LRP1 modulates APP trafficking and APP metabolism within compartments of the secretory pathway. Increased AICD generation is ineffective in nuclear translocation and transcriptional activation

LRP1 modulates APP trafficking and metabolism within compartments of the secretory pathway The amyloid precursor protein (APP) is the parent protein to the amyloid beta peptide (Abeta) and is a central player in Alzheimer’s disease (AD) pathology. Abeta liberation depends on APP cleavage by beta- and gamma-secretases. To date, only a unilateral view of APP processing exists, excluding other proteins, which might be transported together and/or processed dependent on each other by the secretases described above. The low density lipoprotein receptor related protein 1 (LRP1) was shown to function as such a mediator of APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. Therefore, we wanted to investigate whether LRP1 can mediate APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, we demonstrate that APP trafficking is strongly influenced by LRP1 transport through the endoplasmic reticulum (ER) and Golgi compartments. LRP1-constructs with ER- and Golgi-retention motifs (LRP-CT KKAA, LRP-CT KKFF) had the capacity to retard APP trafficking at the respective steps in the secretory pathway. Here, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases. Increased AICD generation is ineffective in nuclear translocation and transcriptional activity A sequence of amyloid precursor protein (APP) cleavages gives rise to the APP intracellular domain (AICD) together with amyloid beta peptide (Abeta) and/or p3 fragment. One of the environmental factors identified favouring the accumulation of AICD appears to be a rise in intracellular pH. This accumulation is a result of an abrogated cleavage event and does not extend to other secretase substrates. AICD can activate the transcription of artificially expressed constructs and many downstream gene targets have been discussed. Here we further identified the metabolism and subcellular localization of the constructs used in this well documented gene reporter assay. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely that cleaved from C83. Furthermore, the AICD surplus is not transcriptionally active but rather remains membrane tethered and free in the cytosol where it interacts with Fe65. However, Fe65 is still essential in AICD mediated transcriptional transactivation although its exact role in this set of events is unclear.

Der Wirkungsmechanismus von Neurosteroiden auf das Cytoskelett neuronaler Zellen

Neurosteroide können langsame genomische und schnelle nicht-genomische Effekte zeigen. Die Synthese und der Metabolismus von Neurosteroiden werden entwicklungsbedingt reguliert. In den letzten Jahren sind immer mehr schnelle Steroideffekte bekannt geworden, die sowohl über klassische als auch über nicht-klassische Rezeptoren laufen. Zum heutigen Stand der Forschung sind die morphologischen Effekte von Neurosteroiden auf das neuronale Cytoskelett und die involvierten Signalkaskaden noch weitgehend unerforscht. In diesem Zusammenhang stellen sich auch die Fragen nach den verantwortlichen Rezeptoren und dem Transportmechanismus sowie der subzellulären Lokalisation der Steroide. Die im Rahmen meiner Promotion erhaltenen Ergebnisse zeigen, dass die Steroide DHEA und Testosteron eine Reorganisation des Aktincytoskeletts in neuronalen Zellen induzieren und dass diese Effekte diesen Steroiden und nicht ihren Folgemetaboliten zuzuordnen sind. DHEA bewirkt die Kontraktion der Zellen, eine erhöhte Ausbildung von Stressfasern und fokalen Adhäsionskomplexen sowie die Bildung von Filopodien. Der diesen Effekten zu Grunde liegende Signalweg konnte eindeutig identifiziert werden. DHEA induziert in neuronalen Zellen die Aktivierung des Rho-Signalwegs. Diese Aktivierung führt zu einem erhöhten Phosphorylierungsstatus der regulatorischen leichten Kette von Myosin II (MRLC) an Serin 19 und der damit verbundenen erhöhten Myosin-Aktin-Interaktion. Die Ausbildung von Filopodien wird vermutlich über eine Aktivierung der GTPase Cdc42 vermittelt. Testosteron induziert das Auswachsen langer Neuriten sowie eine Verminderung von Stressfasern in neuronalen Zellen. Diese Effekte sind abhängig von der Aktivität der PI3-Kinase. Die im Rahmen dieser Arbeit gewonnenen Erkenntnisse deuten darauf hin, dass Testosteron über die PI3-Kinase und FAK den Rac-Signalweg induziert, da es zu einer Inhibierung des Rho-Signalwegs kommt. Zahlreiche Erkenntnisse weisen darauf hin, dass DHEA und Testosteron die Aktivierung der beteiligten Signalwege über einen G-Protein gekoppelten Rezeptor induzieren. DHEA und Testosteron beeinflussen auch die Expression und die Lokalisation der regulatorischen leichten Ketten von Myosin II. Im Gegensatz zu DHEA (Lokalisation der MRLC in der kortikalen Region der Zelle), induziert Testosteron eine Umlokalisation der MRLC in den Zellkern. Daher ist es denkbar, dass die MRLCs, wie auch Aktin, als Transkriptionsfaktoren wirken können. Die Synthese eines funktionalen, fluoreszierenden DHEA-Derivats (DHEA-Bodipy) ermöglichte erstmals, den Transport und die subzelluläre Lokalisation von DHEA in neuronalen Zellen zu beobachten. DHEA-Bodipy wird in neuronalen Zellen in den Mitochondrien lokalisiert. Diese Lokalisation ergibt völlig neue Ansätze im Verständnis zellulärer Wirkungsorte von Steroiden und beteiligter Rezeptoren. Das in meiner Arbeit vorgestellte Verfahren zur Fluoreszenzmarkierung von Steroiden bietet vielfältige Möglichkeiten im Einsatz zellbiologischer Methoden. Nach diesem Verfahren hergestellte, fluoreszierende Steroide eignen sich aufgrund ihrer Stabilität sehr gut für die Untersuchung des Transports und der subzellulären Lokalisation von Steroiden an fixierten und lebenden Zellen sowie für Colokalisationsexperimente. Diese Methode grenzt somit auch die Anzahl möglicher molekularer Interaktionspartner ein. Für Testosteron konnte ebenfalls ein fluoreszierendes Testosteron-Derivat (Testosteron-Bodipy) synthetisiert werden. Die Aufklärung der Effekte von Steroiden auf das neuronale Cytoskelett und der beteiligten Signalkaskaden sowie die Identifizierung der zellulären Wirkungsorte ermöglichen therapeutische Ansätze zur Behandlung neurodegenerativer Erkrankungen, deren Ursachen in Abnormitäten des Cytoskeletts oder fehlregulierter Neurosteroidogenese zu begründen sind.

