F-actin has a very high calorimetric unfolding enthalpy.

The thermal unfolding of F-actin was studied using differential scanning calorimetry. Heat denatures F-actin in two steps. The first is endothermic and corresponds to the unfolding of the peptide chain, while the second is exothermic and is due to the aggregation of the unfolded molecules. The aspect of the thermogram is influenced by the concentration of the protein. For concentrations around 1mg/ml, the steps are superimposed, while the two steps are separated at very low concentrations. It thus becomes possible to estimate the calorimetric enthalpy for the unfolding step. The enthalpy of unfolding is 64 MJ/mol, or 1400 J/g. This value is considerably higher than those mentioned in the literature for the denaturation of actin and other proteins, which are in the range of 25-30 J/g. The large amount of energy required to unfold the molecule of F-actin could be an adaptation of its role as a protein that transmits forces, and consequently must be very resistant to mechanical constraints.

Effect of the polar headgroup of phospholipids on their interaction with actin.

It is generally admitted that actin filaments are anchored to a membrane by membranar actin-binding-proteins. However, we found that actin may also interact directly with membrane phospholipids. The actin-phospholipid complex has been investigated at the air-water interface using a film balance technique. In order to probe the effect of the phospholipid headgroup on the actin-phospholipid interaction, we focus mainly on phospholipids that have the same acyl chain length but different headgroups. For all the phospholipids, the apparent area per molecule (the total surface divided by the number of lipid molecules) increases after the injection of the protein into the subphase, which suggests an intercalation of actin between the phospholipid molecules. This effect seems to be more important for DMPE and DMPS than for DMPG, suggesting that the headgroup plays an important role in this intercalation. The critical surface pressure associated to the liquid expanded-liquid condensed (LE-LC) phospholipid transition increases with the concentration of G-actin and thus suggests that G-actin acts as an impurity, simply competing as a surfactant at the air-water interface. On the other hand, F-actin affects the LE to LC transition of phospholipids differently. In this case, the LE to LC transition is broader and F-actin slightly decreases the critical surface pressure, which suggests that electrostatic interactions are involved.

Adsorption of actin at the air-water interface: a monolayer study.

The intrinsic surface activity of the contractile protein actin has been determined from surface tension measurements using the Wilhelmy hanging-plate method. Actin, a very soluble protein, moves from the subphase to the air-water interface to make a film. In the absence of magnesium, actin is monomeric and is known as G-actin. During the compression the monomers change their conformation or orientation at the interface and they are then pushed reversibly into the subphase upon further compression. No collapse occurs. Actin monomers in the presence of magnesium become activated; at concentrations greater than some critical value, actin polymerizes to form filaments of F-actin. The actin filaments have a higher surface activity than the actin monomers either because they are more hydrophobic or because F-actin, a rigid polymer, is much more efficient at creating excluded volume. The actin filaments then form a rigid film at the interface that collapses when the surface area is decreased. At less than the critical concentration, the actin monomers are present in the subphase in their activated form. However, their concentration increases at the interface during film compression until the critical concentration is reached. The surface pressure isotherm in this case has the characteristics of a G-actin film at the beginning of the compression and of an F-actin film at the end of the compression process.

Surface film pressure of actin: interactions with lipids in mixed monolayers.

The interactions of actin with neutral lipid films made from DLPC, and with positively charged films built from DLPC and stearylamine (SA), have been characterized by the monolayer technique. Injection of actin underneath an expanded lipid film produces an increase in the surface pressure that is consistent with a penetration of the lipid molecules by actin. This adsorption of actin to the lipid is more pronounced either with positively charged films or with Mg(2+) present in the sub-phase, suggesting that the mechanism involves an electrostatic attraction. During compression, the actin molecules are squeezed out into the sub-phase, carrying along some lipid molecules; this suggests a strong affinity of the lipids for actin. An analysis of the dilational modulus shows that when actin is found as monomers at the interface, the mixed actin-lipid film undergoes three phase changes upon compression. On the other hand, when actin is polymerized at the interface, the actin and the lipid form a rigid film for which the compressibility is mostly dominated by actin.

Nanoerythrosomes, a new derivative of erythrocyte ghost: IV. Fate of reinjected nanoerythrosomes.

