generation-of-transgenic-c-elegans-by-biolistic-transformation

Generation of Transgenic <em>C. elegans</em> by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Nur factors are critical for proopiomelanocortin (POMC) induction by CRH in corticotrophs, but the pathways linking CRH to Nur are unknown. In this study we show that in AtT-20 corticotrophs CRH and cAMP induce Nur77 and Nurr1 expression and transcription at the NurRE site by protein kinase A (PKA) and calcium-dependent and -independent mechanisms. Calcium pathways depend on calmodulin kinase II (CAMKII) activity, and calcium-independent pathways are accounted for in part by MAPK activation (Rap1/B-Raf/MAPK-ERK kinase/ERK1/2), demonstrated by the use of molecular and pharmacological tools. AtT-20 corticotrophs express B-Raf, as do other cells in which cAMP stimulates MAPK. CRH/cAMP stimulated ERK2 activity and increased transcriptional activity of a Gal4-Elk1 protein, which was blocked by overexpression of dominant negative mutants and kinase inhibitors and stimulated by expression of B-Raf. The MAPK kinase inhibitors did not affect Nur77 and Nurr1 mRNA induction but blocked CRH or cAMP-stimulated Nur transcriptional activity. Moreover, MAPK stimulated phosphorylation and transactivation of Nur77. The functional impact of these pathways was confirmed at the POMC promoter. In conclusion, in AtT-20 corticotrophs the CRH/cAMP signaling that leads to Nur77/Nurr1 mRNA induction and transcriptional activation, and thus POMC expression, is dependent on protein kinase A and involves calcium/calmodulin kinase II (Nur induction/activation) and MAPK calcium-dependent and -independent (Nur phosphorylation-activation) pathways.

Activation and induction of NUR77/NURR1 in corticotrophs by CRH/cAMP: involvement of calcium, protein kinase A, and MAPK pathways.

Aktivierung und Induktion der NUR77/NURR1 in kortikotropen von CRH / cAMP: Beteiligung von Calcium, Proteinkinase A und MAPK.

Guanine nucleotide exchange factors (GEFs) and their associated GTP-binding proteins (G-proteins) are key regulatory elements in the signal transduction machinery that relays information from the extracellular environment into specific intracellular responses. Among them, the MAPK cascades represent ubiquitous downstream effector pathways. We have previously described that, analogous to the Ras-dependent activation of the Erk-1/2 pathway, members of the Rho family of small G-proteins activate the JNK cascade when GTP is loaded by their corresponding GEFs. Searching for novel regulators of JNK activity we have identified Epac (exchange protein activated by cAMP) as a strong activator of JNK-1. Epac is a member of a growing family of GEFs that specifically display exchange activity on the Rap subfamily of Ras small G-proteins. We report here that while Epac activates the JNK severalfold, a constitutively active (G12V) mutant of Rap1b does not, suggesting that Rap-GTP is not sufficient to transduce Epac-dependent JNK activation. Moreover, Epac signaling to the JNKs was not blocked by inactivation of endogenous Rap, suggesting that Rap activation is not necessary for this response. Consistent with these observations, domain deletion mutant analysis shows that the catalytic GEF domain is dispensable for Epac-mediated activation of JNK. These studies identified a region overlapping the Ras exchange motif domain as critical for JNK activation. Consistent with this, an isolated Ras exchange motif domain from Epac is sufficient to activate JNK. We conclude that Epac signals to the JNK cascade through a new mechanism that does not involve its canonical catalytic action, i.e. Rap-specific GDP/GTP exchange. This represents not only a novel way to activate the JNKs but also a yet undescribed mechanism of downstream signaling by Epac.

Activation of JNK by Epac is independent of its activity as a Rap guanine nucleotide exchanger.

Die Aktivierung von JNK durch Epac ist unabh&#xE4;ngig von seiner T&#xE4;tigkeit als Rap Guanin-Nukleotid-W&#xE4;rmetauscher.