Isolierung und Charakterisierung der ER-residenten Aminopeptidase ERMP1 und Untersuchung ihrer Funktion in der Prozessierung MHC I restringierter CTL Epitope

Die effiziente Generierung von Peptid-Epitopen aus zelleigenen oder viralen Proteinen für die Präsentation auf „Major Histocompatibility Complex I“ (MHC I) Molekülen ist essentiell für die Aktivierung des adaptiven Immunsystems und die Effektorfunktion der CD8+ zytotoxischen T-Zellen (CTLs). CTLs erkennen diese Peptide in Kontext mit MHC I Molekülen über ihren spezifischen T-Zellrezeptor (TCR). Die Generierung dieser Epitope ist das Resultat eines komplexen proteolytischen Prozesses, der im Zytosol und im endoplasmatischen Retikulum (ER) stattfindet. Im Zytosol generiert das Proteasom N-terminal verlängerte Epitop-Vorläufer. Diese werden durch weitere zytosolische Proteasen abgebaut, es sei denn, sie werden durch den „transporter associated with antigen processing“ (TAP) in das ER transportiert. Dort werden sie durch Aminopeptidasen getrimmt, um den Bindungsvoraussetzungen der MHC I Moleküle zu genügen. Im murinen System ist die „ER aminopeptidase associated with antigen processing“ (ERAAP) die bislang einzige beschriebene Aminopeptidase, die dieses N-terminale Trimming von CTL Epitopen vermitteln kann. Das Profil der proteolytischen Aktivität in angereichertem murinen ER kann jedoch nicht allein durch die Aktivität von ERAAP erklärt werden, was auf die Anwesenheit weiterer Aminopeptidasen mit einer potentiellen Funktion in der Antigenprozessierung hinweist. In dieser Arbeit konnte die immunologisch bislang noch nicht beschriebene Aminopeptidase ERMP1 (endoplasmic reticulum metallopeptidase 1) im murinen ER identifiziert werden. Nach Aufreinigung muriner Mikrosomen und anschließender Anionenaustausch-Chromatographie wurden die gesammelten Fraktionen mit fluorogenen Substraten auf Aminopeptidase-Aktivität getestet. Durch massenspektrometrische Analyse konnten in den beobachteten Peaks die schon beschriebenen Aminopeptidasen ERAAP, die „insulin regulated aminopeptidase“ IRAP und die immunologisch bislang nicht beschriebene Aminopeptidase ERMP1 identifiziert werden. Durch Fluoreszenzmikroskopie konnte die intrazelluläre Lokalisation von ERMP1 im ER durch Kolokalisation mit TAP verifiziert werden. Wie viele Komponenten des MHC I Prozessierungsweges wird auch die Expression von ERMP1 durch IFN-γ stimuliert. Dies macht ERMP1 zu einer potentiellen zweiten trimmenden Aminopeptidase im murinen ER. Überexpression von ERMP1 hat einen allelspezifischen Einfluss auf die globale MHC I Präsentation auf der Zelloberfläche und durch Überexpression und shRNA vermitteltes gene silencing konnte außerdem ein epitopspezifischer Effekt nachgewiesen werden. Da N-terminales Trimming durch ERAAP mit der Evasion von Tumoren und veränderter Immundominanz assoziiert wird, ist die detaillierte Charakterisierung der Aminopeptidase ERMP1 ein wichtiger Schritt zum Verständnis der MHC I Antigen-Prozessierung und der Generierung von CTL Epitopen im ER.

