Analyse der Lokalisation und Funktion uncharakterisierter Mitglieder der Zuckertransporterfamilie in Saccharomyces cerevisiae

Die Zuckertransporterfamilie ist eine Unterfamilie der MFS („major facilitator superfamily“), wobei die MFS wiederum als Überfamilie von Transportproteinen definiert wurde, die sich aus Proteinen mit 12 Transmembran-Domänen zusammensetzt. Im Rahmen dieser Doktorarbeit sollte die subzelluläre Lokalisation und physiologische Funktion der uncharakterisierten Mitglieder der Zuckertransporterfamilie Ybr241 und Ygl104 untersucht werden. Mittels Zellfraktionierung durch Saccharosedichtegradienten-Zentrifugation und Fluoreszenzmikroskopie konnte eine Lokalisation von Ybr241 und Ygl104 in der vakuolären Membran festgestellt werden. Da Plasmamembran-Proteine zur Degradation ubiquitiniert, über Endocytose internalisiert und in der Vakuole abgebaut werden, wurden weitere Lokalisationsstudien sowohl in Endocytose-Mutanten als auch in einer Mutante mit Defekten in der Ubiquitinierung durchgeführt. Diese ergaben, daß die vakuoläre Lokalisation nicht auf Degradation zurückzuführen war. Somit handelt es sich bei Ybr241 und Ygl104 um residente vakuoläre Membranproteine. Lokalisationsstudien in vps-Mutanten erbrachten Hinweise darauf, daß zumindest Ygl104, wie die meisten vakuolären Proteine, über den CPY-Weg zur Vakuole befördert wird. Weder durch Wachstumsanalysen noch mit Hilfe von Phenotype MicroArrays™ (Biolog, Inc.) konnten Phänotypen der Deletionsmutanten von Ybr241 und Ygl104 identifiziert werden. Allerdings zeigte sich im Verlauf der Arbeit, daß die Deletionsmutanten einen Vorteil beim Wachstum mit geringen Glucosekonzentrationen bei 37°C haben. Des weiteren bestanden aufgrund von Datenbankanalysen Anhaltspunkte auf eine Beteiligung am Trehalosestoffwechsel. Durch Hitzeschockexperimente konnte eine essentielle Rolle von Ybr241 und Ygl104 bei der Resistenz von Zellen gegenüber schwerem Hitzestreß identifiziert werden. Die verminderte Thermotoleranz der Deletionsmutanten war aber nicht auf einen geringeren Gehalt der Zellen am Streßschutzmolekül Trehalose zurückzuführen. Zudem deckte ein SGA („synthetic genetic array“) eine synthetisch kranke Interaktion von YBR241C und YGL104C mit dem Gen der Trehalose-6-Phosphat-Synthase TPS1 auf. Diese Interaktion sprach gegen eine Beteiligung der Genprodukte am Trehalosestransport, da tps1-Mutanten keine Trehalose enthalten. tps1-Mutanten haben einen Wachstumsdefekt mit schnell fermentierbaren Kohlenstoffquellen, der höchstwahrscheinlich auf einen Mangel an freiem Phosphat zurückzuführen ist. Somit scheinen die Proteine Ybr241 und Ygl104 die intrazelluläre Phosphatkonzentration zu beeinflussen. Eine Analyse ergab, daß der Phosphat- und Polyphosphatgehalt der Mutanten teilweise stark herabgesetzt war. Der Einfluß könnte direkt durch Phosphatimport in die Vakuole stattfinden oder sekundär über eine Verminderung der Glycerinproduktion, da durch die Synthese von Glycerin wieder Phosphat freigesetzt wird. Somit handelt es sich bei Ybr241 und Ygl104 möglicherweise um vakuoläre Phosphat- oder Glycerintransporter. Ferner konnte gezeigt werden, daß die saure Trehalase Ath1 sekretiert wird und Trehalose extrazellulär in Glucose hydrolysiert. Die Glucosemoleküle werden dann von der Hefezelle aufgenommen und verstoffwechselt. Somit spielt Ath1 eine essentielle Rolle beim Wachstum der Hefe mit Trehalose als Kohlenstoffquelle. Ziel des zweiten Teils dieser Doktorarbeit war die Entwicklung eines genomweiten Screens nach ER-Verpackungschaperonen, durch den bisher unbekannte Verpackungschaperone identifiziert werden sollten. Durch Testen verschiedener Varianten des Screens konnte ein Verfahren entwickelt werden, das prinzipiell funktionierte. Für den Einsatz im genomweiten Maßstab war es jedoch ungeeignet, da mit einer hohen Rate an falsch negativen Ergebnissen zu rechnen gewesen wäre