Exposure to sources of UV radiation, such as sunlight, induces a number of cellular alterations that are highly dependent on its ability to affect gene expression. Among them, the rapid activation of genes coding for two subfamilies of proto-oncoproteins, Fos and Jun, which constitute the AP-1 transcription factor, plays a key role in the subsequent regulation of expression of genes involved in DNA repair, cell proliferation, cell cycle arrest, death by apoptosis, and tissue and extracellular matrix remodeling proteases. Besides being regulated at the transcriptional level, Jun and Fos transcriptional activities are also regulated by phosphorylation as a result of the activation of intracellular signaling cascades. In this regard, the phosphorylation of c-Jun by UV-induced JNK has been readily documented, whereas a role for Fos proteins in UV-mediated responses and the identification of Fos-activating kinases has remained elusive. Here we identify p38 MAPKs as proteins that can associate with c-Fos and phosphorylate its transactivation domain both in vitro and in vivo. This phosphorylation is transduced into changes in its transcriptional ability as p38-activated c-Fos enhances AP1-driven gene expression. Our findings indicate that as a consequence of the activation of stress pathways induced by UV light, endogenous c-Fos becomes a substrate of p38 MAPKs and, for the first time, provide evidence that support a critical role for p38 MAPKs in mediating stress-induced c-Fos phosphorylation and gene transcription activation. Using a specific pharmacological inhibitor for p38alpha and -beta, we found that most likely these two isoforms mediate UV-induced c-Fos phosphorylation in vivo. Thus, these newly described pathways act concomitantly with the activation of c-Jun by JNK/MAPKs, thereby contributing to the complexity of AP1-driven gene transcription regulation.

Phosphorylation of c-Fos by members of the p38 MAPK family. Role in the AP-1 response to UV light.

Die Phosphorylierung von c-Fos durch Mitglieder der p38-MAPK-Familie. Rolle in der AP-1-Reaktion auf UV-Licht.

cAMP stimulates proliferation in many cell types. For many years, cAMP-dependent protein kinase (PKA) represented the only known cAMP effector. PKA, however, does not fully mimic the action of cAMP, indicating the existence of a PKA-independent component. Since cAMP-mediated activation of the G-protein Rap1 and its phosphorylation by PKA are strictly required for the effects of cAMP on mitogenesis, we hypothesized that the Rap1 activator Epac might represent the PKA-independent factor. Here we report that Epac acts synergistically with PKA in cAMP-mediated mitogenesis. We have generated a new dominant negative Epac mutant that revealed that activation of Epac is required for thyroid-stimulating hormone or cAMP stimulation of DNA synthesis. We demonstrate that Epac's action on cAMP-mediated activation of Rap1 and cAMP-mediated mitogenesis depends on the subcellular localization of Epac via its DEP domain. Disruption of the DEP-dependent subcellular targeting of Epac abolished cAMP-Epac-mediated Rap1 activation and thyroid-stimulating hormone-mediated cell proliferation, indicating that an Epac-Rap-PKA signaling unit is critical for the mitogenic action of cAMP.

Epac, in synergy with cAMP-dependent protein kinase (PKA), is required for cAMP-mediated mitogenesis.

Lager stimuliert Verbreitung in vielen Zelltypen. Seit vielen Jahren vertreten Lager-abh&auml;ngige Proteinkinase (PKA) den einzigen bekannten cAMP Effektor. PKA, jedoch nicht vollst&auml;ndig die Wirkung von cAMP, nachbilden, die Existenz einer PKA-unabh&auml;ngige Komponente angibt. Da Lager-vermittelte Aktivierung von G-Protein-Rap1 und die Phosphorylierung von PKA sind unbedingt erforderlich, um die Auswirkungen des Lagers auf Mitogenesis, Hypothese wir, dass der Rap1-Aktivator Epac den PKA-unabh&auml;ngige-Faktor darstellen k&ouml;nnte. Hier berichten wir, dass Epac synergistisch mit PKA im Lager-vermittelte Mitogenesis wirkt. Wir haben eine neue dominante negative Epac Mutante generiert, die ergab, dass die Aktivierung der Epac f&uuml;r Thyreoidea - stimulierendes Hormon oder cAMP Stimulation der DNA-Synthese ben&ouml;tigt. Wir zeigen, dass Epac Aktion auf Lager-vermittelte Aktivierung der Rap1 und Lager-vermittelte Mitogenesis h&auml;ngt die subzellulare Lokalisation der Epac &uuml;ber seine DEP-Dom&auml;ne. St&ouml;rung der DEP-abh&auml;ngige subzellulare Ausrichtung der Epac Lager-Epac-vermittelter Rap1 Aktivierung und Schilddr&uuml;sen - Hormon-vermittelte Zellproliferation, darauf hinweist, dass eine Epac-Rap-PKA Signalling Unit kritisch f&uuml;r die mitogenen Aktion des Lagers ist abgeschafft.

