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

DNA DAMAGE RESPONSE OF EX-VIVO PORCINE EYE LENSES IN ORGAN-CULTURE AND IN-VITRO CULTURED LENS EPITHELIAL CELLS TO IONIZING RADIATION.

Astronauts on space missions, especially on long-term missions to Moon or Mars have a higher risk for the expression of radiation late effects such as cancer or sub-capsular cortical eye lens opacities. This is due to higher dose and different patterns of cellular energy deposition from high-linear-energy-transfer (LET) components of galactic cosmic radiation in space than that of terrestrial low-LET radiation on Earth. The eye lens is considered to be a radiation sensitive organ with radiation induced cataract to occur with a threshold absorbed dose of 0.5 Gy of sparsely ionizing radiation. For terrestrial occupational radiation lens exposure limit is set to yearly 20 mSv by the International Commission on Radiological Protection (1). Doses perceived by astronauts are much higher: in average 150 mSv per year on the International Space Station (ISS) and 1.2 to 1.4 mSv per day on Apollo and Skylab missions (2)

Validation of biological recognition elements for signal transduction as first step in the development of whole cell biosensors

Choosing the proper combination of receptor element, cell type and measurable signal requires major consideration for developing cell-based biosensors. In order to use physiologically relevant cellular responses towards (geno)toxic conditions, information on the mechanism of action and of the expected outcome of exposure needs to be considered

Radiation Response of Murine Embryonic Stem Cells

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies

The Use of ProteoTuner Technology to Study Nuclear Factor κB Activation by Heavy Ions

Nuclear factor κB (NF-κB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (¹⁶O, 95 MeV/n, linear energy transfer—LET 51 keV/µm; ¹²C, 95 MeV/n, LET 73 keV/μm; ³⁶Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of ¹⁶O ions and ≈45 hits of ¹²C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of ³⁶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of ³⁶Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits’ effects

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

Transcription Factors in the Cellular Response to Charged Particle Exposure

Charged particles, such as carbon ions, bear the promise of a more effective cancer therapy. In human spaceflight, exposure to charged particles represents an important risk factor for chronic and late effects such as cancer. Biological effects elicited by charged particle exposure depend on their characteristics, e.g., on linear energy transfer (LET). For diverse outcomes (cell death, mutation, transformation, and cell-cycle arrest), an LET dependency of the effect size was observed. These outcomes result from activation of a complex network of signaling pathways in the DNA damage response, which result in cell-protective (DNA repair and cell-cycle arrest) or cell-destructive (cell death) reactions. Triggering of these pathways converges among others in the activation of transcription factors, such as p53, nuclear factor κB (NF-κB), activated protein 1 (AP-1), nuclear erythroid-derived 2-related factor 2 (Nrf2), and cAMP responsive element binding protein (CREB). Depending on dose, radiation quality, and tissue, p53 induces apoptosis or cell-cycle arrest. In low LET radiation therapy, p53 mutations are often associated with therapy resistance, while the outcome of carbon ion therapy seems to be independent of the tumor’s p53 status. NF-κB is a central transcription factor in the immune system and exhibits pro-survival effects. Both p53 and NF-κB are activated after ionizing radiation exposure in an ataxia telangiectasia mutated (ATM)-dependent manner. The NF-κB activation was shown to strongly depend on charged particles’ LET, with a maximal activation in the LET range of 90–300 keV/μm. AP-1 controls proliferation, senescence, differentiation, and apoptosis. Nrf2 can induce cellular antioxidant defense systems, CREB might also be involved in survival responses. The extent of activation of these transcription factors by charged particles and their interaction in the cellular radiation response greatly influences the destiny of the irradiated and also neighboring cells in the bystander effect

An in-vitro approach for water quality determination: activation of NF-κB as marker for cancer-related stress responses induced by anthropogenic pollutants of drinking water

Epidemiological studies show that there is a link between urban water pollution and increase in human morbidity and mortality. With the increase in number of new substances arising from the chemical, pharmaceutical, and agricultural industries, there is an urgent need to develop biological test systems for fast evaluation of potential risks to humans and the environmental ecosystems. Here, a combined cellular reporter assay based on the cellular survival and the stress-induced activation of the survival-promoting factor nuclear factor κB (NF-κB) and its use for the detection of cytotoxicity and cancer-related stress responses is presented. A total of 14 chemicals that may be found in trace-amounts in ground water levels are applied and tested with the presented assay. The project is embedded within the joint research project TOX-BOX which aims to develop a harmonized testing strategy for risk management of anthropogenic trace substances in potable water. The assay identified carbendazim as a NF-κB-activating agent in mammalian cells

RADIATION RESPONSE OF PORCINE LENS EPITHELIAL CELLS AND EYE LENSES IN ORGAN-CULTURE

Astronauts on long-term space missions have a higher risk for the expression of radiation late effects such as cancer or sub-capsular cortical eye lens opacities. This is due to higher dose and different patterns of cellular energy deposition from high-linear-energy-transfer (LET) components of galactic cosmic radiation in space than that of terrestrial low-LET radiation on Earth. The eye lens is a radiation sensitive organ with radiation induced cataract to occur with a threshold absorbed dose of 0.5 Gy (0 - 1 Gy) of sparsely ionizing radiation. Doses perceived by astronauts on the International Space Station (ISS) are in average 150 mSv per year (Cucinotta et al. (2001) Radiat Res. 156:460-466). Radiationinduced lens opacification is assumed to initiate from post irradiation proliferative activity of genetically damaged lens epithelial cells with alterations in cell cycle control, apoptosis, differentiation, and cellular disorganization, or other pathways controlling lens fiber cells’ differentiation. As the porcine eye lens is similar to the human lens in size and anatomy, DNA damage response was investigated in ex-vivo porcine lenses in organ culture, in in-vitro cultivated lens epithelial slabs (ES) and in porcine lens epithelial cells (pLEC). Cell survival of proliferative cells was calculated from colony forming ability (CFA) assay. The phosphorylated form of H2AX (γH2AX) was used as a molecular marker to visualize DNA double strand breaks (DSB) and their repair. Propidium iodide based DNA staining for cellular DNA content marked radiation-induced cell cycle disturbances. In pLEC the cell survival curve of immediate plated cells and after a recovery period of 24 h follow the equation S=1.40xD+ln 1.47 and S=1.59xD+ln 1.79, respectively. DNA DSB are induced in a dose-dependent manner ( 18 DSB/cell/Gy) and repaired during successive recovery ( 5 DSB/cell/Gy residual damage after 24 h). For doses >2 Gy a cell cycle arrest in G2 phase occurred 24 h after X-irradiation and persisted up to 72 h post-irradiation. DNA DSB induction and repair could as well be documented for ES and whole lenses after X-irradiation. In whole lenses, the amount of residual damage (after 24 h and 48 h) was highest in the equatorial zone while in the central epithelial zone DSB repair seemed to proceed with time in a manner comparable to in-vitro cultivated pLEC. Lens organ culture allows cellular metabolism and DNA synthesis in whole lenses. Repair of DNA DSB takes place in the central epithelial layer and is reduced in the equatorial region of cultivated lenses