ABSTRACT

The quantification of colocalizing signals in multi-channel fluorescence microscopy images depends on the reliable segmentation of corresponding regions of interest (ROIs), on the selection of appropriate colocalization coefficients, and on a robust statistical criterion to discriminate true from random colocalization. Here, we introduce a confined displacement algorithm (CDA) based on image correlation spectroscopy in combination with Manders Colocalization Coefficients M1ROI and M2ROI to quantify true and random colocalization of a given florescence pattern. We show that existing algorithms based on block scrambling exaggerate the randomization of fluorescent pattern with resulting inappropriately narrow probability density functions (PDFs) and false significance of true colocalization in terms of p values. We further confine our approach to sub-cellular compartments and show that true and random colocalization can be analyzed for model dendrites and for GABAB receptor subunits GABABR1/2 in cultured hippocampal neurons. Together, we demonstrate that the CDA detects true colocalization of specific fluorescence patterns down to sub-cellular levels.

Acknowledgments The authors thank Diego Díaz-Espinoza and Maria Osorio-Reich for the ImageJ plugin, N. Contreras from Area Kreativa for support with the figures and J. Cowan for insightful comments on the paper. Research in SCIAN-Lab (SH) is funded by FONDECYT 1090246, FONDEF D07I1019, and PBCT ACT47 (CONICYT, Chile). AG is funded by D07I1019. OR is funded by FONDECYT 1090246. AC is funded by FONDECYT 1071001. SH and AC are members of NEMO, ICM-P04-068-F and the Advanced Imaging & Bioinformatics Initiative AI•BI (www.aibi.cl).

ABSTRACT

In recent years, new evidence in biomembrane research brought about a holistic, supramolecular view on membrane-mediated signal transduction. The consequences of sphingomyelinase (SMase) driven formation of ceramide (Cer) at the membrane interface involves reorganization of the lateral membrane structure of lipids and proteins from the nm to the ym level. In this review, we present recent insights about mechanisms and features of the SMase-mediated formation of Cer-enriched domains in model membranes, which have been elucidated through a combination of microscopic techniques with advanced image processing algorithms. This approach extracts subtle morphological and pattern information beyond the visual perception: since domain patterns are the consequences of subjacent biophysical properties, a reliable quantitative description of the supramolecular structure of the membrane domains yields a direct readout of biophysical properties which are difficult to determine otherwise. Most of the information about SMase action on simple lipid interfaces has arisen from monolayer studies, but the correspondence to lipid bilayer systems will also be discussed. Furthermore, the structural changes induced by sphingomyelinase action are not fully explained just by the presence of ceramide but by out-of equilibrium surface dynamics forcing the lipid domains to adopt transient supramolecular pattern with explicit interaction potentials. This rearrangement responds to a few basic physical properties like lipid mixing/demixing kinetics, electrostatic repulsion and line tension. The possible implications of such transient codes for signal transduction are discussed for SMase controlled action on lipid interfaces.

Acknowledgments: This work was supported in part by: SECyT-UNC, MinCyT (Prov. Córdoba), CONICET and FONCyT (Argentina); some aspects of this investigation are inscribed within the PAE 22642 network in Nanobiosciences. B.M., R.G.O. and M.L.F. are Career Investigators of CONICET; L.D. is a Doctoral Fellow of CONICET. Research in SCIAN-Lab (SH) is funded by FONDECYT (1090246) and FONDEF (D07I1019), the Millennium Scientific Initiative (ICM P04-068-F). SCIAN-Lab is a selected member of the German-Chilean Center of Excellence Initiative for Medical Informatics (DAAD). JJ is funded by a PhD scholarship from CONICYT (Chile). FO is funded by FONDECYT (1090246), FONDEF (D07I1019) and Proyecto Bicentenario R18. The Authors thank N. Contreras from Area Kreativa for support with the figures.

ABSTRACT

The receptors for the second messenger InsP(3) comprise a family of closely related ion channels that release Ca(2+) from intracellular stores, most prominently the endoplasmic reticulum and its extension into the nuclear envelope. The precise sub-cellular localization of InsP(3)Rs and the spatial relationships among them are important for the initiation, spatial and temporal properties and propagation of local and global Ca(2+) signals, but the spatial organization of InsP(3)Rs in Ca(2+) stores is poorly characterized. Using nuclei isolated from insect Sf9 cells and freeze-dry rotary shadowing; we have addressed this by directly visualizing the cytoplasmic domain of InsP(3)R located on the cytoplasmic side of the nuclear envelope. Identification of approximately 15nm structures as the cytoplasmic domain of InsP(3)R was indirectly supported by a marked increase in their frequency after transient transfections with cDNAs for rat types 1 and 3 InsP(3)R, and directly confirmed by gold labeling either with heparin or a specific anti-InsP(3)R antibody. Over-expression of InsP(3)R did not result in the formation of arrays or clusters with channels touching each other. Gold-labeling suggests that the channel amino terminus resides near the center of the cytoplasmic tetrameric quaternary structure. The combination of nuclear isolation with freeze-drying and rotary shadow techniques allows direct visualization of InsP(3)Rs in native nuclear envelopes and can be used to determine their spatial distribution and density. Copyright © 2010 Elsevier Inc. All rights reserved.