EPAC, ist in Synergie mit Lager-abh&auml;ngige Proteinkinase (PKA), Lager-vermittelte Mitogenesis erforderlich.

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser(179) at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser(179) phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.

Phosphorylation-induced conformational changes in Rap1b: allosteric effects on switch domains and effector loop.

Rap1b hat in der Signaltransduktion der cAMP mitogenen Antwort verwickelt. Agonisten, die intrazellul&auml;ren cAMP sehr schnell ansteigen zu aktivieren (d.h. GTP-Bindung) und phosphoryliert Rap1b auf Ser(179) seine C-Terminus. Lager-abh&auml;ngige Proteinkinase (PKA)-vermittelten Phosphorylierung von Rap1b ist f&uuml;r Lager-abh&auml;ngige Mitogenesis, Tumorentstehung und Hemmung der AKT-Aktivit&auml;t erforderlich. Die Rolle der Phosphorylierung ist jedoch noch unbekannt. In dieser Studie benutzt wir Amid Wasserstoff/Deuterium Austausch Massenspektroskopie (DXMA) zur Bewertung der potenzieller Konformationsver&auml;nderungen und/oder Mobilit&auml;t induziert durch Phosphorylierung. Wir berichten hier DXMA Daten vergleichen Wechselkurse f&uuml;r PKA phosphoryliert (Rap1-P) und S179D Phosphomimetic (Rap1-D) Rap1b Proteine. Genau das gleiche, verhielt offenbart einen erh&ouml;hten Wechselkurs in diskrete Regionen entlang dem Protein, Rap1-P und Rap1-D; Diese Regionen geh&ouml;ren eine Domain rund um die Phosphorylierung-Website und unerwartet wechseln die beiden Schleifen. So werden lokale Effekte induziert durch Ser(179) Phosphorylierung phosphorylierter mit Effektor-Wechselwirkung beteiligt distale Dom&auml;nen kommunizieren. Diese Ergebnisse liefern eine mechanistische Erkl&auml;rung f&uuml;r die Wirkungen von Rap1 Phosphorylierung von PKA auf Effektor-Protein-Interaktion.

Induziert durch Phosphorylierung Konformationsver&auml;nderungen in Rap1b: allosterische Effekte auf Switch Domains und Effektor-Schleife.

Thyroid cancer is the most common type of endocrine malignancy, encompassing tumors with various levels of invasive growth and aggressiveness. Rap1GAP, a Rap1 GTPase-activating protein, inhibits the RAS superfamily protein Rap1 by facilitating hydrolysis of GTP to GDP. In this study, we analyzed 197 thyroid tumor samples and showed that Rap1GAP was frequently lost or downregulated in various types of tumors, particularly in the most invasive and aggressive forms of thyroid cancer. The downregulation was due to promoter hypermethylation and/or loss of heterozygosity, found in the majority of thyroid tumors. Treatment with demethylating agent 5-aza-deoxycytidine and/or histone deacetylation inhibitor trichostatin A induced gene reexpression in thyroid cells. A genetic polymorphism, Y609C, was seen in 7% of thyroid tumors but was not related to gene downregulation. Loss of Rap1GAP expression correlated with tumor invasiveness but not with specific mutations activating the mitogen-activated protein kinase pathway. Rap1GAP downregulation was required in vitro for cell migration and Matrigel invasion. Recovery of Rap1GAP expression inhibited thyroid cell proliferation and colony formation. Overall, our findings indicate that epigenetic or genetic loss of Rap1GAP is very common in thyroid cancer, where these events are sufficient to promote cell proliferation and invasion.

Downregulation of Rap1GAP through epigenetic silencing and loss of heterozygosity promotes invasion and progression of thyroid tumors.