Unmittelbare, dauerhafte sowie evolutionäre Anpassung an Fremdstoffexpositionen

Eine der Hauptursachen für unerwünschte oder reduzierte Wirkungen von Medikamenten ist die Induktion von Enzymen und Transportern des Medikamentenstoffwechsels. Diese Induktion stellt ursprünglich eine physiologische Reaktion auf die Aufnahme von potentiell schädlichen Fremdstoffen aus der Umwelt dar und sichert so die Gesundheit und Fortpflanzungsfähigkeit von Lebewesen. Beim Menschen sowie anderen Säugetieren werden Fremdstoffe hauptsächlich von den nukleären Rezeptoren PXR und CAR in der Leber und im Dünndarm detektiert. Zu den Medikamenten, welche über PXR und CAR wirken, gehören unter anderem Antikonvulsiva, Statine, antiretrovirale Medikamente, Glucocorticoide sowie Antimykotika. Die durch Fremdstoffe aktivierten Transkriptionsfaktoren PXR und CAR steigern die Menge der Enzyme und Transporter des Fremdstoffmetabolismus. Hierzu zählen vor allem die Cytochrom P450-Enzyme (Cyp-Enzyme) mit breitem Substratspektrum oder der Transporter MDR1, welcher eine Vielzahl von Substraten über Membranen transportiert. Durch die Biotransformation werden die induzierenden, lipophilen Substanzen so modifiziert, dass sie leichter über den Urin oder die Galle ausgeschieden werden können. \r\nDie Dauer der Induktion sollte auf die Zeit der Fremdstoffexposition beschränkt sein, um Störungen des endogenen Stoffwechsels zu vermindern. In dieser Arbeit werden jedoch Hinweise auf dauerhafte und sogar generationsübergreifende Effekte von Medikamenten in Mäusen geliefert. Nachkommen von Müttern, welche bereits vor ihrer Verpaarung einmalig mit TCPOBOP, einem Liganden des murinen CAR, injiziert wurden, hatten eine ungefähr 100-fach gesteigerte Genexpression von Cyp2b10. Auch gab es Expressionsänderungen von Genen, deren Produkte eine Rolle im Lipidstoffwechsel sowie bei Immunkrankheiten spielen. Eine Hochdurchsatz-RNA-Sequenzierung der injizierten Elterngeneration ergab außerdem dauerhafte Expressionsveränderungen anderer Gene des Medikamentenstoffwechsels sowie von Genen mit Verbindung zum Energiemetabolismus. \r\nBerücksichtigt man die enge evolutionäre Verwandtschaft der nukleären Rezeptoren CAR und PXR, sind Langzeitveränderungen auch für PXR möglich und wurden im Verlauf dieser Arbeit ebenfalls untersucht. Eine Hochdurchsatz-Sequenzierung ergab für Mäuse, welche mit dem PXR-Aktivator PCN induziert wurden, dass selbst noch drei Monate nach der Exposition Gene verändert exprimiert waren, welche im Zusammenhang mit Lebernekrosen stehen. Bei Nachkommen von PCN-injizierten Müttern wurden Gene unterschiedlich exprimiert, welche eine Rolle bei der Energiehomöostase sowie im Glukosestoffwechsel spielen. Im Erwachsenenalter sind bei diesen Nachkommen darüber hinaus noch Gene unterschiedlich exprimiert, deren Produkte eine Funktion in der Immunantwort haben. \r\nDa