The mammalian target of rapamycin (mTOR) governs cell growth and proliferation by mediating the mitogen- and nutrient-dependent signal transduction that regulates messenger RNA translation. We identified phosphatidic acid (PA) as a critical component of mTOR signaling. In our study, mitogenic stimulation of mammalian cells led to a phospholipase D-dependent accumulation of cellular PA, which was required for activation of mTOR downstream effectors. PA directly interacted with the domain in mTOR that is targeted by rapamycin, and this interaction was positively correlated with mTOR's ability to activate downstream effectors. The involvement of PA in mTOR signaling reveals an important function of this lipid in signal transduction and protein synthesis, as well as a direct link between mTOR and mitogens. Furthermore, these studies suggest a potential mechanism for the in vivo actions of the immunosuppressant rapamycin.

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Phosphorylation of the ribosomal S6 subunit is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery, which is an important early event that controls mammalian cell growth and proliferation. The recently identified S6 kinase 2 (S6K2), together with its homologue S6K1, is likely responsible for the mitogen-stimulated phosphorylation of S6. Like S6K1, the activation of S6K2 requires signaling from both the phosphatidylinositol 3-kinase and the mammalian target of rapamycin (mTOR). Here we report the investigation of the mechanisms of S6K2 regulation by mTOR. We demonstrate that similar to S6K1 the serum activation of S6K2 in cells is dependent on mTOR kinase activity, amino acid sufficiency, and phosphatidic acid. Previously we have shown that mTOR is a cytoplasmic-nuclear shuttling protein. As a predominantly nuclear protein, S6K2 activation was facilitated by enhanced mTOR nuclear import with the tagging of an exogenous nuclear localization signal and diminished by enhanced mTOR nuclear export with the tagging of a nuclear export sequence. However, further increase of mTOR nuclear import by the tagging of four copies of nuclear localization signal resulted in its decreased ability to activate S6K2, suggesting that mTOR nuclear export may also be an integral part of the activation process. Consistently, the nuclear export inhibitor leptomycin B inhibited S6K2 activation. Taken together, our observations suggest a novel regulatory mechanism in which an optimal cytoplasmicnuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.One of the critical events involved in mitogenic stimulation of mammalian cell growth and proliferation is the increased translation initiation of 5Ј-terminal oligopyrimidine tract-containing mRNAs, which encode components of the protein synthesis machinery (1). Phosphorylation of the ribosomal S6 subunit is correlated with 5Ј-terminal oligopyrimidine tractdependent translation initiation, and the 70-kDa S6 kinase 1 (S6K1) 1 is a serine/threonine protein kinase responsible for mitogen-stimulated S6 phosphorylation (2). In addition to playing an essential role in regulating cell growth, S6K1 appears to be a multifunctional protein involved in other cellular processes such as anti-apoptosis (3) and RNA processing (4). Two parallel pathways are both required for activation of S6K1, and they are mediated by the phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR), respectively (2, 5). While the PI3K pathway transduces mitogenic signals to S6K1, the mTOR pathway is believed to sense amino acid sufficiency and play a permissive role to govern S6K1 activation by PI3K signals (6 -9). mTOR is a serine/threonine protein kinase that belongs to the family of phosphatidylinositol kinase-related kinases (10). The kinase activity of mTOR is required, but not sufficient, for signaling to downstream effectors including S6K1 (11, 12). Most recently we hav...

The mammalian target of rapamycin (mTOR) regulates nutrientdependent cell growth and proliferation through cytoplasmic targets, such as S6 kinase 1 (S6K1). Consistent with its main function in the cytoplasm, mTOR is predominantly cytoplasmic. However, previously we have found that mTOR shuttles between the nucleus and cytoplasm, and we have proposed that the nucleocytoplasmic shuttling of mTOR is required for the maximal activation of S6K1. The intrinsic signals directing mTOR nuclear transport and the underlying mechanisms are unknown. In this study we initially set out to identify nuclear export signals in mTOR. A systematic scan of the mTOR sequence revealed 16 peptides conforming to the canonical leucine-rich nuclear export signal, of which 3 were found by reporter assays to contain leptomycin B-sensitive and leucine-dependent nuclear export activity. Unexpectedly, mTOR proteins with those conserved leucines mutated to alanines were unable to enter the nucleus. Further investigation revealed that the L982A/ L984A and L1287A/L1289A mutations likely induced a global structural change in mTOR, whereas the L545A/L547A mutation directly impaired the nuclear import of the protein, potentially regulated by a nucleocytoplasmic shuttling signal. The loss of nuclear import was accompanied by the significantly reduced ability of the L545A/L547A mutant to activate S6K1 in cells. Most importantly, when nuclear import was restored in the L545A/L547A mutant by the addition of an exogenous nuclear import signal, signaling to S6K1 was rescued. Taken together, our observations suggest the existence of a nuclear shuttling signal in mTOR and provide definitive evidence for the requirement of mTOR nuclear import in its cytoplasmic signaling to S6K1.