Acknowledgments We thank Dr. Suresh Joseph for the type 1 InsP3R antibody. Cesar Cardenas would like to thank the ‘‘Fundación Ciencias Para la Vida” (Chile). This work was support by a MDA grant to CFA and 614 NIH GM065830 to JKF. SCIAN-Lab is a member of the German-Chilean Center of Excellence Initiative for Medical Informatics (DAAD) and the Advanced Imaging & Bioinformatics Initiative AI·BI (www.aibi.cl. Research in SCIAN-Lab (SH) is funded by FONDECYT 1090246, FONDEF (D07I1019), and ICM-P04-068-F (NEMO).

ABSTRACT

We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks and reconstruction of 3D domain morphology using active surface models. This method permits the reconstruction of the spherical surface of GUVs and determination of the area fractions of coexisting lipid domains at the level of single vesicles. Obtaining area fractions enables the scrutiny of the lever rule along lipid phase diagram's tie lines and to test whether or not the coexistence of lipid domains in GUVs correspond to equilibrium thermodynamic phases. The analysis was applied to DLPC/DPPC GUVs displaying coexistence of lipid domains. Our results confirm the lever rule, demonstrating that the observed membrane domains correspond to equilibrium thermodynamic phases (i.e., solid ordered and liquid disordered phases). In addition, the fact that the lever rule is validated from 11 to 14 randomly selected GUVs per molar fraction indicates homogeneity in the lipid composition among the explored GUV populations. In conclusion, our study shows that GUVs are reliable model systems to perform equilibrium thermodynamic studies of membranes.

Acknowledgments: The authors thank N. Contreras from Area Kreativa for support with the figures and Dr. J. Cowan for his help revising English style and grammar. Research in the laboratory of L.A.B. is funded by grants from Forskningsrådet for Natur og Univers (FNU, Denmark), Forskningsrådet for Sundhed og Sygdom (FSS), and the Danish National Research Foundation (which supports MEMPHYS-Center for Biomembrane Physics). Research in SCIAN-Lab (S.H.) is funded by FONDECYT (1060890 and 1090246) and FONDEF (D07I1019) both CONICYT (Chile), and the Millennium Scientific Initiative (ICMP04-068-F). The interdisciplinary collaboration between L.A.B.'s and S.H.'s laboratories was supported by FONDECYT (7060265 and 7080031), CONICYT Chile and MEMPHYS, Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark.

ABSTRACT

Understanding the mechanisms that control synaptic efficacy through the availability of neurotransmitter receptors depends on uncovering their specific intracellular trafficking routes. Gamma-Aminobutyric acid type B (GABAB) receptors (GABABRs) are obligatory heteromers present at dendritic excitatory and inhibitory postsynaptic sites. It is unknown whether synthesis and assembly of GABABRs occur in the somatic endoplasmic reticulum (ER) followed by vesicular transport to dendrites or whether somatic synthesis is followed by independent transport of the subunits for assembly and ER export throughout the somatodendritic compartment. To discriminate between these possibilities we studied the association of GABABR subunits in dendrites of hippocampal neurons combining live fluorescence microscopy, biochemistry, quantitative colocalization, and bimolecular fluorescent complementation. We demonstrate that GABABR subunits are segregated and differentially mobile in dendritic intracellular compartments and that a high proportion of non-associated intracellular subunits exist in the brain. Assembled heteromers are preferentially located at the plasma membrane, but blockade of ER exit results in their intracellular accumulation in the cell body and dendrites. We propose that GABABR subunits assemble in the ER and are exported from the ER throughout the neuron prior to insertion at the plasmamembrane. Our results are consistent with a bulk flow of segregated subunits through the ER and rule out a post-Golgi vesicular transport of preassembled GABABRs.

Acknowledgments We thank C. Morales, M. Calegaro, L. Sandoval for technical assistance and A. Buonanno for carefully reading the manuscript. O.A.R and R.L.V. funded by MECESUP and CONICYT respectively. S.H. funded by FONDECYT 1060890. A.C funded by FONDECYT 1071001, ICM P07-048-F and HFSPO ST00105/2005-C.

