Peptidases compartmentalized to the Ascaris suum intestinal lumen and apical intestinal membrane

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine

Tumor protein D54 defines a new class of intracellular transport vesicles

Transport of proteins and lipids from one membrane compartment to another is via intracellular vesicles. We investigated the function of tumor protein D54 (TPD54/TPD52L2) and found that TPD54 was involved in multiple membrane trafficking pathways: anterograde traffic, recycling, and Golgi integrity. To understand how TPD54 controls these diverse functions, we used an inducible method to reroute TPD54 to mitochondria. Surprisingly, this manipulation resulted in the capture of many small vesicles (30 nm diameter) at the mitochondrial surface. Super-resolution imaging confirmed the presence of similarly sized TPD54-positive structures under normal conditions. It appears that TPD54 defines a new class of transport vesicle, which we term intracellular nanovesicles (INVs). INVs meet three criteria for functionality. They contain specific cargo, they have certain R-SNAREs for fusion, and they are endowed with a variety of Rab GTPases (16 out of 43 tested). The molecular heterogeneity of INVs and the diverse functions of TPD54 suggest that INVs have various membrane origins and a number of destinations. We propose that INVs are a generic class of transport vesicle that transfer cargo between these varied locations

Entwicklung von „Fluorescence Activated Synaptosome Sorting“ (FASS) und die Analyse von VGLUT1-Synapsen des Mäusehirns

Im Gehirn werden Signale zwischen Nervenzellen an chemischen Synapsen übermittelt. Ob eine Synapse dabei erregende, hemmende oder modulatorische Signale überträgt, hängt von den beteiligten Neurotransmittern und deren Neurotransmitter-Rezeptoren ab. Um die physiologische Funktion dieser unterschiedlichen synaptischen Subtypen und deren Beteiligung an pathologischen Prozessen im Gehirn zu untersuchen, ist die detaillierte Kenntnis ihrer biochemischen Zusammensetzung eine notwendige Vorraussetzung. Um die Proteinzusammensetzung und Funktion von Synapsen zu erforschen, fanden in den letzten Jahrzehnten Synaptosomenpräparationen breite Anwendung in der Wissenschaft. Als Synaptosomen bezeichnet man funktionelle synaptische Einheiten, die sowohl die Prä- als auch die Postsynapse enthalten und die seit 1960 routinemäßig mittels differentieller und Dichtegradientenzentrifugation aus Hirngewebe isoliert werden. Die Interpretation der von Synaptosomenpräparationen gewonnen Daten wird allerdings dadurch erschwert, dass die konventionelle Synaptosomenpräparation ein Gemisch von synaptischen Partikel aller synaptischen Subtypen enthält und durch einen beträchtlichen Anteil nicht-synaptischer Partikel, die von Glia und Neuronen stammen, verunreinigt ist. In der vorliegenden Arbeit habe ich zur weiteren Spezifizierung eine neue Methode entwickelt, welche die konventionelle Synaptosomenpräparation um die spezifische Selektion von fluoureszenten Synaptosomen mittels eines FACS-Gerätes erweitert. Hierbei diente eine vor kurzem in unserer Arbeitsgruppe entwickelte fluoreszente VGLUT1VENUS knock-in Mauslinie als Quelle fluoreszenter VGLUT1-Synapsen. Im Folgenden zeige ich, dass durch die von mir als „Flourescent Activated Synaptosome Sorting“ (FASS) bezeichnete Methode äußerst reine VGLUT1-Synaptosomen isoliert werden, die sowohl prä- als auch postsynaptische Bestandteile enthalten. Die weitere Untersuchung der FASS-aufgereinigten VGLUT1VENUS-Synaptosomen liefert Erkenntnisse über die synaptische Verteilung der Neuroligine und zeigt Unterschiede in der Verteilung von Glutamatrezeptoren zwischen synaptischen und extrasynaptischen Zonen. Eine Proteomanalyse zeigt die spezifische Anreicherung von 163 Proteinen in VGLUT1-Synapsen. Darunter sind viele bekannte synaptische Proteine, aber auch eine Vielzahl von Proteinen, für die noch keine Daten zur Lokalisation an VGLUT1-Synapsen vorliegen. Für drei dieser Proteine, nämlich FXYD6, Ly6H und TPD52, wird die Expression und Lokalisation an VGLUT1-Synapsen mit unabhängigen Methoden bestätigt. Zusammengenommen zeigt die vorliegende Arbeit, dass FASS ein neues und adäquates Verfahren für die Anreicherung von höchst reinem synaptischen Material ist, welches verwendet werden kann, um spezifische synaptische Subtypen in biochemischen, physiologischen oder pathophysiologischen Studien zu untersuchen