Erwachsene aufgrund ihrer Lebensdauer sowie der absoluten Krankheitshäufigkeit wesentlich öfter Kontakt mit Fremdstoffen haben, war medizinisch von besonderem Interesse, ob anhaltende Genexpressionsänderungen auch bei Erwachsenen zu beobachten sind. So konnte im Rahmen dieser Arbeit gezeigt werden, dass auch einmalig exponierte Adulttiere Gene dauerhaft verändert exprimieren und die Veränderungen im Medikamentenstoffwechsel an die nächste Generation übertrugen. \r\n\r\nBisher sind klinische Studien zur Risikobewertung von Medikamenten (Pharmakovigilanz) nicht generationsübergreifend angelegt. Diese Arbeit gibt Anstöße dafür, dass dies in Zukunft für viel mehr Medikamente notwendig werden könnte. Neben Veränderungen im Medikamentenstoffwechsel ergeben sich Nebenwirkungen von PXR- und CAR-Liganden vor allem aus ihrer Beteiligung an endogenen Stoffwechselwegen. Nach Aktivierung von CAR, welcher viele metabolische Stoffwechselwege steuert, treten beispielsweise Störungen des Energiestoffwechsels auf. Ein tieferes Verständnis der Rezeptoraktivität von CAR samt einer gezielten Modulierung seiner Aktivität würde wichtige Beiträge zum Verständnis der Regulation des Fremdstoffmetabolismus sowie der Entstehung von Nebenwirkungen durch eine Behandlung mit CAR-Liganden leisten. Dauerhafte Veränderungen endogener Stoffwechselwege könnten dann möglicherweise über eine pharmakologische Modulierung der CAR-Aktivität reduziert werden. \r\nZu diesem Zweck wurden im Verlauf dieser Arbeit die CAR-Rezeptoren der Amphibien (Xenopus tropicalis, Xenopus laevis) und Reptilien (Anolis carolinensis) erstmals kloniert, als Proteine exprimiert und charakterisiert. Vergleiche zwischen Tierarten ermöglichen ein besseres Verständnis von humanen Proteinen. Funktionelle Analysen ergaben Ähnlichkeiten des Xenopus laevis-CAR mit dem PXR der Säugetiere: eine niedrige basale Aktivität sowie eine starke Induzierbarkeit durch Liganden. In weiteren funktionellen Analysen wurden die Determinanten der basalen Aktivität des Xenopus laevis-CAR untersucht. Die basale Aktivität war nicht abhängig von der subzellulären Lokalisation, sondern ergab sich aus der Proteinstruktur, welche nur beim CAR der Landvertebraten in einer aktiven Konformation fixiert ist. Ähnlich dem PXR der Säugetiere besitzt CAR der Amphibien eine Aktivierungsdomäne, welche erst durch Ligandenbindung in eine aktive Konformation gebracht wird. Mutationen einzelner Aminosäuren zum jeweils humanen Homolog erhöhten die basale Aktivität des Xenopus laevis-CAR auf die des humanen Rezeptors. Diese Mutanten mit erhöhter basalen Aktivität zeigten eine verstärkte Interaktion mit dem Kofaktor PGC-1a, einem Regulator des Energiestoffwechsels bei Säugetieren. Die hepatischen Zielgene des CAR der Amphibien überlappen zum Teil mit den humanen Zielgenen und spielen ebenfalls eine Rolle im Energiestoffwechsel.