ABSTRACT

ABSTRACT Sphingomyelinase (SMase)-induced ceramide (Cer)-enriched domains in a lipid monolayer are shown to result from an out-of-equilibrium situation. This is induced by a change of composition caused by the enzymatic production of Cer in a sphingomyelin (SM) monolayer that leads to a fast SM/Cer demixing into a liquid-condensed (LC), Cer-enriched and a liquid- expanded, SM-enriched phases. The morphological evolution and kinetic dependence of Cer-enriched domains is studied under continuous observation by epifluorescence microscopy. Domain shape annealing is observed from branched to rounded shapes after SMase activity quenching by EDTA, with a decay halftime of ~10 min. An out-of-equilibrium fast domain growth is not the determinant factor for domain morphology. Domain shape rearrangement in nearly equilibrium conditions result from the counteraction of intradomain dipolar repulsion and line tension, according to McConnell’s shape transition theory. Phase separation causes a transient compositional overshoot within the LC phase that implies an increased out-of-equilibrium enrichment of Cer into the LC domains. As a consequence, higher intradomain repulsion leads to transient branched structures that relax to rounded shapes by lowering the proportion of Cer in the domain to equilibrium values. The fast action of SMase can be taken as a compositional perturbation that brings about important consequences for the surface organization.

ABSTRACT

Skeletal muscle cells are a main physiological target of insulin. This hormone triggers production of reactive species of oxygen (ROS) in different cell types. ROS are considered a second messenger and can produce redox modifications in proteins such as ion channels inducing changes in their probability of opening. Insulin can produce intracellular calcium release from intracellular stores in myotubes. In this work we studied the relationshipt between ROS generation and calcium increase induced by insulin and the mechanism involved in both events. Using the probe 2', 7'-dichlorofluorescein diacetate (DCFH-DA) we show ROS generation by insulin in myotubes wich could be abolished by the NADPH oxidase inhibitor apocynin and by siRNA against p47phox; a regulatory subunit of NADPH oxidase. We studied the translocation of p47phox from cytoplasm to plasma membrane as a marker of the activation of NADPH oxidase. In order to study the main pathway involved in the activation of this enzyme, we tested the effects of bisindolylmaleimide (BIM); a non specific inhibitor of PKC and Ly290042; an inhibitor of PI3K. Both drugs modified the generation of ROS by insulin. We measured intracellular calcium increase in the presence of BIM, LY290042, apocynin, siRNA against p47phox and two drugs that interfere in IP3 pathway; xestospongin B and U73122. Calcium signals were inhibited by all of these treatments. Results indicate that insulin induces ROS generation trough NADPH activation. ROS participate in calcium increase dependent on IP3 receptors. Both PI3K and PKC are also involved in ROS and calcium increases induced by insulin.

ABSTRACT

AIMS: In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. METHODS AND RESULTS: Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. CONCLUSION: Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

ABSTRACT

AIM: Our objective was to investigate in cardiac muscle the contribution of NADPH oxidase to (a) ryanodine receptor-2 (RyR2) S-glutathionylation and (b) the preconditioning effects of exercise and tachycardia on infarct size following coronary artery occlusion. METHODS AND RESULTS: We measured NADPH oxidase activity, RyR2 S-glutathionylation, and calcium release kinetics in sarcoplasmic reticulum (SR) vesicles isolated from dog ventricular muscle after exercise and tachycardia, plus or minus prior administration of the NADPH oxidase inhibitor apocynin. In ventricular muscle sections, we studied the colocalization of NADPH oxidase and RyR2 by confocal microscopy using fluorescent antibodies. We determined the effect of apocynin on the infarct size produced by occlusion of the descendent anterior coronary artery in animals preconditioned by exercise or tachycardia. Exercise and tachycardia increased NADPH oxidase activity, RyR2 S-glutathionylation, and calcium release rates in isolated SR vesicles. Cardiac muscle sections displayed significant colocalization of NADPH oxidase and RyR2, suggesting direct and specific effects of reactive oxygen species (ROS) produced by NADPH oxidase on RyR2 activation. The NADPH oxidase inhibitor apocynin prevented the increase in RyR2 S-glutathionylation, reduced calcium release activity, and completely prevented the protective effects of exercise and tachycardia on infarct size. CONCLUSIONS: The loss of cardioprotection induced by the NADPH oxidase inhibitor suggests that ROS generated by this enzyme are important mediators of the preconditioning response, which presumably involves NADPH oxidase-induced RyR2 S-glutathionylation.

