Syndecan, a heparan sulphate proteoglycan, is involved in myotube and axonal guidance in Drosophila melanogaster

Slit ist ein extrazelluläres Signalmolekül, welches von Glia-Zellen der ventralen Mittellinie sezerniert als ein abstoßendes Signal wirkt, das Axone an einer Überquerung der Mittellinie hindert. Zusätzlich wirkt Slit als ein regulierender Faktor bei Migrationsprozessen von Muskelvorläuferzellen und ventralen Zweigen des Tracheensystems. Slit bindet an Rezeptoren der Robo-Familie. Syndecan ist ein Transmembranprotein, in dessen extrazellulärem Bereich Heparansulfat-Glykosaminoglykan-Ketten (HSGAG) kovalent gebunden sind. Heparansulfat-Proteoglykane spielen eine Rolle in der Regulation extrazellulärer Signalwege. In der vorliegenden Arbeit wurde die Funktion von Sdc während gerichteter Migrationsprozesse in den Muskeln, im Nerven- und im Tracheensystem näher charakterisiert. sdc-Mutanten zeigen ähnliche Phänotypen, wie Mutanten in Genen des Slit/Robo-Signalwegs und Sdc kolokalisiert mit dem Robo-Rezeptor auf den longitudinalen Axonen des ZNS und an den Apodemen. Genetische Interaktionsstudien konnten Sdc als Teil des Slit/Robo-Signalwegs identifizieren: sdc-Allele zeigen genetische Interaktionen mit slit- sowie mit robo- und robo2-Allelen. In gewebespezifischen Normalisierungsexperimenten konnte ferner gezeigt werden, dass Sdc ausschließlich an den Slit-Rezipientenzellen notwendig ist. Eine eigenständige Rezeptorfunktion von Sdc mit einer Signalfunktion auf cytoplasmatische Faktoren über die konservierte cytoplasmatische Region konnte ausgeschlossen werden, da eine carboxyterminal deletierte Form von Sdc volle Sdc-Aktivität im Slit/Robo-Signalweg bereitstellt. Diese Ergebnisse zeigen, daß Syndecan ein Teil des Slit/Robo-Sigalwegs ist. Die Funktion von Syndecan auf den Slit-Rezipientenzellen besteht wahrscheinlich in der Präsentation membrangebundener HSGAG-Ketten, die die Interaktion des Slit-Liganden mit dem Robo-Rezeptor erleichtern.Slit, the ligand for the Roundabout (Robo) receptors is secreted from midline cells of the Drosophila central nervous system (CNS). It acts as a short-range repellent that controls midline crossing of axons and allows growth cones to select specific pathways along each side of the midline. In addition, Slit directs the migration of muscle precursors and of ventral branches of the tracheal system, showing that it provides long-range activity beyond the limit of the developing CNS . Biochemical studies on the mammalian homologues of Slit and Robo suggested that guidance activity requires cell-surface heparan sulphate to promote Slit/Robo-binding. The invertebrate homolog of Syndecan in the fly was identified in a screen for novel factors that are involved in myotube guidance. In this work I show that this heparan sulphate proteoglycan is required for proper Slit-signalling. Syndecan colocalizes with the Robo-Receptor at the longitudinal axon tracts and at the muscle attachment sites. Mutations of the Drosophila gene syndecan affect all aspects of Slit activity and cause robo-like phenotypes. sdc genetically interacts with robo and slit, and double mutations cause a synergistic strengthening of the single mutant phenotypes. Additionally, tissue specific rescue experiments show that Syndecan is required at the Slit-receiving cells. Rescue experiments with C-terminal truncated versions of Syndecan show that the highly conserved cytoplasmic domain of Syndecan is not important but an attachment of Syndecan to the cell membrane is important for the function of Syndecan in Slit/Robo-signaling. The results suggest that Syndecan is a necessary component of Slit/Robo-signaling. Syndecan is needed at the Slit-receiving cells and most likely acts to present membrane-bound heparan sulphate glycosaminoglycan sugar-chains to facilitate Slit/Robo-signaling

Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas

The Cul3–KLHL21 E3 ubiquitin ligase targets Aurora B to midzone microtubules in anaphase and is required for cytokinesis

Selective ubiquitination of Aurora B by different Cul3 adaptors targets it at the correct time to the correct place during mitosis

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility. © 2021, The Author(s)

Reception of Slit requires only the chondroitin–sulphate-modified extracellular domain of Syndecan at the target cell surface