Schilddr&uuml;senkrebs ist die h&auml;ufigste endokrine Hyperkalz&auml;mie, umfassende Tumoren mit unterschiedlichem invasiven Wachstum und Aggressivit&auml;t. Rap1GAP, ein Protein Rap1 GTPase-Aktivierung hemmt das RAS &Uuml;berfamilie Protein Rap1 durch Hydrolyse von GTP zum BIP zu erleichtern. In dieser Studie, die wir analysiert 197 Schilddr&uuml;sen Tumor Proben und zeigte, dass Rap1GAP h&auml;ufig verloren oder Downregulated in verschiedenen Arten von Tumoren, besonders in invasiven und aggressiven Formen von Schilddr&uuml;senkrebs. Das Medikament wurde durch Promotor Hypermethylierung und/oder Verlust der Heterozygotie, gefunden in der Mehrzahl der Tumoren der Schilddr&uuml;se. Behandlung mit demethylating Agent 5-Aza-Deoxycytidine und/oder Histon Deacetylierung Inhibitor Trichostatin A induzierte gen Reexpression in den Zellen der Schilddr&uuml;se. Eine genetische Polymorphie, Y609C, wurde in 7 % der Tumoren der Schilddr&uuml;se gesehen aber war nicht im Zusammenhang mit Gen-Medikament. Verlust des Rap1GAP Ausdrucks korreliert mit Tumor Invasivit&auml;t aber nicht mit spezifischen Mutationen der Mitogen-aktivierte Protein-Kinase-Signalweg aktivieren. Rap1GAP Medikament wurde in-vitro f&uuml;r Zellmigration und Matrigel Invasion erforderlich. Wiederherstellung der Rap1GAP Ausdruck gehemmt Schilddr&uuml;se Zellproliferation und Kolonie-Gr&uuml;ndung. Unsere Ergebnisse zeigen insgesamt, dass epigenetische oder genetischen Verlust der Rap1GAP sehr h&auml;ufig in Schilddr&uuml;senkrebs, wo diese Ereignisse zur F&ouml;rderung der Zellproliferation und Invasion ausreichen.

Heraufregulation des Rap1GAP durch epigenetische silencing und Verlust der Heterozygotie f&ouml;rdert Invasion und Progression von Tumoren der Schilddr&uuml;se.

cAMP is an ubiquitous second messenger. Localized areas with high cAMP concentration, i.e. cAMP microdomains, provide an elegant mechanism to generate signaling specificity and transduction efficiency. However, the mechanisms underlying cAMP effector targeting into these compartments is still unclear. Here we report the identification of radixin as a scaffolding unit for both cAMP effectors, Epac and PKA. This complex localizes in a submembrane compartment where cAMP synthesis occurs. Compartment disruption by shRNA and dominant negative approaches negatively affects cAMP action. Inhibition can be rescued by expression of Rap1b, a substrate for both Epac1 and PKA, but only in its GTP-bound and phosphorylated state. We propose that radixin scaffolds both cAMP effectors in a functional cAMP-sensing compartment for efficient signal transduction, using Rap1 as a downstream signal integrator.

Radixin assembles cAMP effectors Epac and PKA into a functional cAMP compartment: role in cAMP-dependent cell proliferation.

cAMP ist eine allgegenw&auml;rtige second Messenger. Lokalisierte Bereiche mit hohen cAMP-Konzentration, d.h. Lager Microdomains, bieten einen eleganten Mechanismus zur Signalisierung Spezifit&auml;t und Transduktion Effizienz zu generieren. Die Mechanismen, die cAMP-Effektor gezielt in diese F&auml;cher ist jedoch noch unklar. Hier berichten wir &uuml;ber die Identifizierung von Radixin als ein Ger&uuml;stbau-Einheit f&uuml;r beide Lager Effektoren, Epac und PKA. Dieser Komplex lokalisiert in einem Submembrane Fach in dem cAMP-Synthese erfolgt. Fach St&ouml;rung durch ShRNA und dominierenden negativen Ans&auml;tze wirkt sich nachteilig auf cAMP-Aktion. Hemmung von Ausdruck der Rap1b, ein Substrat f&uuml;r Epac1 und PKA, aber nur in seine GTP-gebundenen gerettet und phosphoryliert werden kann Zustand. Wir schlagen vor, dass Radixin Ger&uuml;ste beide Lager Effektoren in einem funktionalen Lager-Sensorik-Fach f&uuml;r effiziente Signaltransduktion, mithilfe von Rap1 als Integrator nachgeschalteten Signal.

Radixin assembliert cAMP Effektoren Epac und PKA in einem funktionalen cAMP Fach: Rolle im Lager-abh&auml;ngige Zellproliferation.

Bacteria have many voltage- and ligand-gated ion channels, and population-level measurements indicate that membrane potential is important for bacterial survival. However, it has not been possible to probe voltage dynamics in an intact bacterium. Here we developed a method to reveal electrical spiking in Escherichia coli. To probe bacterial membrane potential, we engineered a voltage-sensitive fluorescent protein based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) in E. coli revealed electrical spiking at up to 1 hertz. Spiking was sensitive to chemical and physical perturbations and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated.

Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein.

Bakterien haben viele Spannung und ligandengesteuerte Ionenkan&auml;le und Bev&ouml;lkerungsebene Messungen zeigen, dass potenzielle Membran f&uuml;r bakterielle &Uuml;berleben wichtig ist. Jedoch war es nicht m&ouml;glich, die Spannung Dynamik in einer intakten Bakterium Pr&uuml;fpunkt. Hier haben wir eine Methode zu zeigen, elektrische spiking in Escherichia coli entwickelt. Um m&ouml;gliche bakterielle Membran-Sonde entwickelt wir eine Spannung-empfindlichen fluoreszierenden Proteins basierend auf gr&uuml;n-absorbierenden Proteorhodopsin. Ausdruck des Proteorhodopsin optische Proton Sensors (PROPS) in E. Coli offenbart elektrische spiking bis 1 Hertz. Spiking war empfindlich auf chemische und physikalische Perturbationen und fiel mit rapid Efflux von ein klein-Molek&uuml;l-Fluorophor, was darauf hindeutet, dass bakterielle Efflux Maschinen elektrisch geregelt werden kann.

Elektrische spiking in Escherichia coli untersucht mit einem fluoreszierenden Protein der Spannung-Angabe.

The nuclear receptor DAF-12 has roles in normal development, the decision to pursue dauer development in unfavorable conditions, and the modulation of adult aging. Despite the biologic importance of DAF-12, target genes for this receptor are largely unknown. To identify DAF-12 targets, we performed chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays. We identified 1,175 genomic regions to be bound in vivo by DAF-12, and these regions are enriched in known DAF-12 binding motifs and act as DAF-12 response elements in transfected cells and in transgenic worms. The DAF-12 target genes near these binding sites include an extensive network of interconnected heterochronic and microRNA genes. We also identify the genes encoding components of the miRISC, which is required for the control of target genes by microRNA, as a target of DAF-12 regulation. During reproductive development, many of these target genes are misregulated in daf-12(0) mutants, but this only infrequently results in developmental phenotypes. In contrast, we and others have found that null daf-12 mutations enhance the phenotypes of many miRISC and heterochronic target genes. We also find that environmental fluctuations significantly strengthen the weak heterochronic phenotypes of null daf-12 alleles. During diapause, DAF-12 represses the expression of many heterochronic and miRISC target genes, and prior work has demonstrated that dauer formation can suppress the heterochronic phenotypes of many of these target genes in post-dauer development. Together these data are consistent with daf-12 acting to ensure developmental robustness by committing the animal to adult or dauer developmental programs despite variable internal or external conditions.

DAF-12 regulates a connected network of genes to ensure robust developmental decisions.

Die Kernempf&auml;nger DAF-12 hat Rollen in der normalen Entwicklung, die Entscheidung zur Entwicklung der Dauer in ung&uuml;nstigen Bedingungen und die Beeinflussung der Erwachsenen Altern zu verfolgen. Trotz der biologischen Bedeutung der DAF-12 sind Zielgene f&uuml;r diesen Rezeptor weitgehend unbekannt. Um DAF-12 Ziele zu identifizieren, f&uuml;hrten wir Chromatin Immunopr&auml;zipitation gefolgt von Hybridisierung in ganz-Genom-Fliesen-Arrays. Wir identifiziert 1.175 genomische Regionen von DAF-12 in-vivo gebunden zu sein, und diese Regionen sind in bekannten DAF-12 Bindung Motive bereichert und fungieren als DAF-12-Response-Elemente in transfected Zellen und transgenen W&uuml;rmer. Die DAF-12-Zielgene nahe diese Bindungsstellen enthalten ein dichtes Netz von miteinander verbundenen Heterochronic und MicroRNA Gene. Wir ermitteln auch die Genen Komponenten von der MiRISC, die als Ziel der DAF-12 Verordnung zur Bek&auml;mpfung der Zielgene von MicroRNA, erforderlich ist. W&auml;hrend der reproduktiven Entwicklung viele diese Zielgene sind misregulated in daf-12(0)-Mutanten, aber dies f&uuml;hrt nur selten zu Entwicklungsst&ouml;rungen Ph&auml;notypen. Im Gegensatz dazu haben wir u.a. festgestellt, dass null Daf-12 Mutationen die Ph&auml;notypen viele MiRISC und Heterochronic Zielgene erh&ouml;hen. Wir finden auch, dass die Umwelt bedingte Schwankungen die schwachen Heterochronic Ph&auml;notypen null Daf-12 Allele deutlich st&auml;rken. W&auml;hrend Diapause DAF-12 unterdr&uuml;ckt den Ausdruck viele Heterochronic und MiRISC Zielgene und vorherige Arbeit hat gezeigt, dass Dauer-Gr&uuml;ndung die Heterochronic-Ph&auml;notypen vieler dieser Zielgene in Post-Dauer Entwicklung unterdr&uuml;cken kann. Zusammen sind diese Daten konsistent mit Daf-12 handeln, um Entwicklungsst&ouml;rungen Robustheit zu gew&auml;hrleisten, indem Sie das Tier zu Erwachsenen oder Dauer Entwicklungs-Programme trotz variabler interne oder externe Bedingungen verpflichten.