ABSTRACT

The cytoskeleton and cytoskeletal motors play a fundamental role in neurotransmitter receptor trafficking, but proteins that link GABAB receptors (GABABRs) to the cytoskeleton have not been described.We recently identified Marlin-1, a protein that interacts with GABABR1. Here, we explore the association of GABABRs and Marlin-1 to the cytoskeleton using a combination of biochemistry, microscopy and live cell imaging. Our results indicate that Marlin-1 is associated to microtubules and the molecular motor kinesin-I. We demonstrate that a fraction of Marlin-1 is mobile in dendrites of cultured hippocampal neurons and that mobility is microtubule-dependent. We also show that GABABRs interact robustly with kinesin-I and that intracellular membranes containing GABABRs are sensitive to treatments that disrupt a protein complex containing Marlin-1, kinesin-I and tubulin. Finally, we report that a kinesin-I mutant severely impairs receptor transport. We conclude that Marlin-1 and kinesin-1 link GABABRs to the tubulin cytoskeleton in neurons.

ABSTRACT

Changes of the initial composition and topography of mixed monolayers of Sphingomyelin and Ceramide modulate the degradation of Sphingomyelin by Bacillus cereus Sphingomyelinase. The presence of initial lateral phase boundary due to coexisting condensed and expanded phase domains favors the precatalytic steps of the reaction. The amount and quality of the domain lateral interface, defined by the type of boundary undulation, appears as a modulatory supramolecular code which regulates the catalytic efficiency of the enzyme. The long range domain lattice structuring is determined by the Sphingomyelinase activity.

ABSTRACT

We applied in vivo confocal microscopy of GFP-transgenic zebrafish in combination with 3D image analyses to study the asymmetric morphogenesis of the diencephalic parapineal organ on a supra-cellular, cellular, and sub-cellular level. Following a rough manual segmentation of the respective regions of interest (ROIs), the morphology of generated surface meshes was refined by an active surface model which it-eratively adjusts the mesh towards the morphology of the cellular structures. This procedure is essential for a precise morpho-topological analysis, mostly because of the adversarial diffraction limited resolution in the z-dimension of confocal image stacks. 3D Morphology and topology of the reconstructed cellular and supra-cellular structures during morphogenesis was quantified by principal axis transformations and 3D moment in-variants. Our data indicates that migration of the parapineal organ is accompanied by a rapid transition be-tween predominantly parallel cell orientations towards predominantly perpendicular orientations, a phenome-non which requires a precise control of cell shape and polarity. The orientational transition is followed by a phase of polarized cell motility in which membrane protrusions in the form of blebs and filopodia become ori-ented in the direction of the asymmetric migration. The morpho-topological descriptors unveil information that is not perceptible for a direct visual analysis of the microscopical data sets. This approach becomes essen-tial to access morphogenetic mechanisms which control asymmetry and migration.

ABSTRACT

In the field of image processing, the segmentation techniques allow to identify and to define regions of interest (ROIs) within an image. Applied to Biology and Biophysics, the segmentation of structures in microscopic images is indispensable for its later morphologic analysis.
This work applies to methods for the segmentation and morphologic characterization of biological structures, by means of parametric active contours that interact with gradient vector flow fields (GVF, generalized gradient vector flow fields, GGVF). The active contours are mathematical models that control the deformation of curves (two dimensions) or surfaces (three dimensions), by means of the action of intrinsic parameters (internal forces), and vector fields calculated as a function of the variations of intensities in the images (external forces). A functional of internal and external energy is defined that is aimed to be minimized by means of the equation of Euler-Lagrange; adding a time parameter to the model a solution with iterative methods can be approximated. Active contours in two dimensions were implemented first, and then extended in their formulation and application as active surfaces in three-dimensional ROIs. A criterion was settled down to optimize the resolution of contours in two dimensions, on the basis of the evaluation of curvature, inflection points and perimeter. For three dimensions surface meshes were evaluated, based on the precision in the calculation of area and regularity of curvature.
The developed methods were integrated to a computational application for image processing, being combined with other techniques of segmentation, reconstruction, visualization and parametrization of biological structures.

ABSTRACT

For the quantification of surface bound phosphomonoesterase activity (SBPA) of fungi, roots, or mycorrhiza, a colorimetric method based on p-nitrophenyl-phosphate (pNPP) is widely used. Unfortunately, this method does not reveal information about the localisation of the surface bound phosphomonoesterase (SBP). We introduce a method which localises and quantifies SBPA in living hyphae of ectomycorrhizal (EM) fungi using confocal laser scanning microscopy (LSM) of the hydrophilic substrate ELF-97 (enzyme-labeled fluorescence) and compare it to the pNPP assay. ELF-97 turns into a strongly-fluorescent precipitate upon activation by SBPA and forms bright fluorescent centres on the outer cell wall of the hyphae. Our data shows that the enzymatic reaction is not substrate limited during an incubation period of 15 min in fungal hyphae of Pisolithus tinctorius, Cenococcum geophilum, and Paxillus involutus. Image processing routines determined the total intensity and the average number of the fluorescent ELF-97 centres / µm fungal hyphae of C. geophilum and P. involutus. ELF-97 and pNPP detected similar variations of the SBPA at different pH values (3-7) during the measurement and different phosphorus (P) concentrations during the growth period of the fungi. The ELF-97 method revealed that C. geophilum and P. involutus adapt in different ways to the variation of the P concentrations during the growth period by varying the number, the activity, or both properties of the SBP centres. The phosphatases show peak activities at different pH values, so the response of the fungal mycelium to varying P concentrations in soils is pH selective. In conclusion, ELF-97 is a promising substrate to reveal SBPA and adaptation strategies on a structural-physiological level.

