Phosphatidylcholine is organized in long-lived plasmalemmal platforms

All living cells are enclosed by a membrane that is mainly made up of proteins and lipids. The lateral organization of these constituents is a subject in current research. It has been discussed since two decades whether lipids are able to form stable assemblies, domains or platforms in the plasma membrane. A major issue in this field is the visualization of lipid structures. The lipid phosphatidylcholine (PC) is one of the most common lipids in the plasma membrane. Recently, PC was visualized in membranes via a non-invasive metabolic labeling followed by fluorescent labeling. In the present work, the arrangement of this lipid within the plasma membrane was studied using a combination of this elegant, noninvasive labeling and a variety of modern fluorescent microscopy techniques. Both whole cells as well as cell body free plasma membrane preparations, so-called “membrane sheets”, were used for the analyses. It was demonstrated that PC is not only homogenously found within the plasma membrane, but also organized into locally restricted lipid platforms. The PC domains were characterized by determining their size and calculating the enrichment factor of the lipid in these spots in comparison to their homogenous surrounding. Furthermore, it could be demonstrated that although these PC-enriched structures were not fluctuating in their number of molecules, they exchanged lipids with their surroundings. Based on this study and together with acquired results from collaborators, a model was developed that broadens the current view of the organization of PC within the plasma membrane. PC platforms have been calculated to have a diameter of 120 nm, consist of about 20,000 lipids and PC comprises 50 % of the platform. So far, lipid platforms on the plasma membrane could not be visualized and characterized. Hence, this work is of essential importance for the cell biological field validating the existence of lipid platforms.Lebende Zellen sind umhüllt von einer Zellmembran, welche aus Proteinen und Lipiden besteht. Die laterale Organisation dieser Bausteine innerhalb der Zellmembran ist Gegenstand aktueller Forschung. So wird z.B. seit zwei Dekaden diskutiert ob Lipide stabile Assemblate, Domänen oder Plattformen in der Zellmembran bilden können. Ein grundsätzliches Problem bei der Aufklärung dieser Fragen besteht in der Visualisierung von Lipidstrukturen. Das Lipid Phosphatidylcholin (PC) gehört zu den verbreitesten Lipiden in Zellmembranen. Seit kurzem besteht die Möglichkeit, PC nach nicht-invasiver metabolischer Markierung gefolgt von Fluoreszenzmarkierung in Membranen sichtbar zu machen. In der vorliegenden Arbeit wurde eine Kombination dieser eleganten, nicht invasiven Markierung und verschiedene moderne Methoden aus der Mikroskopie angewendet, um die Verteilung des Lipides innerhalb der Plasmamembran zu untersuchen. Für die Analysen wurden sowohl ganze Zellen als auch zellkörperfreie Plasmamembranpräparationen, sogenannte „Membrane Sheets“ untersucht. Es konnte nicht nur wie erwartet eine homogene Verteilung von PC innerhalb der Plasmamembran beobachtet werden, sondern es wurden auch lokal begrenzte PC-Plattformen entdeckt. Die PC-Domänen wurden charakterisiert durch Vermessung ihrer Größe und der Berechnung des Anreicherungsfaktors vom Lipid gegenüber der homogenen Membranumgebung. Ferner konnte auch gezeigt werden, dass obwohl diese PC-Domänen im Mittel nicht in der Molekülanzahl fluktuieren dennoch Lipide mit ihrer Umgebung austauschen. Anhand von den erhobenen Ergebnissen und den erarbeiteten Resultaten aus Kollaborationen wurde ein Modell entwickelt, welches einen konzeptionellen Rahmen für die Organisation von PC innerhalb der Plasmamembran liefert. Das Modell suggeriert, dass PC 15 Lipidplattfomen einen Durchmesser von 120 nm besitzen und aus rund 20000 PC-Molekülen bestehen, wobei PC 50 % der Plattformoberfläche bedeckt. Die Ergebnisse dieser Arbeit sind aus zellbiologischem Standpunkt weitreichend bedeutend, da bis jetzt keine Lipidplattformen innerhalb von Zellmembranen auf diesem Niveau charakterisiert werden konnten. Somit kann zu der oben genannten Diskussion ein essentieller Beitrag geliefert werden, indem bestätigt wird, dass Lipidplattformen existieren

Activation of NF-κB-Dependent Gene Expression by Accelerated Light and Heavy Ions

During space missions, astronauts are exposed to not only greater amounts of natural radiation than they receive on earth but also to a differing radiation quality, which can result in immediate and long-term risks. To estimate the cancer risk, the molecular and the cellular mechanisms of the response to cosmic radiation have to be uncovered. As the transcription factor Nuclear Factor κB (NF-κB) is involved in cell regulation of apoptosis (programmed cell death), it could influence the cellular outcome (survival, mutation, apoptosis) after radiation exposure and therefore the cancer risk. Human Embryonic Kidney (HEK/293) cells stably transfected with a receptor-reporter-construct carrying the destabilized variant of Enhanced Green Fluorescent Protein (d2EGFP) under the control of the NF-κB response element were used in this work to analyse the NF-κB-dependent gene expression in response to accelerated light and heavy ions. Irradiation was performed either with X-rays (150 kV), with 75 MeV/nucleon carbon or 29 MeV/nucleon lead ions at GANIL (LET ~30 or 8800 keV/μm, respectively), Caen, or with 2.1 MeV α particles (LET ~160 keV/μm) at PTB, Braunschweig. After irradiation the following biological endpoints were determined (i) cell survival via the colony forming ability test, (ii) time-dependent activation of NF-κB dependent d2EGFP gene expression using flow cytometry, (iii) quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) of selected NF-κB target genes. High X-ray doses induce the expression of IκBα and GADD45β. After exposure with 1 nuclear hit of 2.1 MeV α particles, d2EGFP fluorescence can already be seen. After exposure of HEK cells with 5 nuclear hits, maximal NF-κB activation is achieved. The NF-κB target genes IκBα and GADD45β are upregulated shortly after α-particle exposure. Exposure to carbon ions results in a slight activation of the NF-κB pathway. Lead ions induced the expression of the IκBα gene. The NF-κB pathway is activated by particle radiation, and the extent of activation seems to correlate with the LET of the applied radiation. As activation of the NF-κB pathway is supposed to play a role in the negative regulation of apoptosis, survival of cells with DNA damage might be favoured especially after low doses of densely ionising radiation