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 Cerenriched domains which drive structural reorganization in lipid membranes.Wepreviously 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 (P ¼ 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 speci c out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-speci c 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 elds 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

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

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

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.