ABSTRACT

We set out to identify molecular mechanisms underlying the onset of necrotic Ca2+ overload, triggered in two epithelial cell lines by oxidative stress or metabolic depletion. As reported earlier, the overload was inhibited by extracellular Ca2+ chelation and the cation channel blocker gadolinium. However, the surface permeability to Ca2+ was reduced by 60%, thus discarding a role for Ca2+ channel/carrier activation. Instead, we registered a collapse of the plasma membrane Ca2+ ATPase (PMCA). Remarkably, inhibition of the Na+/K+ ATPase rescued the PMCA and reverted the Ca2+ rise. Thermodynamic considerations suggest that the Ca2+ overload develops when the Na+/K+ ATPase, by virtue of the Na+ overload, clamps the ATP phosphorylation potential below the minimum required by the PMCA. In addition to providing the mechanism for the onset of Ca2+ overload, the crosstalk between cation pumps offers a novel explanation for the role of Na+ in cell death.

ABSTRACT

Sphingomyelinases (SMases) hydrolyze the membrane constituent sphingomyelin (SM) to phosphocholine and ceramide (Cer). Growing evidence supports that SMase-induced SM/Cer conversion leads to the formation of lateral Cer-enriched domains which drive structural reorganization in lipid membranes. We previously provided visual evidence in real-time for the formation of Cer-enriched domains in SM monolayers through the action of the neutral Bacillus cereus SMase. In this work, we disclose a succession of discrete morphologic transitions and lateral organization of Cer-enriched domains that underlay the SMase-generated surface topography. We further reveal how these structural parameters couple to the generation of twodimensional electrostatic elds, based upon the species or orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epi uorescence microscopy on Langmuir monolayers revealed: 1), spontaneous nucleation and circular growth of Cer-enriched domains after injection of SMase into the subphase of the SM monolayer; 2), domain-intrinsic discrete transitions from circular to periodically undulating shapes followed by a second transition toward increasingly branched morphologies; 3), lateral superstructure organization into predominantly hexagonal domain lattices; 4), formation of super-superstructures by the hexagonal lattices; and 5), rotationally and laterally coupled domain movement before domain border contact. All patterns proved to be speci c for the SMase-driven system since they could not be observed with Cer-enriched domains generated by de ned mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (pi = 10 mN/m). Following the theories of lateral shape transitions, dipolar electrostatic interactions of lipid domains, and direct determinations of the monolayer dipole potential, our data show that SMase induces a domain-speci c packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In consequence, protein-driven generation of specic out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-specific out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (mm) scales. The cross-talk between lateral domain structures and dipolar electrostatic fields adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes.

ABSTRACT

In roots and mycorrhizae, the activity of phosphatases is an important parameter to characterise the efficiency of plants to access non-soluble phosphate pools in soils. We have quantified surface bound phosphatase (SBP) activity in non-mycorrhizal short roots and ectomycorrhizae of Nothofagus obliqua with image processed confocal laser scanning microscopy (LSM) using the fluorogenic substrate ELF-97 (enzyme-labelled fluorescence). Through interactive segmentation of root cells, mantle, and SBP-active centres, this method revealed a precise anatomical-physiological description of the SBP activity in cross-sections of short roots of N. obliqua, and of the mycorrhizal associations of N. obliqua with Pisolithus tinctorius and with Cenococcum geophilum in a controlled pH range (3-7). Our method revealed that the strategy of the examined species to vary the SBP activity was based primarily on the variation of the number and of the extension of the SBP-active centres. Fluctuations of the activities inside individual SBP-active centres were small. It was observed that non-mycorrhizal short roots of N. obliqua focus the distribution of SBP-active centres on the rhizodermis cells. In these cells, the SBP-active centres are distributed heterogeneously, and not preferentially in contact with the soil interface. The distribution of the SBP-active centres between the root cells and the mantle depended on the symbiont and on the pH of the buffer. The mantle hyphae of the N. obliqua-P. tinctorius associations promote direct contact between SBP-active centres and soil particles. In contrast, the mantle hyphae of N. obliqua-C. geophilum associations limit the expression of SBP-active centres to the interface between the mantle and the rhizodermis cells of N. obliqua. At this location, SBP-active centres are not in direct contact with any adjacent soil particles. Our observations of a pH-dependent activity, and of a mycorrhizal association-dependent activity of the SBP-active centres, together with the observed heterogeneity of the location of these centres relative to adjacent soil particles, challenge the general hypothesis that increased contact between mycorrhizae and soil results in higher efficiency of nutrient uptake.

