Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes

The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino aciddependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.Peer reviewe

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites

The influence of V-type H+-ATPases on vesicle fusion processes

The proton pumping activity of the V-type H+-ATPase (V-ATPase) complex is essential to maintain the luminal acidification of defined cellular compartments. A broad range of physiological functions, e.g. the proteolytic degradation within lysosomes, intracellular protein sorting and signalling by the mTORC1 (mechanistic target of rapamycin complex 1) depend on functional V‑ATPase complexes. In addition, there is an ongoing debate about a role for the V‑ATPase V0 sector in promoting membrane fusion processes. The present work addresses the influence of the V-ATPase complex on vesicle fusion events in genetically defined mouse models, lacking either the V-ATPase V0 subunit a3 or the V-ATPase accessory subunit 2 (ATP6AP2), a chaperone for V-ATPase V0 sector assembly. In fibroblasts, a detailed characterisation of endosome and phagosome maturation revealed functional vesicle fusion upon knockout of the V-ATPase V0 subunit a3. Similarly, phagosome maturation proceeded in ATP6AP2-deficient macrophages with significantly decreased V‑ATPase V0 subunit levels. Even the pharmacological reduction of accessible, free V0 sectors or pharmacologically inhibited vesicle acidification did not disturb the acquisition of lysosomal markers to phagosomes. In summary, neither the presence nor the activity of the V‑ATPase complex were required for endocytic and phagosomal fusion processes. Therefore, a general fusion promoting influence of V‑ATPases is not supported by the data obtained in this study [...]Die vesikuläre H + -ATPase (V-ATPase) vermitt elt als Protonenpumpe die Ansäuerung definierter Kompartimente der Zelle. Auf diese Weise ermöglicht die V-ATPase eine Vielzahl physiologischer Funktionen, beispielsweise die lysosomale Proteindegradierung, die intrazelluläre Sortierung von Proteinen und den mTORC1 (mechanistic target of rapamycin complex 1) Signalweg. Darüber hinaus wird eine mögliche Beteiligung des V 0 Komplexes der V-ATPase an der Fusion biologischer Membranen diskutiert. In der vorliegenden Arbeit wurde anhand von Mausmodellen untersucht, inwieweit sich die genomische Deletion der V 0 Untereinheit a3 bzw. des V-ATPase akzessorischen Proteins 2 (ATP6AP2), eines Assemblierungshelfers des V0 Komplexes, auf die Fusion von Vesikeln auswirkt. Die detaillierte Analyse der Endosomen- und Phagosomenreifung in Fibroblasten demonstrierte auch in Abwesenheit der V 0 Untereinheit a3 eine funktionale Vesikelfusion. Ebenso hatten der Knockout von ATP6AP2 und die daraus resultierende, verminderte Expression von V0 Komplex Untereinheiten keinen Einfluss auf die Phagosomenreifung in Makrophagen. Selbst die pharmakologische Reduktion frei zugänglicher V0 Komplexe oder der induzierte Anstieg des vesikulären pH-Wertes konnten den Transfer lysosomaler Markerproteine auf Phagosomen nicht beeinträchtigen. Zusammenfassend war weder die Präsenz der V-ATPase noch ihre Aktivität für den Ablauf der analysierten Fusionsprozesse notwendig. Dementsprechend kann ein fusionsfördernder Einfluss des Komplexes mit den Ergebnissen dieser Arbeit nicht bestätigt werden [...

Organic and conventional farmers differ in their perspectives on cover crop use and breeding

Cover crops play an important role in agricultural sustainability. Unlike commodity cash crops, however, there has been relatively little cover crop breeding research and development. We conducted an online survey to evaluate: (a) the perspectives of organic and conventional farmers in the USA who use cover crops and (b) the specific cover crop traits that are important to farmers. We recruited participants from both organic and conventional agriculture networks and 69% of respondents reported that they farmed organic land. In addition to demographic data and information on management practices, we quantified farmer perspectives on four winter annual cover crops: (1) Austrian winter pea, (2) crimson clover, (3) hairy vetch and (4) cereal rye. Overall, respondents represented a wide range of states, farm sizes, plant hardiness zones and cash crops produced. Of the 417 full responses received, 87% of respondents reported that they used cover crops. The maximum amount farmers were willing to spend on cover crop seed varied by farmer type: 1% of conventional farmers versus 19% of organic farmers were willing to spend over US$185 ha−1 (US$75 acre−1). Organic and conventional farmers differed in terms of the reasons why they grew cover crops, with organic farmers placing greater value on the ecosystem services from cover crops. More organic (63%) than conventional (51%) farmers agreed that participatory breeding was important for cover crop variety development (P = 0.047). Both groups shared strong support for cover crop research and considered many of the same traits to be important for breeding. For the legume cover crops, nitrogen fixation was considered the most important trait, whereas winter hardiness, early vigor, biomass production and weed suppression were the most important traits for cereal rye. Our results illustrate common interests as well as differences in the perspectives between organic and conventional farmers on cover crops and can be used to inform nascent cover crop breeding efforts

Vacuolar ATPase in Phagosome-Lysosome Fusion

Kissing S, Hermsen C, Repnik U, et al. Vacuolar ATPase in Phagosome-Lysosome Fusion. Journal of Biological Chemistry. 2015;290(22):14166-14180.The vacuolar H+ -ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V-0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V-0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V-0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V-0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes

Die jüngerlatènezeitliche Zentralsiedlung Basel-Gasfabrik im Fokus: Ausgewählte Ergebnisse der interdisziplinären Forschung

Vor 110 Jahren wurde die spätkeltische Siedlung Basel-Gasfabrik in der Nähe des heutigen Voltaplatzes bei Aushubarbeiten zum damaligen Gaswerk entdeckt. Seither fanden in unterschiedlichen Intensitäten mehr als 400 archäologische Rettungsgrabungen statt, die immer in Zusammenhang mit Bauprojekten standen. Mit dem Auslaufen der Feldarbeiten konnten zwei grosse, multidisziplinär konzipierte Auswertungsvorhaben mit den Titeln «Über die Toten zu den Lebenden» (Umgang mit sterblichen Überresten) und «Über den Grubenrand geschaut» (Siedlung) durchgeführt werden. Der wissenschaftliche Bericht beleuchtet schlaglichtartig Resultate der beiden Forschungsprojekte