Intraflagellar transport in the developing and mature vertebrate retina

Intraflagellar transport (IFT) is required for the assembly and maintenance of cilia. In this study we analyzed the subcellular localization of IFT proteins in retinal cells by correlative high-resolution immunofluorescence and immunoelectron microscopy. The rod photoreceptor cell was used as a model system to analyze protein distribution in cilia. To date the expression of IFT proteins has been described in the ciliary region without deciphering the precise spatial and temporal subcellular localization of IFT proteins, which was the focus of my work. rnThe establishment of the pre-embedding immunoelectron method was an important first step for the present doctoral thesis. Results of this work reveal the differential localization of IFT20, IFT52, IFT57, IFT88, IFT140 in sub-ciliary compartments and also their presence in non-ciliary compartments of retinal photoreceptor cells. Furthermore, the localization of IFT20, IFT52 and IFT57 in dendritic processes of non-ciliated neurons indicates that IFT protein complexes also operate in non-ciliated cells and may participate in intracellular vesicle trafficking in eukaryotic cells in general.rnIn addition, we have investigated the involvement of IFT proteins in the ciliogenesis of vertebrate photoreceptor cilia. Electron microscopy analyses revealed six morphologically distinct stages. The first stages are characterized by electron dense centriolar satellites and a ciliary vesicle, while the formation of a ciliary shaft and of the light sensitive outer segment disks are features of the later stages. IFT proteins were expressed during all stages of photoreceptor cell development and found to be associated with the ciliary apparatus. In addition to the centriole and basal body IFT proteins are present in the photoreceptor cytoplasm, associated with centriolar satellites, post-Golgi vesicles and with the ciliary vesicle. Therewith the data provide an evidence for the involvement of IFT proteins during ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium of photoreceptor cells. Moreover, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis indicates roles of IFT proteins beyond their well-established function for IFT in mature cilia and flagella. rn

Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells--implications for milk cholesterol secretion

The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used to test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at the end of lactation, during the dry period (DP), and early lactation using immunohistochemical and immunofluorescence approaches. The presence of ABCA1 and ABCG1 in MFG isolated from fresh milk was determined by immunofluorescence. The ABCA1 protein expression in MEC, expressed as arbitrary units, was higher during the end of lactation (12.2±0.24) and the DP (12.5±0.22) as compared with during early lactation (10.2±0.65). In contrast, no significant change in ABCG1 expression existed between the stages. Throughout the cycle, ABCA1 and ABCG1 were detected in the apical (41.9±24.8 and 49.0±4.96% of cows, respectively), basal (56.2±28.1 and 54.6±7.78% of cows, respectively), or entire cytoplasm (56.8±13.4 and 61.6±14.4% of cows, respectively) of MEC, or showed combined localization. Unlike ABCG1, ABCA1 was absent at the apical aspect of MEC during early lactation. Immunolabeling experiments revealed the presence of ABCA1 and ABCG1 in MFG membranes. Findings suggest a differential, functional stage-dependent role of ABCA1 and ABCG1 in cholesterol homeostasis of the MG epithelium. The presence of ABCA1 and ABCG1 in MFG membranes suggests that these proteins are involved in cholesterol exchange between MEC and alveolar milk.

Mining for Class-Specific Motifs in Protein Sequence Classification

Background: In protein sequence classification, identification of the sequence motifs or n-grams that can precisely discriminate between classes is a more interesting scientific question than the classification itself. A number of classification methods aim at accurate classification but fail to explain which sequence features indeed contribute to the accuracy. We hypothesize that sequences in lower denominations (n-grams) can be used to explore the sequence landscape and to identify class-specific motifs that discriminate between classes during classification. Discriminative n-grams are short peptide sequences that are highly frequent in one class but are either minimally present or absent in other classes. In this study, we present a new substitution-based scoring function for identifying discriminative n-grams that are highly specific to a class. Results: We present a scoring function based on discriminative n-grams that can effectively discriminate between classes. The scoring function, initially, harvests the entire set of 4- to 8-grams from the protein sequences of different classes in the dataset. Similar n-grams of the same size are combined to form new n-grams, where the similarity is defined by positive amino acid substitution scores in the BLOSUM62 matrix. Substitution has resulted in a large increase in the number of discriminatory n-grams harvested. Due to the unbalanced nature of the dataset, the frequencies of the n-grams are normalized using a dampening factor, which gives more weightage to the n-grams that appear in fewer classes and vice-versa. After the n-grams are normalized, the scoring function identifies discriminative 4- to 8-grams for each class that are frequent enough to be above a selection threshold. By mapping these discriminative n-grams back to the protein sequences, we obtained contiguous n-grams that represent short class-specific motifs in protein sequences. Our method fared well compared to an existing motif finding method known as Wordspy. We have validated our enriched set of class-specific motifs against the functionally important motifs obtained from the NLSdb, Prosite and ELM databases. We demonstrate that this method is very generic; thus can be widely applied to detect class-specific motifs in many protein sequence classification tasks. Conclusion: The proposed scoring function and methodology is able to identify class-specific motifs using discriminative n-grams derived from the protein sequences. The implementation of amino acid substitution scores for similarity detection, and the dampening factor to normalize the unbalanced datasets have significant effect on the performance of the scoring function. Our multipronged validation tests demonstrate that this method can detect class-specific motifs from a wide variety of protein sequence classes with a potential application to detecting proteome-specific motifs of different organisms.