BackgroundCaenorhabditis elegans sarcomeres have been studied extensively utilizing both forward and reverse genetic techniques to provide insight into muscle development and the mechanisms behind muscle contraction. A previous genetic screen investigating early muscle development produced 13 independent mutant genes exhibiting a Pat (paralyzed and arrested elongation at the two-fold length of embryonic development) muscle phenotype. This study reports the identification and characterization of one of those genes, pat-9.ResultsPositional cloning, reverse genetics, and plasmid rescue experiments were used to identify the predicted C. elegans gene T27B1.2 (recently named ztf-19) as the pat-9 gene. Analysis of pat-9 showed it is expressed early in development and within body wall muscle lineages, consistent with a role in muscle development and producing a Pat phenotype. However, unlike most of the other known Pat gene family members, which encode structural components of muscle attachment sites, PAT-9 is an exclusively nuclear protein. Analysis of the predicted PAT-9 amino acid sequence identified one putative nuclear localization domain and three C2H2 zinc finger domains. Both immunocytochemistry and PAT-9::GFP fusion expression confirm that PAT-9 is primarily a nuclear protein and chromatin immunoprecipitation (ChIP) experiments showed that PAT-9 is present on certain gene promoters.ConclusionsWe have shown that the T27B1.2 gene is pat-9. Considering the Pat-9 mutant phenotype shows severely disrupted muscle attachment sites despite PAT-9 being a nuclear zinc finger protein and not a structural component of muscle attachment sites, we propose that PAT-9 likely functions in the regulation of gene expression for some necessary structural or regulatory component(s) of the muscle attachment sites.

Rebecca Bachmann widmet sich in ihrem Beitrag dem Konzept von Entscheidungen in Videospielen. Dabei geht sie von der häufig angebrachten Kritik aus, diese seien irrelevant, da der Spielverlauf unabhängig von der Einzelentscheidung nahezu identisch verläuft. Dieser Kritik schließt sich Bachmann mittels Beispielen aus »Telltales The Walking Dead - Season Two« an und arbeitet daraus ein bestimmtes Verständnis von Entscheidungen in Videospielen heraus, das diese als ergebnisrelevant charakterisiert. Im Kontrast dazu entwirft Bachmann mithilfe der philosophischen Willensfreiheitstheorie von Robert Kane ein anderes Konzept von Entscheidungen, welches den Fokus auf Charakterbildung legt. Danach konstruieren Spieler*innen mit ihren Entscheidungen den Charakter der Spielfigur, weswegen jede Entscheidung relevant ist. Diese These macht sie erneut am Beispiel von »The Walking Dead« stark und hebt auch hervor, dass genau dieser Aspekt Videospiele mit Entscheidungsoption auszeichnet.

Gedankenexperimente in der Philosophie zeichnen sich durch ein widersprüchliches Verhältnis aus: Sie werden gleichzeitig häufig genutzt und vielfältig kritisiert. Im Zentrum der Kritik steht dabei das Szenario sowie seine teilweise als absurd wahrgenommenen Details. Als Verteidigungsstrategie der Methode wird daher zum einen versucht, realistische Gedankenexperimente zu bevorzugen, zum anderen, das Argument hinter dem Szenario deutlicher in den Fokus zu rücken. Im Zuge dessen wird jedoch das eigentlich Charakteristische an einem Gedankenexperiment – das Szenario – vernachlässigt. Um die Relevanz des Szenarios zu betonen, soll in diesem Aufsatz ein Blick in die Wissenschaftsforschung geworfen werden. Mithilfe von Überlegungen Ludwik Flecks, Bruno Latours und David Kirbys wird die Bedeutsamkeit von Transformationsprozessen wissenschaftlicher Theorien gezeigt. Mit Veränderungsstrategien wie dem Visualisieren und Generieren von Aufmerksamkeit versuchen Wissenschaftler*innen, ihre Theorien sowohl im Kreis der Kolleg*innen als auch in der breiten Öffentlichkeit zu verbreiten. Wissenschaftskommunikation hat dabei einen epistemologischen Stellenwert für die wissenschaftliche Praxis. Die These, die in diesem Aufsatz vertreten wird, ist, dass Gedankenexperimente hierbei die Funktion von Vermittlungsinstrumenten zwischen den beiden Kreisen der wissenschaftsinternen und -externen Kommunikation einnehmen. Dies soll mit der Analyse eines konkreten Beispiels – dem Geiger-Gedankenexperiment aus der angewandten Ethik – plausibel gemacht werden. Bei der Untersuchung, wie dieses Gedankenexperiment wissenschaftlich bzw. populär dargestellt und diskutiert wird, fällt auf, dass dabei das Szenario im Zentrum der Aufmerksamkeit steht. Die eigentliche Argumentation hingegen wird meist nur aus dem Szenario missverständlich abgeleitet. Aus dieser Analyse lassen sich zwei mögliche Reaktionen ableiten: Zum einen kann sie als Begründung dafür verstanden werden, kritisch mit der Methode Gedankenexperiment umzugehen. Zum anderen – und dies ist die Position, die in diesem Aufsatz starkgemacht werden soll – kann die Beobachtung, dass das absurde Szenario des Geiger-Gedankenexperiments so populär ist, die These untermauern, dass Gedankenexperimente strategisch zur Vermittlung genutzt werden können. Absurde Szenarien können dabei hilfreich sein, weil sie Aufmerksamkeit wecken, in Erinnerung bleiben und zur Diskussion anregen. Insbesondere im Bereich der angewandten Ethik, die interdisziplinär diskutierte Themen behandelt, können Gedankenexperimente genutzt werden, um einen Diskurs zu öffnen und eine gemeinsame Diskussionsbasis zu schaffen.

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