DAF-12 regelt ein verbundenes Netzwerk von Genen, die robuste entwicklungspolitischen Entscheidungen sicherzustellen.

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieved this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately tenfold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio >10. Arch(D95N) lacked endogenous proton pumping and had 50% greater sensitivity than wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits and electrophysiology in systems for which electrode-based techniques are challenging.

Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.

Zuverl&auml;ssige Optische Erkennung von einzelnen Aktionspotentiale in S&auml;ugetier-Neuronen war einer der am l&auml;ngsten bestehende Herausforderungen der Neurowissenschaften. Hier erreichten wir dieses Ziel mit die endogene Fluoreszenz eines mikrobiellen Rhodopsin-Proteins, Archaerhodopsin 3 (Arch) von Halorubrum Sodomense, in kultivierten Ratte hippocampal Neuronen exprimiert. Dieses genetisch codierte Spannung-Kennzeichen ausgestellt eine etwa zehnfache Verbesserung Empfindlichkeit und Geschwindigkeit &uuml;ber vorhandene Protein-basierte Spannung Indikatoren, mit einem ungef&auml;hr lineare zweifachen Anstieg der Helligkeit zwischen-150 mV und + 150 mV und eine Reaktionszeit von Buszykluszeiten. Bogen erkannt einzigen elektrisch ausgel&ouml;st Aktionspotentiale mit einem optischen Signal-Rausch-Verh&auml;ltnis &gt; 10. Arch(D95N) fehlte endogene Proton Pumpen und hatte 50 % h&ouml;here Empfindlichkeit als Wildtyp aber eine langsamere Reaktion (41 ms). Dennoch wurde Arch(D95N) einzelne Aktionspotentiale beschlossen. Mikrobielle Rhodopsin-basierte Spannung Indikatoren Versprechen optische Verh&ouml;r komplexe neuronale Schaltkreise und Elektrophysiologie in Systemen aktivieren f&uuml;r die Elektrode-basierte Techniken anspruchsvoll sind.

Article	Year
Activation and induction of NUR77/NURR1 in corticotrophs by CRH/cAMP: involvement of calcium, protein kinase A, and MAPK pathways. Molecular endocrinology (Baltimore, Md.)\| PubMed ID: 12089357	2002
Activation of JNK by Epac is independent of its activity as a Rap guanine nucleotide exchanger. The Journal of biological chemistry\| PubMed ID: 12783872	2003
Phosphorylation of c-Fos by members of the p38 MAPK family. Role in the AP-1 response to UV light. The Journal of biological chemistry\| PubMed ID: 15708845	2005
Epac, in synergy with cAMP-dependent protein kinase (PKA), is required for cAMP-mediated mitogenesis. The Journal of biological chemistry\| PubMed ID: 18063584	2008
Phosphorylation-induced conformational changes in Rap1b: allosteric effects on switch domains and effector loop. The Journal of biological chemistry\| PubMed ID: 19651783	2009
Downregulation of Rap1GAP through epigenetic silencing and loss of heterozygosity promotes invasion and progression of thyroid tumors. Cancer research\| PubMed ID: 20124489	2010
Radixin assembles cAMP effectors Epac and PKA into a functional cAMP compartment: role in cAMP-dependent cell proliferation. The Journal of biological chemistry\| PubMed ID: 21047789	2011
Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein. Science (New York, N.Y.)\| PubMed ID: 21764748	2011
DAF-12 regulates a connected network of genes to ensure robust developmental decisions. PLoS genetics\| PubMed ID: 21814518	2011
Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nature methods\| PubMed ID: 22120467	2012

Daniel Hochbaum

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