Morphology of the differentiation and maturation of LLC-PK1 epithelia

In the present study, a stereologic approach was utilized to quantitatively assess morphological changes during the differentiation of LLC-PK1 cells into an epithelial membrane. This renal epithelial cell line has been described to undergo morphological changes during differentiation and maturation from subconfluent culture to a confluent epithelial layer. An increase in the number of apical microvilli, interpreted as an areal increase in this membrane domain was reported. This morphological differentiation was found to be accompanied by an increase in the expression of apical Na(+)-dependent hexose transport and the activities of certain brush border enzymes. Since no data are available that quantify the morphologic changes during LLC-PK1 differentiation, a quantitative morphologic-stereologic-investigation was performed for an early (6 days) and a late (12 days) state of confluence of LLC-PK1 monolayer cultures. The following morphological parameters were determined by light and electron microscopic morphometry: volume fractions (Vv) of nuclei, mitochondria, and lysosomes, and surface densities (Sv) of the apical and basolateral cell membrane domains. For the apical membrane surface, the microvillous fraction has been measured separately. Since the stereologic approach used in the present study allows the determination of absolute cell volumes, the absolute measures of organelle volumes (V) and membrane surfaces (S) per average cell can be calculated from volume and surface densities. Although no changes in cell density were found for 6 and 12 day old LLC-PK1 monolayers, indicating ceased cell proliferation due to contact inhibition, remarkable changes were found concerning the absolute cell volume and apical membrane surface. The observed increase in the apical cell surface was exclusively due to the enlarged microvillous surface fraction. This finding is in good agreement with the increased number of Na(+)-dependent hexose transporters as well as with the increased expression of apical membrane marker enzymes observed during the differentiation of LLC-PK1 monolayers.     

Lateral diffusion of a human sperm-head antigen during incubation in a capacitation medium and induction of the acrosome reaction in vitro

An integral component of human spermatozoa, a glycoprotein of Mr 143,000 (two subunits of Mr 76,000 and 67,000) was recognized by the a-HS 1A.1 monoclonal antibody. The antigen was localized on the plasma membrane over the sperm head, as demonstrated by transmission electron microscopy. The antigen-antibody binding on gametes during changes in their functional state was followed by an indirect immunofluorescence assay of live human spermatozoa. In freshly ejaculated spermatozoa the antibody binding pattern revealed a patchwork quilt-like topography of the plasma membrane over the acrosome; the percentage of positive cells varied from 20 to 78% with a mean of 50% (n = 82). Incubation in a capacitation medium could increase this percentage up to 98%, revealing new epitopes in an energy-dependent and temperature-independent manner; concomitantly, a part of the antigen migrated in energy-independent and temperature-dependent manner and accumulated in a ring over the postacrosome. When an acrosome reaction was induced in vitro in the presence of Ca2+ with either A23187, ionomycin or human follicular fluid, the HS 1A.1 antigen migrated until immobilization in a well defined pattern around the equatorial segment (single band) or around the equatorial and postacrosomal segments (2 or, seldom, 3 bands). The new antigen localization resulted from a lateral diffusion of pre-existing molecules, occurred in only a few minutes, did not require energy and was temperature-dependent. At the same time, the well outlined large patch burst into a multitude of small spots before vanishing. this veil-like labelling was often observed in spermatozoa kept in the seminal plasma or treated with a metabolic poison. The HS 1A.1 antigen localization reflects surface changes induced by the incubation in a capacitation medium and the acrosome reaction. Apart from the regional heterogeneity of the plasma membrane of a single cell, as noted above, there were differences in the plasma membrane changes in individual spermatozoa from the same ejaculate as well as in semen samples from different donors. The new antibody binding pattern was often alike in successive ejaculates of the same donor. In patients consulting for infertility the percentage of positive cells was often low and migration of the antigen was slight or absent.     

Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus

Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric alpha-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.     

Potential role of the membrane in the development of intestinal cellular damage after whole-body gamma irradiation of the rat

Our study emphasizes the effect of gamma irradiation on intestinal cell membrane fluidity and addresses the potential relationships existing between radiation-induced lipoperoxidation, membrane fluidity, and changes in membrane protein activities. Male Wistar rats were exposed to an 8-Gy total body irradiation (60Co source) and studied 1, 4, and 7 days after irradiation (D1, D4, and D7). Membrane enzyme activities and fluorescence anisotropy were determined on small intestinal crude membrane preparations. The supernatants of membrane preparations as well as plasma were used for malonedialdehyde (MDA) quantification. The effect of carbamylcholine on electrical parameters was estimated on distal ileum placed in Ussing chambers. We observed a decrease in fluorescence anisotropy for at least 7 days, an increase in membrane production of MDA at D4, a decrease in membrane enzyme activities at D4, but an amplification of carbamylcholine-induced increase in short-circuit current at D4 and D7. Furthermore, correlations were observed between the 1,6-diphenyl-1,3,5-hexatriene anisotropy coefficient and sucrase activity and between MDA levels and leucine aminopeptidase activity. Thus, total body irradiation induces changes in intestinal membrane fluidity and an increase in lipoperoxidation. These modifications may have an impact on the activity of membrane proteins involved in intestinal function.     