Recently, we have developed a promising new drug carrier named nanoerythrosome (nEryt). This transporter are small vesicles made with the red blood cell membrane. Anticancer drugs like daunorubicin, linked to these nEryt, have a higher antineoplastic activity than the free drug. In this paper, we first analyzed the biodistribution of 125I-nEryt purified by dialysis following intravenous (i.v.) or intraperitoneal (i.p.) injections in CD1 mice. After i.v. administration, nEryt, are rapidly removed from blood circulation (< 30 min). Mainly the liver and spleen take up the vesicles. I.p. injections of nEryt purified by dialysis, showed a marked activity in the inguinal lymph nodes 2 hours post-injection. nEryt purified by centrifugation have a different biodistribution. They accumulate also in the lungs. We demonstrated that accumulation in the lungs is due to particle aggregation during the preparation procedure. Comparative analysis of size distribution of each nEryt preparation revealed that nEtyt purified by centrifugation has a mean diameter of 1.5 microm which is 10 times higher than its dialyzed counterpart. Light microscopic autoradiographs of dialyzed nEryt, reinjected i.v., showed accumulation of nEryt in the sinusoidal lumen as well as in the parenchymal cells of the liver. Autoradiographs of the spleen revealed that nEryt are distributed specifically near the marginal zone and that some of them have escaped the meshes of the red pulp cords.

Effects of new triphenylethylene platinum(II) complexes on the interaction with phosphatidylcholine liposomes.

In a previous work we synthesized a class of new antineoplastic drugs by coupling a cisplatin derivative to a triphenylethylene moiety similar to the antiestrogen, tamoxifen. These drugs differ in the number of hydroxy functions on the triphenylethylene rings and in the length of the linking arm. To gain more insight into the cellular mechanism by which these new drugs act on cells, we studied, using differential scanning calorimetry, the effects of these compounds on the phase transition of membrane phospholipid (distearoyl phosphatidyl choline (DSPC)), and correlated these effects to drug cytotoxicity. The drugs without hydroxy function showed the highest cytotoxicity and induced little change on the thermogram of DSPC. Contrarily, the drugs bearing two or three hydroxy groups were less toxic, but induced important modifications of the thermogram. We suggest that the drugs with no hydroxy group enter the membrane, with the triphenylethylene moiety localized deep within the hydrophobic core of the bilayer and do not affect the cooperativity region (C2-C8). In contrast, drugs which bear hydroxy groups on the triphenylethylene rings system perturb the phospholipid molecular arrangement; this may be due either to the additional steric hindrance of the hydroxy functions in the core of the bilayer, or to their hydrophilic effect on the polar head of the lipid. In vitro, the cytotoxic effect of these drugs seems not to be related to their affinity for the estrogen receptor. We suggest that the addition of a triphenylethylene moiety to the platinum(II) complexes increases the hydrophobicity, and consequently the resulting drugs become more permeable to the membrane, particularly the non-hydroxylated triphenylethylene derivatives.

Interaction between G-actin and various types of liposomes: A 19F, 31P, and 2H nuclear magnetic resonance study.

We have investigated in the present study the interaction between G-actin and various types of liposomes, zwitterionic, positively charged, and negatively charged. To investigate at the molecular level the conformation of actin in the presence of lipids, we have selectively attached a fluorinated probe, 3-bromo-1,1,1-trifluoropropanone, to the actin cysteine residues 10, 285, and 374 and used high-resolution 19F nuclear magnetic resonance spectroscopy to investigate the probe resonances. The results indicate a change in the mobility of the 19F labels when G-actin is in the presence of positively charged liposomes made of DMPC and stearylamine and in the presence of DMPG, a negatively charged lipid. No conformational change was observed in the actin molecule in the presence of neutral liposomes. Electron micrographs of these systems reveal the formation of paracrystalline arrays of actin filaments at the surface of the positively charged liposomes, while no evidence of actin polymerization or paracrystallization was observed in the presence of DMPG. The interaction between actin and the lipid polar headgroup has also been investigated using solid-state phosphorus and deuterium NMR. The results indicate no evidence of interaction between actin and zwitterionic liposomes but show an interaction between the positively charged liposomes and a negative charge on the actin molecules. Interestingly, the negatively charged liposomes interact with a positive charge, which is most likely associated with the three residues (His-Arg-Lys) preceding the cysteine 374 residue in the protein.

Nanoerythrosomes, a new derivative of erythrocyte ghost: III. Is phagocytosis involved in the mechanism of action?

We have previously developed a new drug carrier, named nanoerythrosome which is prepared by extrusion of erythrocyte ghosts to produce small vesicles having an average diameter of 100 nm. Daunorubicin (DNR) conjugated to these nanoerythrosomes has a higher antineoplastic index than the free drug. Moreover, since nanoerythrosomes are particles, phagocytosis may be involved in their mechanism of potentiation. In the present study, we have compared the mechanism of penetration between free DNR and conjugate DNR linked to nanoerythrosomes, on cells presenting high phagocytic activity, macrophages, and cells lacking phagocytic activity, the P388 D1 cell line. Our results demonstrate that: 1) The nanoerythrosome-DNR complex is rapidly adsorbed and phagocytosed by the macrophages, but not by the P388 D1 cell line. 2) On the contrary, DNR enters both phagocytic and non phagocytic cells. Furthermore, the cellular distribution of DNR is the same in both cell lines, the nucleus being the target organelle. We conclude that phagocytosis of the nanoerythrosome-DNR complex is not involved in its mechanism of action.

Effects of nucleotides on the denaturation of F actin: a differential scanning calorimetry and FTIR spectroscopy study.

We have utilized DSC and high pressure FTIR spectroscopy to study the specificity and mechanism by which ATP protects actin against heat and pressure denaturation. Analysis of the thermograms shows that ATP raises the transition temperature Tm for actin from 69.6 to 75.8 degrees C, and the calorimetric enthalpy, deltaH, from 680 to 990 kJ/mole. Moreover, the peak becomes sharper indicating a more cooperative process. Among the other nucleotide triphosphates, only UTP increases the Tm by 2.5 degrees C, whereas GTP and CTP have negligable effects; ADP and AMP are less active, increasing the Tm by 2.1 and 1.6 degrees C, respectively. Therefore, gamma phosphate plays a key role in this protection, but its hydrolysis is not implicated since the nonhydrolysable analogue of ATP, ATP-PNP have the same activity as ATP. FTIR spectroscopy demonstrates that ATP also protects actin against high pressure denaturation. Analysis of the amide I band during the increase in pressure clearly illustrates that ATP protects particularly a region rich in beta-sheets of the actin molecule.

Nanoerythrosomes, a new derivative of erythrocyte ghost II: identification of the mechanism of action.

We have recently developed a new drug which is a carrier from red blood cell membrane. This carrier, named nanoerythrosome (nEryt), is prepared by extrusion of erythrocyte ghosts to produce small vesicles having an average diameter of 100 nm. Daunorubicin (DNR) was covalently conjugated to the nEryt (nEryt-DNR) using glutaraldehyde as homobifunctional linking arm. This led to a complex that is more active than free DNR both in vitro and in vivo. In this study, we identified the mechanisms that make the complex nEryt-DNR more active than free DNR. Using fluorescence microscopy and cellular-uptake, we observed that the nEryt-DNR complex cannot diffuse through the cell membrane and do not enter the cell by endocytosis. Our results suggest that the nEryt-DNR is rapidly absorbed onto the cell membrane. Free DNR is then slowly released by hydrolysis of the glutaraldehyde linking arm, producing a high concentration of free DNR in the cell's vicinity over a long period of time.

Does actin bind to membrane lipids under conditions compatible with those existing in vivo?

Using an in vitro system composed of liposomes and pure actin, we previously established that actin can interact directly with membrane lipids and suggested that this interaction may also exist in vivo. However, an important potential caveat has been brought to our attention concerning the high concentrations of lipids used in our assays. Indeed, it has been hypothesized that under our experimental conditions, divalent cations may become tightly bound to the lipid bilayers, reducing the free divalent cation concentration to non-physiological levels. The observed actin-lipid interaction has therefore been suggested to be an in vitro artifact. In order to test this hypothesis, we have measured the capture of Mg++ by DSPC liposomes under our experimental conditions. Our results show that only one Mg++ ion is captured for every 40 DSPC molecules. The resulting reduction in the free Mg++ concentration is therefore negligible in our assays. It is concluded that the actin-lipid interaction which we have previously documented indeed occurs under ionic conditions compatible with those found in vivo.

Mechanism of interaction between actin and membrane lipids: a pressure-tuning infrared spectroscopy study.

Using pressure-tuning Fourier transform infrared spectroscopy to study an in vitro system consisting of actin and distearoyl-phosphatidylcholine (DSPC) liposomes, we have determined the mechanism of interaction between actin and membrane lipids. This interaction results in a significant conformational change in actin molecules. Analysis of the amide I band of actin shows an increase in the beta-sheets to alpha-helix ratio, in random turns, and in interactions between actin monomers. In the absence of lipids, the actin molecules are denatured by pressures of 8 x 10(8) Pa and more, which give rise to a random organization of the peptide chain. However, in the presence of DSPC liposomes, pressure greater than 2 x 10(8) Pa induces a change in actin conformation, which is dominated by strongly interacting beta-sheets. As the spectra of the lipid molecules are not changed by the presence of actin, the organization of the lipid molecules in the bilayer is not affected by the protein. It is concluded from these results that this interaction of actin with membrane lipids involves very few lipid molecules. These lipid molecules may interact with actin at a few specific sites on the protein.

The release from metaphase arrest in blue mussel oocytes.

In Mytilus edulis, shed oocytes are arrested at metaphase I of meiosis until fertilization. We previously suggested (Dubé and Dufresne, J. Exp. Zool. 256:323-332, 1990) that such a metaphase arrest depends upon a continuous synthesis of short-lived proteins, the destruction of which is sufficient to induce meiosis resumption. We further investigated the mechanism of metaphase release in blue mussel oocytes as triggered either by fertilization or by inhibition of protein synthesis (emetine) or phosphorylation (6-dimethylaminopurine, 6-DMAP). Treatment of unfertilized oocytes (UF) with emetine induces completion of the first meiotic cycle including extrusion of the polar body, followed by chromosome decondensation and by the formation of large membrane-bound nuclei, as visualized by Hoechst staining and transmission electron microscopy (TEM). Inhibition of protein phosphorylation with 6-DMAP induces directly chromosome decondensation and the formation of multiple nuclei surrounded by nuclear membrane. These interphasic nuclei exhibit continuous 3H-thymidine incorporation. p13 precipitation of p34 and associated proteins reveals "putative" cyclins in UF, no longer detected after metaphase/anaphase transition due to fertilization or emetine treatment. In the presence of 6-DMAP, new migrating forms are observed. The phosphorylated p34cdc2 homolog becomes dephosphorylated after fertilization or emetine treatment, whereas 6-DMAP induces its phosphorylation on tyrosine. Histone H1 kinase activity is reduced after these treatments, compared to the UF sample. Our results suggest that the metaphase/anaphase transition triggered by fertilization in blue mussel oocytes is induced by the rapid destruction of a set of continuously synthesized proteins accompanied by decreased histone H1 kinase activity. These events can be mimicked by inhibiting protein synthesis. Inhibition of protein phosphorylation would drive the cell to interphase without commitment to meiosis I.

Nanoerythrosome, a new derivative of erythrocyte ghost: preparation and antineoplastic potential as drug carrier for daunorubicin.

Liposomes and monoclonal antibodies are used as drug carriers for the optimal delivery of pharmacologic agents. However, they present disadvantages that led us to develop a new model of drug carriers: the nanoerythrosomes. Nanoerythrosomes are vesicles prepared by the extrusion of red blood cell ghosts, the average diameter of these vesicles is 0.1 micron. Daunorubicin was covalently linked to nanoerythrosomes and the cytotoxicity of daunorubicin conjugated to nanoerythrosomes was assessed on P388D1 cell line. The results indicated that the cytotoxicity of conjugated daunorubicin was as high as the free daunorubicin. Daunorubicin--nanoerythrosome conjugates had a higher antineoplastic activity than the free drug on CDF1 leukemia tumors. These results indicate that nonoerythrosomes could be potentially used as drug carriers.

Actin conformation is drastically altered by direct interaction with membrane lipids: a differential scanning calorimetry study.

One of the current dogmas in cytoskeleton research holds that actin filaments are attached to the cell membrane through integral membrane actin-binding proteins. We have challenged this concept, using an in vitro system composed of pure actin and liposomes, and have found that actin may also interact with membrane lipids. Differential scanning calorimetry (DSC) shows that when the actin molecule is in contact with such lipids, it undergoes a major conformational change which results in the complete disappearance of its phase transition. Conversely, DSC scans reveal that the phase transition of the membrane lipids is only weakly affected by the presence of actin. Indeed, the lipids' main transition shows only slight shifts in Tm, from 56.6 to 57 degrees C, and delta Hcal, from 10.1 to 8.8 kcal/mol. In the lipids' pretransition, Tp is shifted from 52.7 to 53.7 degrees C, and delta Hcal is shifted from 0.75 to 0.33 kcal/mol. This interaction between purified actin and membrane lipids is inhibited by high concentrations of KCl, thus indicating that the phenomenon is primarily electrostatic in nature. The ultrastructural consequences of this change in actin conformation were investigated by electron microscopy, which revealed the formation of paracrystalline arrays of actin filaments at the surface of the liposomes. We therefore propose a model in which a limited number of lipid molecules may interact with specific sites on the actin molecule, resulting in the protein's observed conformational change.

Changes in molar volume and heat capacity of actin upon polymerization.

We have used densimetry and microcalorimetry to measure the changes in molar volume and heat capacity of the actin molecule during Mg(2+)-induced polymerization. Molar volume is decreased by 720 ml/mol. This result is in contradiction with previous measurements by Ikkai and Ooi [(1966) Science 152, 1756-1757], and by Swezey and Somero [(1985) Biochemistry 24, 852-860]: both of these groups reported increases in actin volume during polymerization, of 391 ml/mol and 63 ml/mol respectively. We also observed a decrease in heat capacity of about 69.5 kJ.K-1.mol-1 during polymerization. This is in agreement with the concept of conformational fluctuation of proteins proposed by Lumry and Gregory [(1989) J.Mol. Liq. 42, 113-144]whereby either ligand binding by a protein or monomer-monomer interaction decreases the protein's conformational flexibility.

Kinetic study of the thermal denaturation of G actin using differential scanning calorimetry and intrinsic fluorescence spectroscopy.

Previous studies of the thermal denaturation of G actin have yielded conflicting results which have led to contradictory interpretations of the denaturation process. In their interpretations, these authors postulated that the thermal denaturation of G actin can be described by thermodynamical equilibrium laws despite the fact that this transition is irreversible. Using differential scanning calorimetry and fluorescence spectroscopy, we now show that thermal denaturation of G actin does not obey to equilibrium thermodynamics, but rather, is a two-step irreversible phenomenon under kinetic control. It can therefore be analyzed using the recently developed Sanchez-Ruiz equations.

Interaction of actin with positively charged phospholipids: a monolayer study.

The interactions of actin, a negatively charged cytoskeletal protein, with lipids have been studied by the monolayer technique. The lipids (either pure phosphatidylcholine or incorporating 10%, 30% or 50% of a positively charged surfactant, stearylamine) were spread at the air/water interface and actin was allowed to interact with the monolayer after injection in the subphase. The results show that at a given surface pressure, increasing the density of positive charges in the lipid monolayer causes a significant increase in the intercalation of the actin within the lipid molecules, thereby indicating that the adsorption of actin is facilitated by electrostatic interactions. However, this intercalation is only possible up to a critical surface pressure above which actin does not penetrate the lipid surface.

Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus.

Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoeba proteus cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin.

Target cell deformability determines the type of phagocytic mechanism used by Entamoeba histolytica-like, Laredo strain.

Morphological study of red blood cell phagocytosis by Entamoeba histolytica-like (Laredo strain) has shown that this amoeba is able to ingest by two distinct mechanisms. One is classical phagocytosis and the other is by suction or microphagocytosis. Rigidification of red blood cells by treatment with glutaraldehyde shows that there is a correlation between the deformability of the ingested cell and the type of phagocytosis observed. Indeed, as the red cells become more rigid, less microphagocytosis is observed. To demonstrate that this shift in phagocytic mechanisms is not induced by the modification of a surface receptor by the glutaraldehyde treatment, the amoebas were fed with erythrocyte ghosts. Since these have lost most of their hemoglobin content, they are less rigid than the intact erythrocytes. The ghosts, even after glutaraldehyde treatment, are always ingested by microphagocytosis. These results have therefore led us to conclude that the type of erythrocyte phagocytosis used by E histolytica-like (Laredo strain) is determined by the deformability of the targetted red blood cells.