Wirkung und Interaktion der Wachstumsfaktoren Myostatin und IGF-1 im murinen Herzen

Myostatin, ein Mitglied der TGF-β Familie von Wachstumsfaktoren, ist ein negativer Regulator des Skelettmuskelwachstums. Obwohl Myostatin nach einer Vielzahl pathologischer Zustände im Herzen massiv hochreguliert wird, ist die physiologische und pathophysiologische Funktion von Myostatin im Herzen noch kaum erforscht. Deshalb wurde im Rahmen dieser Dissertation die Funktion von Myostatin im adulten Herzen untersucht. Dazu wurden Mausmodelle, in denen Myostatin in Kardiomyozyten deletiert und überexprimiert wird, verwendet. Ich konnte zeigen, dass die akute Deletion von Myostatin in Kardiomyozyten zu einer erhöhten Lethalität, Herzinsuffizienz und Hypertrophie führt. Dabei konnte ich eine Aktivierung der AMP-aktivierten Kinase (AMPK) als Ursache der Hypertrophie identifizieren und mit Hilfe eines AMPK Inhibitors die Entstehung der Hypertrophie in vivo verhindern. Des Weiteren konnte ich in vivo und in vitro zeigen, dass Myostatin AMPK über die TGF-β-aktivierte Kinase 1 (TAK1) und seinen kanonischen Rezeptor inhibiert. Die akute Deletion von Myostatin hemmte auch die Expression von Rgs2, einem Inhibitor der Gq Signalkaskade, und führte dadurch zu einer Aktivierung dieses für Herzinsuffizienz elementaren Signalweges. Außerdem verbesserte die akute adulte Überexpression von Myostatin die Herzkontraktilität leicht, während eine langfristige Überexpression eine interstitielle Fibrose, die über TAK1 und p38 vermittelt wird, induzierte. Hiermit konnte ich Myostatin als neuen Regulator der Hypertrophie und Herzinsuffizienz etablieren.rnMyostatin, a member of the TGF-beta family of growth factors, is a potent negative regulator of skeletal muscle growth. Although it is also massively upregulated under pathological conditions in the heart, its cardiac function is barely understood. The purpose of this dissertation was to delineate the cardiac function of Myostatin in the adult heart. Therefore I used mouse models, in which myostatin was deleted or overexpressed in cardiomyocytes. I could prove that acute deletion of myostatin in cardiomyocytes leads to an increased lethality, heart failure and cardiac hypertrophy. Cardiac hypertrophy was mediated by AMP-activated kinase (AMPK) and AMPK inhibition rescued the development of cardiac hypertrophy in vivo. Furthermore myostatin inhibited AMPK in vivo and in vitro via the TGF-beta activated kinase 1 (TAK1) and its canonical receptor. Acute deletion of myostatin also inhibited expression of Rgs2, an inhibitor of Gq signaling, that protects against heart failure. Furthermore, acute overexpression of myostatin in adult cardiomyocytes tends to increase cardiac contractility, whereas long-term overexpression induces interstitial fibrosis via TAK1 and p38. In summary, I identified myostatin as a novel regulator of cardiac hypertrophy and heart failure that needs to be tightly regulated in the heart.r

Rab35 regulates evoked exocytosis of endothelial Weibel-Palade bodies

Weibel-Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exoyctosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling haemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15 and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase-activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target RabGTPases. Among the inhibitory RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von-Willebrand factor (VWF) and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat and PH domain-containing protein ACAP2 as Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6 supported histamine-evoked WPB exocytosis as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6

Purification of Synaptosome Populations Using Fluorescence-Activated Synaptosome Sorting

International audienceFor several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and some functional features of the cells in the central nervous system. Indeed, brain tissue contains a complex intermingled network of neuronal, glial, and vascular cells. To reduce this complexity biochemists have optimized fractionation protocols that enrich in specific compartments such as synapses (called “synaptosomes”) and synaptic vesicles, for example. However, recently, these approaches suffered from a lack of specificity and purity. In a recent effort, we extended the conventional synaptosome preparation to purify fluorescent synaptosomes on a cell sorter. We could prove that our method allows for the steep enrichment in fluorescent excitatory VGLUT1venus synaptosomes containing the presynaptic element and the tip of the post-synaptic element and a strong depletion in neuronal and glial contaminants. Here, we propose a detailed procedure for the implementation of Fluorescence Activated Synaptosome Sorting

Rab35 regulates evoked exocytosis of endothelial Weibel-Palade bodies

Weibel-Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exoyctosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling haemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15 and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase-activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target RabGTPases. Among the inhibitory RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von-Willebrand factor (VWF) and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat and PH domain-containing protein ACAP2 as Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6 supported histamine-evoked WPB exocytosis as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6