Redox enzymes in the tissues of the large intestine in the postnatal ontogeny of rats]

Using histochemical methods, age-related changes in activity of some redox enzymes in muscular and superficial layers of the mucous membrane, as well as in neurons of the myenteric nervous plexus of the large intestine have been studied in albino rats 5-day-old, 1-, 5-, 13-, 24-month-old. In young animals (1-5-month-old) an essential increase of the enzymatic activity of the energy metabolism takes place, in mature animals--stabilization of these processes, senescence brings about multidirectional changes in them. Manifestation degree of the changes in energy metabolism, occurring in old age are determined by certain metabolic and functional peculiarities in the organ tissue. In old animals certain strain of the energy metabolism develops, resulting from discoordination of energetic cycles in tissue of the large intestine wall.     

Mechanics of the transduction of sound in the tympanal organ of adults and larvae of locusts

Summary The mechanical transmission of sound in the tympanal organ of adults and 5th instar larvae ofLocusta migratoria andSchistocerca gregaria has been investigated by means of stroboscopic measurements within a frequency range from 1–20 kHz.Frequency dependent spatial distributions of amplitudes and phases of oscillation on the tympanal membrane and the Müller's organ could be demonstrated. Cuticular structures on the membrane may act as a lever arm (e.g. elevated process) and cause a transformation of the (unidimensional) membrane motion into components of displacements in the Müller's organ perpendicular, as well as even parallel, to the membrane.Sites of maximum relative displacements at distinct frequencies are found to be correlated to the course of the dendrites of the acoustic receptor cells. Differences in morphology of the tympanal organ between the two species as well as between adults and larvae always correspond to differences in the mechanical properties (resonances etc.). Consequently, differences or changes in the neurophysiological response characteristics of the different receptor cells have been found.Based upon these findings a correlation between the anatomical and physiological classification of the receptor cell groups is presented.Abbreviations T1, T2, T3, T6, T7 reference points on the tympanal membrane - M1, M4 reference points on the ganglion of the Müller's organ - K1, K2 reference points on the elevated process     

The role of retinal in the thermal stability of the purple membrane.

Differential scanning calorimetry demonstrates that the bleached form of the purple membrane does not possess any measurable thermal transition in water, up to 105 degrees C, whereas in 0.1 M phosphate pH 7.5 it shows a transition at about 82 degrees C, with an enthalpy of 110 kJ/mol. In the latter medium, the native membrane shows the main transition at 97 degrees C, with an enthalpy of 390 kJ/mol. The reduced form of the purple membrane shows two small transitions in water, as well as in 0.1 M phosphate, which do not seem to be related to the main thermal transition of the native membrane. Fourier-transform infrared spectra in D2O show that the two modified samples, as well as the native one, undergo similar secondary structural changes upon thermal denaturation. These changes appear to extend through a wide temperature range for both modified forms, particularly for the bleached one. The results suggest that the main thermal transition in the purple membrane is due to a cooperative conformational change involving the disruption of the network of electrostatic and hydrogen-bonding interactions which originate from the protonated Schiff base. In the two modified membranes, these conformational changes appear to proceed smoothly through a rather low or non-cooperative process. The thermal behaviour of the bleached membrane in water resembles that of the molten globule state described for several globular proteins.     

Interaction of a peptide derived from C-terminus of human TRPA1 channel with model membranes mimicking the inner leaflet of the plasma membrane

The transient receptor potential ankyrin 1 channel (TRPA1) belongs to the TRP cation channel superfamily that responds to a panoply of stimuli such as changes in temperature, calcium levels, reactive oxygen and nitrogen species and lipid mediators among others. The TRP superfamily has been implicated in diverse pathological states including neurodegenerative disorders, kidney diseases, inflammation, pain and cancer. The intracellular C-terminus is an important regulator of TRP channel activity. Studies with this and other TRP superfamily members have shown that the C-terminus association with lipid bilayer alters channel sensitivity and activation, especially interactions occurring through basic residues. Nevertheless, it is not yet clear how this process takes place and which regions in the C-terminus would be responsible for such membrane recognition. With that in mind, herein the first putative membrane interacting region of the C-terminus of human TRPA1, (corresponding to a 29 residue peptide, IAEVQKHASLKRIAMQVELHTSLEKKLPL) named H1 due to its potential helical character was chosen for studies of membrane interaction.The affinity of H1 to lipid membranes, H1 structural changes occurring upon this interaction as well as effects of this interaction in lipid organization and integrity were investigated using a biophysical approach. Lipid models systems composed of zwitterionic and anionic lipids, namely those present in the lipid membrane inner leaflet, where H1 is prone to interact, where used. The study reveals a strong interaction and affinity of H1 as well as peptide structuration especially with membranes containing anionic lipids. Moreover, the interactions and peptide structure adoption are headgroup specific.     

A novel viscosity-sensitive fluorescent probe for monitoring the changes of mitochondrial viscosity

As a fundamental physical parameter, viscosity influences the diffusion in biological processes. The changes in intracellular viscosity led to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Here, we devised and synthesized a viscosity-sensitive fluorescent probe LBX-1 . LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement in fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 could localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes in mitochondrial viscosity in complex biological systems.     

Kinetics of electron transport, proton transfer and photophosphorylation in chloroplasts and their relation to temperature-induced structural changes in the thylakoid membrane

Effects of various temperatures on the rates of electron transport between two photosystems, the light-induced uptake of protons, kinetics of proton efflux from the chloroplasts in the dark and photophosphorylation were studied in isolated chloroplasts. There are correlations between the physical state of thylakoid membrane and the rates of electron- and proton transport processes. The temperature dependence of "structural" parameter (fluidity of lipids in membrane) as well as the rates of electron- and proton transport processes reveal the breaks under the same temperatures. Stimulation of photophosphorylation by temperature increasing correlates with the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the protein part of CF0-CF1 complex. The rate of photophosphorylation also correlates with the physical state of membrane lipids. Thermoinduced "melting" of the thylakoid membrane inhibits the ATP formation because of a decrease in photosystem 2 photochemical activity and stimulation of membrane conductivity for protons.     

Changes in cholesterol levels in the plasma membrane modulate cell signaling and regulate cell adhesion and migration on fibronectin

The number and distribution of lipid molecules, including cholesterol in particular, in the plasma membrane, may play a key role in regulating several physiological processes in cells. We investigated the role of membrane cholesterol in regulating cell shape, adhesion and motility. The acute depletion of cholesterol from the plasma membrane of cells that were well spread and motile on fibronectin caused the rounding of these cells and decreased their adhesion to and motility on fibronectin. These modifications were less pronounced in cells plated on laminin, vitronectin or plastic, indicating that cholesterol-mediated changes in adhesion and motility are more specific for adhesion mediated by fibronectin-specific integrins, such as alpha5beta1. These changes were accompanied by remodeling of the actin cytoskeleton, the spatial reorganization of paxillin in the membrane, and changes to the dynamics of alpha5 integrin and paxillin-rich focal adhesions. Levels of tyrosine phosphorylation at position 576/577 of FAK and Erk1/Erk2 MAP-kinase activity levels were both lower in cholesterol-depleted than in control cells. These levels normalized only on fibronectin when cholesterol was reincorporated into the cell membrane. Thus, membrane cholesterol content has a specific effect on certain signaling pathways specifically involved in regulating cell motility on fibronectin and organization of the actin cytoskeleton.     

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.     

Effect of detergents on the ATPase activity and barrier properties of the plasma membrane of smooth muscle cells

The effect of detergents on enzymic and barrier properties of membrane structures is studied in the plasma membrane fraction, postsynaptic membranes of smooth muscle cells and sterine bilipid membranes. The formation of hydrophylic pores in the membrane, as well as changes in the phase state of the lipid matrix and fluidity of lipid microenvironment of membrane enzymes are supposed.     

Reaction of the skin fibroblast cytoskeleton to micromanipulation interventions

Micromanipulation is a strong mechanical intervention into cellular integrity and induces large changes in the fine structure of the treated cells. Human diploid skin fibroblasts (KF1 and KF2 cell lines) were chosen as an experimental model. Special hatching needles were used for defined micromanipulation interventions (deformation of plasma membrane). Changes in cytoskeletal structures were visualized by using fluorescent and confocal microscopy. The actin cytoskeleton showed a more sensitive response to micromanipulation than microtubules. Characteristic changes in microfilaments, i.e., thickenings and knot formation, were visible in treated cells fixed immediately after micromanipulation and were the result of hatching-needle pressure on the plasma membrane as well as a reaction of actin filaments localized near the plasma membrane deformation. These direct changes and also other specific alterations in the actin filament network were detectable 14 to 16 h after treatment, but they were not observed when longer reparation intervals were used.     