ABSTRACT

Growing international competition enforces the traditional salmon farm industry to incorporate innovative techniques into the production process. The use of triploid, all-female breeding populations promises multiple advantages over diploid populations. Currently, different methods are tested in order to maximize triploidization rates. So far, an exact, non hazardous and simple method for the quantification of di- and triploid salmon erythrocytes does not exist. We introduce a method that combines a standard microscopic bright field technique (contrast staining with GIEMSAS) with multi-parameter image analysis and termed it quantitative morphologic microscopy (QMM). We used flow cytometry (FC) as a reference method to determine the DNA content of di- and triplod erythrocytes extracted from immature rainbow trouts (Oncorhynchus mykiss). Additionally, we applied quantitative fluorescence microscopy (QFM), using the DNA stains 4',6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), and acridine orange (AO). Our data shows that (i) QFM yields comparable results to FC when more than one discriminative parameter is considered, (ii) fluorescence dependent parameters can be substituted by pure morphological parameters, and (iii) QMM possess comparable or even superior discriminative capacities than FC or QFM. The developed method opens a perspective for the classification of microscopic objects with many possible applications.

ABSTRACT

We determined the location and the activity of surface-bound phosphomonoesterase (SBP) of five ectomycorrhizal (EM) fungi of Nothofagus obliqua. EM fungal mycelium of Paxillus involutus, Austropaxillus boletinoides, Descolea antartica, Cenococcum geophilum, and Pisolithus tinctorius was grown in media with varying concentrations of dissolved phosphorus. For each growth condition, the SBP activity was detected at different pH (3-7). SBP activity was assessed using a colorimetric method based on the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol phosphate (pNP) + P. A new technique involving confocal laser scanning microscopy (LSM) was used to locate and quantify SBP activity on the hyphal surface. The EM fungi showed two fundamentally different patterns of SBP activity in relation to varying environmental conditions (P-concentrations and pH). For D. antartica, A. boletinoides, and C. geophilum, changes of the SBP activity were induced primarily by changes in the number of SBP-active centers on the hyphae. For P. tinctorius and P. involutus, the number of SBP-active centers per µm hyphal length changed much less than the intensity of the SBP-active centers on the hyphae. The results of this study not only add a new aspect to the present discussion about the role of SBP-active centers in EM fungi, but also introduces LSM as a valuable method for studying EM fungi.

ABSTRACT

Purpose: To evaluate selective effects of ultraviolet (UV) irradiation on spontaneous and induced apoptosis in freshly extracted mice thymocytes. Materials and methods: Cells were exposed to UVA (1620-10200 J/m2), UVB (34-1620 J/m2) or UVC (1.5-1620 J/m2) and incubated 5.5 h with or without hydrocortisone, phorbol-12-myristate-13-acetate (PMA) or anti-Fas antibody (CD95). Additionally cells were irradiated with gamma rays (5 Gy) prior to UVB exposure (408 J/m2) at different time intervals. Apoptosis was quantified by DNA fragmentation. Results: Up to an irradiation of 5000 J/m2, UVA exposure did not show any effects on thymocyte apoptosis, while at 10200 J/m2 considerable DNA fragmentation was observed. In contrast, UVB and UVC irradiation clearly inhibit natural and cortisone-induced apoptosis, and more over, UVB inhibits apoptosis triggered by PMA and gamma irradiation, but not by CD95. Conclusions: The response of freshly extracted mice thymocytes to UV irradiation strongly depends on the wavelength. It is suggested that both a survival and an apoptotic pathway are possible, depending on the physiological state, spectral composition and cell type. Possible involvement of ERK and SAPK/JNK in the apoptotic pathway is discussed.

ABSTRACT

Apoptosis is a an important process in corneal development, homeostasis, and disease. This study was performed to determine for the first time basic temporal apoptotic features of SV-40 immortalized human corneal epithelial (HCE) cells. Additionally, we introduce a sensitive analysis of confocal microscopic images to measure the kinetics of staurosporine (STS) induced phosphatidylserine (PS) membrane translocation and early nuclear morphological changes. Methods: HCE cells were cultured in the presence of STS to induce apoptosis. Caspase-3 activity was measured with the fluorogenic substrate z-DEVD-rhodamine 110. We determined mitochondrial viability with a 4-[3-(4-iodophenyl)- 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate reduction assay, and chromatin degradation with a fluorometric method using 4,6-diamidino-2-phenylindole (DAPI). Membrane translocation of PS and nuclear alterations were assessed by quantitative fluorescence microscopy. Image processing routines were written in interactive data language (IDL).