Physical exercise induces rapid release of small extracellular vesicles into the circulation

Cells secrete extracellular vesicles (EVs) by default and in response to diverse stimuli for the purpose of cell communication and tissue homeostasis. EVs are present in all body fluids including peripheral blood, and their appearance correlates with specific physiological and pathological conditions. Here, we show that physical activity is associated with the release of nano-sized EVs into the circulation. Healthy individuals were subjected to an incremental exercise protocol of cycling or running until exhaustion, and EVs were isolated from blood plasma samples taken before, immediately after and 90 min after exercise. Small EVs with the size of 100-130 nm, that carried proteins characteristic of exosomes, were significantly increased immediately after cycling exercise and declined again within 90 min at rest. In response to treadmill running, elevation of small EVs was moderate but appeared more sustained. To delineate EV release kinetics, plasma samples were additionally taken at the end of each increment of the cycling exercise protocol. Release of small EVs into the circulation was initiated in an early phase of exercise, before the individual anaerobic threshold, which is marked by the rise of lactate. Taken together, our study revealed that exercise triggers a rapid release of EVs with the characteristic size of exosomes into the circulation, initiated in the aerobic phase of exercise. We hypothesize that EVs released during physical activity may participate in cell communication during exercise-mediated adaptation processes that involve signalling across tissues and organs

Physical exercise induces rapid release of small extracellular vesicles into the circulation

Cells secrete extracellular vesicles (EVs) by default and in response to diverse stimuli for the purpose of cell communication and tissue homeostasis. EVs are present in all body fluids including peripheral blood, and their appearance correlates with specific physiological and pathological conditions. Here, we show that physical activity is associated with the release of nano-sized EVs into the circulation. Healthy individuals were subjected to an incremental exercise protocol of cycling or running until exhaustion, and EVs were isolated from blood plasma samples taken before, immediately after and 90 min after exercise. Small EVs with the size of 100–130 nm, that carried proteins characteristic of exosomes, were significantly increased immediately after cycling exercise and declined again within 90 min at rest. In response to treadmill running, elevation of small EVs was moderate but appeared more sustained. To delineate EV release kinetics, plasma samples were additionally taken at the end of each increment of the cycling exercise protocol. Release of small EVs into the circulation was initiated in an early phase of exercise, before the individual anaerobic threshold, which is marked by the rise of lactate. Taken together, our study revealed that exercise triggers a rapid release of EVs with the characteristic size of exosomes into the circulation, initiated in the aerobic phase of exercise. We hypothesize that EVs released during physical activity may participate in cell communication during exercise-mediated adaptation processes that involve signalling across tissues and organs

Oligodendroglial p130Cas is a target of Fyn kinase involved in process formation, cell migration and survival

Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation

Oligodendrocytes support axonal transport and maintenance via exosome secretion.

Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term integrity of axons depends on intrinsic mechanisms including axonal transport and extrinsic support from adjacent glial cells. The mechanisms of support provided by myelinating oligodendrocytes to underlying axons are only partly understood. Oligodendrocytes release extracellular vesicles (EVs) with properties of exosomes, which upon delivery to neurons improve neuronal viability in vitro. Here, we show that oligodendroglial exosome secretion is impaired in 2 mouse mutants exhibiting secondary axonal degeneration due to oligodendrocyte-specific gene defects. Wild-type oligodendroglial exosomes support neurons by improving the metabolic state and promoting axonal transport in nutrient-deprived neurons. Mutant oligodendrocytes release fewer exosomes, which share a common signature of underrepresented proteins. Notably, mutant exosomes lack the ability to support nutrient-deprived neurons and to promote axonal transport. Together, these findings indicate that glia-to-neuron exosome transfer promotes neuronal long-term maintenance by facilitating axonal transport, providing a novel mechanistic link between myelin diseases and secondary loss of axonal integrity

Identification of p130Cas as an oligodendroglial tyrosine-phosphorylated protein in the presence of active Fyn.

<p>(A) Tyrosine-phosphorylated proteins were immunoprecipitated from Oli-<i>neu</i> cells overexpressing active Fyn. A Coomassie-stained SDS-Polyacrylamide gel with first and second elution (E1 and E2), antibody control (control) and MW marker (M) is shown. The indicated band (p130Cas) was excised and analyzed by mass spectrometry. Molecular weights in kilodalton are indicated on the right. (B) Primary structure of p130Cas. The identified peptides are shown in bold red and cover 20% of the protein sequence. (C) p130Cas expression at different developmental stages in cultured oligodendrocytes (DIV, days in vitro, 1, 2, 4, 6, 8) was analyzed by Western blots with antibodies indicated on the left. CNP and MOG represent early and late maturation markers, respectively, and GAPDH serves as a loading control.</p

The effect of p130Cas knockdown on spreading and migration in Oli-<i>neu</i> does not result from changes in cellular viability.

<p>(A) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 hours later, the cells were detached from their culture vessel and 6 h after re-plating a TUNEL assay was performed to test for apoptosis. Data show the mean ± SEM from 3 independent experiments; ns, not significant (Student’s <i>t</i> test). (B) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in A were lysed and p130Cas levels were assessed by Western blotting. α-tubulin serves as loading control. (C) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 h later, the cells were detached from their culture vessel and 0.5 h and 6 h after re-plating MTT and LDH assays were carried out to test for cell viability and membrane integrity, respectively. Data show the mean ± SEM from 3 independent experiments; ns, not significant (Student’s <i>t</i> test). (D) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in C were lysed and p130Cas levels were assessed by Western blotting. α-tubulin serves as loading control.</p

The effect of p130Cas on spreading and migration of oligodendroglial cells.

<p>(A) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 hours later, the cells were detached from their culture vessel and 30 min after re-plating they were subjected to immunofluorescence analysis of cell spreading. Cells were classified as either not spread (arrowhead), showing lamellipodia (asterisk) or lamella (arrow). (B) Statistical evaluation of A. Data represents the mean ± SEM from 3 independent experiments; ** p<0.01 (Student’s <i>t</i> test). (C) Oli-<i>neu</i> cells were treated as in A. Here, immunofluorescence analysis of process thickness was carried out 4 hours after re-plating. Three examples per condition are shown. Areas of measurement are outlined by white dashed rectangles. (D) Statistical evaluation of C. The thickest process for each individual cell was measured and plotted on a chart (from high to low diameters) and the average ratio ± SEM from 3 independent experiments is presented; ** p<0.01 (Student’s <i>t</i> test). (E) Oli-<i>neu</i> cells were treated with siRNA as in A. After 24 hours, they were re-plated in Boyden chambers and allowed to migrate for 6 hours in the presence of 5 ng/ml bFGF before fixation and migration analysis were carried out. Data are expressed as a percentage of basal migration, i.e. the migration of Oli-<i>neu</i> without chemoattractant. Data represent the mean ± SEM from 3 independent experiments; * p<0.05 (Student’s <i>t</i> test). (F) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in A-E were lysed and p130Cas levels were assessed by Western blotting. GAPDH serves as loading control.</p