant-negative form of p38b <i>(p38b<sup>DN-S</sup>)</i> in <i>Drosophila</i> clock neurons and respective controls.
<p>Both, expression of a dominant negative form of p38b in either all clock neurons (<i>UAS-p38b<sup>DN-S</sup>;tim(UAS)-Gal4/+;+</i>) or just in a subset of clock cells, the PDF-positive LN<sub>v</sub>s (<i>UAS</i>-<i>p38b<sup>DN-S</sup>;Pdf-Gal4/+;+</i>), resulted in a diurnal activity profile with a significantly delayed evening activity onset in comparison with respective controls (upper panels in A and B). This delay in evening activity is accompanied by a significantly prolonged free-running period in <i>UAS</i>-<i>p38b<sup>DN-S</sup>;tim(UAS)-Gal4/+;+</i> (lower panels in A) as well as in <i>UAS</i>-<i>p38b<sup>DN-S</sup>;Pdf-Gal4/+;+</i> flies(lower panels in B), when released into constant darkness. For recording and processing of activity data as well as for figure labeling see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004565#pgen-1004565-g003" target="_blank">Figure 3</a>.</p