ABSTRACT

The role of lipids in membranes has changed rapidly from static to dynamic and emphasized their involvement in information transduction, linking temporal and topological structuring through compositionally driven effects. Ceramide has been described as an important modulator of different membrane functions. In mixtures with ganglioside GM1, the condensation induced by ceramide increases intermolecular interactions, leading to an increase of the phase transition temperature and size of the self-assembled structure. In mixtures with phosphatidylcholines, ceramide segregates laterally in the gel state, forming domains whose thickness depend on global concentration and chain asymmetry of the sphingolipid.

ABSTRACT

We present a method to localise and quantify surface bound phosphatase activity (SBPA) in mycorrhiza fungi and mycorrhiza of Nothofagus obliqua (Mirb.) Oerst., using image processed confocal fluorescent microscopy. ELF-97 is a hydrophilic substrate which turns into a strongly fluorescent precipitate upon activation by phosphomonoesterases. In fungal mycelium, this technique has recently been approved by comparison with a standard method (p-nitrophenyl-phosphate). The microscopic technique based on ELF-97 revealed that Paxillus involutus (Batsch: Fr.) Sing. and Austropaxillus boletinoides (Sing.) Brsky. & Jarosch provide different adaptive strategies to changing phosphate concentrations and different pH (3-7). We also analysed SBPA in four mycorrhiza associations of N. obliqua and found that the organisation of the mantle played an essential role regarding the SBPA. In general, mycorrhized roots shifted SBPA from the root to the mantle. In this context, Pisolithus tinctorius (Pers.) Coker & Couch proved to be the most relevant mycorrhiza partner for N. obliqua by increasing significantly the overall SBPA of the mycorrhiza in respect to unmycorrhized roots. In conclusion, ELF-97 fluorescence microscopy in combination with image processing routines determined SBPA and revealed P-adaptation strategies of mycorrhiza fungi and mycorrhiza on a structural-physiological level.

ABSTRACT

Fluorescence spectroscopy and microscopy are powerful techniques to detect dynamic properties in artificial and natural lipid membrane systems. Unfortunately, most fluorescent dyes that sense dynamically relevant membrane parameters are UV sensitive. Their major disadvantage is a high susceptibility to fluorescence bleaching. Additionally, the risk for hazardous damages in biological components generally increases with decreasing excitation wavelength. Therefore the use of non- UV sensitive membrane dyes would provide significant advantage, particularly for applications in fluorescence microscopy, which usually implies high local excitation intensities. We applied steadystate fluorescence spectroscopy techniques to several UV and non-UV membrane dyes to detect and compare dynamically relevant excitation and emission characteristics. Small unilamellar liposomes (composed of egg yolk phosphatidylcholine) served as a model system for biological membranes. The dynamic properties of the membranes were varied by two independent parameters: the intrinsic cholesterol content (0 50 mol%) and temperature (10 508C). We tested four non-UV sensitive membrane dyes: 9-diethylamino-5H-benzophenoxazine-5-one (Nile Red), 4-(dicyanovinyl)julolidine (DCVJ), N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 4-64), and 1,18-dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlorate (DiIC18). We also tested three derivatives of DiIC18: DiIC16 and DiIC12 differ in acyl chain length and Fast-DiIC18 provides double bonds between hydrocarbon atoms. The spectral results were compared to established fluorescence characteristics of four UV membrane dyes: the anisotropy of 1-6-phenyl-1,3,5,-hexatrien (DPH), two derivatives of DPH (TMA-DPH and COO2-DHP), and the generalized polarization of 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan). Our results indicate that the tested non- UV dyes do not reveal dynamically relevant membrane parameters in a direct manner. However, spectral characteristics make DiIC18, Nile Red, and DCVJ promising probes for the microscopic detection of lateral lipid organization, an indirect indicator of membrane dynamics. In particular, DiIC18 showed very selective shifts in the emission spectra at defined temperatures and cholesterol contents that have not been reported elsewhere.