Phosphorylation of sst2 receptors in neuroendocrine tumors after octreotide treatment of patients

Somatostatin analogues, which are used to treat neuroendocrine tumors, target the high levels of somatostatin receptor subtype 2 (SSTR1; alias sst2) expressed in these cancers. However, some tumors are resistant to somatostatin analogues, and it is unknown whether the defect lies in sst2 activation or downstream signaling events. Because sst2 phosphorylation occurs rapidly after receptor activation, we examined whether sst2 is phosphorylated in neuroendocrine tumors. The sst2 receptor phosphorylation was evaluated by IHC and Western blot analysis with the new Ra-1124 antibody specific for the sst2 receptor phosphorylated at Ser341/343 in receptor-positive neuroendocrine tumors obtained from 10 octreotide-treated and 7 octreotide-naïve patients. The specificity, time course, and subcellular localization of sst2 receptor phosphorylation were examined in human embryo kinase-sst2 cell cultures by immunofluorescence and confocal microscopy. All seven octreotide-naïve tumors displayed exclusively nonphosphorylated cell surface sst2 expression. In contrast, 9 of the 10 octreotide-treated tumors contained phosphorylated sst2 that was predominantly internalized. Western blot analysis confirmed the IHC data. Octreotide treatment of human embryo kinase-sst2 cells in culture demonstrated that phosphorylated sst2 was localized at the plasma membrane after 10 seconds of stimulation and was subsequently internalized into endocytic vesicles. These data show, for the first time to our knowledge, that phosphorylated sst2 is present in most gastrointestinal neuroendocrine tumors from patients treated with octreotide but that a striking variability exists in the subcellular distribution of phosphorylated receptors among such tumors.

The Trypanosoma brucei cAMP phosphodiesterases TbrPDEB1 and TbrPDEB2: flagellar enzymes that are essential for parasite virulence

Cyclic nucleotide specific phosphodiesterases (PDEs) are pivotal regulators of cellular signaling. They are also important drug targets. Besides catalytic activity and substrate specificity, their subcellular localization and interaction with other cell components are also functionally important. In contrast to the mammalian PDEs, the significance of PDEs in protozoal pathogens remains mostly unknown. The genome of Trypanosoma brucei, the causative agent of human sleeping sickness, codes for five different PDEs. Two of these, TbrPDEB1 and TbrPDEB2, are closely similar, cAMP-specific PDEs containing two GAF-domains in their N-terminal regions. Despite their similarity, these two PDEs exhibit different subcellular localizations. TbrPDEB1 is located in the flagellum, whereas TbrPDEB2 is distributed between flagellum and cytoplasm. RNAi against the two mRNAs revealed that the two enzymes can complement each other but that a simultaneous ablation of both leads to cell death in bloodstream form trypanosomes. RNAi against TbrPDEB1 and TbrPDEB2 also functions in vivo where it completely prevents infection and eliminates ongoing infections. Our data demonstrate that TbrPDEB1 and TbrPDEB2 are essential for virulence, making them valuable potential targets for new PDE-inhibitor based trypanocidal drugs. Furthermore, they are compatible with the notion that the flagellum of T. brucei is an important site of cAMP signaling.--Oberholzer, M., Marti, G., Baresic, M., Kunz, S., Hemphill, A., Seebeck, T. The Trypanosoma brucei cAMP phosphodiesterases TbrPDEB1 and TbrPDEB2: flagellar enzymes that are essential for parasite virulence.

The use of light- and electron microscopy for studies on the cell- and molecular biology of parasites and parasitic diseases

Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host.

