Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans.

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20 µM with a Vmax of 0.079 nmol/(µg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

Monomeric structure of an active form of bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.

Digitonin-facilitated delivery of imaging probes enables single-cell analysis of AKT signalling activities in suspension cells.

Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.

Nanodisc scaffold peptide (NSPr) replaces detergent by reconstituting acyl-CoA:cholesterol acyltransferase 1 into peptidiscs.

To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the biological activity and/or perturb oligomerization of individual MPs. Nanodisc scaffold peptide (NSPr), an amphipathic peptide analog of ApoA1, was recently shown to reconstitute detergent solubilized MPs into peptidiscs in vitro. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also known as sterol O-acyltransferase 1 (SOAT1), plays a key role in cellular cholesterol storage in various cell types and is a drug target to treat multiple human diseases. ACAT1 contains nine transmembrane domains (TMDs) and primarily forms a homotetramer in vitro and in intact cells; deletion of the N-terminal dimerization domain produces a homodimer with full retention in catalytic activity. ACAT1 is prone to inactivation by numerous detergents. Here we pursued the use of NSPr to overcome the detergent-induced inactivation of ACAT1 by generating near detergent-free ACAT1 peptidiscs. Based on native-PAGE analysis, we showed that NSPr reconstitutes ACAT1 into soluble peptidiscs, in which ACAT1 exists predominantly in oligomeric states greater than a homotetramer. The formation of these higher-order oligomeric states was independent of the N-terminal dimerization domain, suggesting that the oligomerization is mediated through hydrophobic interactions of multiple ACAT1 subunits. ACAT1 peptidiscs were still susceptible to heat-mediated inactivation, presumably due to the residual detergent (CHAPS) bound to ACAT1. We then conditioned ACAT1 with phosphatidylcholine (PC) to replace CHAPS prior to the formation of ACAT1 peptidiscs. The results showed, when PC was included, ACAT1 was present mainly in higher-order oligomeric states with greater enzymatic activity. With PC present, the enzymatic activity of ACAT1 peptidiscs was protected from heat-mediated inactivation. These results support the use of NSPr to create a near detergent-free solution of ACAT1 in peptidiscs for various in vitro studies. Our current results also raise the possibility that, under certain conditions, ACAT1 may form higher-order oligomeric states in vivo.

Interactions of OSW-1 with Lipid Bilayers in Comparison with Digitonin and Soyasaponin.

OSW-1, a unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, has potent cell-growth inhibition activity. In this study, we conducted fluorescence measurements and microscopic observations using palmitoyloleoylphosphatidylcholine (POPC)-cholesterol (Chol) bilayers to evaluate the membrane-binding affinity of OSW-1 in comparison with another steroidal saponin, digitonin, and the triterpenoid saponin, soyasaponin Bb(I). The membrane activities of these saponins were evaluated using calcein leakage assays and fitted to the binding isotherm by changing the ratios of saponin-lipids. Digitonin showed the highest binding affinity for the POPC-Chol membrane (Kapp = 0.38 µM-1) and the strongest membrane disruptivity in the bound saponin-lipid ratio at the point of 50% calcein leakage (r50 = 0.47) occurrence. OSW-1 showed slightly lower activity (Kapp = 0.31 µM-1; r50 = 0.78), and the soyasaponin was the lowest in the membrane affinity and the calcein leakage activity (Kapp = 0.017 µM-1; r50 = 1.66). The effect of OSW-1 was further assessed using confocal microscopy in an experiment utilizing DiI and rhodamine 6G as the fluorescence probes. The addition of 30 µM OSW-1 induced inward membrane curvature in some giant unilamellar vesicles (GUVs). At the higher OSW-1 concentration (58 µM, r50 = 0.78) where the 50% calcein leakage was observed, the morphology of some GUVs became elongated. With digitonin at the corresponding concentration (35 µM, r50 = 0.47), membrane disruption and formation of large aggregates in aqueous solution were observed, probably due to a detergent-type mechanism. These saponins, including OSW-1, required Chol to exhibit their potent membrane activity although their mechanisms are thought to be different. At the effective concentration, OSW-1 preferably binds to the bilayers without prominent disruption of vesicles and exerts its activity through the formation of saponin-Chol complexes, probably resulting in membrane permeabilization.

Dominant negative effect of the loss-of-function γ-secretase mutants on the wild-type enzyme through heterooligomerization.