ABSTRACT

Lipid lateral organization is increasingly found to modulate membrane-bound enzymes. We followed in real time the reaction course of sphingomyelin (SM) degradation by Bacillus cereus sphingomyelinase (SMase) of lipid monolayers by epifluorescence microscopy. There is evidence that formation of ceramide (Cer), a lipid second messenger, drives structural reorganization of membrane lipids. Our results provide visual evidence that SMase activity initially alters surface topography by inducing phase separation into condensed (Cer-enriched) and expanded (SM-enriched) domains. The Cer-enriched phase grows steadily as the reaction proceeds at a constant rate. The surface topography derived from the SMase-driven reaction was compared with, and found to differ from, that of premixed SM/Cer monolayers of the same lipid composition, indicating that substantial information content is stored depending on the manner in which the surface was generated. The long-range topographic changes feed back on the kinetics of Smase, and the onset of condensed-phase percolation is temporally correlated with a rapid drop of reaction rate. These observations reveal a bidirectional influence and communication between effects taking place at the local molecular level and the supramolecular organization. The results suggest a novel biocatalytictopographic mechanism in which a surface enzymatic activity can influence the function of amphitropic proteins important for cell function.

ABSTRACT

We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.

ABSTRACT

Five different carotenoids (b-carotene, lutein, zeaxanthin, canthaxanthin and astaxanthin) at three different doses (1, 2.5 and 5 mol%) were incorporated in phosphatidylcholine multilamellar (MLV) liposomes and their recovery, incorporation rate and effects on membrane light-scattering, phase transition and UV-quenching capacity were comparatively studied. From carotenoid-doped MLV, small unilamellar liposomes (SUV) were obtained by sonication and the carotenoid incorporation into bilayers was comparatively investigated and discussed, together with their effects on UV-quenching. Structure- and dose- related differences were observed and correlated with the capacity of each carotenoid to integrate in the bilayers or to locate outside the bilayers. Lutein and zeaxanthin showed the highest stability and incorporation rate in MLV while in SUV, lutein proved to be the best included in the bilayer, to decrease the transition temperature and to broaden the transition peak, confirming its ability to rigidify membranes. Zeaxanthin behaviour was similar in MLV but inferior in SUV, due to its location outside bilayer in MLV. The carotenoid-dependent volumes of liposomes, correlated with their light-scattering properties were not significantly modified against free liposomes. The UV-quenching capacity was different in carotenoid-charged MLV (where lutein absorbed specifically at 360-460 nm) while in SUV, all carotenoids had quenching capacity against free liposomes. These data suggest a significant dependence of membrane properties on carotenoid stability, location and saturation limit in liposome bilayers.

ABSTRACT

A variety of methods to incorporate cholesterol into lipid membrane systems have been applied with varying success. We tested an incorporation method based on cholesterol-loaded methyl-b-cyclodextrins and compared it to a method that uses cholesterol-loaded liposomes. With methyl-b-cyclodextrin, we increased the cholesterol content in microsomal membranes to almost the fourfold of the original content. With cholesterolloaded liposomes instead, we achieved an elevation of 140%. Short incubation times and well-defined carrier properties favor the b-cyclodextrin method. For direct detection of membrane cholesterol, we slightly modified a microenzymatic fluorescence assay originally developed for precise cholesterol detection in serum. Without the need to perform lipid extraction, this assay was reliable for cholesterol detection in liposomes and in microsomes. Additionally, we compared the sensitivity of the fluidity-sensitive fluorescent dyes pyrene, pyrene methanol, bis-pyrene, 1-6-phenyl-1,3,5,-hexatrien, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5,-hexatrien in order to detect cholesterol indirectly by the dynamically relevant changes exerted on lipid matrices. These dyes differ not only in their membrane location but also in their dynamical behavior. We calibrated the dyes in liposomes of defined cholesterol content and used the most suited ones to follow and quantify the cholesterol incorporation into liposomal and microsomal membranes.

ABSTRACT

PURPOSE: To evaluate the involvement of cholesterol induced variations of membrane dynamics in mouse thymocyte apoptosis. MATERIALS AND METHODS: Membranes of thymocytes of RK mice were enriched with cholesterol using methyl-beta-cyclodextrins as carriers. Spontaneous apoptosis was compared with apoptosis induced either by X-irradiation, by treatment with dexamethasone (DEX), and by phorbol-12-myristate-13-acetate (PMA). Apoptotic cells were quantified by means of flow cytofluorometry. RESULTS: Small amounts of incorporated cholesterol enhance the cellular sensitivity for spontaneous apoptosis whereas larger amounts of incorporated cholesterol protect against spontaneous apoptosis and apoptosis induced by irradiation, DEX, or PMA. CONCLUSIONS: Cholesterol exerts specific rigidity effects on lipid membranes which have been shown to be involved in thymocyte apoptosis. The general effect of higher concentrations of cholesterol protecting against apoptosis hints towards a central protective mechanism. This study believes that either cholesterol paralyses great parts of the cell metabolism or that the apoptotic chain reaction is interrupted at a central point due to protection of membrane lipid regions from oxidative stress.