Temperature Dependence of Vasopressin Action on the Toad Bladder

Toad bladders were challenged with vasopressin at one temperature, fixed on the mucosa with 1% glutaraldehyde, and then subjected to an osmotic gradient at another temperature. Thus, the temperature dependence of vasopressin action on membrane permeability was distinguished from the temperature dependence of osmotic water flux. As the temperature was raised from 20° to 38°C, there was a substantial increase in the velocity of vasopressin action, but osmotic flux was hardly affected. In this range of temperature the apparent energy of activation for net water movement across the bladder amounted to only 1.2 kcal/mole, a value well below the activation energy for bulk water viscosity. It is suggested that osmotic water flux takes place through narrow, nonpolar channels in the membrane. When the temperature was raised from 4° to 20°C, both vasopressin action as well as osmotic water flux were markedly enhanced. Activation energies for net water movement were now 8.5 kcal/mole (4°–9°C) and 4.1 kcal/mole (9°–20°C), indicating that the components of the aqueous channel undergo conformational changes as the temperature is lowered from 20°C. At 43°C bladder reactivity to vasopressin was lost, and irreversible changes in selective permeability were observed. The apparent energy of activation for net water movement across the denatured membrane was 6.6 kcal/mole. Approximately 1 µosmol of NaCl was exchanged for 1 µl of H₂O across the denatured membrane.     

The effects of 20-hydroxyecdysone on the metabolic labeling of membrane proteins in Drosophila imaginal discs

20-Hydroxyecdysone induces evagination of imaginal discs of Drosophila melanogaster cultured in vitro. The possible involvement of cell-surface proteins in this process has prompted us to study the synthesis of membrane proteins in imaginal discs. A procedure is reported for the isolation of membrane vesicle fractions from discs that are enriched for the plasma membrane enzyme, Na+/K+-ATPase, and that label with the surface-labeling reagent [125I]iodosulfanilic acid. 20-Hydroxyecdysone alters the pattern of [35S]methionine incorporation into polypeptides in these membrane vesicle fractions. Increased and decreased incorporation as well as changes in migration on two-dimensional gels of specific polypeptides are caused by the hormone. These changes parallel in time the onset and the continuation of evagination.     

On the molecular mechanism of the blue to purple transition of bacteriorhodopsin. UV-difference spectroscopy and electron spin resonance studies

Conformational changes in the bacteriorhodopsin molecule related to the blue to purple transition have been monitored using UV-difference spectrophotometry. Mn2+ binding to the deionized blue membrane, which restores the purple form, promotes the appearance of a difference spectrum that can be interpreted as arising from tryptophan perturbation. Similar difference spectra were found upon pH increase of the blue membrane suspensions. Such pH increase yields the deionized purple membrane and shows an apparent pK of 5.4. Binding of Hg2+ to the blue membrane does not induce any UV-difference spectrum or change the apparent pK of the transition. ESR studies of Mn2+ binding show that in the pink membrane several high and medium affinity binding sites have been converted to low affinity ones. In the NaBH4-reduced membrane, a medium affinity site has been converted to a low affinity site. Upon Mn2+ binding to the reduced membrane or pH increase, absorption changes were found in the visible region which showed a dependence upon bound Mn2+ as well as an apparent pK similar to those of the nonreduced membrane. It is proposed that the functional form of the membrane depends primarily on the deprotonated state of a control group and that cation binding only affects the pK of this deprotonation through changes in the membrane surface potential.     

Bacillus thuringiensis insecticidal crylab toxin does not affect the membrane integrity of the mammalian intestinal epithelial cells: An in vitro study

Summary The mammalian intestinal epithelium has been found, based on in vivo experiments, to be resistant to insecticidal Cry toxins, which are derived from Bacillus thuringiensis and fatally damage insect midgut cells. Thus, the toxins are commonly used as a genetic resource in insect-resistant transgenic plants for feed. However, Cry toxins bind to the cellular brush border membrane vescle (BBMV) of mammalian intestinal cells. In this study, we investigated the affinity of Cry1Ab toxin, a lepidopteran-specific Cry1-type toxin, to the cellular BBMV of two mammalian intestinal cells as well as the effect of the toxin on the membrane potential of three mammalian intestinal cells compared to its effects on the silkworm midgut cell. We found that Cry1Ab toxin did bind to the bovine and porcine BBMV, but far more weakly than it did to the silkworm midgut BBMV. Furthermore, although the silkworm midgut cells developed severe membrane potential changes within 1 h following the toxin treatment at a final concentration of 2 μg/ml, no such membraneous changes were observed on the bovine, procine, and human intestinal cells. The present in vitro results suggest that, although Cry1Ab toxin may bind weakly or nonspecifically to certain BBMV components in the mammalian intestinal cell, it does not damage the cell’s membrane integrity, thus exerting no subsequent adverse effects on the cell.