γ-secretase is an intramembrane protease complex consisting of nicastrin, presenilin-1/2, APH-1a/b, and Pen-2. Hydrolysis of the 99-residue transmembrane fragment of amyloid precursor protein (APP-C99) by γ-secretase produces ß-amyloid (Aß) peptides. Pathogenic mutations in PSEN1 and PSEN2, which encode the catalytic subunit presenilin-1/2 of γ-secretase, lead to familial Alzheimer's disease in an autosomal dominant manner. However, the underlying mechanism of how the mutant PSEN gene may affect the function of the WT allele remains to be elucidated. Here we report that each of the loss-of-function γ-secretase variants that carries a PSEN1 mutation suppresses the protease activity of the WT γ-secretase on Aß production. Each of these γ-secretase variants forms a stable oligomer with the WT γ-secretase in vitro in the presence of the detergent CHAPSO {3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate}, but not digitonin. Importantly, robust protease activity of γ-secretase is detectable in the presence of CHAPSO, but not digitonin. These experimental observations suggest a dominant negative effect of the γ-secretase, in which the protease activity of WT γ-secretase is suppressed by the loss-of-function γ-secretase variants through hetero-oligomerization. The relevance of this finding to the genesis of Alzheimer's disease is critically evaluated.

Oxidative phosphorylation supercomplexes and respirasome reconstitution of the colorless alga Polytomella sp.

The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F1FO-ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-ß-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V2 and V4) and different respiratory supercomplexes (I/IV6, I/III4, I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V4 ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called "respirasome" was able to perform in-vitro oxygen consumption.

pH regulation in glycosomes of procyclic form Trypanosoma brucei.

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin ß superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle.

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens.

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.

In vitro ovicidal and larvicidal activities of some saponins and flavonoids against parasitic nematodes of goats.

This study assessed the anthelmintic activity of plant-derived compounds against gastrointestinal nematodes of goats using the egg hatch and larval motility assays. The compounds tested were saponins (digitonin and aescin) and their respective sapogenins (aglycones), hecogenin acetate and flavonoids (catechin, hesperidin, isocordoin and a mixture of isocordoin and cordoin). Additionally, cytotoxicity of active substances was analysed on Vero cell through 3-4,5-dimethylthiazol-2-yl,2,5diphenyltetrazolium bromide (MTT) and propidium iodide (PI) tests. Significant reduction on the egg hatching (P 90%). Nevertheless, higher cytotoxicity was observed in the MTT assay, with IC50 of 0.20 mg mL-1 (aescin) and 0.0074 mg mL-1 (digitonin). Aescin and digitonin have a pronounced in vitro anthelmintic effect and the glycone portion of these saponins plays an important role in this activity.

Structural basis of human γ-secretase assembly.

The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer's disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Šresolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase.

Purification of Active Respiratory Supercomplex from Bovine Heart Mitochondria Enables Functional Studies.

To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8-35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.

Soaping the NMDA receptor: Various types of detergents influence differently [(3)H]MK-801 binding to rat brain membranes.

Membranes prepared from rat brain were treated with increasing concentrations of cationic, neutral, anionic and zwitterionic surfactants. Potent inactivation of [(3)H]MK-801 binding to NMDA receptors (NRs) was provided by the cation cetyl pyridinium (IC50 25 µM) and the neutral digitonin (IC50 37 µM). A 2 h incubation of rat brain membranes at 24°C with 100 µM of the neutral Triton X-100 resulted in about 50% reversible inhibition (without inactivation). Reversible inhibition was also effected by the anion deoxycholate (IC50 700 µM), and by the zwitterions N-lauryl sulfobetaine (12-SB(±), 400 µM) and CHAPS (1.5 mM), with inactivation at higher concentrations. Keeping the NR cation channel in the closed state significantly protected against inactivation by cations and by 12-SB(±), but not by the other detergents. Inactivation depended differentially on the amount of the membranes, on the duration of the treatment, and on the temperature. Varying the amount of membranes by a factor 8 yielded for cetyl trimethylammonium (16-NMe3(+)) IC50s of inactivation from 10 to 80 µM, while for deoxycholate the IC50 of inactivation was 1.2 mM for all tissue quantities. Some compounds inactivated within a few min (16-NMe3(+), digitonin, CHAPS), while inactivation by others took at least half an hour (Triton X-100, deoxycholate, 12-SB(±)). These last 3 ones also exhibited the steepest temperature dependence. Knowledge about the influence of various parameters is helpful in selecting appropriate conditions allowing the treatment of brain membranes with amphiphiles without risking irreversible inactivation.