Expression of nitric oxide synthase III in human thyroid follicular cells: evidence for increased expression in hyperthyroidism

Nitric oxide mediates a wide array of cellular functions in many tissues. It is generated by three known isoforms of nitric oxide synthases (NOS). Recently, the endothelial isoform, NOSIII, was shown to be abundantly expressed in the rat thyroid gland and its expression increased in goitrous glands. In this study, we analyzed whether NOSIII is expressed in human thyroid tissue and whether levels of expression vary in different states of thyroid gland function. Semiquantitative RT-PCR was used to assess variations in NOSIII gene expression in seven patients with Graves' disease, one with a TSH-receptor germline mutation and six hypothyroid patients (Hashimoto's thyroiditis). Protein expression and subcellular localization were determined by immunohistochemistry (two normal thyroids, five multinodular goiters, ten hyperthyroid patients and two hypothyroid patients). NOSIII mRNA was detected in all samples: the levels were significantly higher in tissues from hyperthyroid patients compared with euthyroid and hypothyroid patients. NOSIII immunoreactivity was detected in vascular endothelial cells, but was also found in thyroid follicular cells. In patients with Graves' disease, the immunostaining was diffusely enhanced in all follicular cells. A more intense signal was observed in toxic adenomas and in samples obtained from a patient with severe hyperthyroidism due to an activating mutation in the TSH receptor. In multinodular goiters, large follicles displayed a weak signal whereas small proliferative follicles showed intense immunoreactivity near the apical plasma membrane. In hypothyroid patients, NOSIII immunoreactivity was barely detectable. In summary, NOSIII is expressed both in endothelial cells and thyroid follicular cells. The endothelial localization of NOSIII is consistent with a role for nitric oxide in the vascular control of the thyroid. NOSIII expression in thyroid follicular cells and the variations in its immunoreactivity suggest a possible role for nitric oxide in thyrocyte function and/or growth.

Characterization of the first FGFRL1 mutation identified in a craniosynostosis patient

Fibroblast growth factor receptor-like 1 (FGFRL1) is a recently discovered transmembrane protein whose functions remain unclear. Since mutations in the related receptors FGFR1-3 cause skeletal malformations, DNA samples from 55 patients suffering from congenital skeletal malformations and 109 controls were searched for mutations in FGFRL1. One patient was identified harboring a frameshift mutation in the intracellular domain of this novel receptor. The patient showed craniosynostosis, radio-ulnar synostosis and genital abnormalities and had previously been diagnosed with Antley-Bixler syndrome. The effect of the FGFRL1 mutation was studied in vitro. In a reporter gene assay, the wild-type as well as the mutant receptor inhibited FGF signaling. However, the mutant protein differed from the wild-type protein in its subcellular localization. Mutant FGFRL1 was mainly found at the plasma membrane where it interacted with FGF ligands, while the wild-type protein was preferentially located in vesicular structures and the Golgi complex. Two motifs from the intracellular domain of FGFRL1 appeared to be responsible for this differential distribution, a tandem tyrosine based motif and a histidine-rich sequence. Deletion of either one led to the preferential redistribution of FGFRL1 to the plasma membrane. It is therefore likely that mutant FGFRL1 contributes to the skeletal malformations of the patient.

Modeling Framework for the Establishment of the Apical-Basal Embryonic Axis in Plants

The apical-basal axis of the early plant embryo determines the body plan of the adult organism. To establish a polarized embryonic axis, plants evolved a unique mechanism that involves directional, cell-to-cell transport of the growth regulator auxin. Auxin transport relies on PIN auxin transporters 1], whose polar subcellular localization determines the flow directionality. PIN-mediated auxin transport mediates the spatial and temporal activity of the auxin response machinery 2-7] that contributes to embryo patterning processes, including establishment of the apical (shoot) and basal (root) embryo poles 8]. However, little is known of upstream mechanisms guiding the (re)polarization of auxin fluxes during embryogenesis 9]. Here, we developed a model of plant embryogenesis that correctly generates emergent cell polarities and auxin-mediated sequential initiation of apical-basal axis of plant embryo. The model relies on two precisely localized auxin sources and a feedback between auxin and the polar, subcellular PIN transporter localization. Simulations reproduced PIN polarity and auxin distribution, as well as previously unknown polarization events during early embryogenesis. The spectrum of validated model predictions suggests that our model corresponds to a minimal mechanistic framework for initiation and orientation of the apical-basal axis to guide both embryonic and postembryonic plant development.

Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM) and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90) also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA) we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET). We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation.

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments.

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.