Syndecan (Sdc) is a conserved transmembrane heparan sulfate proteoglycan (HSPG) bearing additional chondroitin sulfate (CS) modifications on its extracellular domain. In vertebrates, this extracellular domain of Sdc is shed and acts as a soluble effector of cellular communication events, and its cytoplasmic domain participates in intracellular signaling needed to maintain epithelial integrity. In Drosophila, Sdc has been shown to be necessary for Slit signaling-dependent axon and myotube guidance during CNS development and muscle pattern formation. We report that Sdc acts in a cell-autonomous manner in Slit-receiving cells and that its membrane-anchored extracellular domain is sufficient to mediate Slit signaling. Sdc activity can be replaced by the human homolog hsdc2. However, the HSPG Dally-like protein (Dlp), which lacks CS modifications at its extracellular domain, can only partially substitute for Sdc function, and its activity is not restricted to the Slit target cells. Our results suggest that Sdc and Dlp act in a cooperative but nonredundant fashion in axon and myotube guidance. We propose that Dlp, which lacks CS modifications, participates in the transfer of Slit from its site of expression to the target cells, where CS-modified Sdc concentrates and presents the ligand

Chromosome Missegregation Associated with RUVBL1 Deficiency

RUVBL1 (RuvB-like1) and RUVBL2 (RuvB-like 2) are integral components of multisubunit protein complexes involved in processes ranging from cellular metabolism, transcription and chromatin remodeling to DNA repair. Here, we show that although RUVBL1 and RUVBL2 are known to form heterodimeric complexes in which they stabilize each other, the subunits separate during cytokinesis. In anaphase-to-telophase transition, RUVBL1 localizes to structures of the mitotic spindle apparatus, where it partially co-localizes with polo-like kinase 1 (PLK1). The ability of PLK1 to phosphorylate RUVBL1-but not RUVBL2-in vitro and their physical association in vivo suggest that this kinase differentially regulates the function of the RuvB-like proteins during mitosis. We further show that siRNA-mediated knock-down of RuvB-like proteins causes severe defects in chromosome alignment and segregation. In addition, we show that the ATPase activity of RUVBL1 is indispensable for cell proliferation. Our data thus demonstrate that RUVBL1 is essential for efficient mitosis and proliferation

High Throughput FISH Screening Identifies Small Molecules That Modulate Oncogenic lncRNA MALAT1 via GSK3B and hnRNPs

Traditionally, small molecule-based drug discovery has mainly focused on proteins as the drug target. Opening RNA as an additional target space for small molecules offers the possibility to therapeutically modulate disease-driving non-coding RNA targets as well as mRNA of otherwise undruggable protein targets. MALAT1 is a highly conserved long-noncoding RNA whose overexpression correlates with poor overall patient survival in some cancers. We report here a fluorescence in-situ hybridization-based high-content imaging screen to identify small molecules that modulate the oncogenic lncRNA MALAT1 in a cellular setting. From a library of FDA approved drugs and known bioactive molecules, we identified two compounds, including Niclosamide, an FDA-approved drug, that lead to a rapid decrease of MALAT1 nuclear levels with good potency. Mode-of-action studies suggest a novel cellular regulatory pathway that impacts MALAT1 lncRNA nuclear levels by GSK3B activation and the involvement of the RNA modulating family of heterogenous nuclear ribonucleoproteins (hnRNPs). This study is the basis for the identification of novel targets that lead to a reduction of the oncogenic lncRNA MALAT1 in a cancer setting

RUVBL1 depletion affects the length of mitosis and results in lagging chromosomes.

<p><b>(A)</b> HeLa cells were transfected with the indicated siRNA oligos and analyzed by RT-PCR and immunoblot 48 h post-transfection. <b>(B)</b> Confocal live imaging after siRNA-mediated knock-down was performed. The figure shows stills of control or RUVBL1 siRNA-treated cells. Lagging chromosomes are indicated with arrowheads. DNA is shown in cyan and α-tubulin in red. <b>(C)</b> Early mitotic progression was analyzed by measuring the time from prophase (nuclear envelope breakdown: NEB) to anaphase onset (left panel). Mitotic exit was estimated by measuring the period of time from anaphase onset until accomplished cytokinesis (right panel). The bottom and top of the boxes represent the first and third quartiles, respectively. Horizontal lines inside the boxes represent the median of the data points (n = 100). Whiskers span the 10th and 90th percentiles, with individual dots showing data points that lie outside of these percentiles. Statistical significance was determined by Mann–Whitney test and <i>p</i>-values are indicated above. <b>(D)</b> Occurrence of aberrant mitotic phenotypes was quantified by analyzing 100 cell divisions in each cell line.</p