Sterol-recognition ability and membrane-disrupting activity of Ornithogalum saponin OSW-1 and usual 3-O-glycosyl saponins.

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.

Disordering Effects of Digitonin on Phospholipid Monolayers.

Digitonin, a steroidal saponin obtained from the foxglove plant (Digitalis purpurea), displays a wide spectrum of biological properties and is often used as a model in mechanistic investigations of the biological activity of saponins. In the present study, Langmuir monolayers of zwitterionic (DPPC, DMPE, POPC, POPE, DSPC, DSPE, and DPPE) and ionic (DPPS and DPPG) phospholipids were employed in order to better understand the effect of digitonin on the lipid organization. For this purpose, a combination of surface pressure relaxation, infrared reflection absorption spectroscopy (IRRAS), and fluorescence microscopy measurements was used. The observed increase in surface pressure (Π) suggests that digitonin can adsorb at the air/water interface, both bare and covered with the uncompressed phospholipid monolayers. However, the detailed analysis of IRRAS and fluorescence microscopy data shows that digitonin interacts with the lipid monolayers in a very selective way, and both the headgroup and the lipid tails affect this interaction. Nevertheless, it should be noted that in no case did digitonin cause any disruptive effects on the monolayers. The DPPE and DPPS monolayers get disordered by penetration with digitonin, despite an increase in surface pressure, leading to an unprecedented LC-LE transition. Interestingly, saponin could be easily squeezed out of these monolayers by mechanical compression.

Simultaneous multicolor detection of RNA and proteins using super-resolution microscopy.

A number of non-membranous cellular bodies have been identified in higher eukaryotes, and these bodies contain a specific set of proteins and RNAs that are used to fulfill their functions. The size of these RNA-containing cellular bodies is usually on a submicron scale, making it difficult to observe fine structures using optical microscopy due to the diffraction limitation of visible light. Recently, microscope companies have released super-resolution microscopes that were developed using different principles, enabling the observation of sub-micron structures not resolvable in conventional fluorescent microscopy. Here, we describe multi-color fluorescent in situ hybridization techniques optimized for the simultaneous detection of RNA and proteins using super-resolution microscopy, namely structured illumination microscopy (SIM).

Syntaxin 8 regulates platelet dense granule secretion, aggregation, and thrombus stability.

Platelet secretion not only drives thrombosis and hemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelet. Although several platelet SNAREs have been identified, further members of the SNARE family may be needed to fine-tune platelet secretion. In this study we identified expression of the t-SNARE syntaxin 8 (STX8) (Qc SNARE) in mouse and human platelets. In mouse studies, whereas STX8 was not essential for α-granule or lysosome secretion, Stx8(-/-) platelets showed a significant defect in dense granule secretion in response to thrombin and CRP. This was most pronounced at intermediate concentrations of agonists. They also showed an aggregation defect that could be rescued with exogenous ADP and increased embolization in Stx8(-/-) mice in vivo consistent with an important autocrine and paracrine role for ADP in aggregation and thrombus stabilization. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in regulating granule release from platelets and thus platelet function in thrombosis and hemostasis.

Impact of two different saponins on the organization of model lipid membranes.

Saponins, naturally occurring plant compounds are known for their biological and pharmacological activity. This activity is strongly related to the amphiphilic character of saponins that allows them to aggregate in aqueous solution and interact with membrane components. In this work, Langmuir monolayer techniques combined with polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy were used to study the interaction of selected saponins with lipid model membranes. Two structurally different saponins were used: digitonin and a commercial Merck Saponin. Membranes of different composition, namely, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) were formed at the air/water and air/saponin solution interfaces. The saponin-lipid interaction was characterized by changes in surface pressure, surface potential, surface morphology and PM-IRRAS signal. Both saponins interact with model membranes and change the physical state of membranes by perturbing the lipid acyl chain orientation. The changes in membrane fluidity were more significant upon the interaction with Merck Saponin. A higher affinity of saponins for cholesterol than phosphatidylglycerols was observed. Moreover, our results indicate that digitonin interacts strongly with cholesterol and solubilize the cholesterol monolayer at higher surface pressures. It was shown, that digitonin easily penetrate to the cholesterol monolayer and forms a hydrogen bond with the hydroxyl groups. These findings might be useful in further understanding of the saponin action at the membrane interface and of the mechanism of membrane lysis.

Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel.