Geochemistry of surface sediments from the mid-oceanic Kolbeinsey Ridge, north of Iceland

In order to assess recent submarine volcanic contributions to the sediments from the active Kolbeinsey Ridge, surface samples were analyzed chemically. The contribution of major and trace elements studied differ within the study area. A statistical analysis of the geochemical variables using factor analysis and cluster method allows to distinguish possible sample groups. Cluster method identifies three distinct sediment groups located in different areas of sedimentation. Group 1 is characterized by highest contents of Fe2O3, V, Co, Ni, Cu and Zn demonstrating the input of volcaniclastic material. Group 2 comprises high values of CaCO3, CaO and Sr representing biogenic carbonate. Group 3 is characterized by the elements K, Rb, Cs, La and Pb indicating the terrigenous component. The absolute percentage of the volcanic, biogenic and terrigenous components in the bulk sediments was calculated by using a normative sediment method. The highest volcanic component (> 60% on a carbonate free basis) is found on the ridge crest. The biogenic component is highest (10–30%) in the eastern part of the Spar Fracture Zone influenced by the East Iceland Current. Samples from the western and southeastern region of the study area contain more than 90% of terrigenous component which appears to be mainly controlled by input of ice-rafted debris

A Bayesian method for inferring quantitative information from FRET data

<p>Abstract</p> <p>Background</p> <p>Understanding biological networks requires identifying their elementary protein interactions and establishing the timing and strength of those interactions. Fluorescence microscopy and Förster resonance energy transfer (FRET) have the potential to reveal such information because they allow molecular interactions to be monitored in living cells, but it is unclear how best to analyze FRET data. Existing techniques differ in assumptions, manipulations of data and the quantities they derive. To address this variation, we have developed a versatile Bayesian analysis based on clear assumptions and systematic statistics.</p> <p>Results</p> <p>Our algorithm infers values of the FRET efficiency and dissociation constant, <it>K_d</it>, between a pair of fluorescently tagged proteins. It gives a posterior probability distribution for these parameters, conveying more extensive information than single-value estimates can. The width and shape of the distribution reflects the reliability of the estimate and we used simulated data to determine how measurement noise, data quantity and fluorophore concentrations affect the inference. We are able to show why varying concentrations of donors and acceptors is necessary for estimating <it>K_d</it>. We further demonstrate that the inference improves if additional knowledge is available, for example of the FRET efficiency, which could be obtained from separate fluorescence lifetime measurements.</p> <p>Conclusions</p> <p>We present a general, systematic approach for extracting quantitative information on molecular interactions from FRET data. Our method yields both an estimate of the dissociation constant and the uncertainty associated with that estimate. The information produced by our algorithm can help design optimal experiments and is fundamental for developing mathematical models of biochemical networks.</p

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment

Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM

Fluorescence lifetime imaging (FLIM) is a quantitative, intensity-independent microscopical method for measurement of diverse biochemical and physical properties in cell biology. It is a highly effective method for measurements of Förster resonance energy transfer (FRET), and for quantification of protein-protein interactions in cells. Time-domain FLIM-FRET measurements of these dynamic interactions are particularly challenging, since the technique requires excellent photon statistics to derive experimental parameters from the complex decay kinetics often observed from fluorophores in living cells. Here we present a new time-domain multi-confocal FLIM instrument with an array of 64 visible beamlets to achieve parallelised excitation and detection with average excitation powers of ~ 1–2 μW per beamlet. We exemplify this instrument with up to 0.5 frames per second time-lapse FLIM measurements of cAMP levels using an Epac-based fluorescent biosensor in live HeLa cells with nanometer spatial and picosecond temporal resolution. We demonstrate the use of time-dependent phasor plots to determine parameterisation for multi-exponential decay fitting to monitor the fractional contribution of the activated conformation of the biosensor. Our parallelised confocal approach avoids having to compromise on speed, noise, accuracy in lifetime measurements and provides powerful means to quantify biochemical dynamics in living cells

FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres

In this work we demonstrate for the first time the use of Förster resonance energy transfer (FRET) as an assay to monitor the dynamics of cross-bridge conformational changes directly in single muscle fibres. The advantage of FRET imaging is its ability to measure distances in the nanometre range, relevant for structural changes in actomyosin cross-bridges. To reach this goal we have used several FRET couples to investigate different locations in the actomyosin complex. We exchanged the native essential light chain of myosin with a recombinant essential light chain labelled with various thiol-reactive chromophores. The second fluorophore of the FRET couple was introduced by three approaches: labelling actin, labelling SH1 cysteine and binding an adenosine triphosphate (ATP) analogue. We characterise FRET in rigor cross-bridges: in this condition muscle fibres are well described by a single FRET population model which allows us to evaluate the true FRET efficiency for a single couple and the consequent donor–acceptor distance. The results obtained are in good agreement with the distances expected from crystallographic data. The FRET characterisation presented herein is essential before moving onto dynamic measurements, as the FRET efficiency differences to be detected in an active muscle fibre are on the order of 10–15% of the FRET efficiencies evaluated here. This means that, to obtain reliable results to monitor the dynamics of cross-bridge conformational changes, we had to fully characterise the system in a steady-state condition, demonstrating firstly the possibility to detect FRET and secondly the viability of the present approach to distinguish small FRET variations

Heme Oxygenase Isoforms Differ in Their Subcellular Trafficking during Hypoxia and Are Differentially Modulated by Cytochrome P450 Reductase

Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER) which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans

Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented